Immunogenicity and cross-protective efficacy induced by delayed attenuated Salmonella with the regulated length of lipopolysaccharide in mice

Immunogenicity and cross-protective efficacy induced by delayed attenuated Salmonella with the regulated length of lipopolysaccharide in mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Immunogenicity and cross-protective efficacy induced by delayed attenuated Salmonella with the regulated length of lipopolysaccharide in mice Qingke Kong, Xiaoping Bian, Qing Liu, Yaolin Chen, Wenjin Zhang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3971522/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Non-typhoidal Salmonella enterica serovar (NTS) is a major global foodborne pathogen that poses a major public health concern worldwide, and no vaccines were available for protecting against infection of multiple Salmonella serotypes, therefore, the development of Salmonella vaccines to provide broad protection is valuable. In this work, we aimed to regulate lipopolysaccharide (LPS) synthesis of live Salmonella in vivo for exposing conserved protein antigens on the outer membrane while maintaining smooth LPS patterns in vitro to keep their original ability to invade host cells for inducing cross-protection against infection of multiple Salmonella serotypes. We generated a series of mutants defective in genes to affect the length of LPS. These mutants exhibit in vivo regulated-delayed attenuation and altered length of LPS, and all these mutants were derived from SW067 (D pagL7 D pagP81 ::P lpp lpxE D lpxR9 D fur9 ) containing ∆ pagP81 ::P lpp lpxE mutation to reduce their endotoxic activity. Animal experiments demonstrated that all regulated delayed attenuated mutants exhibited reduced ability to colonize the organs of the mice, and SW114 ( waaI ), SW116 ( waaJ ), SW118 ( waaL ), and SW120 ( wbaP ) induced a significant production of IgG and IgA against OMPs isolated from S . Typhimurium, S . Enteritidis, and S . Choleraesuis. SW114 ( waaI ), SW116 ( waaJ ), and SW118 ( waaL ) were capable of conferring significant protection against infection of wild-type S . Enteritidis and S . Choleraesuis. In conclusion, regulated delayed attenuated Salmonella vaccines with the whole core oligosaccharides of LPS showed a good ability to expose conserved outer antigens and to trigger strong cross-immune responses against both homologous and heterologous Salmonella infections. These results give new insight into the development of the Salmonella vaccine against multiple serotypes of Salmonella . Biological sciences/Microbiology/Vaccines/Live attenuated vaccines Biological sciences/Microbiology/Bacteria/Bacterial development Regulated delayed attenuated Salmonella outer membrane proteins lipopolysaccharide cross-protection Salmonella serotypes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Salmonella enterica is a significant zoonotic foodborne pathogen that causes considerable financial losses to the livestock industry and poses severe human health concerns worldwide [ 1 ]. Salmonella is categorized into typhoidal Salmonella and non-typhoidal Salmonella (NTS) based on their ability to induce specific pathologies in humans [ 2 ]. Typhoidal Salmonella is commonly known as Typhi ( Salmonella enterica serovar Typhi) and Paratyphi ( Salmonella enterica serovar Paratyphi) A, B, or C, whereas other serovars are referred to as NTS [ 3 ]. In 2017, a total of 14.3 million cases of enteric fever were reported. Additionally, there were 53.5 thousand cases of NTS invasive disease reported during the same period [ 4 ]. While enteric fever still often occurs in human infection, NTS is a significant cause of global foodborne diseases through the transmission of Salmonella from animals to humans [ 5 ]. Among the NTS serovars isolated from food, Salmonella enterica serovar Typhimurium ( S . Typhimurium) was the most prevalent one in Africa [ 6 ], Australia [ 7 ], and China [ 2 ], and S . Enteritidis was the dominant serovar in the United States and European countries [ 8 , 9 ]. Other serovars such as S . Anatum ( Salmonella enterica serovar Anatum) and S . Weltevreden ( Salmonella enterica serovar Weltevreden) were also frequently isolated in beef and seafood in the world [ 10 ] and S . Derby, S . Thompson, and S . Aberdeen are the most common serotypes detected in chicken, pig, duck, aquatic products, and soft-shell turtle in China, respectively [ 2 ]. An increasing trend of antimicrobial resistance (AMR) in Salmonella , especially multidrug resistance (MDR), has been on the rise and is a global concern [ 11 ]. Vaccination has been demonstrated as a highly effective strategy to combat infections of antibiotic-resistant Salmonella . There are currently three types of licensed Salmonella vaccines in humans, including the live attenuated oral vaccine Ty21a, the unconjugated Vi polysaccharide vaccine, and the Typhoid conjugated vaccine (TCV) [ 12 ]. Since these vaccines primarily target Salmonella surface polysaccharide antigens, there is limited cross-protection against other serotypes of Salmonella , although Ty21a, derived from S . Typhi, confers moderate cross-protection against S . Paratyphi B [ 13 ]. There are currently no NTS vaccines available for humans, and the use of NST vaccines for animals is limited [ 14 ]. Human NTS vaccine candidates primarily target S . Typhimurium in serogroup B carrying O:4 antigen and/or S . Enteritidis in serogroup D carrying O:9 antigen [ 14 ], and Salmonella vaccines for animals are mainly used to prevent fowl typhoid caused by S . Gallinarum of serogroup D carrying the O:9 antigen, infections in laying hens caused by S . Typhimurium and Enteritidis, and piglet paratyphoid fever caused by S . Choleraesuis of serogroup C carrying the O:7 antigen [ 15 , 16 ]. These NTS vaccine candidates only cover specific Salmonella serotypes and cannot protect against infections caused by other commonly prevalent serotypes. Therefore, it is necessary to develop a universal vaccine that provides broad protection against different Salmonella serotypes to reduce infections of Salmonella . Although the millions of lipopolysaccharide (LPS) molecules present on the surface of Salmonella create a formidable barrier to restrict antibody access to the bacterial surface [ 17 ], previous studies have demonstrated that isolated outer membrane proteins (OMPs) from Salmonella conferred protection against both homologous and heterologous serotype Salmonella challenges in mice [ 17 – 20 ], indicating that certain OMPs are highly conserved in Salmonella to confer cross-protection. These studies imply that the OMPs of Salmonella are promising targets for the development of vaccines against infection with multiple serotypes. The process of purifying OMPs from Salmonella is both labor-intensive and costly, and some conserved OMPs are low or not expressed under laboratory conditions, rendering them unsuitable for large-scale production and application. Hence, live attenuated Salmonella vaccines may be ideal candidates for developing a vaccine with broad protection due to their cost-effectiveness, ease of management, and the capacity to generate long-lasting immunity with just one dosage. Our earlier research has shown that while attenuated Salmonella with permanent truncated LPS induces somewhat cross-immune responses against other Salmonella serotypes and enteric bacteria, mutants do not effectively colonize the host intestine to induce robust immune responses [ 19 , 20 ]. Therefore, attenuated Salmonella with the permanent truncated LPS is not a suitable candidate for vaccine development. Regulated delayed attenuated Salmonella (RDAS) that was developed in Curtiss's lab possesses the critical feature of undergoing regulated delayed attenuation in vivo . RDAS vaccine candidates are allowed to synthesize virulence factors required for colonization and invasion by adding specific substances to the culture medium in vitro , and after colonization in the organs, the mutants gradually undergo attenuation due to the absence of specific inducers after oral immunization in vivo [ 21 , 22 ]. Therefore, we wanted to investigate whether the regulated length of LPS in attenuated Salmonella would expose more conserved antigens, thus inducing more effective cross-immunity. In this study, we constructed a series of RDAS that regulate LPS synthesis, which is capable of synthesizing full LPS lengths in the presence of arabinose in vitro while synthesizing different truncated lengths of LPS in the absence of arabinose in vivo [ 23 ] (Fig. 1 ). Our objective was to develop ideal candidates for live attenuated Salmonella vaccines to trigger cross-immune responses effectively and confer cross-protection against infection of multiple serotypes of Salmonella . Material and methods Growth conditions, medium, plasmids, and strains of bacteria Table 1 provides a comprehensive list of all the strains of bacteria and plasmids used in this study. Salmonella and E. coli were routinely cultivated either in Luria-Bertani (LB) broth (L1010, Solarbio Biotech, China) or on LB agar (L1015, Solarbio Biotech, China) [24] with or without 0.1% arabinose at a temperature of 37˚C. In cases where necessary, the culture media were supplemented with 25 μg/ml of chloramphenicol (A600118, Sangon Biotech, China). To facilitate the growth of Asd- strains, 2,6-diaminopimelic acid (DAP) was included at a final concentration of 50 µg/ml (D1377, Sigma Aldrich, USA) [25]. For the cultivation of strains involving allelic exchange experiments, sacB gene-based counterselection was conducted on LB agar containing 5% sucrose (A610498, Sangon Biotech, China). All Salmonella mutants originated from the S . Typhimurium strain UK-1 [26], and their genotypes were verified through PCR employing the corresponding primer pair outlined in Table 2. Construction of plasmids and mutant strains The allelic exchange method was utilized to introduce gene mutations in Salmonella , employing pYA4278 as described in the previous study [27]. The transformation of E. coli was carried out using a technique known as electroporation. To select the transformants, appropriate antibiotic supplements were added to LB agar plate cultures. Primers utilized in this study were listed in Table 2. For arabinose-regulated LPS synthesis in this study, we applied the same strategy as the D pagL ::TT araC P BAD wbaP deletion-insertion mutation to construct other mutations [28]. Briefly, for the construction of the waaC mutation, in which the complete waaC open-reading frame was deleted, the c3761 genomes were employed as the template for cloning, a DNA fragment of 350-bp containing the region upstream of the waaC gene (from ATG start codon, but not including ATG) was amplified using primers WaaC-Del 1F and WaaC-Del 1R, and another 350-bp DNA fragment containing the region downstream of the waaC gene (from TAA stop codon, but not including TAA) was amplified using primers WaaC-Del 2F and WaaC-Del 2R. Universal primers Vec-F and Vec-R were applied to amplify vector fragments. The 5′ flanking regions of these DNA fragments contain homologous regions for recombination. The PCR products were purified by agarose gel (A620014, Sangon Biotech, China) and ligated to pYA4278 using Gibson Assembly Master Mix (E55510, New England Biolabs, USA) to generate the suicide plasmid pSW012 for deleting the waaC gene. The waaC mutation was introduced into c3761 and SW067 by allelic exchange via conjugation with the c7213, harboring the plasmid of pSW012, to generate SW079 (∆ waaC40 ) and SW107 (D pagL7 D pagP81 ::P lpp lpxE D lpxR9 D fur9 ∆ waaC40 ), respectively. The genotypes of Δ waaC were verified by PCR and LPS profiles. The same strategy also applied to the deletion of the waaF , waaG , waaI , waaJ , waaL, or wbaP genes in the c3761 and SW067 genomes (Table 1). For the construction of the D pagL ::TT araC P BAD waaC deletion-insertion mutation, the vector of pYA3700 [29] was linearized using the primers WaaC-VF and WaaC-VR. The fragment waaC gene was then amplified by WaaC-1F / WaaC-R and WaaC-2F / WaaC-R from the c3761 genomes and inserted into pYA3700 via Gibson Assembly Master Mix to generate intermediate plasmid pSW019 and pSW020, respectively. Subsequently, the fragment TT araC P BAD waaC was amplified using primers P1 and P2-(WaaC), and the gel-purified product was ligated to the linearized vector pYA4284 by Gibson Assembly Master Mix to form the suicide plasmids pSW033 and pSW034, respectively. The pagL mutation was introduced into SW079 (D waaC40 ) by allelic exchange via conjugation with the c7213 harboring the plasmid of pYA4284 to yield the strain SW086 (D waaC40 D pagL7 ). The Δ pagL ::TT araC P BAD waaC mutation was introduced into SW086 by allelic exchange by conjugation with the c7213 harboring the plasmids of pSW033 and pSW034 to yield SW093 (∆ waaC40 D pagL71 ::TT araC P BAD waaC ) and SW094 (∆ waaC40 D pagL72 ::TT araC P BAD waaC ), respectively. PCR and LPS profiles were applied to confirm the genotypes of the Δ pagL ::TT araC P BAD waaC mutations. After the mutation with tightly regulated LPS was successfully screened and identified in the wild-type background, the correct mutation was introduced into the mutant strain SW107 (D pagL7 D pagP81 ::P lpp lpxE D lpxR9 D fur9 ∆ waaC40 ) to generate the mutant strain SW108 (D pagL71 ::TT araC P BAD waaC D pagP81 ::P lpp lpxE D lpxR9 D fur9 ∆ waaC40 ). The other mutant strains were generated using the same procedure. Phenotypic determination of bacteria Bacterial strain phenotypes were identified in vitro , and each experiment was conducted at least twice. The OMPs and LPS were purified from Salmonella following described methods [30-32]. The OMP underwent electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and was subsequently stained with Coomassie brilliant blue. The LPS samples of S . Typhimurium went through silver staining to establish their LPS profile, following the method described by Hitchcock and Brown [33]. Lipid A was extracted from Salmonella after mild acid hydrolysis at pH 4.5 to break the Kdo-lipid A linkage and subjected to Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) [34, 35]. Animals The laboratory animals utilized in this study were female BALB/c mice (18-19 g) sourced from Hunan SJA Laboratory Animal Co., Ltd. The mice received appropriate care and were managed by competent and trained personnel, and the experiments were conducted in accordance with the guidelines provided in the Guide for the Care and Use of Laboratory Animals. The animal house's temperature, humidity, and ventilation were regularly checked to guarantee ideal climatic conditions. Every effort was made to alleviate animal suffering while the experiments were being conducted. A seven-day acclimation period was provided for all animals prior to the commencement of experiments after their arrival. Daily observation of mice Daily observations were made on mice that received vaccinations. Monitoring was conducted for local reactions and adverse effects, including abnormalities, a decline in overall health, and decreased food consumption. Additionally, evidence of unkempt fur, restlessness, diarrhea, morbidity, and mortality was recorded. Immunity of mice Mice were immunized by oral administration. Briefly, 5 ml of appropriate fresh media was used to stationary culture a solitary bacterial strain clone overnight in a 37°C incubator. The following day, the cultures were diluted 1:100 within a 100 ml identical medium and then incubated at 37°C until the bacterial solution's optical density value at 600 nm (OD 600 ) reached 0.85 (approximately 1 × 10 9 CFU / ml). The 100 ml suspension of the bacterial strain was gathered through centrifugation at 5000 rpm under room temperature conditions and resuspended in 2 ml of buffered saline with gelatin (BSG), at which point the bacterial concentration was approximately 1 × 10 9 CFU / 20 µl. The mouse cage was removed from food and water for six hours, and the animals were orally given 20 µl of BSG containing 10 9 CFU of the corresponding mutants. At 4 weeks, mice received a subsequent dose of the identical strain that matched the previous dosage. Colonization of S . Typhimurium mutants in organs To assess the bacterial load in organs, the mice were orally given 20 µl of BSG containing 1 × 10 9 CFU mutant strains. At specific time intervals, three mice from each group were humanely euthanized. The Peyer's patches (PP) were gathered from various locations on the surface of the small intestine. The bacterial load within the PP indicated the average bacterial load in the combined PP from every mouse. Samples from the spleen and liver were obtained and weighed individually. Each sample was added to 1 ml of phosphate buffer saline (PBS) to get a homogenized sample. Subsequently, various dilutions of the samples ranging from 10 −1 to 10 −3 (depending on the specific tissue) were dropped onto MacConkey agar and LB agar to measure the total amount of live bacteria present. MacConkey agar indicator plates are designed to exclude interference from E. coli in tissue homogenates. The bacterial load was calculated based on the bacteria on the indicator plates. If no colonies were observed on the LB plate after culturing the homogenizing 0.1 ml of the organ, the selenite cysteine broth (100212, Sigma, USA) was used to inoculate the remaining PBS solution from each tissue sample the following day. Samples that showed positive results after being enriched in selenite cysteine broth at 37°C overnight were noted to have a CFU count of less than 10 per gram. Tissue damage caused by S. Typhimurium mutants To investigate the liver and spleen tissue damage caused by the mutants after the mice were orally given 20 µl of BSG containing 1 × 10 9 CFU mutant strains, we employed hematoxylin and eosin (H&E) staining for histopathological analysis. On the sixth day following oral inoculation, two mice in each group were humanely euthanized, and the liver and spleen were taken to determine the scope of the organ damage caused by the mutants. The standard procedure was followed for fixing and processing tissues for H&E staining. We observed the morphological aspects of tissues to identify signs of inflammation, including infiltration of macrophages, accumulation, distortion, and abnormal red pulp areas in the liver and spleen. Survival assay The protection rates of the immunized mice were assessed at 9 weeks post-initial immunization by the oral challenge with 10 9 CFU of wild-type S . Typhimurium (c3761) in 20 μl BSG (~10000 × LD 50 ) [19]. Mice were orally challenged with 10 7 CFU /20 μl of S . Enteritidis (c3700) or S . Choleraesuis (c3545) at 9 weeks after the first immunization to assess the cross-protection induced by regulated delayed attenuated Salmonella . The c3700 and c3545 were clinical isolates from the chicken and pig, respectively, both with an LD 50 of 10 5 CFU / 20 μl [19]. Challenged mice were monitored daily for evidence of unkempt fur, restlessness, diarrhea, morbidity, and mortality, which were recorded for 25 days after the challenge. Quantitative enzyme-linked immunosorbent assay (ELISA) Serum was collected by puncturing the mandibular vein to obtain blood, followed by centrifugation at 3500 rpm. To analyze the secretory IgA (S-IgA), the vaginal tract of each mouse was washed with 60 μl of PBS, and the wash fluids were pooled together. The enzyme-linked immunosorbent assay (ELISA) was utilized to quantify the concentration of serum and vaginal wash antibodies against Salmonella LPS or OMPs induced by regulated attenuated Salmonella strains, following established protocols [36, 37]. Briefly, solutions containing 200 ng per well of Salmonella -derived OMP or LPS were suspended in 100 µl of coating buffer composed of sodium carbonate-bicarbonate (pH 9.6). These solutions were then utilized to coat 96-well plates overnight at a temperature of 4°C. To generate standard curves for each antibody isotype, we coated plates with duplicate samples of the purified mouse Ig isotype standard (1010-01, Southern Biotech, USA). Each well was coated with 200 ng of the standard in 100 µl of coating buffer. The plates were washed three times with tris buffered saline with 0.1% Tween 20 (TBST) and then blocked with 3% BSA (A602449, Sangon Biotech, China) for 2 hours at 37°C. Next, a 100 µl volume of the sample, diluted 100-fold or 10-fold, was added to each well in duplicate, the plates were then incubated for 1 hour at 37°C. To creat the standard curve, the unconjugated IgG (0107-01, Southern Biotech, USA), IgA (0106-01, Southern Biotech, USA), IgG1 (0102-01, Southern Biotech, USA), or IgG2a (0103-01, Southern Biotech, USA) obtained from ordinary mice were diluted by serial 2 times in 100 µl PBS of each plate well. The unconjugated IgG was diluted from 500 ng/ml to 0.488 ng/ml, IgG1 and IgG2a from 1 µg/ml to 8 ng/ml, and the IgA was diluted from 500 ng/ml to 0.488 ng/ml. Following TBST washing, each well was added with a 1:5000 dilution of biotinylated goat anti-mouse IgA (1040-08, Southern Biotech, USA), IgG1 (1070-08, Southern Biotech, USA), IgG2a (1080-08, Southern Biotech, USA), or IgG (1030-08, Southern Biotech, USA), the plates were then incubated at 37°C for 1 hour. A 100 µl volume of p-nitrophenyl phosphate (N1891, Sigma, USA) with a final concentration of 1 mg/ml was added to each of the wells for color development after they were previously incubated for an hour at 37°C with a 1:3000 dilution of streptavidin-alkaline phosphatase conjugate (7100-04, Southern Biotech, USA). Color development (absorbance) was read at 405 nm using an automated ELISA plate after appropriate incubation. Using linear regression in Excel (R 2 ≥0.95), the OD values at 405 nm were plotted against the representative concentrations of the diluted unconjugated antibody solutions to generate the standard curves. The corresponding standard curve was applied to calculate the total levels of antibodies in the samples. Flow Cytometry Mice were euthanized on day 72 after the first immunization, with 3 mice per group, the spleens were then collected using aseptic techniques. To obtain spleen cell suspensions, the samples were passed through a sterile cell strainer with a pore size of 40 µm (BS-40-CS, Biosharp, China), and the erythrocytes were lysed using RBC lysis buffer (R1010, Solarbio Biotech, China) for 5 min at 4℃. The resulting splenocytes were then suspended in RPMI 1640 medium, which was supplemented with 10% fetal calf serum (FCS). The single-cell suspension of spleen was stained with immune-cell-specific antibodies for flow cytometry (Beckmann Coulter Inc., Fullerton, CA, USA) as follows: APC anti-IgG antibody (405308, Biolegend, USA), FITC anti-CD80 antibody (104706, Biolegend, USA), Alexa Fluor® 700 anti-IgD antibody (405730, Biolegend, USA), and Pacific Blue™ anti-CD45R/B220 antibody for B M cell (103227, Biolegend, USA). FITC anti-CD4 antibody, APC anti-CD279 (PD-1) antibody (135210, Biolegend, USA), and Brilliant Violet 421™ anti-CD185 (CXCR5) antibody (145512, Biolegend, USA) for T fh cell. Statistical analysis The Graph-Pad Prism 8.0 software was utilized to conduct statistical calculations. Unless otherwise mentioned, numerical data were presented as means ± SEM. To assess differences in the levels of antibodies and the quantities of B M and T fh cells, we conducted either one-way or two-way ANOVA analysis, subsequently applying Tukey's multiple comparisons tests. The log-rank test was implemented to assess differences in mouse survival, with the Kaplan-Meier survival curve serving as the monitoring tool. To compare means, the least significant difference test was employed. A p-value below 0.05 was considered to indicate a meaningful difference. RESULTS Construction of Salmonella mutants To finely regulate the expression of genes related to LPS synthesis to control the synthesis of the native Salmonella LPS, we constructed at least two suicide plasmids carrying the TT araC P BAD cassette and different SD sequences and/or start codons for each gene for LPS synthesis (Table 1, Table 2). These suicide plasmids were then utilized to integrate the TT araC P BAD cassette carrying the candidate genes into the chromosome in place of the pagL gene since pagL deletion did not alter the virulence of Salmonella and maintained the same immunogenicity and colonization ability as the wild-type Salmonella [38]. For an example of the regulated gene waaC , we first introduced the Δ waaC and Δ pagL mutation in the genome of c3761 by allelic exchange via conjugation with the c7213, harboring the plasmid of pSW012 or pYA4284, then two suicide plasmids (pSW033 and pSW034) were utilized to integrate the TT araC P BAD cassette with waaC carrying two different SD sequences into the chromosome of SW086 (D waaC40 D pagL7 ) in place of the pagL gene to generate mutants SW093 (Δ waaC40 Δ pagL71 ::TT araC P BAD waaC ) and SW094 (Δ waaC40 Δ pagL72 ::TT araC P BAD waaC ), respectively. The results of silver staining experiments showed that the mutant SW079 (Δ waaC40 ) was unable to synthesize a typical LPS banding pattern due to the deletion of the glycosyltransferase gene waaC , which is responsible for the addition of L-glycerol-D-mannoheptulose (Hep) residues to the core of LPS (Supplementary Figure 1, Figure 2A). When the mutants SW093 (Δ waaC40 Δ pagL71 ::TT araC P BAD waaC ) and SW094 (Δ waaC40 Δ pagL72 ::TT araC P BAD waaC ) were cultured in vitro without 0.1% arabinose, both SW093 (Δ waaC40 Δ pagL71 ::TT araC P BAD waaC ) and SW094 (Δ waaC40 Δ pagL72 ::TT araC P BAD waaC ) mutants failed to synthesize a typical LPS banding patterns, however only SW093 (Δ waaC40 Δ pagL71 ::TT araC P BAD waaC ) was able to synthesize LPS banding pattern similar to the wild strain when arabinose was present (Supplementary Figure 1). This result suggested that the mutant SW093 (Δ waaC40 Δ pagL71 ::TT araC P BAD waaC ) exhibited the ability to tightly regulate LPS synthesis. The same strategy was also applied to other genes, including waaF, waaG , waaI , waaJ , waaL , and wbaP . The integrations resulted in generating mutant strains with varying lengths of LPS (Supplementary Figure 1, Figure 2A). After mutants with tightly regulated LPS were successfully identified in the wild-type background, these mutations were introduced into the mutant strain SW067 (c9705 D fur9 ) to investigate whether the regulated LPS synthesis of Salmonella has an effect on the induced cross-immune response [38]. Phenotype determination of the mutant strains When the genes waaC , waaF , waaG , waaI , waaJ , waaL , or wbaP were deleted from the genome of SW067 (c9705 D fur9 ), the mutant strains exhibited an inability to produce typical LPS banding pattern, indicating that the absence of any glycosyltransferase in LPS synthesis hinders the formation of a whole LPS (Supplementary Figure 2). When cultured in the presence of 0.1% arabinose, a series of the mutant strains with regulated LPS synthesis including SW108 ( waaC ), SW110 ( waaF ), SW112 ( waaG ), SW114 ( waaI ), SW116 ( waaJ ), SW118 ( waaL ), and SW120 ( waaP ) exhibited typical LPS banding patterns while culturing in the absence of arabinose, these mutants displayed the rough LPS phenotype (Figure 2B). As the mutants will be administered orally and need to colonize the host organ, to evaluate whether they still display the tightly regulated LPS in vivo , these mutants were cultured in vitro in nutrient broth supplemented with varying concentrations of mouse serum, they were unable to synthesize the whole LPS in the absence of free arabinose (Supplementary Figure 3A). These results indicated that arabinose was unavailable in the serum and the synthesis of LPS in the mutants was entirely regulated by exogenously added arabinose. The outer membrane protein profile (Supplementary Figure 3B) demonstrated that this system did not impact the expression of OMPs, and MALDI-TOF MS analysis of lipid A showed that the phosphate group was removed to result in the production of monophosphorylated lipid A (Figure 2C). Colonization of the regulated delayed attenuated Salmonella The colonization levels in the organs of mice reflect the interaction between the attenuated Salmonella and the lymphoid tissue, impacting the capacity of attenuated Salmonella to elicit immune responses. To investigate the dissemination and proliferation capabilities of the regulated delayed attenuated Salmonella in mice, we assessed the bacterial burdens in the liver, spleen, and PP of mice at 3, 6, 14, and 28 days following oral administration with 1×10 9 CFU / 20 µl of the mutant strains, which were grown in the presence of 0.1% arabinose. The average CFU counts of all mutant strains isolated from the liver, spleen, or PP were approximately 10 4 at 3 days after oral inoculation (Figure 3A, 3B, 3C), indicating that attenuated Salmonella maintained the ability to invade and colonize the gastrointestinal tract and were rapidly disseminated to systemic sites and underwent substantial proliferation within the organs. At 6 days post-infection, all mutant strains exhibited significantly decreased colonization levels in the liver, spleen, and PP. All mutant strains colonized and persisted in the organs for at least 28 days, and did not show significant differences. Within these 28 days, the bacterial counts in the liver, spleen, and PP gradually decreased significantly, suggesting that the regulated delayed attenuated Salmonella could be cleared after colonization, and the abnormal appearance of the skin and other symptoms were not observed during the current experiment. Histological analysis after immunization with the mutant strains To further investigate the potential of the regulated delayed attenuated Salmonella to induce tissue inflammation, we conducted histopathology analyses on the liver and spleen of mice inoculated with mutant strains, using BSG as the control. The mice were orally inoculated with 1×10 9 CFU of the mutant strains in the 20µl BSG, which were cultured in LB with 0.1% arabinose, and the examinations were performed 7 days post-infection. The results showed that the hepatocytes of mice in groups SW108 ( waaC ) and SW112 ( waaG ) displayed vacuolation, while those in groups SW110 ( waaF ) and SW120 ( wbaP ) exhibited small focal necrosis in the liver (Figure 4). Mice in the SW112 ( waaG ) group showed signs of inflammatory cell infiltration and small focal necrosis in the splenic tissue. No noticeable lesions were observed in mice from the SW114 ( waaI ), SW116 ( waaJ ), and SW118 ( waaL ) groups. Serum IgG and mucosal IgA responses to OMPs from S. Typhimurium To evaluate the immune responses triggered by the regulated delayed attenuated Salmonella , a total of 8 sets of mice were administered with 20 µl of BSG consisting of 1 × 10 9 CFU strains via the oral route. Subsequently, these mice were boosted with an identical dose of the matching mutants 4 weeks later. The levels of serum IgG and mucosal IgA responses towards OMPs derived from S . Typhimurium were assessed using the ELISA method at 8 weeks after initial immunization. The SW114 ( waaI ), SW116 ( waaJ ), SW118 ( waaL ), and SW120 ( wbaP ) strains elicited considerably increased levels of IgG and IgA specific to S . Typhimurium OMPs compared to the SW108 ( waaC ), SW112 ( waaG ) and BSG groups (Figure 5A, 5B). Moreover, the SW110 ( waaF ) strain, which synthesized native S . Typhimurium LPS in the presence of arabinose in vitro and produced only a partial inner core of LPS in the absence of arabinose in vivo , also triggered mice to produce similar levels of S . Typhimurium OMPs-specific IgG and IgA antibodies as the SW118 ( waaL ) and SW120 ( wbaP ) strains, which both synthesized an intact inner and outer core of LPS in the absence of arabinose in vivo (Figure 5A, 5B). Although the levels of IgA specific to S . Typhimurium OMP triggered by the SW116 ( waaJ ) strain were significantly enhanced compared to the BSG control groups, it was lower than that of the SW110 ( waaF ), SW114 ( waaI ), SW118 ( waaL ), and SW120 ( wbaP ) immunized groups (Figure 5B). We also assessed the levels of anti-OMP IgG isotype subclasses IgG1 and IgG2a in the serum of mice. During the early phase, the IgG1 titers were comparable to the levels of IgG2a (data not presented). However, the SW114 ( waaI ), SW116 ( waaJ ), SW118 ( waaL ), and SW120 ( wbaP ) strains stimulated a more rapid increase in IgG2a titers than IgG1 titers compared to the SW108 ( waaC ), SW112 ( waaG ) and BSG control groups at the eighth week after the initial immunization, suggesting a shift towards a Th1-biased immune response (Figure 5C, 5D). Notably, the SW110 ( waaF ) strain not only provoked significantly increased IgG2a but also elicited S . Typhimurium OMP-specific subclasses IgG1. Evaluation of cross-reactive antibodies against OMPs from other Salmonella serotypes To further analyze the cross-immune response induced by the regulated delayed attenuated Salmonella in mice, we assessed serum IgG and mucosal IgA responses to S . Enteritidis and S . Choleraesuis OMPs in mice at 8 weeks after the initial immunization. Not only did the SW118 ( waaL ) and SW120 ( wbaP ) mutants stimulate S . Enteritidis OMPs-specific IgG production, but also the SW114 ( waaI ) and SW116 ( waaJ ) mutants elicit higher levels of IgG specific to S . Enteritidis OMP than the control groups of BSG (Figure 6A). The SW118 ( waaL ) mutant, but not SW120 ( wbaP ), could elicit the highest S . Enteritidis OMP-specific IgG titer among all groups. The SW118 ( waaL ) and SW120 ( wbaP ) strains induced the production of S . Enteritidis OMP-specific vaginal IgA, whereas the SW114 ( waaI ) and SW116 ( waaJ ) strains failed to elicit vaginal mucosal IgA responses (Figure 6B). The levels of S . Choleraesuis OMP-specific serum IgG and mucosal IgA triggered by the SW114 ( waaI ), SW116 ( waaJ ), SW118 ( waaL ), and SW120 ( wbaP ) strains were significantly enhanced compared to those observed in the other treatment groups (Figure 6C,6D). Further analysis indicated that IgG2a was the major subtype of OMP-specific IgG triggered by the regulated delayed attenuated Salmonella (Figure 6E, 6F, 6G, 6H). The SW114 ( waaI ), SW116 ( waaJ ), SW118 ( waaL ), and SW120 ( wbaP ) strains could stimulate similar levels of S . Enteritidis OMP-specific IgG2a compared to SW108 ( waaC ), SW110 ( waaF ) and SW112 ( waaG ) strains (Figure 6F). Interestingly, in addition to the SW110 ( waaF ) strains triggered S . Choleraesuis OMP-specific IgG1 production, the SW118 ( waaL ) mutants, which synthesize both the complete inner and outer cores of the LPS in the absence of arabinose in vivo , also induced similar levels of S . Choleraesuis OMP-specific IgG1 (Figure 6G). Both the SW114 ( waaI ) and SW116 ( waaJ ) strains induced significantly enhanced levels of S . Choleraesuis OMP-specific IgG2a relative to the SW108 ( waaC ), SW110 ( waaF ), SW112 ( waaG ) strains, but lower than the SW118 ( waaL ) and SW120 ( wbaP ) strains (Figure 6H). Assessment of long-term immune responses induced by regulated delayed attenuated Salmonella A successful vaccine must effectively elicit and establish lasting memory immunity; therefore, we investigated the long-term immune responses triggered in mice following regulated delayed attenuated Salmonella immunization via oral administration. As SW114 ( waaI ), SW116 ( waaJ ), and SW118 ( waaL ) performed well in inducing antibody production, they were chosen to explore their abilities to induce long-term immunity. The IgG antibody levels in the serum of immunized mice were quantified on the 72nd day following the initial immunization. The results showed that S . Typhimurium, S . Enteritidis, and S . Choleraesuis OMP-specific IgG were still observable in all experimental groups compared to the BSG control group on 72 days (Supplementary Figure 4A, 4B, 4C). The levels of IgG antibodies in the SW118 ( waaL ) group exhibited a statistically significant increase compared to both the SW114 ( waaI ) and SW116 ( waaJ ) groups. The establishment of a durable population of memory B cells is crucial for a vaccine to develop sustained immunity [39]. In this study, we conducted flow cytometry analysis on day 72 after the first immunization to evaluate the IgG + B M . The percentage of B220 + CD80 + IgG + IgD - B M cells [28] in the groups immunized with the SW116 ( waaJ ) was considerably increased compared to the SW114 ( waaI ) and BSG groups but less than the SW118 ( waaL ) immunized group (Figure 7A, 7B). These findings indicated that the regulated delayed attenuated Salmonella constructed in this study, particularly the SW118 ( waaL ), which both synthesized an intact inner and outer core of LPS in the absence of arabinose in vivo , could elicit and establish lasting memory immunity in mice. Evaluation of protection against challenge by wild-type S . Typhimurium To investigate the protection rates conferred by the regulated delayed attenuated Salmonella , the immunized mice were orally challenged with 1 × 10 9 CFU (10000 × LD 50 ) of c3761 in 20 µl BSG at 9 weeks after the first immunization. The results showed that all of the mice in the BSG control group died; In contrast, all the vaccinated mice survived (Figure 8A), implying that all of the regulated delayed attenuated Salmonella were able to provide complete protection to the mice against the wild-type S . Typhimurium challenge. Evaluation of cross-protection against infection with heterologous serotype of Salmonella To evaluate protection against the challenges of different serotypes of Salmonella , sixteen groups of BALB/c mice were used to immunize, including fourteen experimental groups and two control groups. The mice in each group were independently challenged by oral administration with 1 × 10 7 CFU (~100 × LD 50 ) of c3700 ( S . Enteritidis) or c3545 ( S . Choleraesuis) in 20 µl BSG, respectively. As shown in Figure 8B, SW116 ( waaJ ) provided 100% protection in mice against the wild-type S . Enteritidis challenge. The SW114 ( waaI ) could confer an 87.5% level of protection in mice. Both the SW118 ( waaL ) and SW120 ( wbaP ) could provide 62.5% protection (Figure 8B). Although the SW108 ( waaC ), SW110 ( waaF ), and SW112 ( waaG ) mutants did not provide significant protection for mice against the wild-type S . Enteritidis challenge, it prolonged their survival time (Figure 8B). The protection rate against the challenge of S . Choleraesuis was inconsistent with the level of protection against S . Enteritidis. SW118 ( waaL ), SW114 ( waaI ), and SW116 ( waaJ ) mutants provided protection rates of 75% and 50% for mice against the wild-type S . Choleraesuis challenge, respectively. Interestingly, although SW108 ( waaC ) did not confer a high level of protection against the S . Enteritidis challenge, it provided the highest level of protection against the S . Choleraesuis challenge with a survival rate of 87.5% (Figure 8C). The SW110 ( waaF ), SW120 ( wbaP ), and SW112 ( waaG ) mutants did not provide significant protection for mice against the wild-type S . Choleraesuis challenge. Evaluation of immunogenicity against OMPs from different enteric bacteria Our previous research has demonstrated that the absence of O-antigen stimulates elevated levels of antibodies against the outer membrane protein antigen in serum, and these antibodies exhibit cross-reactivity with conserved surface epitopes present in other enteric bacteria [19, 20, 40]. Therefore, we determined whether the regulated delayed attenuated Salmonella mutants had a similar effect. The reactivity of immune sera obtained from orally immunized mice belonging to SW114 ( waaI ), SW116 ( waaJ ), and SW118 ( waaL ), as described in the previous experiment (Figure 6), was evaluated using ELISA against OMPs isolated from several wild-type E.coli (O8, O138, O139) strains (Figure 9). The results roughly correlated with that we obtained against OMPs of heterologous serotype Salmonella (Figure 6). The three vaccine strains elicited a significant reactivity response in mice against OMPs from several E. coli strains compared to the BSG control group, and there was no statistical difference between the groups (Figure 9). Discussion This study investigated the immunogenicity and protective efficacy of the regulated delayed attenuated Salmonella mutants in mice. The goal was to develop potentially regulated delayed attenuated Salmonella that could provide protection against both homologous and heterologous serotype Salmonella infections. The serotypes of S . Typhimurium, S . Enteritidis, and S . Choleraesuis in the NTS [ 41 ] belong to serogroups B1, D1, and C1, respectively, and have many natural hosts such as mice, chickens, poultry, and pigs [ 42 , 43 ]. Numerous studies have demonstrated that these serotypes have the ability to establish systemic infections in mice after oral administration [ 17 , 44 – 47 ]. Therefore, the mouse model was chosen to evaluate the immunogenicity of the regulated delayed attenuated Salmonella mutants and their cross-protection efficiency against S . Typhimurium, S . Choleraesuis, and S . Enteritidis infections in this study. These mutant strains were derived from the same genetic background, thus avoiding any potential interference factors to compare immunogenicity and cross-protection efficiency induced by these strains. Successful colonization of attenuated Salmonella is a prerequisite for inducing an effective immune response in mice, and the intact LPS structure is essential for Salmonella adhesion and colonization in the intestine and systemic infection [ 40 ]. Previous studies have reported that while permanent deficiency of the O-antigen reduces host organ damage by Salmonella , it also affects the level of colonization in mice [ 18 , 48 ]. In this study, a series of Salmonella mutants with arabinose-dependent regulated synthesis of LPS were constructed, and the results have shown that at least one of them carrying altered length of LPS was successfully identified to be tightly regulated by arabinose in LB media and Nutrient broth with mice serum (Fig. 2 B), which was consistent with our previous studies [ 18 , 28 ]. The regulated delayed attenuated Salmonella constructed in this study can control the synthesis of Salmonella LPS by adding arabinose to the culture medium, so that Salmonella still has an intact LPS structure at the early stage of colonization in mice. The regulated delayed attenuated Salmonella exhibited a persistent ability of colonization in the initial stages of infection, at least maintaining this level for 6 days (Fig. 3 ), indicating that these mutants with regulated LPS synthesis were able to successfully colonize the liver, spleen, and PP of mice during the early stages of infection due to the presence of the intact LPS, which is consistent with the timeframe required for antigen recognition and lymphocyte activation leading to the development of adaptive immunity. Subsequently, the level of colonization of the mutants gradually decreased in mice, suggesting that the mutants could be cleared after inducing immunity, thus ensuring the safety of candidate vaccines. Live attenuated Salmonella vaccines must balance safety with immunogenicity [ 49 ], which can be achieved by incorporating a minimum of two genetically unlinked attenuating mutations. Hence, we constructed Salmonella mutants with the fur mutation and the arabinose-regulated LPS synthesis coupled with the deletion of pagL , lpxR , and codon-optimized lpxE substitution for pagP to engineer Salmonella synthesizing the 4′-monophosphoryl-hexa-acylated lipid A, which decreases the interaction of lipid A and TLR4 and endotoxic activity while maintaining its good immunogenicity [ 38 ]. Histopathological assay conducted on mice 7 days post-immunization demonstrated that delayed attenuated Salmonella mutants exhibited varying degrees of inflammation. Mice that were immunized with the strains SW108 ( waaC ) and SW110 ( waaF ), which are characterized by the deep rough phenotype and have a partial inner core of LPS in the absence of arabinose in vivo , showed localized damage to liver cells. Among the mutants SW112 ( waaG ), SW114 ( waaI ), and SW116 ( waaJ ), which had an intact inner core or partial outer core of LPS in the absence of arabinose in vivo , only strain SW112 ( waaG ) caused damage to liver and spleen cells (Fig. 4 ). Interestingly, no abnormalities were observed in mice infected with strains SW114 ( waaI ) and SW116 ( waaJ ). While the strains SW118 ( waaL ) and SW120 ( wbaP ) had both the inner and outer core of LPS but lacked the O-antigen in the absence of arabinose in vivo , mice that were immunized with strain SW118 ( waaL ) did not exhibit any observable abnormalities, while those inoculated with strain SW120 ( wbaP ) showed small areas of necrosis in the liver. These results indicated that mutants exhibiting deep rough phenotype may still have significant adverse effects due to slower generation of a rough phenotype in vivo . Additionally, while the process of LPS synthesis is intricate and subtle, even when the degree of truncated LPS is identical, the resulting properties observed in mice may differ. Attenuated live Salmonella vaccines have the ability to induce antibodies against conserved outer membrane proteins that are cross-reactive with other enteric pathogens, particularly in strains engineered to achieve down-regulation of O-antigen synthesis in vivo . The immune responses observed in mice following immunization with regulated delayed attenuated Salmonella were found to be varied. Despite the absence of a significant antibody response against OMPs of S . Typhimurium when mice were administered SW108 ( waaC ) and SW112 ( waaG ), these mutants still provided complete protection in mice when exposed to a lethal dose of highly virulent wild-type S . Typhimurium (Fig. 5 and Fig. 8 A). Considering that the regulated delayed attenuated Salmonella was originated from UK1, this result is not unexpected, and it can be attributed to the presence of various surface antigens in S . Typhimurium, such as flagellins, fimbriae, and enterobacterial common antigens (ECA), and the removal of immunodominant O-antigens from the surface of Salmonella also enhances the immunogenicity of OMPs and other surface antigens. Our aim was to develop a live attenuated Salmonella to induce cross-immune responses effectively and confer cross-protection against infection of multiple serotypes of Salmonella . In this study, mice in groups SW116 ( waaJ ), SW114 ( waaI ), and SW118 ( waaL ) exhibited significant antibody levels and a considerable survival rate when challenged with both wild-type S . Enteritidis and S . Choleraesuis, but mice in the SW108 ( waaC ) group, which exhibited negligible antibodies against OMPs from S . Choleraesuis, had the highest survival rate compared to the other groups (Fig. 6 and Fig. 8 B, 8 C). In addition, mice in group SW120 ( wbaP ), which displayed the identical LPS as SW118 ( waaL ), were characterized by elevated levels of antibodies, but displayed a considerably lower survival rate of only 37.5%, implying that no apparent relationship exists between antibody levels assessed in mice immunized with mutants and protection when challenged with other Salmonella . It seems that the conclusion made in this study was not consistent with other research [ 50 ]. Salmonella possesses many immunologically related cross-reactive OMP antigens, including abundant (OmpA, OmpC, and OmpD) and minor (e.g., NmpC and OmpX) proteins as well as the most copious lipoprotein (murein lipoprotein). These OMPs, although possessing some micro-heterogeneity, nevertheless share antigenic determinants, which can induce antibodies cross-reactive immunity to heterologous Salmonella , but exhibit less protective ability because not all antibodies elicited by OMPs bind effectively to the wild-type Salmonella due to the masking effect of the lipopolysaccharide layer on the surface of Salmonella . For example, OmpA, which is located as a monomer on the outer membrane of Salmonella , produces channels in the lipopolysaccharide layer that are not large enough to allow antibodies to cross the O-Ag into the bacterial surface for binding, and therefore antibodies against OmpA are not protective [ 17 ]. However, certain conserved protein antigens on the outer membrane of Salmonella are immunoprotective because the footprint formed by these antigens is large enough for antibodies to readily cross the lipopolysaccharide layer. Such as OmpD in S . Enteritidis is nearly identical to the S . Typhimurium and differs only by a single amino acid, which is located on the outer membrane and exists as a trimer to generate a tunnel aperture size that allows a single Fab to cross the LPS channel and bind to the bacterial surface [ 17 ]. Although only one Fab can access the LPS channel created by certain trimeric proteins in the surrounding LPS layer, it is also able to confer protection against infection of Salmonella . This explains our findings that cross-protection induced by the regulated delayed attenuated Salmonella is related to the level of effective antibody. Although we do not know which outer membrane proteins other than OmpD are capable of generating larger tunnels that allow antibodies to cross the O-Ag to bind to the outer membrane, we demonstrated that certain conserved antigens on the outer membrane do provide cross-protection to the challenge of Salmonella . In addition to the surface outer membrane proteins, the inner and outer cores of different serotypes of Salmonella are conserved, and only SW118 ( waaL ) and SW120 ( wbaP ) were able to recognize the LPS of wild-type S . Typhimurium in immune sera from mice immunized with the different mutants (Supplementary Fig. 5). This result implies that the Salmonella cores of LPS may also be one of the reasons why the mutants were able to induce cross-protective in mice. The vaccine administration induces the proliferation of long-lived plasma cells and memory B cells responsible for sustaining humoral immunity. In this study, it was observed that on day 72 after the initial immunization, the mice still exhibited significant levels of IgG antibodies. This finding suggests that the regulated delayed attenuated Salmonella effectively stimulated the production of a substantial population of long-lived plasma cells in the mice (Supplementary Fig. 4), particularly in the case of SW118 ( waaL ). Furthermore, regulated delayed attenuated Salmonella can effectively stimulate the generation of IgG + B M in mice (Fig. 7 ). This finding indicates that the regulated delayed attenuated Salmonella can successfully induce a persistent humoral immune response and maintain immune memory in mice. In conclusion, we systematically investigated the immunogenicity and protective efficacy of delayed attenuated Salmonella mutants. Our results indicate that strains containing whole-core oligosaccharides of lipid A not only expose more conserved OMPs but also have the ability to elicit enhanced cross-protective immunity against both homologous and heterologous Salmonella infections. Declarations Ethics approval and consent to participate Animal experiments were conducted in the Laboratory Animal Center of Southwest University after the Review Form for Laboratory Animal Welfare and Ethicals submitted by us was approved by IACUC (No. IACUC-20191120-02). We usually make an appointment in the online system with our registered personal information and carry out animal experiments after approval, and there is no license number for each animal experiment. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analyzed during the current study to reproduce these findings are available from the corresponding author upon request. Competing interests The authors declare that they have no competing interests. Acknowledgments This work was funded by National Natural Science Foundation of China (82241063), National Key Research and Development Program of China (2022YFD1800900), National Center of Technology Innovation for Pigs (NCTIP-XD/B11), and Sichuan Natural Science Foundation (2023YFH0080). Author contributions YH and QK initiated the research. XP and QK led the design of in vitro and in vivo experiments, data acquisition and analysis, and manuscript preparation. QL, YL, WJ, MR, XF, and LY aided in data acquisition. All authors contributed to the article and approved the submitted version. References Duff, N. et al. Global Action for Local Impact: The 11th International Conference on Typhoid and Other Invasive Salmonelloses. Clin. Infect. Dis . 71, S59-S63 (2020). doi: 10.1093/cid/ciaa236 . Wang, Y. et al. The temporal dynamics of antimicrobial-resistant Salmonella enterica and predominant serovars in China. Natl. Sci. Rev. 10, nwac269 (2023). doi: 10.1093/nsr/nwac269 . Gal-Mor, O. et al. Same species, different diseases: how and why typhoidal and non-typhoidal Salmonella enterica serovars differ. Front. Microbiol. 