The Impact of the CpxA on Regulating Polymyxin B Resistance in Salmonella Typhimurium

The Impact of the CpxA on Regulating Polymyxin B Resistance in Salmonella Typhimurium | 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 Research Article The Impact of the CpxA on Regulating Polymyxin B Resistance in Salmonella Typhimurium Xiaoqing Zhang, Yupeng Fang, Chengbin Xue, Ziwei Guo, Cunlei Liu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5322344/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 Polymyxin B is considered as a last-resort antibiotic for multidrug-resistant or extensively drug-resistant gram-negative bacterial infections. Addressing Salmonella resistance to polymyxin B is crucial for global public health. Our study revealed that CpxA/ArcB knockout inhibits the motility, tolerance to acid stress and oxidative stress of S.typhimurium . Most notably, the tolerance of S.typhimurium to polymyxin B could be increased by either CpxA or CpxA/ArcB deletion. In this study, transcriptomic detection and analysis were used to clarify the mechanisms by which CpxA- deleted S.typhimurium is involved in resistance to polymyxin B stress, which may be related to processes such as increased assembly of bacterial flagella. Salmonella typhimurium CpxAR two-component system Polymyxin resistance CpxA Flagellar assembly. Figures Figure 1 Figure 2 Figure 3 Introduction Salmonella typhimurium and Salmonella enteritidis are the most common Salmonella enteritidis that can infect humans, posing a serious threat to public health[ 1 ]. Salmonellosis is a major challenge in animal husbandry, not only seriously affects the health and production performance of animals, but also causes significant losses in the economic development of the livestock industry[ 2 ]. Polymyxin is a naturally occurring antibiotics that have been shown to have antibacterial activity against Salmonella [ 3 ]. Polymyxin B is a clinically available polymyxin used to treat infections caused by multidrug or extensively drug-resistant gram-negative bacteria[ 4 ] and has ultimately been considered the last-resort treatment for such infections[ 5 ]. Therefore, solving the problem of Salmonella resistance to polymyxin B is of great significance to world public health security. CpxAR ( CpxA/CpxR ) is a regulatory two-component system that can contribute to the stress response and virulence in various gram-negative pathogens[ 6 ]. In C. sakazakii , CpxAR deletion strains showed weakened bacterial adhesion and invasion ability, lower levels of tight junction proteins disruption and reduced apoptosis and cytotoxicity in cells cultured in vitro, as well as attenuated virulence in newborn mice[ 6 ]. In Shigella , the invasive phenotype can be repressed at low pH, and this pH-dependent regulation also requires CpxAR[ 7 ]. In enteropathogenic Yersinia pseudotuberculosis , CpxAR could influence biofilm development through its impact on exopolysaccharide production[ 8 ]. In Actinobacillus pleuropneumoniae , CpxAR plays an important role in cold resistance by upregulating CspC expression[ 9 ]. CpxAR contributes to the biofilm formation ability of A. pleuropneumoniae [ 10 ], CpxAR and CpxD mutants exhibited non-capsulated and were strongly impaired in virulence for mice, which suggested that CpxAR is a key modulator of capsule export that facilitates A. pleuropneumoniae survival in the host[ 11 ]. These findings suggest a key role for CpxAR in the development of bacterial virulence, as well as the response to various environment stress. The antibiotic resistance caused by CpxAR deficiency also received wide attention. In Klebsiella pneumoniae , the CpxAR deficient strain was more susceptible to β-lactams and chloramphenicol than the wild-type strain, which reveals its direct involvement in conferring antimicrobial resistance against clinically significant antibiotics for the very first time[ 12 ]. In a Proteus mirabilis clinical isolate, alterations in CpxA might confer imipenem and amikacin resistance without significant defects in the growth rate[ 13 ]. The antibiotic resistance formation in Salmonella enterica serovar Enteritidis could also be impaired by CpxAR, for the development of multidrug resistance through the regulation of cell membrane permeability and efflux pump activity[ 14 ]. For example, CpxR contributes to the resistance of S.enterica serovar Typhimurium to aminoglycosides and β-lactams by influencing the expression level of multidrug resistance-related