5, 391 (2014). doi: 10.3389/fmicb.2014.00391 . Stanaway J. D. et al. The global burden of non-typhoidal Salmonella invasive disease: a systematic analysis for the Global Burden of Disease Study 2017. Lancet. Infect. Dis. 19, 1312–1324. doi: 10.1016/S1473-3099(19)30418-9 . Galán-Relaño, Á. et al. Salmonella and Salmonellosis: An Update on Public Health Implications and Control Strategies. Animals. 13, 3666 (2023). doi: 10.3390/ani13233666 . Reddy, E. A. et al. Community-acquired bloodstream infections in Africa: a systematic review and meta-analysis. Lancet Infect Dis. 10, 417–432 (2010). doi: 10.1016/S1473-3099(10)70072-4 . Williamson, D. A. et al. Increasing antimicrobial resistance in Nontyphoidal Salmonella isolates in Australia from 1979 to 2015. Antimicrob. Agents. Ch. 62, e02012-2017 (2018). doi: 10.1128/AAC.02012-17 . Scallan, E. et al. Foodborne illness acquired in the United States–major pathogens. Emerg. Infect. Dis. 17, 7–15 (2011). doi: 10.3201/eid1701.P11101 . Authority, E. F. S. et al. The European Union One Health 2018 Zoonoses Report. EFSA. J. 17, e05926 (2019). doi: 10.2903/j.efsa.2019.5926 . Ferrari, R. G. et al. Worldwide Epidemiology of Salmonella serovars in animal-1 based foods: a meta-analysis. Appl. Environ. Microbiol. 85, (2019). doi: 10.1128/AEM.00591-19 . Yang, X. et al. Prevalence, bacterial load, and antimicrobial resistance of Salmonella serovars isolated from retail mmeat and meat products in China. Front. Microbiol. 10, 2121 (2019). doi: 10.3389/fmicb.2019.02121 . Frost, I. et al. The role of bacterial vaccines in the fight against antimicrobial resistance: an analysis of the preclinical and clinical development pipeline. Lancet. Microbe. 4, e113-e125 (2023). doi: 10.1016/S2666-5247(22)00303-2 . MacLennan, C. A. et al. Vaccines against invasive Salmonella disease current status and future directions. Hum. Vaccin. Immunother. 10, 1478–1493 (2014). doi: 10.4161/hv.29054 . Fuche, F. J. et al. Salmonella serogroup C: current status of vaccines and why they are needed. Clin. Vaccine. Immunol. 23, 737–745 (2016). doi: 10.1128/CVI.00243-16 . Wigley, P. Salmonella enterica serovar Gallinarum: addressing fundamental questions in bacteriology sixty years on from the 9R vaccine. Avian. Pathol. 46, 119–124 (2017). doi: 10.1016/j.mib.2022.102262 . Li, Q. et al. Complete genome sequence of Salmonella enterica serovar Choleraesuis vaccine strain C500 attenuated by chemical mutation. Genome. Announc. 2, e01022-14 (2014). doi: 10.1128/genomeA.01022-14 . Dominguez-Medina, C. C. et al. Outer membrane protein size and LPS O-antigen define protective antibody targeting to the Salmonella surface. Nat. Commun. 11, 851 (2020). doi: 10.1038/s41467-020-14655-9 . Kong, Q. et al. Regulated delayed expression of rfaH in an attenuated Salmonella enterica serovar typhimurium vaccine enhances immunogenicity of outer membrane proteins and a heterologous antigen. Infect. Immun. 77, 5572–5582 (2009). doi: 10.1128/IAI.00831-09 . Liu, Q. et al. Outer membrane vesicles derived from Salmonella Typhimurium mutants with truncated LPS induce cross-protective immune responses against infection of Salmonella enterica serovars in the mouse model. Int. J. Med. Microbiol. 306, 697–706 (2016). doi: 10.1016/j.ijmm.2016.08.004 . Liu, Q. et al. Immunogenicity and cross-protective efficacy induced by outer membrane proteins from Salmonella Typhimurium mutants with truncated LPS in Mice. Int. J. Mol. Sci. 17, 416 (2016). doi: 10.3390/ijms17030416 . Curtiss, R. et al. Salmonella enterica serovar Typhimurium strains with regulated delayed attenuation in vivo. Infect. Immun. 77, 1071–1082 (2009). doi: 10.1128/IAI.00693-08 . Kong, Q. et al. Regulated delayed expression of rfc enhances the immunogenicity and protective efficacy of a heterologous antigen delivered by live attenuated Salmonella enterica vaccines. Vaccine. 28, 6094–6103 (2010). doi: 10.1016/j.vaccine.2010.06.074 . Guzman, L.-M. et al. Tight regulation, modulation, and high-level expression by vectors containing the arabinose P BAD promoter. J. Bacteriol. 177, 4121–4130 (1995). doi: 10.1128/jb.177.14.4121-4130.1995 . Bertani, G. Studies on lysogenesis I: the mode of phage liberation by lysogenic Escherichia coli . J Bacteriol. 62, 293–300 (1951). doi: 10.1128/jb.62.3.293-300.1951 . Nakayama, K. et al. Construction of an Asd + expression-cloning vector: stable maintenance and high level expression of cloned genes in a Salmonella vaccine strain. Bio/technology. 6, 693–697 (1988). doi: 10.1038/nbt0688-693 . Curtiss, R. et al. Nonrecombinant and recombinant avirulent Salmonella vaccines for poultry. Vet. Immunol. Immunopathol. 54, 365–372 (1996). doi: 10.1016/S0165-2427(96)05683-8 . Edwards, R. A. et al. Improved allelic exchange vectors and their use to analyze 987P fimbria gene expression. Gene. 207, 149–157 (1998). doi: 10.1016/S0378-1119(97)00619-7 . Su, H. et al. Regulated delayed Shigella flexneri 2a O-antigen synthesis in live recombinant Salmonella enterica serovar Typhimurium induces comparable levels of protective immune responses with constitutive antigen synthesis system. Theranostics. 9, 3565–3579 (2019). doi: 10.7150/thno.33046 . Wei, et al. Highly efficient method for introducing successive multiple scarless gene deletions and markerless gene insertions into the Yersinia pestis chromosome. Appl. Environ. Microbiol. 74, 4241–4245 (2008). doi: 10.1128/AEM.00940-08 . Kang, et al. Immune responses to recombinant pneumococcal PspA antigen delivered by live attenuated Salmonella enterica serovar typhimurium vaccine. Infect. Immun. 70, 1739 (2002). doi: 10.1128/IAI.70.4.1739-1749.2002 . Yi, E. C. et al. Rapid isolation method for lipopolysaccharide and lipid A from Gram-negative bacteria. Analyst. 125, 651–656 (2000). doi: 10.1039/B000368I . Hirschfeld, M. et al. Cutting Edge: Repurification of lipopolysaccharide eliminates signaling through both human and murine Toll-Like receptor 2. J. Immunol. 165, 618–622 (2000). doi: 10.4049/jimmunol.165.2.618 . Hitchcock, P. J. et al. Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J. Bacteriol. 154, 269–277 (1983). doi: 10.1128/jb.154.1.269-277.1983 . Gibbons, H. S. et al. An inner membrane dioxygenase that generates the 2-hydroxymyristate moiety of Salmonella lipid A. Biochemistry. 47, 2814–2825 (2008). doi: 10.1021/bi702457c . Bligh, E. L. G. et al. A rapid method of total lipid extraction and purification. Can. J. Biochem Physiol. 37, 911–917 (1959). doi: https://doi.org/10.1139/o59-099 . Li, Y. et al. A sopB deletion mutation enhances the immunogenicity and protective efficacy of a heterologous antigen delivered by live attenuated Salmonella enterica vaccines. Infect. Immun. 76, 5238–5246 (2008). doi: 10.1128/IAI.00720-08 . Takahashi, K. et al. Detection of lipopolysaccharide (LPS) and identification of its serotype by an enzyme-linked immunosorbent assay (ELISA) using poly-L-lysine. J. Immunol. Methods. 153, 67–71 (1992). doi: 10.1016/0022-1759(92)90306-E . Kong, Q. et al. Salmonella synthesizing 1-monophosphorylated lipopolysaccharide exhibits low endotoxic activity while retaining its immunogenicity. J. Immunol. 187, 412–423 (2011). doi: 10.4049/jimmunol.1100339 . Wang, S. W. et al. Mechanism of antigen presentation and specificity of antibody cross-reactivity elicited by an oligosaccharide-conjugate cancer vaccine. J. Am. Chem. Soc. 145, 9840–9849 (2023). doi: 10.1021/jacs.3c02003 . Kong, Q. et al. Effect of deletion of genes involved in lipopolysaccharide core and O-antigen synthesis on virulence and immunogenicity of Salmonella enterica serovar typhimurium . Infect. Immun. 79, 4227–4239 (2011). doi: 10.1128/IAI.05398-11 . Fierer, J. Invasive Non-typhoidal Salmonella (iNTS) Infections. Clin. Infect. Dis. 75, 732–738 (2022). doi: 10.1093/cid/ciac035 . Santos, R. L. et al. Animal models of Salmonella infections: enteritis versus typhoid fever. Microbes. Infect. 3, 1335–1344 (2001). doi: 10.1016/s1286-4579(01)01495-2 . Wang, B. X. et al. One species, different diseases: the unique molecular mechanisms that underlie the pathogenesis of typhoidal Salmonella infections. Curr. Opin. Microbiol. 72, 102262 (2023). doi: 10.1016/j.mib.2022.102262 . Perez Jorge, G. et al. p53 gene delivery via a recombinant Salmonella enterica Typhimurium leads to human bladder carcinoma cell death in vitro. Lett. Appl. Microbiol. 75, 1010–1020 (2022). doi: 10.1111/lam.13777 . Li, Q. et al. Live attenuated Salmonella enterica serovar Choleraesuis vector delivering a conserved surface protein enolase induces high and broad protection against Streptococcus suis serotypes 2, 7, and 9 in mice. Vaccine. 38, 6904–6913 (2020). doi: 10.1016/j.vaccine.2020.08.062 . Felgner, S. et al. aroA -deficient Salmonella enterica serovar Typhimurium is mmore than a metabolically attenuated mutant. mBio . 7, e01220-16 (2016). doi: 10.1128/mBio.01220-16 . Sears, K. T. et al. Evaluation of three candidate live-attenuated Salmonella enterica serovar Typhimurium vaccines to prevent Non-Typhoidal Salmonella infection in an infant mouse model. Vaccines. 11, 1562 (2023). doi: 10.3390/vaccines11101562 . Senevirathne, A. et al. Assessing an O-antigen deficient, live attenuated Salmonella Gallinarium strain that is DIVA compatible, environmentally safe, and protects chickens against fowl typhoid. Dev. Comp. Immunol. 133, 104433 (2022). doi: https://doi.org/10.1016/j.dci.2022.104433 . Ji, H. J. et al. Development of live attenuated. Front. Immunol. 13, 931052 (2022). doi: 10.3389/fimmu.2022.931052 . Wu, Y. et al. Endogenous stimulus-powered antibiotic release from nanoreactors for a combination therapy of bacterial infections. Nat. Commun. 10, 4464 (2019). doi: 10.1038/s41467-019-12233-2 . Roland, K. et al. Construction and evaluation of a delta cya delta crp Salmonella typhimurium strain expressing avian pathogenic Escherichia coli O78 LPS as a vaccine to prevent airsacculitis in chickens. Avian. diseases. 43, 429–441 (1999). doi: 10.2307/1592640 . Tables Table 1. Bacterial strains and plasmids used in this study Strains or plasmids Descriptions Source Salmonella strains c3761 Wild type S . Typhimurium, UK-1 [26] c3700 S . Enteritidis, a clinical isolate from chicken, S246 [20] c3545 S . Choleraesuis, a clinical isolate from pig, S340 [20] c9705 D pagL7 D pagP81 ::P lpp lpxE D lpxR9 [38] SW067 c9705 D fur9 Lab stock SW079 D waaC40 This study SW080 D waaF41 This study SW081 D waaG42 , the same strain as c11308 [40] SW082 D waaI43 , the same strain as c11309 [40] SW083 D waaJ44 , the same strain as c11310 [40] SW084 D waaL46 , the same strain as c11312 [40] SW085 D wbaP45 , the same strain as c11311 [40] SW086 D waaC40 D pagL7 This study SW087 D waaF41 D pagL7 This study SW088 D waaG42 D pagL7 This study SW089 D waaI43 D pagL7 This study SW090 D waaJ44 D pagL7 This study SW091 D waaL46 D pagL7 This study SW092 D wbaP45 D pagL7 This study SW093 D waaC40 D pagL71 ::TT araC P BAD waaC This study SW094 D waaC40 D pagL72 ::TT araC P BAD waaC This study SW095 D waaF41 D pagL73 ::TT araC P BAD waaF This study SW096 D waaF41 D pagL74 ::TT araC P BAD waaF This study SW097 D waaG42 D pagL75 ::TT araC P BAD waaG This study SW098 D waaG42 D pagL76 ::TT araC P BAD waaG This study SW099 D waaI43 D pagL77 ::TT araC P BAD waaI This study SW100 D waaI43 D pagL78 ::TT araC P BAD waaI This study SW101 D waaJ44 D pagL79 ::TT araC P BAD waaJ This study SW102 D waaJ44 D pagL80 ::TT araC P BAD waaJ This study SW103 D waaL46 D pagL81 ::TT araC P BAD waaL This study SW104 D waaL46 D pagL82 ::TT araC P BAD waaL This study SW105 D wbaP45 D pagL83 ::TT araC P BAD D wbaP This study SW106 D wbaP45 D pagL84 ::TT araC P BAD D wbaP This study SW107 D pagL7 D pagP81 ::P lpp lpxE D lpxR9 D fur9 ∆ waaC40 This study SW108 D pagL71 ::TT araC P BAD waaC D pagP81 ::P lpp lpxE D lpxR9 D fur9 ∆ waaC40 This study SW109 D pagL7 D pagP81 ::P lpp lpxE D lpxR9 D fur9 ∆ waaF41 This study SW110 D pagL74 ::TT araC P BAD waaF D pagP81 ::P lpp lpxE D lpxR9 D fur9 ∆ waaF41 This study SW111 D pagL7 D pagP81 ::P lpp lpxE D lpxR9 D fur9 ∆ waaG42 This study SW112 D pagL76 ::TT araC P BAD waaG D pagP81 ::P lpp lpxE D lpxR9 D fur9 ∆ waaG42 This study SW113 D pagL7 D lpxR9 D pagP81 ::P lpp lpxE D fur9 ∆waaI43 This study SW114 D pagL77 ::TT araC P BAD waaI D pagP81 ::P lpp lpxE D lpxR9 D fur9 ∆ waaI43 This study SW115 D pagL7 D pagP81 ::P lpp lpxE D lpxR9 D fur9 ∆ waaJ44 This study SW116 D pagL79 ::TT araC P BAD waaJ D pagP81 ::P lpp lpxE D lpxR9 D fur9 ∆ waaJ44 This study SW117 D pagL7 D pagP81 ::P lpp lpxE D lpxR9 D fur9 ∆ waaL46 This study SW118 D pagL81 ::TT araC P BAD waaL D pagP81 ::P lpp lpxE D lpxR9 D fur9 ∆ waaL46 This study SW119 D pagL7 D pagP81 ::P lpp lpxE D lpxR9 D fur9 ∆ waaP45 This study SW120 D pagL83 ::TT araC P BAD D wbaP D pagP81 ::P lpp lpxE D lpxR9 D fur9 ∆ waaP45 This study E. coli strains c7232 endA1 hsdR17 ( r K -, m k + ) glnV44 thi-1 recA1 gyrA relA1 ∆( lacZYA-argF ) U169 λ pir deoR (f 80dlac ∆( lacZ ) M15 ) [51] c7213 thi-1 thr-1 leuB6 glnV44 fhuA21 lacY1 recA1 RP4-2-Tc::Mu[ λ pir] ∆ asdA4 ∆( zhf-2 ::Tn 10 ) [51] Plasmids pYA4278 sacB mobRP4 R6K ori Cm + , derived from pRE112 [27] pYA3700 Cloning vector containing TT araC P BAD cassette [29] pYA4284 ∆ pagL7 [38] pSW012 for ∆ waaC40 mutation, suicide plasmid This study pSW013 for D waaF41 mutation, suicide plasmid This study pSW014 for D waaG42 mutation, the same suicide plasmid as pYA4896 [40] pSW015 for D waaI43 mutation, the same suicide plasmid as pYA4897 [40] pSW016 for D waaJ44 mutation, the same suicide plasmid as pYA4898 [40] pSW017 for D waaL46 mutation, the same suicide plasmid as pYA4900 [40] pSW018 for D wbaP45 mutation, the same suicide plasmid as pYA4899 [40] pSW019 Insert waaC1 gene to pYA3700 This study pSW020 Insert waaC2 gene to pYA3700 This study pSW021 Insert waaF1 gene to pYA3700 This study pSW022 Insert waaF2 gene to pYA3700 This study pSW023 Insert waaG1 gene to pYA3700 This study pSW024 Insert waaG2 gene to pYA3700 This study pSW025 Insert waaI1 gene to pYA3700 This study pSW026 Insert waaI2 gene to pYA3700 This study pSW027 Insert waaJ1 gene to pYA3700 This study pSW028 Insert waaJ2 gene to pYA3700 This study pSW029 Insert waaL1 gene to pYA3700 This study pSW030 Insert waaL2 gene to pYA3700 This study pSW031 Insert waaP1 gene to pYA3700 This study pSW032 Insert waaP2 gene to pYA3700 This study pSW033 for D pagL71 ::TT araC P BAD waaC mutation This study pSW034 for D pagL72 ::TT araC P BAD waaC mutation This study pSW035 for D pagL73 ::TT araC P BAD waaF mutation This study pSW036 for D pagL74 ::TT araC P BAD waaF mutation This study pSW037 for D pagL75 ::TT araC P BAD waaG mutation This study pSW038 for D pagL76 ::TT araC P BAD waaG mutation This study pSW039 for D pagL77 ::TT araC P BAD waaI mutation This study pSW040 for D pagL78 ::TT araC P BAD waaI mutation This study pSW041 for D pagL79 ::TT araC P BAD waaJ mutation This study pSW042 for D pagL80 ::TT araC P BAD waaJ mutation This study pSW043 for D pagL81 ::TT araC P BAD waaL mutation This study pSW044 for D pagL82 ::TT araC P BAD waaL mutation This study pSW045 for D pagL83 ::TT araC P BAD wbaP mutation This study pSW046 for D pagL84 ::TT araC P BAD wbaP mutation This study Table 2. Prime used in this study Primer name Sequence (5’-3’) (the sequences highlighted by red color contain SD and start codon) WaaC-Del 1F cattctgaaatgagccggcgctgaatagcgagcag WaaC-Del 1R cagagtctctttaaacgccctcttccgaca WaaC-Del 2F gggcgtttaaagagactctgtctcatccca WaaC-Del 2R agcatttatcagggtaagccctccagtaccgtatt Vec-F accctgataaatgcttcaataa Vec-R gctcatttcagaatggaaggtc WaaF-Del 1F cattctgaaatgagcgaaagcccgaaactgtttga WaaF-Del 1R aaacccgcatacttacgcgtcgcggttcag WaaF-Del 2F acgcgtaagtatgcgggttttgatcgttaa WaaF-Del 2R agcatttatcagggtctccagtccataccgtgctttat WaaC-1F tttctccata aaggctctata tgcgggttttgatcgttaa WaaC-2F tttctccata aaggctctatg tgcgggttttgatcgttaa WaaC-R aaaaaacgggttaatgaatctttccaaatac WaaC-VF gattcattaacccgtttttttgggctagcc WaaC-VR atagagcctttatggagaaacagtagagagt WaaF-1F gtttctccata aaggctctata tgaaaattttggtcattggc WaaF-2F gtttctccata aaggctctatg tgaaaattttggtcattggc WaaF-R caaaaaaacgggttaaacgccctcttccgacaa WaaF-VF gggcgtttaacccgtttttttgggctagcc WaaF-VR atagagcctttatggagaaacagtagagagt WaaG-1F gtttctccata aaggctctata tgagagttgccttttgcttat WaaG-2F gtttctccata aaggctctatg tgagagttgccttttgcttat WaaG-R caaaaaaacgggtcaaccatctaaatcacctg WaaG-VF agatggttgacccgtttttttgggctagcc WaaG-VR atagagcctttatggagaaacagtagagagt WaaI-1F gtttctccata aaggctctata tgagcagaaaatattttgaag WaaI-2F gtttctccata aaggctctatg tgagcagaaaatattttgaag WaaI-R aaaaaacgggttattcaagaagtttacgttt WaaI-VF cttgaataacccgtttttttgggctagcc WaaI-VR atagagcctttatggagaaacagtagagagt WaaJ-1F gtttctccata aaggctctata tggattcatttcctgagata WaaJ-2F gtttctccata aaggctctatg tggattcatttcctgagata WaaJ-R aaaaaacgggttatttgtggaaaagtttac WaaJ-VF ccacaaataacccgtttttttgggctagcc WaaJ-VR atagagcctttatggagaaacagtagagagt WaaL-1F gtttctccata aaggctctata tgctaaccacatcattaac WaaL-2F gtttctccata aaggctctatg tgctaaccacatcattaac WaaL-R aaaaaacgggttatctatttcttagcgccag WaaL-VF gaaatagataacccgtttttttgggctagcc WaaL-VR atagagcctttatggagaaacagtagagagt WbaP -1F gtttctccata aaggctctata tggttgagctgaaagcgccg WbaP -2F gtttctccata aaggctctatg tggttgagctgaaagcgccg WbaP -R caaaaaaacgggttacagattttttcttattg WbaP -VF aaatctgtaacccgtttttttgggctagcc WbaP -VR atagagcctttatggagaaacagtagagagt P1 ttgaaatggtggtggatttattattctatcctagaat P2-(WaaC) aattgttattcaactttaatgaatctttccaaatac P-VF agttgaataacaattagcgag P-VR tccaccaccatttcaatgtcaa P3-(WaaF) ctaattgttattcaactttaaacgccctcttccgacaa P4-(WaaG) ctaattgttattcaacttcaaccatctaaatcacctgt P5-(WaaI) ctaattgttattcaactttattcaagaagtttacgttt P6-(WaaJ) taattgttattcaactttatttgtggaaaagtttacg P7-(WaaL) taattgttattcaactttatctatttcttagcgccag P8-(WbaP) aattgttattcaactttacagattttttcttattgtc Additional Declarations (Not answered) Supplementary Files SupplementaryFigure1.tif SupplementaryFigure2.tif SupplementaryFigure3.tif SupplementaryFigure4.tif SupplementaryFigure5.tif supplementarymaterials.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3971522","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":275829084,"identity":"631b3df1-fd57-4817-b5a4-8749d3e34075","order_by":0,"name":"Qingke Kong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYBACCTBpkMDDz8x8+AFpWiTb2dIMSNDCkMBgcJ5HQYIoLZLtvYdfvClIkzE+zMNgwFBjE01QizTPuTTLOQY5PGaHeQ88YDiWlttASIucRI6ZMY9BBVALX4IBY8NhErQYN/MYSBClRVoix/gxD9BhBszEapHsOWPGOMcgjUfiMDCQE4jxi8TxHuMPb/4k2/P3Hz784EONDWEtQMAmwQNjJhChHASYP/AQVjQKRsEoGAUjGQAAC1s3dCyMQXEAAAAASUVORK5CYII=","orcid":"","institution":"Southwest University","correspondingAuthor":true,"prefix":"","firstName":"Qingke","middleName":"","lastName":"Kong","suffix":""},{"id":275829085,"identity":"cba27a0c-0f12-472a-b0b9-2e1dc70f6be4","order_by":1,"name":"Xiaoping Bian","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoping","middleName":"","lastName":"Bian","suffix":""},{"id":275829086,"identity":"f1c24a78-f3e2-4fb7-b503-41de73b875d6","order_by":2,"name":"Qing Liu","email":"","orcid":"","institution":"College of animal science, Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Qing","middleName":"","lastName":"Liu","suffix":""},{"id":275829087,"identity":"20216471-1ecd-4418-b738-d4db055e9945","order_by":3,"name":"Yaolin Chen","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Yaolin","middleName":"","lastName":"Chen","suffix":""},{"id":275829088,"identity":"77355c51-ab9e-4854-a6a5-6306c70ad619","order_by":4,"name":"Wenjin Zhang","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Wenjin","middleName":"","lastName":"Zhang","suffix":""},{"id":275829089,"identity":"2c0fc721-8c6b-4a00-b4e6-b82af839f236","order_by":5,"name":"Mengru Li","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Mengru","middleName":"","lastName":"Li","suffix":""},{"id":275829090,"identity":"2a12e914-ea33-48ac-9c16-ff6a5ce9d10f","order_by":6,"name":"Xiaofen Zhang","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Xiaofen","middleName":"","lastName":"Zhang","suffix":""},{"id":275829091,"identity":"8f3dc721-0382-459a-a1d7-154bd2a9bedf","order_by":7,"name":"Liu Yang","email":"","orcid":"","institution":"National Center of Technology Innovation for Pigs","correspondingAuthor":false,"prefix":"","firstName":"Liu","middleName":"","lastName":"Yang","suffix":""},{"id":275829092,"identity":"82f4d374-ec23-4657-8521-33e81ef4fcbb","order_by":8,"name":"Yonghong Liao","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Yonghong","middleName":"","lastName":"Liao","suffix":""}],"badges":[],"createdAt":"2024-02-20 03:45:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3971522/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3971522/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51946822,"identity":"e7d25395-0224-4a72-a428-0561c553a526","added_by":"auto","created_at":"2024-03-04 11:14:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":224501,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe characteristics of the regulated delayed attenuated \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSalmonella\u003c/strong\u003e\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThe regulated delayed attenuated \u003cem\u003eSalmonella \u003c/em\u003eare capable of synthesizing full LPS lengths by utilizing arabinose and maintaining smooth LPS patterns to keep their original ability to colonize the small intestines at the initial stage of the infection after immunization of mice via the oral route. Subsequently, different truncated lengths of LPS were synthesized in the absence of arabinose \u003cem\u003ein vivo\u003c/em\u003e, thereby exposing conserved protein antigens on the outer membrane for inducing the production of specific antibodies.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3971522/v1/fac8fbd0803bd358d7aad456.png"},{"id":51946821,"identity":"3e789ac8-74a7-407d-91ef-b2f82e7a82b8","added_by":"auto","created_at":"2024-03-04 11:14:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":274574,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConstruction of mutants and phenotypic determination.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) All these mutants were derived from SW067 (D\u003cem\u003epagL7 \u003c/em\u003eD\u003cem\u003epagP81\u003c/em\u003e::P\u003csub\u003elpp \u003c/sub\u003e\u003cem\u003elpxE \u003c/em\u003eD\u003cem\u003elpxR9\u003c/em\u003e D\u003cem\u003efur9\u003c/em\u003e). The genes \u003cem\u003ewaaC\u003c/em\u003e, \u003cem\u003ewaaF\u003c/em\u003e, \u003cem\u003ewaaG\u003c/em\u003e, \u003cem\u003ewaaI\u003c/em\u003e, \u003cem\u003ewaaJ\u003c/em\u003e, \u003cem\u003ewaaL\u003c/em\u003e, or \u003cem\u003ewbaP\u003c/em\u003e were deleted from the genome of SW067, respectively. The gene \u003cem\u003epagL\u003c/em\u003e was replaced by TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaC\u003c/em\u003e, TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaF\u003c/em\u003e, TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaG\u003c/em\u003e, TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaI\u003c/em\u003e, TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaJ\u003c/em\u003e, TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaL,\u003c/em\u003e or TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewbaP\u003c/em\u003e to generate a set of genetically modified strains capable of producing smooth LPS in response to arabinose regulation, respectively. (B) Upon the addition of arabinose in laboratory conditions, these mutants successfully synthesized a typical LPS pattern. In contrast, these mutants exhibited rough phenotypes in the absence of arabinose due to the loss of their ability the form LPS. (C) Lipid A structure of wild-type and \u003cem\u003elpxE\u003c/em\u003e-expressing SW111 (D\u003cem\u003epagL7\u003c/em\u003e D\u003cem\u003epagP81\u003c/em\u003e::P\u003csub\u003elpp\u003c/sub\u003e \u003cem\u003elpxE \u003c/em\u003eD\u003cem\u003elpxR9 \u003c/em\u003eD\u003cem\u003efur9 \u003c/em\u003e∆\u003cem\u003ewaaG42\u003c/em\u003e) and SW112 (D\u003cem\u003epagL76\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaG\u003c/em\u003e D\u003cem\u003epagP81\u003c/em\u003e::P\u003csub\u003elpp\u003c/sub\u003e \u003cem\u003elpxE \u003c/em\u003eD\u003cem\u003elpxR9 \u003c/em\u003eD\u003cem\u003efur9 \u003c/em\u003e∆\u003cem\u003ewaaG42\u003c/em\u003e). In the negative-ion mode, using MALDI-TOF-MS, one can detect the lipid A species. The unaltered lipid A is represented by the peak at m/z 1796.3.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3971522/v1/6a449f69a1200b06b8b60968.png"},{"id":51946813,"identity":"2ed2ba3a-9713-4b98-9ef7-6f2e02ce1bf7","added_by":"auto","created_at":"2024-03-04 11:14:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":104241,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBacterial burden in liver, spleen, and PP of mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGroups of mice were given 20 µl of BSG including 1 × 10\u003csup\u003e9\u003c/sup\u003e CFU mutants via oral route, and three mice from each group were humanely euthanized at 3, 6, 14, and 28d postinoculation. The liver (A), spleen (B), and PP (C) were collected. After treating with tissue homogenizer, homogenized tissues were dropped on the LB and MacConkey agar after appropriate dilutions with phosphate buffer saline (PBS), and the bacterial loading was calculated based on the bacteria on the plates. The standard differences between the mice in each group were shown by the error bars. Data are presented as the means ± SEM (n = 3).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3971522/v1/327b8679a518c5c0ec72ed3d.png"},{"id":51946834,"identity":"69fcfb36-a3e0-4241-aa8f-761183b574e6","added_by":"auto","created_at":"2024-03-04 11:14:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1027528,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistopathological analysis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effects of regulated delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003eon the liver and spleen were assessed by histopathological examination at 6 days post-inoculation. The black arrow symbolizes hepatocytes. The blue circles depicted in the image signify areas of focal necrosis. The red circles depict the infiltration of neutrophils. Images were captured with a magnification of 10 × (scale bars 250 μm) and an inset magnification of 40 × (scale bars 50 μm).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3971522/v1/ac244bb095cfe79faed8a4be.png"},{"id":51946817,"identity":"a584cb27-3567-4ab1-9117-f68333a99de2","added_by":"auto","created_at":"2024-03-04 11:14:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":172954,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmune response to OMPs from \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. Typhimurium induced by the regulated delayed attenuated \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSalmonella\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum and vaginal secretions of mice were gathered at week 8 post-initial vaccination. Quantitative ELISA was applied to analyze specific IgG (A), IgA (B), IgG1 (C), and IgG2a (D) against \u003cem\u003eS\u003c/em\u003e. Typhimurium OMPs. The results displayed the precise levels of antibodies, as measured by a standard curve, in mice orally inoculated with the regulated delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e at the scheduled weeks. The standard differences between the mice in each group were shown by the error bars. Data are presented as the means ± SEM (n = 8), superscript letters a, b, and c indicate P \u0026lt; 0.05 for comparisons with the BSG, SW112 (\u003cem\u003ewaaG\u003c/em\u003e), and SW116 (\u003cem\u003ewaaJ\u003c/em\u003e) groups, respectively.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3971522/v1/ab68672b523d60c031d0a0c2.png"},{"id":51946815,"identity":"c641fdd5-73b6-4552-a4b6-a50bb3d08f11","added_by":"auto","created_at":"2024-03-04 11:14:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":269953,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmune response to OMPs from \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. Choleraesuis and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. Enteritidis induced by the regulated delayed attenuated \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSalmonella\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum and vaginal secretions of mice were gathered at week 8 post-initial vaccination. Quantitative ELISA was applied to analyze specific IgG (A, C) and IgA (B, D) against OMPs from \u003cem\u003eS\u003c/em\u003e. Choleraesuis and \u003cem\u003eS\u003c/em\u003e. Enteritidis, as well as the subtypes IgG1 (E, G) and IgG2a (F, H). The results displayed the precise levels of antibodies, as measured by a standard curve, in mice orally inoculated with the regulated delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e at the scheduled weeks. The standard differences between the mice in each group were shown by the error bars. Data are presented as the means ± SEM (n = 8), superscript letters a, b, and c indicate P \u0026lt; 0.05 for comparisons with the BSG, SW114 (\u003cem\u003ewaaI\u003c/em\u003e), and SW120 (\u003cem\u003ewbaP\u003c/em\u003e) groups, respectively.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3971522/v1/cc013a91e61d630e016e1f6f.png"},{"id":51946832,"identity":"9138a9af-b74a-4187-8687-349d61ddbaf9","added_by":"auto","created_at":"2024-03-04 11:14:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":238845,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe assessment of long-term\u003c/strong\u003e \u003cstrong\u003eimmune responses induced by regulated delayed attenuated \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSalmonella\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe percentage of the B\u003csub\u003eM\u003c/sub\u003e cells from immunized mice was evaluated by flow cytometry on day 72 after the initial immunization. (A) B\u003csub\u003eM\u003c/sub\u003e cells were gated as the IgG\u003csup\u003e+ \u003c/sup\u003eB220\u003csup\u003e+ \u003c/sup\u003eCD80\u003csup\u003e+\u003c/sup\u003e IgD\u003csup\u003e-\u003c/sup\u003e cells. (B) The percentage of B\u003csub\u003eM\u003c/sub\u003e cells was calculated in the sample. The representative dot plots derived from the flow cytometry analysis were illustrated in the figure, with the indicated gating and cell populations. The standard differences between the mice in each group were shown by the error bars. Data are presented as the means ± SEM (n = 3), superscript letters a and b indicate P \u0026lt; 0.05 for comparisons with the BSG and SW116 (\u003cem\u003ewaaJ\u003c/em\u003e) groups, respectively.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3971522/v1/157647fbe93fd7ef6a99efc1.png"},{"id":51946816,"identity":"69b1ca47-8082-4d70-a7af-2ab74da03bec","added_by":"auto","created_at":"2024-03-04 11:14:46","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":130925,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of protection against challenge by multiple serotypes\u003c/strong\u003e \u003cem\u003e\u003cstrong\u003eSalmonella\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe immunized mice underwent an oral challenge with 10\u003csup\u003e9\u003c/sup\u003e CFU (10000 × LD\u003csub\u003e50\u003c/sub\u003e) of χ3761 (A), 10\u003csup\u003e7\u003c/sup\u003e CFU (100 LD\u003csub\u003e50\u003c/sub\u003e) of χ3700 (B), or 10\u003csup\u003e7\u003c/sup\u003e CFU (100 LD\u003csub\u003e50\u003c/sub\u003e) of χ3545 (C) in 20 µl BSG at week 9 after the initial immunization, respectively. Following the challenge, mortality was recorded for 25 days. Data from three independent experiments were summarized (n = 8).\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-3971522/v1/f17ec3adecf36a86a2d7138a.png"},{"id":51946819,"identity":"dcef1bb0-610f-4e90-af59-5bf10fc1b853","added_by":"auto","created_at":"2024-03-04 11:14:47","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":115964,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmune response to OMPs from other enteric bacteria induced by the regulated delayed attenuated \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSalmonella\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sera were gathered from mice (n = 8) in SW114 (\u003cem\u003ewaaI\u003c/em\u003e), SW116 (\u003cem\u003ewaaJ\u003c/em\u003e), and SW118 (\u003cem\u003ewaaL\u003c/em\u003e) groups on days 42 after the primary immunization. Quantitative ELISA was applied to analyze specific IgG against OMPs from \u003cem\u003eE. coli\u003c/em\u003e O8 strain (A), \u003cem\u003eE. coli\u003c/em\u003e O138 strain (B), and \u003cem\u003eE. coli\u003c/em\u003e O139 strain (C). The results displayed the precise levels of antibodies, as measured by a standard curve. The standard differences between the mice in each group were shown by the error bars. Data are presented as the means ± SEM (n = 8), superscript letters a indicate P \u0026lt; 0.05 for comparisons with the BSG groups.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-3971522/v1/8a46d27addd3df31fba70c8b.png"},{"id":53448003,"identity":"1b2f5eaa-ec12-4d21-8f56-05633ca24713","added_by":"auto","created_at":"2024-03-26 05:53:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3199047,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3971522/v1/774aefa2-72f1-4b97-b224-d752826d7cb4.pdf"},{"id":51947201,"identity":"9e5dd630-1d58-4a42-8fcf-13d4a4133ca2","added_by":"auto","created_at":"2024-03-04 11:22:46","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":152069,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-3971522/v1/9059fea61a79c968ca61d08b.tif"},{"id":51946833,"identity":"67d79812-0674-4d62-9801-beda21a71cf4","added_by":"auto","created_at":"2024-03-04 11:14:48","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":69238,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-3971522/v1/d960fb94c9b7ff29bf1a7d48.tif"},{"id":51946818,"identity":"d898ab48-5976-41ef-816d-0a68b417d303","added_by":"auto","created_at":"2024-03-04 11:14:47","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":300023,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-3971522/v1/810eac0af5f372055a4ea537.tif"},{"id":51946824,"identity":"bad18769-c6f7-4fc6-b6ab-8cb70b9835a9","added_by":"auto","created_at":"2024-03-04 11:14:47","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":115493,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure4.tif","url":"https://assets-eu.researchsquare.com/files/rs-3971522/v1/857cf4b48b9c45d685bb432b.tif"},{"id":51946823,"identity":"c6d054a1-c0b6-4577-8189-fa42e3832c32","added_by":"auto","created_at":"2024-03-04 11:14:47","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":68947,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure5.tif","url":"https://assets-eu.researchsquare.com/files/rs-3971522/v1/4c879a1b7bc254c251f93027.tif"},{"id":51946826,"identity":"15060edc-6c71-45d4-9a96-32a928b120f1","added_by":"auto","created_at":"2024-03-04 11:14:48","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":17873,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-3971522/v1/6c77e465e02b814e584d0818.docx"}],"financialInterests":"(Not answered)","formattedTitle":"Immunogenicity and cross-protective efficacy induced by delayed attenuated Salmonella with the regulated length of lipopolysaccharide in mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eSalmonella enterica\u003c/em\u003e is a significant zoonotic foodborne pathogen that causes considerable financial losses to the livestock industry and poses severe human health concerns worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. \u003cem\u003eSalmonella\u003c/em\u003e is categorized into typhoidal \u003cem\u003eSalmonella\u003c/em\u003e and non-typhoidal \u003cem\u003eSalmonella\u003c/em\u003e (NTS) based on their ability to induce specific pathologies in humans [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Typhoidal \u003cem\u003eSalmonella\u003c/em\u003e is commonly known as Typhi (\u003cem\u003eSalmonella enterica\u003c/em\u003e serovar Typhi) and Paratyphi (\u003cem\u003eSalmonella enterica\u003c/em\u003e serovar Paratyphi) A, B, or C, whereas other serovars are referred to as NTS [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In 2017, a total of 14.3\u0026nbsp;million cases of enteric fever were reported. Additionally, there were 53.5 thousand cases of NTS invasive disease reported during the same period [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. While enteric fever still often occurs in human infection, NTS is a significant cause of global foodborne diseases through the transmission of \u003cem\u003eSalmonella\u003c/em\u003e from animals to humans [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Among the NTS serovars isolated from food, \u003cem\u003eSalmonella enterica\u003c/em\u003e serovar Typhimurium (\u003cem\u003eS\u003c/em\u003e. Typhimurium) was the most prevalent one in Africa [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], Australia [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and China [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], and \u003cem\u003eS\u003c/em\u003e. Enteritidis was the dominant serovar in the United States and European countries [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Other serovars such as \u003cem\u003eS\u003c/em\u003e. Anatum (\u003cem\u003eSalmonella enterica\u003c/em\u003e serovar Anatum) and \u003cem\u003eS\u003c/em\u003e. Weltevreden (\u003cem\u003eSalmonella enterica\u003c/em\u003e serovar Weltevreden) were also frequently isolated in beef and seafood in the world [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and \u003cem\u003eS\u003c/em\u003e. Derby, \u003cem\u003eS\u003c/em\u003e. Thompson, and \u003cem\u003eS\u003c/em\u003e. Aberdeen are the most common serotypes detected in chicken, pig, duck, aquatic products, and soft-shell turtle in China, respectively [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAn increasing trend of antimicrobial resistance (AMR) in \u003cem\u003eSalmonella\u003c/em\u003e, especially multidrug resistance (MDR), has been on the rise and is a global concern [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Vaccination has been demonstrated as a highly effective strategy to combat infections of antibiotic-resistant \u003cem\u003eSalmonella\u003c/em\u003e. There are currently three types of licensed \u003cem\u003eSalmonella\u003c/em\u003e vaccines in humans, including the live attenuated oral vaccine Ty21a, the unconjugated Vi polysaccharide vaccine, and the Typhoid conjugated vaccine (TCV) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Since these vaccines primarily target \u003cem\u003eSalmonella\u003c/em\u003e surface polysaccharide antigens, there is limited cross-protection against other serotypes of \u003cem\u003eSalmonella\u003c/em\u003e, although Ty21a, derived from \u003cem\u003eS\u003c/em\u003e. Typhi, confers moderate cross-protection against \u003cem\u003eS\u003c/em\u003e. Paratyphi B [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. There are currently no NTS vaccines available for humans, and the use of NST vaccines for animals is limited [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Human NTS vaccine candidates primarily target \u003cem\u003eS\u003c/em\u003e. Typhimurium in serogroup B carrying O:4 antigen and/or \u003cem\u003eS\u003c/em\u003e. Enteritidis in serogroup D carrying O:9 antigen [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and \u003cem\u003eSalmonella\u003c/em\u003e vaccines for animals are mainly used to prevent fowl typhoid caused by \u003cem\u003eS\u003c/em\u003e. Gallinarum of serogroup D carrying the O:9 antigen, infections in laying hens caused by \u003cem\u003eS\u003c/em\u003e. Typhimurium and Enteritidis, and piglet paratyphoid fever caused by \u003cem\u003eS\u003c/em\u003e. Choleraesuis of serogroup C carrying the O:7 antigen [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. These NTS vaccine candidates only cover specific \u003cem\u003eSalmonella\u003c/em\u003e serotypes and cannot protect against infections caused by other commonly prevalent serotypes. Therefore, it is necessary to develop a universal vaccine that provides broad protection against different \u003cem\u003eSalmonella\u003c/em\u003e serotypes to reduce infections of \u003cem\u003eSalmonella\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAlthough the millions of lipopolysaccharide (LPS) molecules present on the surface of \u003cem\u003eSalmonella\u003c/em\u003e create a formidable barrier to restrict antibody access to the bacterial surface [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], previous studies have demonstrated that isolated outer membrane proteins (OMPs) from \u003cem\u003eSalmonella\u003c/em\u003e conferred protection against both homologous and heterologous serotype \u003cem\u003eSalmonella\u003c/em\u003e challenges in mice [\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], indicating that certain OMPs are highly conserved in \u003cem\u003eSalmonella\u003c/em\u003e to confer cross-protection. These studies imply that the OMPs of \u003cem\u003eSalmonella\u003c/em\u003e are promising targets for the development of vaccines against infection with multiple serotypes. The process of purifying OMPs from \u003cem\u003eSalmonella\u003c/em\u003e is both labor-intensive and costly, and some conserved OMPs are low or not expressed under laboratory conditions, rendering them unsuitable for large-scale production and application. Hence, live attenuated \u003cem\u003eSalmonella\u003c/em\u003e vaccines may be ideal candidates for developing a vaccine with broad protection due to their cost-effectiveness, ease of management, and the capacity to generate long-lasting immunity with just one dosage. Our earlier research has shown that while attenuated \u003cem\u003eSalmonella\u003c/em\u003e with permanent truncated LPS induces somewhat cross-immune responses against other \u003cem\u003eSalmonella\u003c/em\u003e serotypes and enteric bacteria, mutants do not effectively colonize the host intestine to induce robust immune responses [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Therefore, attenuated \u003cem\u003eSalmonella\u003c/em\u003e with the permanent truncated LPS is not a suitable candidate for vaccine development.\u003c/p\u003e \u003cp\u003eRegulated delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e (RDAS) that was developed in Curtiss's lab possesses the critical feature of undergoing regulated delayed attenuation \u003cem\u003ein vivo\u003c/em\u003e. RDAS vaccine candidates are allowed to synthesize virulence factors required for colonization and invasion by adding specific substances to the culture medium \u003cem\u003ein vitro\u003c/em\u003e, and after colonization in the organs, the mutants gradually undergo attenuation due to the absence of specific inducers after oral immunization \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Therefore, we wanted to investigate whether the regulated length of LPS in attenuated \u003cem\u003eSalmonella\u003c/em\u003e would expose more conserved antigens, thus inducing more effective cross-immunity. In this study, we constructed a series of RDAS that regulate LPS synthesis, which is capable of synthesizing full LPS lengths in the presence of arabinose \u003cem\u003ein vitro\u003c/em\u003e while synthesizing different truncated lengths of LPS in the absence of arabinose \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Our objective was to develop ideal candidates for live attenuated \u003cem\u003eSalmonella\u003c/em\u003e vaccines to trigger cross-immune responses effectively and confer cross-protection against infection of multiple serotypes of \u003cem\u003eSalmonella\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Material and methods","content":"\u003ch2\u003eGrowth conditions, medium, plasmids, and strains of bacteria\u003c/h2\u003e\n\u003cp\u003eTable 1 provides a comprehensive list of all the strains of bacteria and plasmids used in this study.\u0026nbsp;\u003cem\u003eSalmonella\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e were routinely cultivated either in Luria-Bertani (LB) broth (L1010, Solarbio Biotech, China) or on LB agar (L1015, Solarbio Biotech, China)\u0026nbsp;[24]\u0026nbsp;with or without 0.1% arabinose\u0026nbsp;at a temperature of 37˚C. In cases where necessary, the culture media were supplemented with 25 \u0026mu;g/ml of chloramphenicol (A600118, Sangon Biotech, China). To facilitate the growth of Asd- strains, 2,6-diaminopimelic acid (DAP) was included at a final concentration of 50 \u0026micro;g/ml (D1377, Sigma Aldrich, USA)\u0026nbsp;[25]. For the cultivation of strains involving allelic exchange experiments, \u003cem\u003esacB\u003c/em\u003e gene-based counterselection was conducted on LB agar containing 5% sucrose (A610498, Sangon Biotech, China). All \u003cem\u003eSalmonella\u003c/em\u003e mutants\u0026nbsp;originated from the \u003cem\u003eS\u003c/em\u003e. Typhimurium strain UK-1\u0026nbsp;[26],\u0026nbsp;and their genotypes were verified through PCR employing the corresponding primer\u0026nbsp;pair outlined\u0026nbsp;in Table 2.\u003c/p\u003e\n\u003ch2\u003eConstruction of plasmids and mutant strains\u003c/h2\u003e\n\u003cp\u003eThe allelic exchange method was utilized to introduce gene mutations in \u003cem\u003eSalmonella\u003c/em\u003e, employing pYA4278 as described in the previous study\u0026nbsp;[27]. The transformation of \u003cem\u003eE. coli\u003c/em\u003e was carried out using a technique known as electroporation. To select the transformants, appropriate antibiotic supplements were added to LB agar plate cultures. Primers utilized in this study were\u0026nbsp;listed\u0026nbsp;in Table 2. For arabinose-regulated LPS synthesis in this study, we applied the same strategy as the\u0026nbsp;D\u003cem\u003epagL\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewbaP\u003c/em\u003e deletion-insertion mutation to construct other mutations\u0026nbsp;[28]. Briefly, for the construction of the \u003cem\u003ewaaC\u003c/em\u003e mutation, in which the complete \u003cem\u003ewaaC\u003c/em\u003e open-reading frame was deleted, the\u0026nbsp;c3761\u0026nbsp;genomes were employed as the template for cloning, a DNA fragment of 350-bp containing the region upstream of the \u003cem\u003ewaaC\u003c/em\u003e gene (from ATG start codon, but not including ATG) was amplified using primers WaaC-Del 1F and WaaC-Del 1R, and another 350-bp DNA fragment containing the region downstream of the \u003cem\u003ewaaC\u003c/em\u003e gene (from TAA stop codon, but not including TAA) was amplified using primers WaaC-Del 2F and WaaC-Del 2R. Universal primers Vec-F and Vec-R were applied to amplify vector fragments. The 5\u0026prime; flanking regions of these DNA fragments contain homologous regions for recombination. The PCR products were purified by agarose gel (A620014, Sangon Biotech, China) and ligated to pYA4278 using Gibson Assembly Master Mix (E55510, New England Biolabs, USA)\u0026nbsp;to generate the suicide plasmid\u0026nbsp;pSW012 for\u0026nbsp;deleting the \u003cem\u003ewaaC\u003c/em\u003e gene.\u0026nbsp;The \u003cem\u003ewaaC\u0026nbsp;\u003c/em\u003emutation was introduced into\u0026nbsp;c3761\u0026nbsp;and SW067 by allelic exchange via conjugation with the\u0026nbsp;c7213, harboring the plasmid of\u0026nbsp;pSW012, to generate SW079 (∆\u003cem\u003ewaaC40\u003c/em\u003e) and SW107 (D\u003cem\u003epagL7\u003c/em\u003e D\u003cem\u003epagP81\u003c/em\u003e::P\u003csub\u003elpp\u003c/sub\u003e \u003cem\u003elpxE\u0026nbsp;\u003c/em\u003eD\u003cem\u003elpxR9\u003c/em\u003e D\u003cem\u003efur9\u0026nbsp;\u003c/em\u003e∆\u003cem\u003ewaaC40\u003c/em\u003e), respectively. The genotypes of \u0026Delta;\u003cem\u003ewaaC\u0026nbsp;\u003c/em\u003ewere verified by PCR and LPS profiles. The same strategy\u0026nbsp;also applied to\u0026nbsp;the deletion of the\u0026nbsp;\u003cem\u003ewaaF\u003c/em\u003e,\u003cem\u003e\u0026nbsp;waaG\u003c/em\u003e,\u003cem\u003e\u0026nbsp;waaI\u003c/em\u003e,\u003cem\u003e\u0026nbsp;waaJ\u003c/em\u003e,\u003cem\u003e\u0026nbsp;waaL,\u003c/em\u003e or\u0026nbsp;\u003cem\u003ewbaP\u003c/em\u003e genes in the\u0026nbsp;c3761\u0026nbsp;and SW067 genomes (Table 1).\u003c/p\u003e\n\u003cp\u003eFor the construction of the\u0026nbsp;D\u003cem\u003epagL\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaC\u003c/em\u003e deletion-insertion mutation,\u0026nbsp;the vector of\u0026nbsp;pYA3700\u0026nbsp;[29]\u0026nbsp;was linearized using the primers WaaC-VF and WaaC-VR. The fragment\u003cem\u003e\u0026nbsp;waaC\u003c/em\u003e gene was then amplified by WaaC-1F / WaaC-R and WaaC-2F / WaaC-R from the\u0026nbsp;c3761\u0026nbsp;genomes and inserted into\u0026nbsp;pYA3700\u0026nbsp;via Gibson Assembly Master Mix\u0026nbsp;to generate intermediate plasmid pSW019 and pSW020,\u0026nbsp;respectively. Subsequently,\u0026nbsp;the fragment TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaC\u003c/em\u003e was amplified using primers P1 and P2-(WaaC), and the gel-purified product was ligated to the\u0026nbsp;linearized\u0026nbsp;vector pYA4284 by\u0026nbsp;Gibson Assembly Master Mix\u0026nbsp;to form the suicide plasmids\u0026nbsp;pSW033 and pSW034,\u0026nbsp;respectively.\u0026nbsp;The \u003cem\u003epagL\u0026nbsp;\u003c/em\u003emutation was introduced into\u0026nbsp;SW079 (D\u003cem\u003ewaaC40\u003c/em\u003e)\u0026nbsp;by allelic exchange via conjugation with the\u0026nbsp;c7213 harboring the plasmid of pYA4284 to yield the strain SW086 (D\u003cem\u003ewaaC40\u003c/em\u003e D\u003cem\u003epagL7\u003c/em\u003e). The \u0026Delta;\u003cem\u003epagL\u003c/em\u003e::TT\u0026nbsp;\u003cem\u003earaC\u0026nbsp;\u003c/em\u003eP\u003csub\u003eBAD\u003c/sub\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003ewaaC\u003c/em\u003e mutation was introduced into\u0026nbsp;SW086 by allelic exchange by conjugation with the\u0026nbsp;c7213 harboring the plasmids of\u0026nbsp;pSW033 and pSW034\u0026nbsp;to yield SW093\u0026nbsp;(∆\u003cem\u003ewaaC40\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagL71\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaC\u003c/em\u003e) and SW094 (∆\u003cem\u003ewaaC40\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagL72\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaC\u003c/em\u003e),\u0026nbsp;respectively. PCR and LPS profiles were applied to confirm the genotypes of the \u0026Delta;\u003cem\u003epagL\u003c/em\u003e::TT\u0026nbsp;\u003cem\u003earaC\u0026nbsp;\u003c/em\u003eP\u003csub\u003eBAD\u003c/sub\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003ewaaC\u003c/em\u003e mutations.\u0026nbsp;After the mutation with tightly regulated LPS was successfully screened and identified in the wild-type background, the correct mutation was introduced into the mutant strain SW107 (D\u003cem\u003epagL7\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagP81\u003c/em\u003e::P\u003csub\u003elpp\u003c/sub\u003e \u003cem\u003elpxE\u003c/em\u003e D\u003cem\u003elpxR9\u0026nbsp;\u003c/em\u003eD\u003cem\u003efur9\u0026nbsp;\u003c/em\u003e∆\u003cem\u003ewaaC40\u003c/em\u003e) to generate the mutant strain SW108 (D\u003cem\u003epagL71\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaC\u003c/em\u003e D\u003cem\u003epagP81\u003c/em\u003e::P\u003csub\u003elpp\u003c/sub\u003e \u003cem\u003elpxE\u003c/em\u003e D\u003cem\u003elpxR9\u0026nbsp;\u003c/em\u003eD\u003cem\u003efur9\u0026nbsp;\u003c/em\u003e∆\u003cem\u003ewaaC40\u003c/em\u003e). The other mutant strains were generated using the same procedure.\u003c/p\u003e\n\u003ch2\u003ePhenotypic determination of bacteria\u003c/h2\u003e\n\u003cp\u003eBacterial strain phenotypes were identified \u003cem\u003ein vitro\u003c/em\u003e, and each experiment was conducted at least twice. The OMPs and LPS were purified from \u003cem\u003eSalmonella\u003c/em\u003e following described\u0026nbsp;methods\u0026nbsp;[30-32]. The OMP underwent electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and was subsequently stained with Coomassie brilliant blue. The\u0026nbsp;LPS samples\u0026nbsp;of \u003cem\u003eS\u003c/em\u003e.\u0026nbsp;Typhimurium\u0026nbsp;went through silver staining to establish their LPS profile, following the method\u0026nbsp;described by Hitchcock and Brown\u0026nbsp;[33]. Lipid A was extracted from \u003cem\u003eSalmonella\u003c/em\u003e after mild acid hydrolysis at pH 4.5 to break the Kdo-lipid A linkage\u0026nbsp;and subjected to Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry\u0026nbsp;(MALDI-TOF MS)\u0026nbsp;[34, 35].\u003c/p\u003e\n\u003ch2\u003eAnimals\u003c/h2\u003e\n\u003cp\u003eThe laboratory animals utilized in this study were female BALB/c mice (18-19 g) sourced from Hunan SJA Laboratory Animal Co., Ltd. The mice received appropriate care and were managed by competent and trained personnel, and the experiments were conducted in accordance with the guidelines provided in the Guide for the Care and Use of Laboratory Animals. The animal house\u0026apos;s temperature, humidity, and ventilation were regularly checked to guarantee ideal climatic conditions. Every effort was made to alleviate animal suffering while the experiments were being conducted. A seven-day acclimation period was provided for all animals prior to the commencement of experiments\u0026nbsp;after\u0026nbsp;their arrival.