genes including AcrB [ 15 ]. In Salmonella enterica serovar Typhimurium, the CpxA/CpxR gene deletion mutant also presented decreased ceftriaxone resistance[ 16 ]. However, the regulatory mechanism of antibiotic resistance regulated by CpxAR in a variety of gram-negative bacteria is still unclear, and its influence on polymyxin B resistance is still not well understood. In this research, we offered a new perspective for understanding the function of the CpxAR two-component system in response to polymyxin B through the knockout of CpxA in S. enterica serovar Typhimurium. Materials and methods Bacterial strains and plasmids The S. enterica serovar Typhimurium strainand plasmids, including pKD46, pKD4, pCP20 and pBR322 used for gene modification, were maintained in our laboratory. All strains including SL1344, SL1344 Δ CpxA , SL1344 Δ CpxA / pBR322- CpxA , SL1344 Δ CpxA Δ ArcB , SL1344 Δ CpxA Δ ArcB / pBR322- CpxA-ArcB were validated via PCR or gene sequencing. Growth curve detection The strains cultured for 12 h were inoculated into LB broth liquid medium at a ratio of 1:100, then cultured at 37 ℃ and 180 rpm. Their absorbance (OD 600 ) was tested every two hours. Bacterial motility determination 2 mL of the corresponding bacterial suspension with OD 600 = 1.0 was washed with ice-cold PBS and resuspended with 1mL PBS. Then 2µL of the resuspended bacteria were absorbed and dropped on the center of LB agar semisolid medium, then cultured at 37℃ for 12h. Colony diameters were measured and recorded. Biofilm formation ability assay 200 µL of the corresponding bacterial solution with OD 600 = 0.2 was taken and cultured in a 96-well plate at 37 ℃ for 36 h. Then fixed with methanol for 15 min, washed with PBS, dyed with 0.5% crystal violet for 30 min, and washed three times with PBS. Then 200 µL of ethanol was added to dissolve the crystal violet for 30 min, and the absorbance (OD 570 ) of each sample was determined. Resistance to environmental stress The strains cultured overnight were inoculated into the corresponding LB broth at a ratio of 1:100. For the acid stress experiments, medium with pH = 5.5/4.5 was used. For the hypertonic stress test, 2.5 M NaCl was added to the medium. For the oxidative stress experiments, 10 mM H 2 O 2 was added to the medium. After being culture at 37℃, 180 rpm for 3h and dilute at a ratio of 10 − 7 , 100 µL was added to an LB agar plate. Then the bacteria were cultured overnight, and make bacterium colony computation. The relative survival rate of each sample was calculated by comparing the colony count results of each sample with those of the corresponding strain cultured under normal conditions. Resistance to polymyxin B The bacterial suspension, which was precultured with the corresponding concentration of polymyxin B, was diluted to 2×10 5 CFU/mL with Mueller‒Hinton (MH) broth. The mixture was added to a 96-well plate, and the antibiotic concentration was adjusted to the corresponding value. The plate was incubated at 37°C for 16–18 h, and the MICs of each strain were observed and recorded. After serial dilution, the suspension was spread on polymyxin LB agar plates, and the control group was spread on LB agar plates. Plates were incubated overnight at 37°C and record with photographs. Antibiotic Susceptibility Test After culture for 12 h, the bacterial strains were washed with PBS and adjusted to a McFarland standard turbidity of 0.5. The strains were spread on LB agar plates, and antimicrobial discs were applied. Plates were incubated at 37°C for 24 hours, and the diameters of the antimicrobial circles were measured. Transcriptome Sequencing S.typhimurium and its CpxA -delated strain were inoculated into LB broth containing 0.125 µg/ml polymyxin B, and cultured overnight at 37°C, followed by dilution and subculture. After growing to the logarithmic phase, the bacteria were washed twice with PBS, collected, quickly frozen in liquid nitrogen, and then sent to Beijing Novogene Bioinformatics Technology Co., Ltd. for transcriptome sequencing and analysis. Statistical analysis Three biological replicates were set for each group (n = 3) in all the experiments above. Statistical analysis was performed with GraphPad Prism 7. The results are presented as the means ± SEM. The data were analyzed by Student’s t test. F test was used to compare the variances within each group, and the data without similar variance were adjusted by Welch’s correction. p < 0.05 was considered significant. Results The purity of SL1344 kept in our laboratory was determined, which meets experimental standards without other bacterial contamination. On this basis, we constructed a CpxA deletion strain SL1344∆ CpxA , and a CpxA/ArcB deletion strain SL1344Δ CpxA Δ ArcB , via the Red homologous recombination system. Concurrently, we also constructed their gene-complemented strains SL1344Δ CpxA /pBR322- CpxA and SL1344Δ CpxA Δ ArcB /pBR322- CpxA-ArcB . 