\u003c/p\u003e\n\u003ch2\u003eDaily observation of mice\u003c/h2\u003e\n\u003cp\u003eDaily observations were made on mice that received vaccinations. Monitoring was conducted for local reactions and adverse effects, including abnormalities, a decline in overall health, and decreased food consumption. Additionally, evidence of unkempt fur, restlessness, diarrhea, morbidity, and mortality was recorded.\u003c/p\u003e\n\u003ch2\u003eImmunity of mice\u003c/h2\u003e\n\u003cp\u003eMice were immunized by oral administration. Briefly, 5 ml of appropriate fresh media was used to stationary culture a solitary bacterial strain clone overnight in a 37\u0026deg;C incubator. The following day, the cultures were diluted 1:100 within a 100 ml identical medium and then incubated at 37\u0026deg;C until the bacterial solution\u0026apos;s optical density value at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) reached 0.85 (approximately 1 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e CFU / ml). The 100 ml suspension of the bacterial strain was gathered through centrifugation at 5000 rpm under room temperature conditions and resuspended in 2 ml of buffered saline with gelatin (BSG), at which point the bacterial concentration was approximately 1 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e CFU / 20 \u0026micro;l. The mouse cage was removed from food and water for six hours, and the animals were orally given 20 \u0026micro;l of BSG containing 10\u003csup\u003e9\u003c/sup\u003e CFU of the corresponding mutants. At 4 weeks, mice received a subsequent dose of the identical strain that matched the previous dosage.\u003c/p\u003e\n\u003ch2\u003eColonization of \u003cem\u003eS\u003c/em\u003e. Typhimurium mutants in organs\u003c/h2\u003e\n\u003cp\u003eTo assess the bacterial load in organs, the mice were orally given 20 \u0026micro;l of BSG containing 1 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e CFU mutant strains. At specific time intervals, three mice from each group were humanely euthanized. The Peyer\u0026apos;s patches (PP) were gathered from various locations on the surface of the small intestine. The bacterial load within the PP indicated the average bacterial load in the combined PP from every mouse. Samples from the spleen and liver were obtained and weighed individually. Each sample was added to 1 ml of phosphate buffer saline (PBS) to get a homogenized sample. Subsequently, various dilutions of the samples ranging from 10\u003csup\u003e\u0026minus;1\u003c/sup\u003e to 10\u003csup\u003e\u0026minus;3\u003c/sup\u003e (depending on the specific tissue) were dropped onto MacConkey agar and LB agar to measure the total amount of live bacteria present. MacConkey agar indicator plates are designed to exclude interference from \u003cem\u003eE. coli\u003c/em\u003e in tissue homogenates. The bacterial load was calculated based on the bacteria on the indicator plates. If no colonies were observed on the LB plate after culturing the homogenizing 0.1 ml of the organ, the selenite cysteine broth (100212, Sigma, USA) was used to inoculate the remaining PBS solution from each tissue sample the following day. Samples that showed positive results after being enriched in selenite cysteine broth at 37\u0026deg;C overnight were noted to have a CFU count of less than 10 per gram.\u003c/p\u003e\n\u003ch2\u003eTissue damage caused by \u003cem\u003eS.\u0026nbsp;\u003c/em\u003eTyphimurium mutants\u003c/h2\u003e\n\u003cp\u003eTo investigate the liver and spleen tissue damage caused by the mutants after the mice were orally given 20 \u0026micro;l of BSG containing 1 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e CFU mutant strains, we employed hematoxylin and eosin (H\u0026amp;E) staining for histopathological analysis. On the sixth day following oral inoculation, two mice in each group were humanely euthanized, and the liver and spleen were taken to determine the scope of the organ damage caused by the mutants. The standard procedure was followed for fixing and processing tissues for H\u0026amp;E staining. We observed the morphological aspects of tissues to identify signs of inflammation, including infiltration of macrophages, accumulation, distortion, and abnormal red pulp areas in the liver and spleen.\u003c/p\u003e\n\u003ch2\u003eSurvival assay\u003c/h2\u003e\n\u003cp\u003eThe protection rates of the immunized mice\u0026nbsp;were assessed at 9 weeks post-initial immunization by the oral challenge with 10\u003csup\u003e9\u003c/sup\u003e CFU of wild-type \u003cem\u003eS\u003c/em\u003e. Typhimurium (c3761) in 20\u0026nbsp;\u0026mu;l BSG (~10000\u0026nbsp;\u0026times;\u0026nbsp;LD\u003csub\u003e50\u003c/sub\u003e)\u0026nbsp;[19]. Mice were orally challenged with 10\u003csup\u003e7\u003c/sup\u003e CFU /20\u0026nbsp;\u0026mu;l of \u003cem\u003eS\u003c/em\u003e. Enteritidis (c3700) or \u003cem\u003eS\u003c/em\u003e. Choleraesuis (c3545) at 9 weeks after the first immunization to\u0026nbsp;assess\u0026nbsp;the cross-protection induced by\u0026nbsp;regulated delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e. The\u0026nbsp;c3700\u0026nbsp;and\u0026nbsp;c3545 were clinical isolates from the chicken and pig, respectively, both with an LD\u003csub\u003e50\u003c/sub\u003e of 10\u003csup\u003e5\u003c/sup\u003e CFU / 20\u0026nbsp;\u0026mu;l\u0026nbsp;[19]. Challenged mice were monitored daily for evidence of unkempt fur, restlessness, diarrhea, morbidity, and mortality, which were recorded for 25 days after the challenge.\u003c/p\u003e\n\u003ch2\u003eQuantitative enzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e\n\u003cp\u003eSerum was collected by puncturing the mandibular vein to obtain blood, followed by centrifugation at 3500 rpm. To analyze the secretory IgA (S-IgA), the vaginal tract of each mouse was washed with 60 \u0026mu;l of PBS, and the wash fluids were pooled together. The enzyme-linked immunosorbent assay (ELISA) was utilized to quantify the concentration of serum and vaginal wash antibodies against \u003cem\u003eSalmonella\u003c/em\u003e LPS or OMPs induced by\u0026nbsp;regulated\u0026nbsp;attenuated \u003cem\u003eSalmonella\u003c/em\u003e strains, following established protocols\u0026nbsp;[36, 37]. Briefly, solutions containing 200 ng per well of \u003cem\u003eSalmonella\u003c/em\u003e-derived OMP or LPS were suspended in 100 \u0026micro;l of coating buffer composed of sodium carbonate-bicarbonate (pH 9.6). These solutions were then utilized to coat 96-well plates overnight at a temperature of 4\u0026deg;C. To generate standard curves for each antibody isotype, we coated plates with duplicate samples of the purified mouse Ig isotype standard (1010-01, Southern Biotech, USA). Each well was coated with 200 ng of the standard in 100 \u0026micro;l of coating buffer. The plates were washed three times with tris buffered saline with 0.1% Tween 20 (TBST) and then blocked with 3% BSA (A602449, Sangon Biotech, China) for 2 hours at 37\u0026deg;C. Next, a 100 \u0026micro;l volume of the sample, diluted 100-fold or 10-fold, was added to each well in duplicate, the plates were then incubated for 1 hour at 37\u0026deg;C. To creat the standard curve, the unconjugated IgG (0107-01, Southern Biotech, USA), IgA (0106-01, Southern Biotech, USA), IgG1 (0102-01, Southern Biotech, USA), or IgG2a (0103-01, Southern Biotech, USA) obtained from ordinary mice were diluted by serial 2 times in 100 \u0026micro;l PBS of each plate well. The unconjugated IgG was diluted from 500 ng/ml to 0.488 ng/ml, IgG1 and IgG2a from 1 \u0026micro;g/ml to 8 ng/ml, and the IgA was diluted from 500 ng/ml to 0.488 ng/ml. Following TBST washing, each well was added with a 1:5000 dilution of biotinylated goat anti-mouse IgA (1040-08, Southern Biotech, USA), IgG1 (1070-08, Southern Biotech, USA), IgG2a (1080-08, Southern Biotech, USA), or IgG (1030-08, Southern Biotech, USA), the plates were then incubated at 37\u0026deg;C for 1 hour. A 100 \u0026micro;l volume of p-nitrophenyl phosphate (N1891, Sigma, USA) with a final concentration of 1 mg/ml was added to each of the wells for color development after they were previously incubated for an hour at 37\u0026deg;C with a 1:3000 dilution of streptavidin-alkaline phosphatase conjugate (7100-04, Southern Biotech, USA). Color development (absorbance) was read at 405 nm using an automated ELISA plate after appropriate incubation. Using linear regression in Excel (R\u003csup\u003e2\u003c/sup\u003e\u0026ge;0.95), the OD values at 405 nm were plotted against the representative concentrations of the diluted unconjugated antibody solutions to generate the standard curves. The corresponding standard curve was applied to calculate the total levels of antibodies in the samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow Cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were euthanized on day 72 after the first immunization, with 3 mice per group, the spleens were then collected using aseptic techniques. To obtain spleen cell suspensions, the samples were passed through a sterile cell strainer with a pore size of 40 \u0026micro;m (BS-40-CS, Biosharp,\u0026nbsp;China), and the erythrocytes were lysed using RBC lysis buffer (R1010,\u0026nbsp;Solarbio Biotech, China) for 5 min at 4℃. The resulting splenocytes were then suspended in RPMI 1640 medium, which was supplemented with 10% fetal calf serum (FCS). The single-cell suspension of spleen was stained with immune-cell-specific antibodies for flow cytometry (Beckmann Coulter Inc., Fullerton, CA, USA) as follows: APC anti-IgG antibody (405308,\u0026nbsp;Biolegend, USA), FITC anti-CD80 antibody (104706,\u0026nbsp;Biolegend, USA), Alexa Fluor\u0026reg; 700 anti-IgD antibody (405730,\u0026nbsp;Biolegend, USA), and Pacific Blue\u0026trade; anti-CD45R/B220 antibody for B\u003csub\u003eM\u003c/sub\u003e cell (103227,\u0026nbsp;Biolegend, USA). FITC anti-CD4 antibody, APC anti-CD279 (PD-1) antibody (135210,\u0026nbsp;Biolegend, USA), and Brilliant Violet 421\u0026trade; anti-CD185 (CXCR5) antibody (145512,\u0026nbsp;Biolegend, USA) for T\u003csub\u003efh\u003c/sub\u003e cell.\u003c/p\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eThe Graph-Pad Prism 8.0 software was utilized to conduct statistical calculations. Unless otherwise mentioned, numerical data were presented as means \u0026plusmn; SEM. To assess differences in the levels of antibodies and the quantities of B\u003csub\u003eM\u003c/sub\u003e and T\u003csub\u003efh\u003c/sub\u003e cells, we conducted either one-way or two-way ANOVA analysis, subsequently applying Tukey\u0026apos;s multiple comparisons tests. The log-rank test was implemented to assess differences in mouse survival, with the Kaplan-Meier survival curve serving as the monitoring tool. To compare means, the least significant difference test was employed. A p-value below 0.05 was considered to indicate a meaningful difference.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eConstruction of \u003cem\u003eSalmonella\u003c/em\u003e mutants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo finely regulate the expression of genes related to LPS synthesis to control the synthesis of the native \u003cem\u003eSalmonella\u003c/em\u003e LPS, we constructed at least two suicide plasmids carrying the TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e cassette and different SD sequences and/or start codons\u0026nbsp;for each gene for LPS synthesis\u0026nbsp;(Table 1, Table 2). These suicide plasmids were then utilized to integrate the TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e cassette carrying the candidate genes into the chromosome in place of the \u003cem\u003epagL\u003c/em\u003e gene since \u003cem\u003epagL\u003c/em\u003e deletion did not alter the virulence of \u003cem\u003eSalmonella\u003c/em\u003e and maintained the same immunogenicity and colonization ability as the wild-type \u003cem\u003eSalmonella\u003c/em\u003e [38]. For an example of the regulated gene \u003cem\u003ewaaC\u003c/em\u003e, we first introduced the\u0026nbsp;\u0026Delta;\u003cem\u003ewaaC\u003c/em\u003e and\u0026nbsp;\u0026Delta;\u003cem\u003epagL\u003c/em\u003e mutation in the genome of\u0026nbsp;c3761 by allelic exchange via conjugation with the\u0026nbsp;c7213, harboring the plasmid of\u0026nbsp;pSW012\u0026nbsp;or\u0026nbsp;pYA4284,\u0026nbsp;then two suicide plasmids (pSW033 and pSW034) were utilized to integrate the TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e cassette with \u003cem\u003ewaaC\u003c/em\u003e carrying two different SD sequences into the chromosome of SW086 (D\u003cem\u003ewaaC40\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagL7\u003c/em\u003e)\u0026nbsp;in place of the \u003cem\u003epagL\u003c/em\u003e gene to generate mutants SW093 (\u0026Delta;\u003cem\u003ewaaC40\u0026nbsp;\u003c/em\u003e\u0026Delta;\u003cem\u003epagL71\u003c/em\u003e::TT\u003cem\u003e\u0026nbsp;araC\u0026nbsp;\u003c/em\u003eP\u003csub\u003eBAD\u003c/sub\u003e\u003cem\u003e\u0026nbsp;waaC\u003c/em\u003e) and SW094 (\u0026Delta;\u003cem\u003ewaaC40\u0026nbsp;\u003c/em\u003e\u0026Delta;\u003cem\u003epagL72\u003c/em\u003e::TT\u003cem\u003e\u0026nbsp;araC\u0026nbsp;\u003c/em\u003eP\u003csub\u003eBAD\u003c/sub\u003e\u003cem\u003e\u0026nbsp;waaC\u003c/em\u003e), respectively. The results of silver staining experiments showed that\u0026nbsp;the mutant\u0026nbsp;SW079 (\u0026Delta;\u003cem\u003ewaaC40\u003c/em\u003e) was unable to synthesize a\u0026nbsp;typical LPS banding pattern\u0026nbsp;due to the deletion of the glycosyltransferase gene \u003cem\u003ewaaC\u003c/em\u003e, which is responsible for the addition of L-glycerol-D-mannoheptulose (Hep) residues to the core of LPS (Supplementary Figure 1, Figure 2A). When the mutants SW093 (\u0026Delta;\u003cem\u003ewaaC40\u0026nbsp;\u003c/em\u003e\u0026Delta;\u003cem\u003epagL71\u003c/em\u003e::TT\u003cem\u003e\u0026nbsp;araC\u0026nbsp;\u003c/em\u003eP\u003csub\u003eBAD\u003c/sub\u003e\u003cem\u003e\u0026nbsp;waaC\u003c/em\u003e) and SW094 (\u0026Delta;\u003cem\u003ewaaC40\u0026nbsp;\u003c/em\u003e\u0026Delta;\u003cem\u003epagL72\u003c/em\u003e::TT\u003cem\u003e\u0026nbsp;araC\u0026nbsp;\u003c/em\u003eP\u003csub\u003eBAD\u003c/sub\u003e\u003cem\u003e\u0026nbsp;waaC\u003c/em\u003e) were cultured \u003cem\u003ein vitro\u003c/em\u003e without 0.1% arabinose, both SW093 (\u0026Delta;\u003cem\u003ewaaC40\u0026nbsp;\u003c/em\u003e\u0026Delta;\u003cem\u003epagL71\u003c/em\u003e::TT\u003cem\u003e\u0026nbsp;araC\u0026nbsp;\u003c/em\u003eP\u003csub\u003eBAD\u003c/sub\u003e\u003cem\u003e\u0026nbsp;waaC\u003c/em\u003e) and SW094 (\u0026Delta;\u003cem\u003ewaaC40\u0026nbsp;\u003c/em\u003e\u0026Delta;\u003cem\u003epagL72\u003c/em\u003e::TT\u003cem\u003e\u0026nbsp;araC\u0026nbsp;\u003c/em\u003eP\u003csub\u003eBAD\u003c/sub\u003e\u003cem\u003e\u0026nbsp;waaC\u003c/em\u003e) mutants failed to synthesize a\u0026nbsp;typical LPS banding patterns, however only SW093 (\u0026Delta;\u003cem\u003ewaaC40\u0026nbsp;\u003c/em\u003e\u0026Delta;\u003cem\u003epagL71\u003c/em\u003e::TT\u003cem\u003e\u0026nbsp;araC\u0026nbsp;\u003c/em\u003eP\u003csub\u003eBAD\u003c/sub\u003e\u003cem\u003e\u0026nbsp;waaC\u003c/em\u003e) was able to synthesize\u0026nbsp;LPS banding pattern\u0026nbsp;similar to the wild strain when arabinose was present (Supplementary Figure 1). This result suggested that the mutant SW093 (\u0026Delta;\u003cem\u003ewaaC40\u0026nbsp;\u003c/em\u003e\u0026Delta;\u003cem\u003epagL71\u003c/em\u003e::TT\u003cem\u003e\u0026nbsp;araC\u0026nbsp;\u003c/em\u003eP\u003csub\u003eBAD\u003c/sub\u003e\u003cem\u003e\u0026nbsp;waaC\u003c/em\u003e)\u0026nbsp;exhibited the ability to\u0026nbsp;tightly\u0026nbsp;regulate LPS synthesis.\u0026nbsp;The same strategy was also applied to other genes, including \u003cem\u003ewaaF,\u003c/em\u003e \u003cem\u003ewaaG\u003c/em\u003e, \u003cem\u003ewaaI\u003c/em\u003e, \u003cem\u003ewaaJ\u003c/em\u003e, \u003cem\u003ewaaL\u003c/em\u003e,\u0026nbsp;and\u0026nbsp;\u003cem\u003ewbaP\u003c/em\u003e. The integrations resulted in generating mutant strains with varying lengths of LPS (Supplementary Figure 1, Figure 2A).\u003c/p\u003e\n\u003cp\u003eAfter mutants with tightly regulated LPS were successfully\u0026nbsp;identified in the wild-type background, these mutations were introduced into the mutant strain\u0026nbsp;SW067 (c9705\u0026nbsp;D\u003cem\u003efur9\u003c/em\u003e)\u0026nbsp;to investigate\u0026nbsp;whether\u0026nbsp;the regulated LPS synthesis\u0026nbsp;of\u0026nbsp;\u003cem\u003eSalmonella\u0026nbsp;\u003c/em\u003ehas an\u0026nbsp;effect on the\u0026nbsp;induced\u0026nbsp;cross-immune response\u0026nbsp;[38].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhenotype determination of the mutant strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhen the\u0026nbsp;genes\u0026nbsp;\u003cem\u003ewaaC\u003c/em\u003e, \u003cem\u003ewaaF\u003c/em\u003e, \u003cem\u003ewaaG\u003c/em\u003e, \u003cem\u003ewaaI\u003c/em\u003e, \u003cem\u003ewaaJ\u003c/em\u003e, \u003cem\u003ewaaL\u003c/em\u003e, or \u003cem\u003ewbaP\u003c/em\u003e were deleted from the genome of\u0026nbsp;SW067 (c9705\u0026nbsp;D\u003cem\u003efur9\u003c/em\u003e), the mutant strains exhibited an inability to produce typical LPS banding pattern, indicating that the absence of any glycosyltransferase in LPS synthesis hinders the formation of a whole LPS (Supplementary Figure 2). When cultured in the presence of 0.1% arabinose, a series of the mutant strains with regulated LPS synthesis including SW108 (\u003cem\u003ewaaC\u003c/em\u003e), SW110 (\u003cem\u003ewaaF\u003c/em\u003e), SW112 (\u003cem\u003ewaaG\u003c/em\u003e), SW114 (\u003cem\u003ewaaI\u003c/em\u003e), SW116 (\u003cem\u003ewaaJ\u003c/em\u003e), SW118 (\u003cem\u003ewaaL\u003c/em\u003e), and SW120 (\u003cem\u003ewaaP\u003c/em\u003e) exhibited typical LPS banding patterns while culturing in the absence of arabinose, these mutants displayed the rough LPS phenotype (Figure 2B). As the mutants will be administered orally and need to colonize the host organ, to evaluate whether they still display the tightly regulated LPS \u003cem\u003ein vivo\u003c/em\u003e, these mutants were cultured \u003cem\u003ein vitro\u003c/em\u003e in nutrient broth supplemented with varying concentrations of mouse serum, they were unable to synthesize the whole LPS in the absence of free arabinose (Supplementary Figure 3A). These results indicated that arabinose was unavailable in the serum and the synthesis of LPS in the mutants was entirely regulated by exogenously added arabinose. The outer membrane protein profile (Supplementary Figure 3B) demonstrated that this system did not impact the expression of OMPs, and MALDI-TOF MS analysis of lipid A showed that the phosphate group was removed to result in the production of monophosphorylated lipid A (Figure 2C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColonization of the regulated delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe colonization levels in the organs of mice reflect the interaction between the attenuated \u003cem\u003eSalmonella\u003c/em\u003e and the lymphoid tissue, impacting the capacity of attenuated\u0026nbsp;\u003cem\u003eSalmonella\u0026nbsp;\u003c/em\u003eto elicit immune responses.\u0026nbsp;To investigate the dissemination and proliferation capabilities of the regulated delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e in mice, we assessed the bacterial burdens in the liver, spleen, and PP of mice at 3, 6, 14, and 28 days following oral administration with 1\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU / 20\u0026nbsp;\u0026micro;l of the mutant strains, which were grown in the presence of 0.1% arabinose. The average CFU counts of all mutant strains isolated from the liver, spleen, or PP were approximately 10\u003csup\u003e4\u0026nbsp;\u003c/sup\u003eat 3 days after oral inoculation (Figure 3A, 3B, 3C), indicating that attenuated \u003cem\u003eSalmonella\u0026nbsp;\u003c/em\u003emaintained the ability to invade and colonize the gastrointestinal tract and were rapidly disseminated to systemic sites and underwent substantial proliferation within the organs. At 6 days post-infection, all mutant strains exhibited significantly decreased colonization levels in the liver, spleen, and PP. All mutant strains colonized and persisted in the organs for at least 28 days, and did not show significant differences. Within these 28 days, the bacterial counts in the liver, spleen, and PP gradually decreased significantly, suggesting that the regulated delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e could be cleared after colonization, and the abnormal appearance of the skin and other symptoms were not observed during the current experiment.\u003c/p\u003e\n\u003cp\u003eHistological analysis after immunization with the mutant strains\u003c/p\u003e\n\u003cp\u003eTo further investigate the potential of the\u0026nbsp;regulated\u0026nbsp;delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e to induce tissue inflammation, we conducted histopathology analyses on the liver and spleen of mice inoculated with mutant strains, using BSG as the control. The mice were orally inoculated with 1\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU of the mutant strains in the 20\u0026micro;l BSG,\u0026nbsp;which\u0026nbsp;were cultured in LB with 0.1% arabinose, and the examinations were performed 7 days post-infection. The results showed that the hepatocytes of mice in groups SW108 (\u003cem\u003ewaaC\u003c/em\u003e) and SW112 (\u003cem\u003ewaaG\u003c/em\u003e) displayed vacuolation, while those in groups SW110 (\u003cem\u003ewaaF\u003c/em\u003e) and SW120 (\u003cem\u003ewbaP\u003c/em\u003e) exhibited small focal necrosis in the liver\u0026nbsp;(Figure 4). Mice in the SW112 (\u003cem\u003ewaaG\u003c/em\u003e) group showed signs of inflammatory cell infiltration and small focal necrosis in the splenic tissue. No noticeable lesions were observed in mice from the SW114 (\u003cem\u003ewaaI\u003c/em\u003e), SW116 (\u003cem\u003ewaaJ\u003c/em\u003e), and SW118 (\u003cem\u003ewaaL\u003c/em\u003e) groups.\u003c/p\u003e\n\u003cp\u003eSerum IgG and mucosal IgA responses to OMPs from \u003cem\u003eS.\u0026nbsp;\u003c/em\u003eTyphimurium\u003c/p\u003e\n\u003cp\u003eTo evaluate the immune responses triggered by the\u0026nbsp;regulated\u0026nbsp;delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e, a total of 8 sets of mice were administered with 20 \u0026micro;l of BSG consisting of 1 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e CFU strains via the oral route. Subsequently, these mice were boosted with an identical dose of the matching mutants 4 weeks later. The levels of serum IgG and mucosal IgA responses towards OMPs derived from \u003cem\u003eS\u003c/em\u003e. Typhimurium were assessed using the ELISA method at\u0026nbsp;8 weeks after\u0026nbsp;initial immunization.\u0026nbsp;The SW114 (\u003cem\u003ewaaI\u003c/em\u003e), SW116 (\u003cem\u003ewaaJ\u003c/em\u003e), SW118 (\u003cem\u003ewaaL\u003c/em\u003e), and SW120 (\u003cem\u003ewbaP\u003c/em\u003e) strains elicited considerably increased levels of\u0026nbsp;IgG and IgA specific to \u003cem\u003eS\u003c/em\u003e. Typhimurium OMPs\u0026nbsp;compared\u0026nbsp;to the\u0026nbsp;SW108 (\u003cem\u003ewaaC\u003c/em\u003e), SW112 (\u003cem\u003ewaaG\u003c/em\u003e)\u0026nbsp;and BSG groups\u0026nbsp;(Figure 5A, 5B).\u0026nbsp;Moreover, the SW110 (\u003cem\u003ewaaF\u003c/em\u003e) strain, which synthesized native \u003cem\u003eS\u003c/em\u003e. Typhimurium LPS in the presence of arabinose \u003cem\u003ein vitro\u003c/em\u003e and produced only a partial inner core of LPS in the absence of arabinose \u003cem\u003ein vivo\u003c/em\u003e, also triggered mice to produce similar levels of\u003cem\u003e\u0026nbsp;S\u003c/em\u003e. Typhimurium\u0026nbsp;OMPs-specific\u0026nbsp;IgG and IgA antibodies as the SW118 (\u003cem\u003ewaaL\u003c/em\u003e) and SW120 (\u003cem\u003ewbaP\u003c/em\u003e) strains, which both\u0026nbsp;synthesized\u0026nbsp;an intact inner and outer core of LPS in the absence of arabinose\u0026nbsp;\u003cem\u003ein vivo\u003c/em\u003e (Figure 5A, 5B).\u0026nbsp;Although the levels of IgA specific to\u003cem\u003e\u0026nbsp;S\u003c/em\u003e. Typhimurium OMP triggered by the\u0026nbsp;SW116 (\u003cem\u003ewaaJ\u003c/em\u003e) strain\u0026nbsp;were significantly enhanced compared to the\u0026nbsp;BSG control groups, it was\u0026nbsp;lower than that of the\u0026nbsp;SW110 (\u003cem\u003ewaaF\u003c/em\u003e), SW114 (\u003cem\u003ewaaI\u003c/em\u003e), SW118 (\u003cem\u003ewaaL\u003c/em\u003e), and SW120 (\u003cem\u003ewbaP\u003c/em\u003e)\u0026nbsp;immunized groups (Figure 5B).\u0026nbsp;We also assessed the levels of anti-OMP IgG isotype subclasses IgG1 and IgG2a in the serum of mice. During the early phase, the IgG1 titers were comparable to the levels of IgG2a (data not presented). However, the SW114 (\u003cem\u003ewaaI\u003c/em\u003e), SW116 (\u003cem\u003ewaaJ\u003c/em\u003e), SW118 (\u003cem\u003ewaaL\u003c/em\u003e), and SW120 (\u003cem\u003ewbaP\u003c/em\u003e)\u0026nbsp;strains stimulated\u0026nbsp;a more rapid increase in IgG2a titers than IgG1 titers compared to\u0026nbsp;the\u0026nbsp;SW108 (\u003cem\u003ewaaC\u003c/em\u003e), SW112 (\u003cem\u003ewaaG\u003c/em\u003e)\u0026nbsp;and BSG control groups\u0026nbsp;at the eighth week after the initial immunization, suggesting a shift towards a Th1-biased immune response\u0026nbsp;(Figure 5C, 5D). Notably, the SW110 (\u003cem\u003ewaaF\u003c/em\u003e) strain not only provoked significantly increased IgG2a but also elicited\u0026nbsp;\u003cem\u003eS\u003c/em\u003e. Typhimurium OMP-specific\u0026nbsp;subclasses IgG1.\u003c/p\u003e\n\u003cp\u003eEvaluation of\u0026nbsp;cross-reactive antibodies\u0026nbsp;against OMPs from other \u003cem\u003eSalmonella\u0026nbsp;\u003c/em\u003eserotypes\u003c/p\u003e\n\u003cp\u003eTo further analyze the cross-immune response induced by the\u0026nbsp;regulated\u0026nbsp;delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e in mice, we assessed serum IgG and mucosal IgA responses to \u003cem\u003eS\u003c/em\u003e. Enteritidis and \u003cem\u003eS\u003c/em\u003e. Choleraesuis OMPs in mice at 8 weeks after the initial immunization.\u0026nbsp;Not only did the\u0026nbsp;SW118 (\u003cem\u003ewaaL\u003c/em\u003e) and SW120 (\u003cem\u003ewbaP\u003c/em\u003e)\u0026nbsp;mutants stimulate\u0026nbsp;\u003cem\u003eS\u003c/em\u003e. Enteritidis OMPs-specific IgG\u0026nbsp;production, but also the\u0026nbsp;SW114 (\u003cem\u003ewaaI\u003c/em\u003e)\u0026nbsp;and SW116 (\u003cem\u003ewaaJ\u003c/em\u003e)\u0026nbsp;mutants\u0026nbsp;elicit\u0026nbsp;higher levels\u0026nbsp;of IgG\u0026nbsp;specific to\u003cem\u003e\u0026nbsp;S\u003c/em\u003e. Enteritidis\u0026nbsp;OMP\u0026nbsp;than\u0026nbsp;the control groups of BSG\u0026nbsp;(Figure 6A).\u0026nbsp;The\u0026nbsp;SW118 (\u003cem\u003ewaaL\u003c/em\u003e)\u0026nbsp;mutant, but not\u0026nbsp;SW120 (\u003cem\u003ewbaP\u003c/em\u003e), could elicit\u0026nbsp;the highest\u0026nbsp;\u003cem\u003eS\u003c/em\u003e.\u0026nbsp;Enteritidis\u0026nbsp;OMP-specific\u0026nbsp;IgG titer among all groups. The\u0026nbsp;SW118 (\u003cem\u003ewaaL\u003c/em\u003e) and SW120 (\u003cem\u003ewbaP\u003c/em\u003e) strains\u0026nbsp;induced the production of\u0026nbsp;\u003cem\u003eS\u003c/em\u003e. Enteritidis OMP-specific vaginal IgA, whereas the\u0026nbsp;SW114 (\u003cem\u003ewaaI\u003c/em\u003e) and SW116 (\u003cem\u003ewaaJ\u003c/em\u003e) strains\u0026nbsp;failed to elicit vaginal mucosal IgA responses\u0026nbsp;(Figure 6B).\u0026nbsp;The levels of \u003cem\u003eS\u003c/em\u003e. Choleraesuis OMP-specific serum IgG and mucosal IgA triggered by the SW114 (\u003cem\u003ewaaI\u003c/em\u003e), SW116 (\u003cem\u003ewaaJ\u003c/em\u003e), SW118 (\u003cem\u003ewaaL\u003c/em\u003e), and SW120 (\u003cem\u003ewbaP\u003c/em\u003e) strains were significantly enhanced\u0026nbsp;compared\u0026nbsp;to those observed in the other treatment groups (Figure 6C,6D).\u003c/p\u003e\n\u003cp\u003eFurther analysis indicated that IgG2a was the major subtype of OMP-specific IgG triggered by the regulated delayed attenuated \u003cem\u003eSalmonella\u0026nbsp;\u003c/em\u003e(Figure 6E, 6F, 6G, 6H). The\u0026nbsp;SW114 (\u003cem\u003ewaaI\u003c/em\u003e), SW116 (\u003cem\u003ewaaJ\u003c/em\u003e), SW118 (\u003cem\u003ewaaL\u003c/em\u003e), and SW120 (\u003cem\u003ewbaP\u003c/em\u003e) strains\u0026nbsp;could stimulate similar levels of\u0026nbsp;\u003cem\u003eS\u003c/em\u003e. Enteritidis\u0026nbsp;OMP-specific IgG2a\u0026nbsp;compared to\u0026nbsp;SW108 (\u003cem\u003ewaaC\u003c/em\u003e), SW110 (\u003cem\u003ewaaF\u003c/em\u003e) and SW112 (\u003cem\u003ewaaG\u003c/em\u003e)\u0026nbsp;strains\u0026nbsp;(Figure 6F).\u0026nbsp;Interestingly, in addition to the\u0026nbsp;SW110 (\u003cem\u003ewaaF\u003c/em\u003e)\u0026nbsp;strains triggered\u0026nbsp;\u003cem\u003eS\u003c/em\u003e. Choleraesuis\u0026nbsp;OMP-specific IgG1 production, the\u0026nbsp;SW118 (\u003cem\u003ewaaL\u003c/em\u003e)\u0026nbsp;mutants, which synthesize both the complete inner and outer cores of the LPS\u0026nbsp;in the absence of arabinose \u003cem\u003ein vivo\u003c/em\u003e, also induced similar levels of\u0026nbsp;\u003cem\u003eS\u003c/em\u003e. Choleraesuis\u0026nbsp;OMP-specific IgG1\u0026nbsp;(Figure 6G). Both the\u0026nbsp;SW114 (\u003cem\u003ewaaI\u003c/em\u003e) and SW116 (\u003cem\u003ewaaJ\u003c/em\u003e)\u0026nbsp;strains induced significantly\u0026nbsp;enhanced\u0026nbsp;levels of\u0026nbsp;\u003cem\u003eS\u003c/em\u003e. Choleraesuis\u0026nbsp;OMP-specific IgG2a\u0026nbsp;relative to the\u0026nbsp;SW108 (\u003cem\u003ewaaC\u003c/em\u003e), SW110 (\u003cem\u003ewaaF\u003c/em\u003e),\u0026nbsp;SW112 (\u003cem\u003ewaaG\u003c/em\u003e)\u0026nbsp;strains,\u0026nbsp;but lower than the\u0026nbsp;SW118 (\u003cem\u003ewaaL\u003c/em\u003e) and SW120 (\u003cem\u003ewbaP\u003c/em\u003e) strains (Figure 6H).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of long-term\u003c/strong\u003e \u003cstrong\u003eimmune responses induced by regulated delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA successful vaccine must effectively elicit and establish lasting memory immunity; therefore, we investigated the long-term immune responses triggered in mice following regulated delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e immunization via oral administration. As SW114 (\u003cem\u003ewaaI\u003c/em\u003e), SW116 (\u003cem\u003ewaaJ\u003c/em\u003e), and SW118 (\u003cem\u003ewaaL\u003c/em\u003e) performed well in inducing antibody production, they were chosen to explore their abilities to induce long-term immunity. The IgG antibody levels in the serum of immunized mice were quantified on the 72nd day following the initial immunization. The results showed that\u0026nbsp;\u003cem\u003eS\u003c/em\u003e. Typhimurium,\u003cem\u003e\u0026nbsp;S\u003c/em\u003e. Enteritidis, and \u003cem\u003eS\u003c/em\u003e. Choleraesuis OMP-specific\u0026nbsp;IgG were still observable in all experimental groups compared to the BSG control group on 72 days (Supplementary Figure 4A, 4B, 4C). The levels of IgG antibodies in the SW118 (\u003cem\u003ewaaL\u003c/em\u003e) group exhibited a statistically significant increase compared to both the SW114 (\u003cem\u003ewaaI\u003c/em\u003e) and SW116 (\u003cem\u003ewaaJ\u003c/em\u003e) groups.\u003c/p\u003e\n\u003cp\u003eThe establishment of a durable population of memory B cells is crucial for a vaccine to develop sustained immunity\u0026nbsp;[39]. In this study, we conducted flow cytometry analysis on day 72 after the first immunization to evaluate the IgG\u003csup\u003e+\u003c/sup\u003e B\u003csub\u003eM\u003c/sub\u003e. The percentage of\u003csup\u003e\u0026nbsp;\u003c/sup\u003eB220\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eCD80\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eIgG\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eIgD\u003csup\u003e-\u003c/sup\u003e B\u003csub\u003eM\u003c/sub\u003e cells\u0026nbsp;[28]\u0026nbsp;in the groups immunized with the SW116 (\u003cem\u003ewaaJ\u003c/em\u003e) was considerably increased compared to the SW114 (\u003cem\u003ewaaI\u003c/em\u003e) and BSG groups but less than the SW118 (\u003cem\u003ewaaL\u003c/em\u003e) immunized group\u0026nbsp;(Figure 7A, 7B). These findings indicated that the regulated delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e constructed in this study, particularly the SW118 (\u003cem\u003ewaaL\u003c/em\u003e),\u0026nbsp;which both\u0026nbsp;synthesized\u0026nbsp;an intact inner and outer core of LPS in the absence of arabinose\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003ein vivo\u003c/em\u003e, could\u0026nbsp;elicit and establish lasting memory immunity in mice.\u003c/p\u003e\n\u003cp\u003eEvaluation of protection against challenge by wild-type\u003cem\u003e\u0026nbsp;S\u003c/em\u003e. Typhimurium\u003c/p\u003e\n\u003cp\u003eTo investigate the protection rates conferred by the\u0026nbsp;regulated\u0026nbsp;delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e, the immunized mice were orally challenged with 1 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e CFU (10000\u0026nbsp;\u0026times;\u0026nbsp;LD\u003csub\u003e50\u003c/sub\u003e) of\u0026nbsp;c3761 in 20 \u0026micro;l BSG at 9 weeks after the first immunization. The results showed that all of the mice in the BSG control group died; In contrast, all the vaccinated mice survived (Figure 8A), implying that all of the regulated\u0026nbsp;delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e were able to provide complete protection to the mice against the wild-type \u003cem\u003eS\u003c/em\u003e. Typhimurium challenge.\u003c/p\u003e\n\u003cp\u003eEvaluation of\u0026nbsp;cross-protection against infection with heterologous serotype of \u003cem\u003eSalmonella\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate protection against the challenges of different serotypes of \u003cem\u003eSalmonella\u003c/em\u003e, sixteen groups of BALB/c mice were used to immunize, including fourteen experimental groups and two control groups. The mice in each group were independently challenged by oral administration with 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e CFU (~100 \u0026times; LD\u003csub\u003e50\u003c/sub\u003e) of\u0026nbsp;c3700 (\u003cem\u003eS\u003c/em\u003e. Enteritidis) or\u0026nbsp;c3545 (\u003cem\u003eS\u003c/em\u003e. Choleraesuis) in 20\u0026nbsp;\u0026micro;l BSG, respectively. As shown in Figure 8B, SW116\u0026nbsp;(\u003cem\u003ewaaJ\u003c/em\u003e)\u0026nbsp;provided 100% protection in mice against the wild-type \u003cem\u003eS\u003c/em\u003e. Enteritidis challenge. The SW114 (\u003cem\u003ewaaI\u003c/em\u003e) could confer an 87.5% level of protection in mice. Both the\u0026nbsp;SW118 (\u003cem\u003ewaaL\u003c/em\u003e) and SW120 (\u003cem\u003ewbaP\u003c/em\u003e)\u0026nbsp;could provide\u0026nbsp;62.5% protection (Figure 8B). Although the SW108\u0026nbsp;(\u003cem\u003ewaaC\u003c/em\u003e), SW110\u0026nbsp;(\u003cem\u003ewaaF\u003c/em\u003e), and SW112\u0026nbsp;(\u003cem\u003ewaaG\u003c/em\u003e) mutants\u0026nbsp;did not provide significant protection for mice against the wild-type \u003cem\u003eS\u003c/em\u003e. Enteritidis challenge, it prolonged their survival time (Figure 8B).\u003c/p\u003e\n\u003cp\u003eThe protection rate against the challenge of\u003cem\u003e\u0026nbsp;S\u003c/em\u003e. Choleraesuis was inconsistent with the level of protection against \u003cem\u003eS\u003c/em\u003e. Enteritidis.\u0026nbsp;SW118\u0026nbsp;(\u003cem\u003ewaaL\u003c/em\u003e), SW114\u0026nbsp;(\u003cem\u003ewaaI\u003c/em\u003e), and SW116\u0026nbsp;(\u003cem\u003ewaaJ\u003c/em\u003e)\u0026nbsp;mutants provided protection rates of 75% and 50% for mice against the wild-type \u003cem\u003eS\u003c/em\u003e. Choleraesuis challenge, respectively. Interestingly, although SW108\u0026nbsp;(\u003cem\u003ewaaC\u003c/em\u003e)\u0026nbsp;did not confer a high level of protection against the \u003cem\u003eS\u003c/em\u003e. Enteritidis challenge, it provided the highest level of protection against the \u003cem\u003eS\u003c/em\u003e. Choleraesuis challenge with a survival rate of 87.5% (Figure 8C). The SW110\u0026nbsp;(\u003cem\u003ewaaF\u003c/em\u003e), SW120\u0026nbsp;(\u003cem\u003ewbaP\u003c/em\u003e), and SW112\u0026nbsp;(\u003cem\u003ewaaG\u003c/em\u003e) mutants\u0026nbsp;did not provide significant protection for mice against the wild-type \u003cem\u003eS\u003c/em\u003e. Choleraesuis challenge.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of immunogenicity against OMPs from different enteric bacteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur previous research has demonstrated that the absence of O-antigen stimulates elevated levels of antibodies against the outer membrane protein antigen in serum, and these antibodies exhibit cross-reactivity with conserved surface epitopes present in other enteric bacteria [19, 20, 40]. Therefore, we determined whether the regulated delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e mutants had a similar effect. The reactivity of immune sera obtained from orally immunized mice belonging to SW114 (\u003cem\u003ewaaI\u003c/em\u003e), SW116 (\u003cem\u003ewaaJ\u003c/em\u003e), and SW118 (\u003cem\u003ewaaL\u003c/em\u003e), as described in the previous experiment (Figure 6), was evaluated using ELISA against OMPs isolated from several\u003cem\u003e\u0026nbsp;\u003c/em\u003ewild-type \u003cem\u003eE.coli\u003c/em\u003e (O8, O138, O139) strains (Figure 9). The results roughly correlated with that we obtained against OMPs of heterologous serotype \u003cem\u003eSalmonella\u0026nbsp;\u003c/em\u003e(Figure 6). The three vaccine strains elicited a significant reactivity response in mice against OMPs from several \u003cem\u003eE. coli\u003c/em\u003e strains compared to the BSG control group, and there was no statistical difference between the groups (Figure 9).\u003cstrong\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study investigated the immunogenicity and protective efficacy of the regulated delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e mutants in mice. The goal was to develop potentially regulated delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e that could provide protection against both homologous and heterologous serotype \u003cem\u003eSalmonella\u003c/em\u003e infections. The serotypes of \u003cem\u003eS\u003c/em\u003e. Typhimurium, \u003cem\u003eS\u003c/em\u003e. Enteritidis, and \u003cem\u003eS\u003c/em\u003e. Choleraesuis in the NTS [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] belong to serogroups B1, D1, and C1, respectively, and have many natural hosts such as mice, chickens, poultry, and pigs [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Numerous studies have demonstrated that these serotypes have the ability to establish systemic infections in mice after oral administration [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR45 CR46\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Therefore, the mouse model was chosen to evaluate the immunogenicity of the regulated delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e mutants and their cross-protection efficiency against \u003cem\u003eS\u003c/em\u003e. Typhimurium, \u003cem\u003eS\u003c/em\u003e. Choleraesuis, and \u003cem\u003eS\u003c/em\u003e. Enteritidis infections in this study. These mutant strains were derived from the same genetic background, thus avoiding any potential interference factors to compare immunogenicity and cross-protection efficiency induced by these strains.\u003c/p\u003e \u003cp\u003eSuccessful colonization of attenuated \u003cem\u003eSalmonella\u003c/em\u003e is a prerequisite for inducing an effective immune response in mice, and the intact LPS structure is essential for \u003cem\u003eSalmonella\u003c/em\u003e adhesion and colonization in the intestine and systemic infection [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Previous studies have reported that while permanent deficiency of the O-antigen reduces host organ damage by \u003cem\u003eSalmonella\u003c/em\u003e, it also affects the level of colonization in mice [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. In this study, a series of \u003cem\u003eSalmonella\u003c/em\u003e mutants with arabinose-dependent regulated synthesis of LPS were constructed, and the results have shown that at least one of them carrying altered length of LPS was successfully identified to be tightly regulated by arabinose in LB media and Nutrient broth with mice serum (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), which was consistent with our previous studies [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The regulated delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e constructed in this study can control the synthesis of \u003cem\u003eSalmonella\u003c/em\u003e LPS by adding arabinose to the culture medium, so that \u003cem\u003eSalmonella\u003c/em\u003e still has an intact LPS structure at the early stage of colonization in mice.\u003c/p\u003e \u003cp\u003eThe regulated delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e exhibited a persistent ability of colonization in the initial stages of infection, at least maintaining this level for 6 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), indicating that these mutants with regulated LPS synthesis were able to successfully colonize the liver, spleen, and PP of mice during the early stages of infection due to the presence of the intact LPS, which is consistent with the timeframe required for antigen recognition and lymphocyte activation leading to the development of adaptive immunity. Subsequently, the level of colonization of the mutants gradually decreased in mice, suggesting that the mutants could be cleared after inducing immunity, thus ensuring the safety of candidate vaccines.\u003c/p\u003e \u003cp\u003eLive attenuated \u003cem\u003eSalmonella\u003c/em\u003e vaccines must balance safety with immunogenicity [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], which can be achieved by incorporating a minimum of two genetically unlinked attenuating mutations. Hence, we constructed \u003cem\u003eSalmonella\u003c/em\u003e mutants with the \u003cem\u003efur\u003c/em\u003e mutation and the arabinose-regulated LPS synthesis coupled with the deletion of \u003cem\u003epagL\u003c/em\u003e, \u003cem\u003elpxR\u003c/em\u003e, and codon-optimized \u003cem\u003elpxE\u003c/em\u003e substitution for \u003cem\u003epagP\u003c/em\u003e to engineer \u003cem\u003eSalmonella\u003c/em\u003e synthesizing the 4\u0026prime;-monophosphoryl-hexa-acylated lipid A, which decreases the interaction of lipid A and TLR4 and endotoxic activity while maintaining its good immunogenicity [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Histopathological assay conducted on mice 7 days post-immunization demonstrated that delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e mutants exhibited varying degrees of inflammation. Mice that were immunized with the strains SW108 (\u003cem\u003ewaaC\u003c/em\u003e) and SW110 (\u003cem\u003ewaaF\u003c/em\u003e), which are characterized by the deep rough phenotype and have a partial inner core of LPS in the absence of arabinose \u003cem\u003ein vivo\u003c/em\u003e, showed localized damage to liver cells. Among the mutants SW112 (\u003cem\u003ewaaG\u003c/em\u003e), SW114 (\u003cem\u003ewaaI\u003c/em\u003e), and SW116 (\u003cem\u003ewaaJ\u003c/em\u003e), which had an intact inner core or partial outer core of LPS in the absence of arabinose \u003cem\u003ein vivo\u003c/em\u003e, only strain SW112 (\u003cem\u003ewaaG\u003c/em\u003e) caused damage to liver and spleen cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Interestingly, no abnormalities were observed in mice infected with strains SW114 (\u003cem\u003ewaaI\u003c/em\u003e) and SW116 (\u003cem\u003ewaaJ\u003c/em\u003e). While the strains SW118 (\u003cem\u003ewaaL\u003c/em\u003e) and SW120 (\u003cem\u003ewbaP\u003c/em\u003e) had both the inner and outer core of LPS but lacked the O-antigen in the absence of arabinose \u003cem\u003ein vivo\u003c/em\u003e, mice that were immunized with strain SW118 (\u003cem\u003ewaaL\u003c/em\u003e) did not exhibit any observable abnormalities, while those inoculated with strain SW120 (\u003cem\u003ewbaP\u003c/em\u003e) showed small areas of necrosis in the liver. These results indicated that mutants exhibiting deep rough phenotype may still have significant adverse effects due to slower generation of a rough phenotype \u003cem\u003ein vivo\u003c/em\u003e. Additionally, while the process of LPS synthesis is intricate and subtle, even when the degree of truncated LPS is identical, the resulting properties observed in mice may differ.\u003c/p\u003e \u003cp\u003eAttenuated live \u003cem\u003eSalmonella\u003c/em\u003e vaccines have the ability to induce antibodies against conserved outer membrane proteins that are cross-reactive with other enteric pathogens, particularly in strains engineered to achieve down-regulation of O-antigen synthesis \u003cem\u003ein vivo\u003c/em\u003e. The immune responses observed in mice following immunization with regulated delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e were found to be varied. Despite the absence of a significant antibody response against OMPs of \u003cem\u003eS\u003c/em\u003e. Typhimurium when mice were administered SW108 (\u003cem\u003ewaaC\u003c/em\u003e) and SW112 (\u003cem\u003ewaaG\u003c/em\u003e), these mutants still provided complete protection in mice when exposed to a lethal dose of highly virulent wild-type \u003cem\u003eS\u003c/em\u003e. Typhimurium (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Considering that the regulated delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e was originated from UK1, this result is not unexpected, and it can be attributed to the presence of various surface antigens in \u003cem\u003eS\u003c/em\u003e. Typhimurium, such as flagellins, fimbriae, and enterobacterial common antigens (ECA), and the removal of immunodominant O-antigens from the surface of \u003cem\u003eSalmonella\u003c/em\u003e also enhances the immunogenicity of OMPs and other surface antigens.\u003c/p\u003e \u003cp\u003eOur aim was to develop a live attenuated \u003cem\u003eSalmonella\u003c/em\u003e to induce cross-immune responses effectively and confer cross-protection against infection of multiple serotypes of \u003cem\u003eSalmonella\u003c/em\u003e. In this study, mice in groups SW116 (\u003cem\u003ewaaJ\u003c/em\u003e), SW114 (\u003cem\u003ewaaI\u003c/em\u003e), and SW118 (\u003cem\u003ewaaL\u003c/em\u003e) exhibited significant antibody levels and a considerable survival rate when challenged with both wild-type \u003cem\u003eS\u003c/em\u003e. Enteritidis and \u003cem\u003eS\u003c/em\u003e. Choleraesuis, but mice in the SW108 (\u003cem\u003ewaaC\u003c/em\u003e) group, which exhibited negligible antibodies against OMPs from \u003cem\u003eS\u003c/em\u003e. Choleraesuis, had the highest survival rate compared to the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). In addition, mice in group SW120 (\u003cem\u003ewbaP\u003c/em\u003e), which displayed the identical LPS as SW118 (\u003cem\u003ewaaL\u003c/em\u003e), were characterized by elevated levels of antibodies, but displayed a considerably lower survival rate of only 37.5%, implying that no apparent relationship exists between antibody levels assessed in mice immunized with mutants and protection when challenged with other \u003cem\u003eSalmonella\u003c/em\u003e. It seems that the conclusion made in this study was not consistent with other research [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. \u003cem\u003eSalmonella\u003c/em\u003e possesses many immunologically related cross-reactive OMP antigens, including abundant (OmpA, OmpC, and OmpD) and minor (e.g., NmpC and OmpX) proteins as well as the most copious lipoprotein (murein lipoprotein). These OMPs, although possessing some micro-heterogeneity, nevertheless share antigenic determinants, which can induce antibodies cross-reactive immunity to heterologous \u003cem\u003eSalmonella\u003c/em\u003e, but exhibit less protective ability because not all antibodies elicited by OMPs bind effectively to the wild-type \u003cem\u003eSalmonella\u003c/em\u003e due to the masking effect of the lipopolysaccharide layer on the surface of \u003cem\u003eSalmonella\u003c/em\u003e. For example, OmpA, which is located as a monomer on the outer membrane of \u003cem\u003eSalmonella\u003c/em\u003e, produces channels in the lipopolysaccharide layer that are not large enough to allow antibodies to cross the O-Ag into the bacterial surface for binding, and therefore antibodies against OmpA are not protective [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, certain conserved protein antigens on the outer membrane of \u003cem\u003eSalmonella\u003c/em\u003e are immunoprotective because the footprint formed by these antigens is large enough for antibodies to readily cross the lipopolysaccharide layer. Such as OmpD in \u003cem\u003eS\u003c/em\u003e. Enteritidis is nearly identical to the \u003cem\u003eS\u003c/em\u003e. Typhimurium and differs only by a single amino acid, which is located on the outer membrane and exists as a trimer to generate a tunnel aperture size that allows a single Fab to cross the LPS channel and bind to the bacterial surface [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Although only one Fab can access the LPS channel created by certain trimeric proteins in the surrounding LPS layer, it is also able to confer protection against infection of \u003cem\u003eSalmonella\u003c/em\u003e. This explains our findings that cross-protection induced by the regulated delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e is related to the level of effective antibody. Although we do not know which outer membrane proteins other than OmpD are capable of generating larger tunnels that allow antibodies to cross the O-Ag to bind to the outer membrane, we demonstrated that certain conserved antigens on the outer membrane do provide cross-protection to the challenge of \u003cem\u003eSalmonella\u003c/em\u003e. In addition to the surface outer membrane proteins, the inner and outer cores of different serotypes of \u003cem\u003eSalmonella\u003c/em\u003e are conserved, and only SW118 (\u003cem\u003ewaaL\u003c/em\u003e) and SW120 (\u003cem\u003ewbaP\u003c/em\u003e) were able to recognize the LPS of wild-type \u003cem\u003eS\u003c/em\u003e. Typhimurium in immune sera from mice immunized with the different mutants (Supplementary Fig.\u0026nbsp;5). This result implies that the \u003cem\u003eSalmonella\u003c/em\u003e cores of LPS may also be one of the reasons why the mutants were able to induce cross-protective in mice.\u003c/p\u003e \u003cp\u003eThe vaccine administration induces the proliferation of long-lived plasma cells and memory B cells responsible for sustaining humoral immunity. In this study, it was observed that on day 72 after the initial immunization, the mice still exhibited significant levels of IgG antibodies. This finding suggests that the regulated delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e effectively stimulated the production of a substantial population of long-lived plasma cells in the mice (Supplementary Fig.\u0026nbsp;4), particularly in the case of SW118 (\u003cem\u003ewaaL\u003c/em\u003e). Furthermore, regulated delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e can effectively stimulate the generation of IgG\u003csup\u003e+\u003c/sup\u003e B\u003csub\u003eM\u003c/sub\u003e in mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This finding indicates that the regulated delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e can successfully induce a persistent humoral immune response and maintain immune memory in mice.\u003c/p\u003e \u003cp\u003eIn conclusion, we systematically investigated the immunogenicity and protective efficacy of delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e mutants. Our results indicate that strains containing whole-core oligosaccharides of lipid A not only expose more conserved OMPs but also have the ability to elicit enhanced cross-protective immunity against both homologous and heterologous \u003cem\u003eSalmonella\u003c/em\u003e infections.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal experiments were conducted in the Laboratory Animal Center of Southwest University after the Review Form for Laboratory Animal Welfare and Ethicals submitted by us was approved by IACUC (No. IACUC-20191120-02). We usually make an appointment in the online system with our registered personal information and carry out animal experiments after approval, and there is no license number for each animal experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study to reproduce these findings are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by\u0026nbsp;National Natural Science Foundation of China (82241063),\u0026nbsp;National Key Research and Development Program of China (2022YFD1800900), National Center of Technology Innovation for Pigs (NCTIP-XD/B11),\u0026nbsp;and\u0026nbsp;Sichuan Natural Science Foundation (2023YFH0080).