1. Biological characteristics of the CpxA and CpxA/ArcB deficient strains The biological characteristics of these strains were then analyzed. Strains were cultured under the same conditions in LB broth, and growth curves were generated and plotted (Fig. 1 A). Results showed the deletion of CpxA had no significant effect on the growth of SL1344, whereas combined deletion of CpxA/ArcB inhibited its proliferation ability, indicating that CpxA /ArcB may have influence on the growth metabolism of S.typhimurium . The bacterial motility identification experiment (Fig. 1 B-C) revealed that the deletion of CpxA had no significant effect on the motility of SL1344, but the motility of the CpxA/ArcB deletion strain was significantly reduced, indicating that the double deletion of CpxA/ArcB could inhibit the motility of Salmonella Typhimurium . Meanwhile, the results of the biofilm formation ability assay showed no difference among SL1344 and all the gene deletion strains used above (Fig. 1 D, p > 0.05), indicating that CpxA and ArcB may not affect the biofilm formation ability of Salmonella Typhimurium . In the experiment assessing resistance to environmental stress, the deletion of CpxA/ArcB resulted in a decrease in tolerance to H 2 O 2 treatment (Fig. 1 E). In the hypertonic stress test, there were no significant differences between the SL1344 and gene deletion strains (Fig. 1 F). When pH = 5.5, both the CpxA and CpxA/ArcB deletion strains exhibited significantly decreased growth (Fig. 1 G, p < 0.01). However, the resistance to acidic environments at pH = 4.5 was enhanced when CpxA was deleted, while decreased in CpxA/ArcB deletion strain (Fig. 1 H, p < 0.01). To determine the tolerance of these strains to polymyxin B, the broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) of the strains. Compared with that of SL1344, the MIC of the CpxA deletion strain increased by 2-fold, whereas the MIC of the CpxA/ArcB deletion strain increased by 4-fold (Fig. 1 I). Meanwhile, the agar spot test also showed a certain advantage in polymyxin B resistance for both CpxA and CpxA/ArcB deletion strains compaired with the original strain SL1344. These results indicate that the deletion of CpxA and ArcB increased the resistance of SL1344 to polymyxin B. Finally, we used the K-B disk diffusion method (Table 1) to test the antibiotic susceptibility of these strains. The results showed that the CpxA deletion strain had increased susceptibility to amikacin, netilmicin , and cephalothin , with further increased susceptibility observed in the CpxA/ArcB deletion strain. This indicates the complex role of the CpxAR two-component system and the ArcB gene in regulating Salmonella 's response to antibiotics. 2. Transcriptomic sequencing analysis of CpxA -knockout strains Based on the understanding of the CpxAR system and ArcB in regulating the response of Salmonella to antibiotics, we chose to use transcriptome sequencing analysis to explore the changes in molecular signaling pathways in SL1344 after the knockout of CpxA under polymyxin B stress. In this way, the role of CpxA and its related pathways under polymyxin B stress could be elucidated. The Illumina sequencing platform was used in our study to analyze the gene expression levels of S.enterica serovar Typhimurium pretreated with polymyxin B (SL1344- PolB ), and its CpxA -knockout strain pretreated with polymyxin B (SL1344 ΔCpxA-PolB ). The sequencing fragments obtained from the high-throughput sequencer were transformed into sequence data (reads) via CASAVA base recognition and filtered to obtain clean reads. The Q30 percentages of the clean data for all samples were higher than 95.47%, and the GC contents of the clean data for all samples ranged between 52.12% and 53.03% (Table 2). The filtered sequencing reads were subjected to genomic location analysis via Bowtie2 software. Approximately 93.32%-95.47% of the clean reads were successfully mapped to the reference Salmonella genome, indicating that the reference genome was appropriately selected and that there was no