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYH and QK initiated the research. XP and QK led the design of \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments, data acquisition and analysis, and manuscript preparation. QL, YL, WJ, MR, XF, and LY aided in data acquisition. All authors contributed to the article and approved the submitted version.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDuff, N. et al. Global Action for Local Impact: The 11th International Conference on Typhoid and Other Invasive Salmonelloses. \u003cem\u003eClin. Infect. Dis\u003c/em\u003e. 71, S59-S63 (2020). doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/cid/ciaa236\u003c/span\u003e\u003cspan address=\"10.1093/cid/ciaa236\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, Y. et al. The temporal dynamics of antimicrobial-resistant \u003cem\u003eSalmonella enterica\u003c/em\u003e and predominant serovars in China. Natl. Sci. Rev. 10, nwac269 (2023). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/nsr/nwac269\u003c/span\u003e\u003cspan address=\"10.1093/nsr/nwac269\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGal-Mor, O. et al. Same species, different diseases: how and why typhoidal and non-typhoidal \u003cem\u003eSalmonella enterica\u003c/em\u003e serovars differ. Front. Microbiol. 5, 391 (2014). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fmicb.2014.00391\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2014.00391\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStanaway J. D. et al. The global burden of non-typhoidal \u003cem\u003eSalmonella\u003c/em\u003e invasive disease: a systematic analysis for the Global Burden of Disease Study 2017. Lancet. Infect. Dis. 19, 1312\u0026ndash;1324. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S1473-3099(19)30418-9\u003c/span\u003e\u003cspan address=\"10.1016/S1473-3099(19)30418-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGal\u0026aacute;n-Rela\u0026ntilde;o, \u0026Aacute;. et al. \u003cem\u003eSalmonella\u003c/em\u003e and Salmonellosis: An Update on Public Health Implications and Control Strategies. Animals. 13, 3666 (2023). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ani13233666\u003c/span\u003e\u003cspan address=\"10.3390/ani13233666\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReddy, E. A. et al. Community-acquired bloodstream infections in Africa: a systematic review and meta-analysis. Lancet Infect Dis. 10, 417\u0026ndash;432 (2010). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S1473-3099(10)70072-4\u003c/span\u003e\u003cspan address=\"10.1016/S1473-3099(10)70072-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilliamson, D. A. et al. Increasing antimicrobial resistance in Nontyphoidal \u003cem\u003eSalmonella\u003c/em\u003e isolates in Australia from 1979 to 2015. Antimicrob. Agents. Ch. 62, e02012-2017 (2018). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/AAC.02012-17\u003c/span\u003e\u003cspan address=\"10.1128/AAC.02012-17\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScallan, E. et al. Foodborne illness acquired in the United States\u0026ndash;major pathogens. Emerg. Infect. Dis. 17, 7\u0026ndash;15 (2011). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3201/eid1701.P11101\u003c/span\u003e\u003cspan address=\"10.3201/eid1701.P11101\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAuthority, E. F. S. et al. The European Union One Health 2018 Zoonoses Report. EFSA. J. 17, e05926 (2019). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2903/j.efsa.2019.5926\u003c/span\u003e\u003cspan address=\"10.2903/j.efsa.2019.5926\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerrari, R. G. et al. Worldwide Epidemiology of \u003cem\u003eSalmonella\u003c/em\u003e serovars in animal-1 based foods: a meta-analysis. Appl. Environ. Microbiol. 85, (2019). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/AEM.00591-19\u003c/span\u003e\u003cspan address=\"10.1128/AEM.00591-19\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, X. et al. Prevalence, bacterial load, and antimicrobial resistance of \u003cem\u003eSalmonella\u003c/em\u003e serovars isolated from retail mmeat and meat products in China. Front. Microbiol. 10, 2121 (2019). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fmicb.2019.02121\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2019.02121\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrost, I. et al. The role of bacterial vaccines in the fight against antimicrobial resistance: an analysis of the preclinical and clinical development pipeline. Lancet. Microbe. 4, e113-e125 (2023). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S2666-5247(22)00303-2\u003c/span\u003e\u003cspan address=\"10.1016/S2666-5247(22)00303-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMacLennan, C. A. et al. Vaccines against invasive \u003cem\u003eSalmonella\u003c/em\u003e disease current status and future directions. Hum. Vaccin. Immunother. 10, 1478\u0026ndash;1493 (2014). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4161/hv.29054\u003c/span\u003e\u003cspan address=\"10.4161/hv.29054\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFuche, F. J. et al. \u003cem\u003eSalmonella\u003c/em\u003e serogroup C: current status of vaccines and why they are needed. Clin. Vaccine. Immunol. 23, 737\u0026ndash;745 (2016). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/CVI.00243-16\u003c/span\u003e\u003cspan address=\"10.1128/CVI.00243-16\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWigley, P. \u003cem\u003eSalmonella enterica\u003c/em\u003e serovar Gallinarum: addressing fundamental questions in bacteriology sixty years on from the 9R vaccine. Avian. Pathol. 46, 119\u0026ndash;124 (2017). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.mib.2022.102262\u003c/span\u003e\u003cspan address=\"10.1016/j.mib.2022.102262\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, Q. et al. Complete genome sequence of \u003cem\u003eSalmonella enterica\u003c/em\u003e serovar Choleraesuis vaccine strain C500 attenuated by chemical mutation. Genome. Announc. 2, e01022-14 (2014). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/genomeA.01022-14\u003c/span\u003e\u003cspan address=\"10.1128/genomeA.01022-14\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDominguez-Medina, C. C. et al. Outer membrane protein size and LPS O-antigen define protective antibody targeting to the \u003cem\u003eSalmonella\u003c/em\u003e surface. Nat. Commun. 11, 851 (2020). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41467-020-14655-9\u003c/span\u003e\u003cspan address=\"10.1038/s41467-020-14655-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKong, Q. et al. Regulated delayed expression of \u003cem\u003erfaH\u003c/em\u003e in an attenuated \u003cem\u003eSalmonella enterica\u003c/em\u003e serovar \u003cem\u003etyphimurium\u003c/em\u003e vaccine enhances immunogenicity of outer membrane proteins and a heterologous antigen. \u003cem\u003eInfect. Immun.\u003c/em\u003e 77, 5572\u0026ndash;5582 (2009). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/IAI.00831-09\u003c/span\u003e\u003cspan address=\"10.1128/IAI.00831-09\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, Q. et al. Outer membrane vesicles derived from \u003cem\u003eSalmonella\u003c/em\u003e Typhimurium mutants with truncated LPS induce cross-protective immune responses against infection of \u003cem\u003eSalmonella enterica\u003c/em\u003e serovars in the mouse model. Int. J. Med. Microbiol. 306, 697\u0026ndash;706 (2016). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ijmm.2016.08.004\u003c/span\u003e\u003cspan address=\"10.1016/j.ijmm.2016.08.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, Q. et al. Immunogenicity and cross-protective efficacy induced by outer membrane proteins from \u003cem\u003eSalmonella\u003c/em\u003e Typhimurium mutants with truncated LPS in Mice. Int. J. Mol. Sci. 17, 416 (2016). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms17030416\u003c/span\u003e\u003cspan address=\"10.3390/ijms17030416\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCurtiss, R. et al. \u003cem\u003eSalmonella enterica\u003c/em\u003e serovar Typhimurium strains with regulated delayed attenuation in vivo. Infect. Immun. 77, 1071\u0026ndash;1082 (2009). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/IAI.00693-08\u003c/span\u003e\u003cspan address=\"10.1128/IAI.00693-08\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKong, Q. et al. Regulated delayed expression of \u003cem\u003erfc\u003c/em\u003e enhances the immunogenicity and protective efficacy of a heterologous antigen delivered by live attenuated \u003cem\u003eSalmonella enterica\u003c/em\u003e vaccines. Vaccine. 28, 6094\u0026ndash;6103 (2010). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.vaccine.2010.06.074\u003c/span\u003e\u003cspan address=\"10.1016/j.vaccine.2010.06.074\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuzman, L.-M. et al. Tight regulation, modulation, and high-level expression by vectors containing the arabinose P\u003csub\u003eBAD\u003c/sub\u003e promoter. J. Bacteriol. 177, 4121\u0026ndash;4130 (1995). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/jb.177.14.4121-4130.1995\u003c/span\u003e\u003cspan address=\"10.1128/jb.177.14.4121-4130.1995\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBertani, G. Studies on lysogenesis I: the mode of phage liberation by lysogenic \u003cem\u003eEscherichia coli\u003c/em\u003e. J Bacteriol. 62, 293\u0026ndash;300 (1951). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/jb.62.3.293-300.1951\u003c/span\u003e\u003cspan address=\"10.1128/jb.62.3.293-300.1951\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakayama, K. et al. Construction of an Asd\u0026thinsp;+\u0026thinsp;expression-cloning vector: stable maintenance and high level expression of cloned genes in a \u003cem\u003eSalmonella\u003c/em\u003e vaccine strain. Bio/technology. 6, 693\u0026ndash;697 (1988). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nbt0688-693\u003c/span\u003e\u003cspan address=\"10.1038/nbt0688-693\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCurtiss, R. et al. Nonrecombinant and recombinant avirulent \u003cem\u003eSalmonella\u003c/em\u003e vaccines for poultry. Vet. Immunol. Immunopathol. 54, 365\u0026ndash;372 (1996). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S0165-2427(96)05683-8\u003c/span\u003e\u003cspan address=\"10.1016/S0165-2427(96)05683-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEdwards, R. A. et al. Improved allelic exchange vectors and their use to analyze 987P fimbria gene expression. Gene. 207, 149\u0026ndash;157 (1998). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S0378-1119(97)00619-7\u003c/span\u003e\u003cspan address=\"10.1016/S0378-1119(97)00619-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSu, H. et al. Regulated delayed \u003cem\u003eShigella flexneri\u003c/em\u003e 2a O-antigen synthesis in live recombinant \u003cem\u003eSalmonella enterica\u003c/em\u003e serovar Typhimurium induces comparable levels of protective immune responses with constitutive antigen synthesis system. Theranostics. 9, 3565\u0026ndash;3579 (2019). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7150/thno.33046\u003c/span\u003e\u003cspan address=\"10.7150/thno.33046\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei, et al. Highly efficient method for introducing successive multiple scarless gene deletions and markerless gene insertions into the \u003cem\u003eYersinia pestis\u003c/em\u003e chromosome. Appl. Environ. Microbiol. 74, 4241\u0026ndash;4245 (2008). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/AEM.00940-08\u003c/span\u003e\u003cspan address=\"10.1128/AEM.00940-08\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKang, et al. Immune responses to recombinant \u003cem\u003epneumococcal\u003c/em\u003e PspA antigen delivered by live attenuated \u003cem\u003eSalmonella enterica\u003c/em\u003e serovar \u003cem\u003etyphimurium\u003c/em\u003e vaccine. Infect. Immun. 70, 1739 (2002). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/IAI.70.4.1739-1749.2002\u003c/span\u003e\u003cspan address=\"10.1128/IAI.70.4.1739-1749.2002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYi, E. C. et al. Rapid isolation method for lipopolysaccharide and lipid A from Gram-negative bacteria. Analyst. 125, 651\u0026ndash;656 (2000). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/B000368I\u003c/span\u003e\u003cspan address=\"10.1039/B000368I\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHirschfeld, M. et al. Cutting Edge: Repurification of lipopolysaccharide eliminates signaling through both human and murine Toll-Like receptor 2. J. Immunol. 165, 618\u0026ndash;622 (2000). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4049/jimmunol.165.2.618\u003c/span\u003e\u003cspan address=\"10.4049/jimmunol.165.2.618\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHitchcock, P. J. et al. Morphological heterogeneity among \u003cem\u003eSalmonella\u003c/em\u003e lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J. Bacteriol. 154, 269\u0026ndash;277 (1983). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/jb.154.1.269-277.1983\u003c/span\u003e\u003cspan address=\"10.1128/jb.154.1.269-277.1983\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGibbons, H. S. et al. An inner membrane dioxygenase that generates the 2-hydroxymyristate moiety of \u003cem\u003eSalmonella\u003c/em\u003e lipid A. Biochemistry. 47, 2814\u0026ndash;2825 (2008). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/bi702457c\u003c/span\u003e\u003cspan address=\"10.1021/bi702457c\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBligh, E. L. G. et al. A rapid method of total lipid extraction and purification. Can. J. Biochem Physiol. 37, 911\u0026ndash;917 (1959). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1139/o59-099\u003c/span\u003e\u003cspan address=\"10.1139/o59-099\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, Y. et al. A \u003cem\u003esopB\u003c/em\u003e deletion mutation enhances the immunogenicity and protective efficacy of a heterologous antigen delivered by live attenuated \u003cem\u003eSalmonella enterica\u003c/em\u003e vaccines. Infect. Immun. 76, 5238\u0026ndash;5246 (2008). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/IAI.00720-08\u003c/span\u003e\u003cspan address=\"10.1128/IAI.00720-08\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakahashi, K. et al. Detection of lipopolysaccharide (LPS) and identification of its serotype by an enzyme-linked immunosorbent assay (ELISA) using poly-L-lysine. J. Immunol. Methods. 153, 67\u0026ndash;71 (1992). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/0022-1759(92)90306-E\u003c/span\u003e\u003cspan address=\"10.1016/0022-1759(92)90306-E\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKong, Q. et al. \u003cem\u003eSalmonella\u003c/em\u003e synthesizing 1-monophosphorylated lipopolysaccharide exhibits low endotoxic activity while retaining its immunogenicity. J. Immunol. 187, 412\u0026ndash;423 (2011). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4049/jimmunol.1100339\u003c/span\u003e\u003cspan address=\"10.4049/jimmunol.1100339\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, S. W. et al. Mechanism of antigen presentation and specificity of antibody cross-reactivity elicited by an oligosaccharide-conjugate cancer vaccine. J. Am. Chem. Soc. 145, 9840\u0026ndash;9849 (2023). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/jacs.3c02003\u003c/span\u003e\u003cspan address=\"10.1021/jacs.3c02003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKong, Q. et al. Effect of deletion of genes involved in lipopolysaccharide core and O-antigen synthesis on virulence and immunogenicity of \u003cem\u003eSalmonella enterica\u003c/em\u003e serovar \u003cem\u003etyphimurium\u003c/em\u003e. Infect. Immun. 79, 4227\u0026ndash;4239 (2011). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/IAI.05398-11\u003c/span\u003e\u003cspan address=\"10.1128/IAI.05398-11\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFierer, J. Invasive Non-typhoidal \u003cem\u003eSalmonella\u003c/em\u003e (iNTS) Infections. Clin. Infect. Dis. 75, 732\u0026ndash;738 (2022). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/cid/ciac035\u003c/span\u003e\u003cspan address=\"10.1093/cid/ciac035\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSantos, R. L. et al. Animal models of \u003cem\u003eSalmonella\u003c/em\u003e infections: enteritis versus typhoid fever. Microbes. Infect. 3, 1335\u0026ndash;1344 (2001). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/s1286-4579(01)01495-2\u003c/span\u003e\u003cspan address=\"10.1016/s1286-4579(01)01495-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, B. X. et al. One species, different diseases: the unique molecular mechanisms that underlie the pathogenesis of typhoidal \u003cem\u003eSalmonella\u003c/em\u003e infections. Curr. Opin. Microbiol. 72, 102262 (2023). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.mib.2022.102262\u003c/span\u003e\u003cspan address=\"10.1016/j.mib.2022.102262\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerez Jorge, G. et al. p53 gene delivery via a recombinant \u003cem\u003eSalmonella enterica\u003c/em\u003e Typhimurium leads to human bladder carcinoma cell death in vitro. Lett. Appl. Microbiol. 75, 1010\u0026ndash;1020 (2022). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/lam.13777\u003c/span\u003e\u003cspan address=\"10.1111/lam.13777\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, Q. et al. Live attenuated \u003cem\u003eSalmonella enterica\u003c/em\u003e serovar Choleraesuis vector delivering a conserved surface protein enolase induces high and broad protection against \u003cem\u003eStreptococcus suis\u003c/em\u003e serotypes 2, 7, and 9 in mice. Vaccine. 38, 6904\u0026ndash;6913 (2020). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.vaccine.2020.08.062\u003c/span\u003e\u003cspan address=\"10.1016/j.vaccine.2020.08.062\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFelgner, S. et al. \u003cem\u003earoA\u003c/em\u003e-deficient \u003cem\u003eSalmonella enterica\u003c/em\u003e serovar Typhimurium is mmore than a metabolically attenuated mutant. \u003cem\u003emBio\u003c/em\u003e. 7, e01220-16 (2016). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/mBio.01220-16\u003c/span\u003e\u003cspan address=\"10.1128/mBio.01220-16\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSears, K. T. et al. Evaluation of three candidate live-attenuated \u003cem\u003eSalmonella enterica\u003c/em\u003e serovar Typhimurium vaccines to prevent Non-Typhoidal \u003cem\u003eSalmonella\u003c/em\u003e infection in an infant mouse model. Vaccines. 11, 1562 (2023). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/vaccines11101562\u003c/span\u003e\u003cspan address=\"10.3390/vaccines11101562\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSenevirathne, A. et al. Assessing an O-antigen deficient, live attenuated \u003cem\u003eSalmonella\u003c/em\u003e Gallinarium strain that is DIVA compatible, environmentally safe, and protects chickens against fowl typhoid. Dev. Comp. Immunol. 133, 104433 (2022). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.dci.2022.104433\u003c/span\u003e\u003cspan address=\"10.1016/j.dci.2022.104433\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJi, H. J. et al. Development of live attenuated. Front. Immunol. 13, 931052 (2022). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2022.931052\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2022.931052\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, Y. et al. Endogenous stimulus-powered antibiotic release from nanoreactors for a combination therapy of bacterial infections. Nat. Commun. 10, 4464 (2019). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41467-019-12233-2\u003c/span\u003e\u003cspan address=\"10.1038/s41467-019-12233-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoland, K. et al. Construction and evaluation of a delta \u003cem\u003ecya\u003c/em\u003e delta \u003cem\u003ecrp Salmonella typhimurium\u003c/em\u003e strain expressing avian pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e O78 LPS as a vaccine to prevent airsacculitis in chickens. Avian. diseases. 43, 429\u0026ndash;441 (1999). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2307/1592640\u003c/span\u003e\u003cspan address=\"10.2307/1592640\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. Bacterial strains and plasmids used in this study\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"107%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrains or plasmids\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDescriptions\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eSalmonella\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;strains\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003ec3761\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eWild type \u003cem\u003eS\u003c/em\u003e. Typhimurium, UK-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003e[26]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003ec3700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003e\u003cem\u003eS\u003c/em\u003e. Enteritidis, a clinical isolate from chicken, S246\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003e[20]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003ec3545\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003e\u003cem\u003eS\u003c/em\u003e. Choleraesuis, a clinical isolate from pig, S340\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003e[20]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003ec9705\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003eD\u003cem\u003epagL7\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagP81\u003c/em\u003e::P\u003csub\u003elpp\u0026nbsp;\u003c/sub\u003e\u003cem\u003elpxE\u0026nbsp;\u003c/em\u003eD\u003cem\u003elpxR9\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\" valign=\"top\"\u003e\n \u003cp\u003e[38]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003eSW067\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003ec9705\u0026nbsp;D\u003cem\u003efur9\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\" valign=\"top\"\u003e\n \u003cp\u003eLab stock\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW079\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003ewaaC40\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW080\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003ewaaF41\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW081\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003ewaaG42\u003c/em\u003e, the\u003cem\u003e\u0026nbsp;\u003c/em\u003esame strain as\u003cem\u003e\u0026nbsp;\u003c/em\u003ec11308\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\" valign=\"top\"\u003e\n \u003cp\u003e[40]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW082\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003ewaaI43\u003c/em\u003e,\u003cem\u003e\u0026nbsp;\u003c/em\u003ethe\u003cem\u003e\u0026nbsp;\u003c/em\u003esame strain as\u0026nbsp;c11309\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\" valign=\"top\"\u003e\n \u003cp\u003e[40]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW083\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003ewaaJ44\u003c/em\u003e, the\u003cem\u003e\u0026nbsp;\u003c/em\u003esame strain as\u0026nbsp;c11310\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\" valign=\"top\"\u003e\n \u003cp\u003e[40]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW084\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003ewaaL46\u003c/em\u003e,\u003cem\u003e\u0026nbsp;\u003c/em\u003ethe\u003cem\u003e\u0026nbsp;\u003c/em\u003esame strain as\u0026nbsp;c11312\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\" valign=\"top\"\u003e\n \u003cp\u003e[40]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW085\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003ewbaP45\u003c/em\u003e, the\u003cem\u003e\u0026nbsp;\u003c/em\u003esame strain as\u0026nbsp;c11311\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003e[40]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW086\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003ewaaC40\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagL7\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW087\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003ewaaF41\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagL7\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW088\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003ewaaG42\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagL7\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW089\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003ewaaI43\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagL7\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW090\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003ewaaJ44\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagL7\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW091\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003ewaaL46\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagL7\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW092\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003ewbaP45\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagL7\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW093\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003ewaaC40\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagL71\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaC\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW094\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003ewaaC40\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagL72\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaC\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW095\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003ewaaF41\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagL73\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaF\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW096\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003ewaaF41\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagL74\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaF\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW097\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003ewaaG42\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagL75\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaG\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW098\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003ewaaG42\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagL76\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaG\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW099\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003ewaaI43\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagL77\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaI\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003ewaaI43\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagL78\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaI\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW101\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003ewaaJ44\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagL79\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaJ\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003ewaaJ44\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagL80\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaJ\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW103\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003ewaaL46\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagL81\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaL\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW104\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003ewaaL46\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagL82\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaL\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW105\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003ewbaP45\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagL83\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e D\u003cem\u003ewbaP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW106\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003ewbaP45\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagL84\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e D\u003cem\u003ewbaP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW107\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003epagL7\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagP81\u003c/em\u003e::P\u003csub\u003elpp\u003c/sub\u003e \u003cem\u003elpxE\u0026nbsp;\u003c/em\u003eD\u003cem\u003elpxR9\u003c/em\u003e D\u003cem\u003efur9\u0026nbsp;\u003c/em\u003e∆\u003cem\u003ewaaC40\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW108\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003epagL71\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaC\u003c/em\u003e D\u003cem\u003epagP81\u003c/em\u003e::P\u003csub\u003elpp\u003c/sub\u003e \u003cem\u003elpxE\u0026nbsp;\u003c/em\u003eD\u003cem\u003elpxR9\u003c/em\u003e D\u003cem\u003efur9\u0026nbsp;\u003c/em\u003e∆\u003cem\u003ewaaC40\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003epagL7\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagP81\u003c/em\u003e::P\u003csub\u003elpp\u003c/sub\u003e \u003cem\u003elpxE\u0026nbsp;\u003c/em\u003eD\u003cem\u003elpxR9\u0026nbsp;\u003c/em\u003eD\u003cem\u003efur9\u0026nbsp;\u003c/em\u003e∆\u003cem\u003ewaaF41\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW110\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003epagL74\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaF\u003c/em\u003e D\u003cem\u003epagP81\u003c/em\u003e::P\u003csub\u003elpp\u003c/sub\u003e \u003cem\u003elpxE\u0026nbsp;\u003c/em\u003eD\u003cem\u003elpxR9\u003c/em\u003e D\u003cem\u003efur9\u0026nbsp;\u003c/em\u003e∆\u003cem\u003ewaaF41\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW111\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003epagL7\u003c/em\u003e D\u003cem\u003epagP81\u003c/em\u003e::P\u003csub\u003elpp\u003c/sub\u003e \u003cem\u003elpxE\u0026nbsp;\u003c/em\u003eD\u003cem\u003elpxR9\u0026nbsp;\u003c/em\u003eD\u003cem\u003efur9\u0026nbsp;\u003c/em\u003e∆\u003cem\u003ewaaG42\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW112\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003epagL76\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaG\u003c/em\u003e D\u003cem\u003epagP81\u003c/em\u003e::P\u003csub\u003elpp\u003c/sub\u003e \u003cem\u003elpxE\u0026nbsp;\u003c/em\u003eD\u003cem\u003elpxR9\u003c/em\u003e D\u003cem\u003efur9\u0026nbsp;\u003c/em\u003e∆\u003cem\u003ewaaG42\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW113\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003epagL7\u003c/em\u003e D\u003cem\u003elpxR9\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagP81\u003c/em\u003e::P\u003csub\u003elpp\u003c/sub\u003e \u003cem\u003elpxE\u0026nbsp;\u003c/em\u003eD\u003cem\u003efur9 ∆waaI43\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW114\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003epagL77\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaI\u003c/em\u003e D\u003cem\u003epagP81\u003c/em\u003e::P\u003csub\u003elpp\u003c/sub\u003e \u003cem\u003elpxE\u0026nbsp;\u003c/em\u003eD\u003cem\u003elpxR9\u003c/em\u003e D\u003cem\u003efur9\u0026nbsp;\u003c/em\u003e∆\u003cem\u003ewaaI43\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW115\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003epagL7\u0026nbsp;\u003c/em\u003eD\u003cem\u003epagP81\u003c/em\u003e::P\u003csub\u003elpp\u003c/sub\u003e \u003cem\u003elpxE\u0026nbsp;\u003c/em\u003eD\u003cem\u003elpxR9\u003c/em\u003e D\u003cem\u003efur9\u0026nbsp;\u003c/em\u003e∆\u003cem\u003ewaaJ44\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW116\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003epagL79\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaJ\u003c/em\u003e D\u003cem\u003epagP81\u003c/em\u003e::P\u003csub\u003elpp\u003c/sub\u003e \u003cem\u003elpxE\u0026nbsp;\u003c/em\u003eD\u003cem\u003elpxR9\u003c/em\u003e D\u003cem\u003efur9\u0026nbsp;\u003c/em\u003e∆\u003cem\u003ewaaJ44\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW117\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003epagL7\u003c/em\u003e D\u003cem\u003epagP81\u003c/em\u003e::P\u003csub\u003elpp\u003c/sub\u003e \u003cem\u003elpxE\u0026nbsp;\u003c/em\u003eD\u003cem\u003elpxR9\u0026nbsp;\u003c/em\u003eD\u003cem\u003efur9\u0026nbsp;\u003c/em\u003e∆\u003cem\u003ewaaL46\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW118\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003epagL81\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaL\u003c/em\u003e D\u003cem\u003epagP81\u003c/em\u003e::P\u003csub\u003elpp\u003c/sub\u003e \u003cem\u003elpxE\u0026nbsp;\u003c/em\u003eD\u003cem\u003elpxR9\u003c/em\u003e D\u003cem\u003efur9\u0026nbsp;\u003c/em\u003e∆\u003cem\u003ewaaL46\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW119\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003epagL7\u003c/em\u003e D\u003cem\u003epagP81\u003c/em\u003e::P\u003csub\u003elpp\u003c/sub\u003e \u003cem\u003elpxE\u0026nbsp;\u003c/em\u003eD\u003cem\u003elpxR9\u0026nbsp;\u003c/em\u003eD\u003cem\u003efur9\u0026nbsp;\u003c/em\u003e∆\u003cem\u003ewaaP45\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eSW120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.62626262626262%\" colspan=\"4\"\u003e\n \u003cp\u003eD\u003cem\u003epagL83\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e D\u003cem\u003ewbaP\u003c/em\u003e D\u003cem\u003epagP81\u003c/em\u003e::P\u003csub\u003elpp\u003c/sub\u003e \u003cem\u003elpxE\u0026nbsp;\u003c/em\u003eD\u003cem\u003elpxR9\u003c/em\u003e D\u003cem\u003efur9\u0026nbsp;\u003c/em\u003e∆\u003cem\u003ewaaP45\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.19191919191919%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/strong\u003e \u003cstrong\u003estrains\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003ec7232\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cem\u003eendA1 hsdR17\u0026nbsp;\u003c/em\u003e(\u003cem\u003er\u003csub\u003eK\u003c/sub\u003e-, m\u003csub\u003ek\u003c/sub\u003e+\u003c/em\u003e)\u003cem\u003e\u0026nbsp;glnV44 thi-1 recA1 gyrA relA1\u0026nbsp;\u003c/em\u003e∆(\u003cem\u003elacZYA-argF\u003c/em\u003e)\u003cem\u003eU169 \u0026lambda;\u003c/em\u003epir \u003cem\u003edeoR\u0026nbsp;\u003c/em\u003e(f\u003cem\u003e80dlac\u003c/em\u003e ∆(\u003cem\u003elacZ\u003c/em\u003e)\u003cem\u003eM15\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\"\u003e\n \u003cp\u003e[51]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003ec7213\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cem\u003ethi-1 thr-1 leuB6 glnV44 fhuA21 lacY1 recA1\u0026nbsp;\u003c/em\u003eRP4-2-Tc::Mu[\u003cem\u003e\u0026lambda;\u003c/em\u003e pir] ∆\u003cem\u003easdA4\u0026nbsp;\u003c/em\u003e∆(\u003cem\u003ezhf-2\u003c/em\u003e::Tn\u003cem\u003e10\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\"\u003e\n \u003cp\u003e[51]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePlasmids\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003epYA4278\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cem\u003esacB mobRP4\u0026nbsp;\u003c/em\u003eR6K \u003cem\u003eori\u0026nbsp;\u003c/em\u003eCm\u003csup\u003e+\u003c/sup\u003e, derived from pRE112\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\"\u003e\n \u003cp\u003e[27]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003epYA3700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\"\u003e\n \u003cp\u003eCloning vector containing TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e cassette\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\"\u003e\n \u003cp\u003e[29]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003epYA4284\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\"\u003e\n \u003cp\u003e∆\u003cem\u003epagL7\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\"\u003e\n \u003cp\u003e[38]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003efor ∆\u003cem\u003ewaaC40\u003c/em\u003e mutation, suicide plasmid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW013\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003efor\u0026nbsp;D\u003cem\u003ewaaF41\u0026nbsp;\u003c/em\u003emutation, suicide plasmid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW014\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003efor\u0026nbsp;D\u003cem\u003ewaaG42\u003c/em\u003e mutation, the same suicide plasmid as pYA4896\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e[40]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW015\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003efor\u0026nbsp;D\u003cem\u003ewaaI43\u0026nbsp;\u003c/em\u003emutation, the same suicide plasmid as pYA4897\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e[40]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW016\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003efor\u0026nbsp;D\u003cem\u003ewaaJ44\u003c/em\u003e mutation, the same suicide plasmid as pYA4898\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e[40]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW017\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003efor\u0026nbsp;D\u003cem\u003ewaaL46\u0026nbsp;\u003c/em\u003emutation, the same suicide plasmid as pYA4900\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e[40]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003efor\u0026nbsp;D\u003cem\u003ewbaP45\u0026nbsp;\u003c/em\u003emutation, the same suicide plasmid as pYA4899\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e[40]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW019\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eInsert \u003cem\u003ewaaC1\u003c/em\u003e gene to pYA3700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eInsert \u003cem\u003ewaaC2\u003c/em\u003e gene to pYA3700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eInsert \u003cem\u003ewaaF1\u003c/em\u003e gene to pYA3700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eInsert \u003cem\u003ewaaF2\u003c/em\u003e gene to pYA3700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eInsert \u003cem\u003ewaaG1\u003c/em\u003e gene to pYA3700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eInsert \u003cem\u003ewaaG2\u003c/em\u003e gene to pYA3700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW025\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eInsert \u003cem\u003ewaaI1\u003c/em\u003e gene to pYA3700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW026\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eInsert \u003cem\u003ewaaI2\u003c/em\u003e gene to pYA3700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW027\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eInsert \u003cem\u003ewaaJ1\u003c/em\u003e gene to pYA3700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW028\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eInsert \u003cem\u003ewaaJ2\u003c/em\u003e gene to pYA3700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW029\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eInsert \u003cem\u003ewaaL1\u003c/em\u003e gene to pYA3700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW030\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eInsert \u003cem\u003ewaaL2\u003c/em\u003e gene to pYA3700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW031\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eInsert \u003cem\u003ewaaP1\u003c/em\u003e gene to pYA3700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW032\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eInsert \u003cem\u003ewaaP2\u003c/em\u003e gene to pYA3700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW033\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003efor\u0026nbsp;D\u003cem\u003epagL71\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaC\u003c/em\u003e mutation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW034\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003efor\u0026nbsp;D\u003cem\u003epagL72\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaC\u003c/em\u003e mutation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW035\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003efor\u0026nbsp;D\u003cem\u003epagL73\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaF\u003c/em\u003e mutation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW036\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003efor\u0026nbsp;D\u003cem\u003epagL74\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaF\u003c/em\u003e mutation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW037\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003efor\u0026nbsp;D\u003cem\u003epagL75\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaG\u003c/em\u003e mutation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW038\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003efor\u0026nbsp;D\u003cem\u003epagL76\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaG\u003c/em\u003e mutation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW039\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003efor\u0026nbsp;D\u003cem\u003epagL77\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaI\u003c/em\u003e mutation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW040\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003efor\u0026nbsp;D\u003cem\u003epagL78\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaI\u003c/em\u003e mutation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW041\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003efor\u0026nbsp;D\u003cem\u003epagL79\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaJ\u003c/em\u003e mutation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW042\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003efor\u0026nbsp;D\u003cem\u003epagL80\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaJ\u003c/em\u003e mutation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW043\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003efor\u0026nbsp;D\u003cem\u003epagL81\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaL\u003c/em\u003e mutation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW044\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003efor\u0026nbsp;D\u003cem\u003epagL82\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewaaL\u003c/em\u003e mutation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW045\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003efor\u0026nbsp;D\u003cem\u003epagL83\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewbaP\u003c/em\u003e mutation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003epSW046\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"59.5959595959596%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003efor\u0026nbsp;D\u003cem\u003epagL84\u003c/em\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ewbaP\u003c/em\u003e mutation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp style=\"margin:0in;text-align:;line-height:normal;font-size:16px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eTable 2. Prime used in this study\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"float: ;width:95.66%;border-collapse:collapse;border:none;margin-left:6.75pt;margin-right:6.75pt;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:36.42%;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:25.5pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:justify;line-height:normal;font-size:16px;font-family:DengXian;margin-top:.05pt;margin-right:0in;margin-bottom: .05pt;margin-left:0in;\"\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ePrimer name\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.58%;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:25.5pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:justify;line-height:normal;font-size:16px;font-family:DengXian;margin-top:.05pt;margin-right:0in;margin-bottom: .05pt;margin-left:0in;\"\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eSequence (5\u0026rsquo;-3\u0026rsquo;) (the sequences highlighted by red color contain SD and start codon)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:36.42%;border:none;padding:0in 5.4pt 0in 5.4pt;height:25.5pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:justify;line-height:normal;font-size:16px;font-family:DengXian;margin-top:.05pt;margin-right:0in;margin-bottom: .05pt;margin-left:0in;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eWaaC-Del 1F\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.58%;border:none;padding:0in 5.4pt 0in 5.4pt;height:25.5pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:justify;line-height:normal;font-size:16px;font-family:DengXian;margin-top:.05pt;margin-right:0in;margin-bottom: .05pt;margin-left:0in;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ecattctgaaatgagccggcgctgaatagcgagcag\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:36.42%;border:none;padding:0in 5.4pt 0in 5.4pt;height:25.5pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:justify;line-height:normal;font-size:16px;font-family:DengXian;margin-top:.05pt;margin-right:0in;margin-bottom: .05pt;margin-left:0in;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eWaaC-Del 1R\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.58%;border:none;padding:0in 5.4pt 0in 5.4pt;height:25.5pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:justify;line-height:normal;font-size:16px;font-family:DengXian;margin-top:.05pt;margin-right:0in;margin-bottom: .05pt;margin-left:0in;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ecagagtctctttaaacgccctcttccgaca\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:36.42%;border:none;padding:0in 5.4pt 0in 5.4pt;height:25.5pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:justify;line-height:normal;font-size:16px;font-family:DengXian;margin-top:.05pt;margin-right:0in;margin-bottom: .05pt;margin-left:0in;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eWaaC-Del 2F\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.58%;border:none;padding:0in 5.4pt 0in 5.4pt;height:25.5pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:justify;line-height:normal;font-size:16px;font-family:DengXian;margin-top:.05pt;margin-right:0in;margin-bottom: .05pt;margin-left:0in;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003egggcgtttaaagagactctgtctcatccca\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:36.42%;border:none;padding:0in 5.4pt 0in 5.4pt;height:25.5pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:justify;line-height:normal;font-size:16px;font-family:DengXian;margin-top:.05pt;margin-right:0in;margin-bottom: .05pt;margin-left:0in;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eWaaC-Del 2R\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.58%;border:none;padding:0in 5.4pt 0in 5.4pt;height:25.5pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:justify;line-height:normal;font-size:16px;font-family:DengXian;margin-top:.05pt;margin-right:0in;margin-bottom: .05pt;margin-left:0in;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eagcatttatcagggtaagccctccagtaccgtatt\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:36.42%;border:none;padding:0in 5.4pt 0in 5.4pt;height:25.5pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:justify;line-height:normal;font-size:16px;font-family:DengXian;margin-top:.05pt;margin-right:0in;margin-bottom: .05pt;margin-left:0in;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eVec-F\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.58%;border:none;padding:0in 5.4pt 0in 5.4pt;height:25.5pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:justify;line-height:normal;font-size:16px;font-family:DengXian;margin-top:.05pt;margin-right:0in;margin-bottom: .05pt;margin-left:0in;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eaccctgataaatgcttcaataa\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:36.42%;border:none;padding:0in 5.4pt 0in 5.4pt;height:25.5pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:justify;line-height:normal;font-size:16px;font-family:DengXian;margin-top:.05pt;margin-right:0in;margin-bottom: .05pt;margin-left:0in;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eVec-R\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.58%;border:none;padding:0in 5.4pt 0in 5.4pt;height:25.5pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:justify;line-height:normal;font-size:16px;font-family:DengXian;margin-top:.05pt;margin-right:0in;margin-bottom: .05pt;margin-left:0in;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003egctcatttcagaatggaaggtc\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:36.42%;border:none;padding:0in 5.4pt 0in 5.4pt;height:25.5pt;\"\u003e\n \u003cp 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outer membrane proteins, lipopolysaccharide, cross-protection, Salmonella serotypes","lastPublishedDoi":"10.21203/rs.3.rs-3971522/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3971522/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNon-typhoidal \u003cem\u003eSalmonella\u003c/em\u003e \u003cem\u003eenterica\u003c/em\u003e serovar (NTS) is a major global foodborne pathogen that poses a major public health concern worldwide, and no vaccines were available for protecting against infection of multiple \u003cem\u003eSalmonella\u003c/em\u003e serotypes, therefore, the development of \u003cem\u003eSalmonella\u003c/em\u003e vaccines to provide broad protection is valuable. In this work, we aimed to regulate lipopolysaccharide (LPS) synthesis of live \u003cem\u003eSalmonella\u003c/em\u003e \u003cem\u003ein vivo\u003c/em\u003e for exposing conserved protein antigens on the outer membrane while maintaining smooth LPS patterns \u003cem\u003ein vitro\u003c/em\u003e to keep their original ability to invade host cells for inducing cross-protection against infection of multiple \u003cem\u003eSalmonella\u003c/em\u003e serotypes. We generated a series of mutants defective in genes to affect the length of LPS. These mutants exhibit \u003cem\u003ein vivo\u003c/em\u003e regulated-delayed attenuation and altered length of LPS, and all these mutants were derived from SW067 (D\u003cem\u003epagL7 \u003c/em\u003eD\u003cem\u003epagP81\u003c/em\u003e::P\u003csub\u003elpp\u003c/sub\u003e \u003cem\u003elpxE\u003c/em\u003e D\u003cem\u003elpxR9 \u003c/em\u003eD\u003cem\u003efur9\u003c/em\u003e) containing ∆\u003cem\u003epagP81\u003c/em\u003e::P\u003csub\u003elpp\u003c/sub\u003e \u003cem\u003elpxE\u003c/em\u003e mutation to reduce their endotoxic activity. Animal experiments demonstrated that all regulated delayed attenuated mutants exhibited reduced ability to colonize the organs of the mice, and SW114 (\u003cem\u003ewaaI\u003c/em\u003e), SW116 (\u003cem\u003ewaaJ\u003c/em\u003e), SW118 (\u003cem\u003ewaaL\u003c/em\u003e), and SW120 (\u003cem\u003ewbaP\u003c/em\u003e) induced a significant production of IgG and IgA against OMPs isolated from \u003cem\u003eS\u003c/em\u003e. Typhimurium, \u003cem\u003eS\u003c/em\u003e. Enteritidis, and \u003cem\u003eS\u003c/em\u003e. Choleraesuis. SW114 (\u003cem\u003ewaaI\u003c/em\u003e), SW116 (\u003cem\u003ewaaJ\u003c/em\u003e), and SW118 (\u003cem\u003ewaaL\u003c/em\u003e) were capable of conferring significant protection against infection of wild-type \u003cem\u003eS\u003c/em\u003e. Enteritidis and \u003cem\u003eS\u003c/em\u003e. Choleraesuis. In conclusion, regulated delayed attenuated \u003cem\u003eSalmonella\u003c/em\u003e vaccines with the whole core oligosaccharides of LPS showed a good ability to expose conserved outer antigens and to trigger strong cross-immune responses against both homologous and heterologous \u003cem\u003eSalmonella \u003c/em\u003einfections. These results give new insight into the development of the \u003cem\u003eSalmonella\u003c/em\u003e vaccine against multiple serotypes of \u003cem\u003eSalmonella\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Immunogenicity and cross-protective efficacy induced by delayed attenuated Salmonella with the regulated length of lipopolysaccharide in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-04 11:14:35","doi":"10.21203/rs.3.rs-3971522/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f61b9c64-45a2-4f1b-880a-3595d7c7f365","owner":[],"postedDate":"March 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":29081774,"name":"Biological sciences/Microbiology/Vaccines/Live attenuated vaccines"},{"id":29081775,"name":"Biological sciences/Microbiology/Bacteria/Bacterial development"}],"tags":[],"updatedAt":"2024-03-26T05:45:21+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-04 11:14:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3971522","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3971522","identity":"rs-3971522","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}