contamination in the relevant experiments. Using the featureCounts function in the Subread software, we filtered out reads with low alignment quality, not properly paired, or aligned to multiple regions of the genome. In total, 4717 compounds were obtained from the sequenced sequences, and 140 features with a constant or single value across samples were identified and removed. After the expression values (FPKM) of all the genes in each sample were calculated, we presented a boxplot (Fig. 2 A) which revealed good uniformity in the gene expression distribution. To ensure the rational selection of biological replicates, we analyzed the correlation of gene expression levels between samples and created a heatmap, which revealed that all the R 2 values between biological replicates were greater than 0.8 (Fig. 2 B), indicating good reproducibility of the biological samples. Principal component analysis (PCA) was then performed using Metaboanalysis, and the results showed that the data grouping was acceptable. The transcripts obtained above were subsequently used for subsequent experiments. After gene expression quantification, we conducted a statistical analysis to identify genes whose expression levels significantly differed under various conditions. We used DESeq2 to normalize the raw readcounts, adjust for sequencing depth, and calculate p -values. Differentially expressed genes (DEGs) were obtained by filtering for |log2(FoldChange)| > 1 and p < 0.05. 223 DEGs were found, of which 139 were upregulated and 84 were down-regulated (Fig. 2 D). Subsequently, we performed Gene Ontology (GO) enrichment analysis (Fig. 2 E) on the aforementioned DEGs. These DEGs were enriched in 287 GO terms, with Cellular Components (CC) mainly enriched in components related to movement of cell or subcellular level, and cilium or flagellum-dependent cell movement; Biological Processes (BP) were primarily enriched in components of membranes and intrinsic components; Molecular Functions (MF) were mainly enriched in oxidoreductase activity and antioxidant activity. Concurrently, we also conducted KEGG pathway enrichment analysis for these DEGs, which were enriched across 60 KEGG pathways (Fig. 2 F). The enrichment was primarily observed in pathways such as flagellar components, bacterial chemotaxis, arginine biosynthesis, ABC transporters, and microbial metabolism in diverse environments. 3. PPI network analysis and hub gene cluster identification These DEGs were further analyzed to find out the key pathways affected by the knockout of CpxA in S.typhimurium . The mainstream hierarchical clustering method was used to analyze gene expression values. After the rows of the expression data were normalized, the clustering results were plotted, which revealed distinct intergroup differences (Fig. 3 A). Then the DEGs were subsequently analyzed using the STRING[ 17 ] database. The protein-protein interaction (PPI) network was exported and processed via Cytoscape software to display their interactive relationships (Fig. 3 B). A prominent cluster with all nodes closely related to each other was observed, which was also closely associated with CpxA . Therefore, we used the MCODE plugin in Cytoscape to extract this cluster. We successfully obtained a cluster containing 22 nodes (Fig. 3 C). GO-BP enrichment analysis of these 22 nodes via the STRING online tool revealed that the functions of this cluster are focused on Bacterial-type flagellum organization and cell motility (Fig. 3 D). Additionally, KEGG enrichment analysis of these 22 nodes also showed significant enrichment in the flagellar assembly (stm02040) pathway: of the 38 nodes in the Flagellar assembly pathway, 21 were in this cluster (Fig. 3 E). This result demonstrates the significant impact of CpxA dysfunction on the structure and function of flagella in Salmonella typhimurium under polymyxin B stress. These findings provide us important insights into the role of the CpxAR two-component system in the resistance of S.typhimurium to polymyxin B. Discussion Polymyxin B is ultimately considered as the last-resort treatment for infections caused by multidrug or extensively drug-resistant gram-negative bacteria. Solving the problem of Salmonella resistance to polymyxin B is of great significance to world public health security. The CpxAR two-component system has been proven to be associated with antibiotic resistance in various bacteria, and this study further demonstrated that the deletion of CpxA/ArcB could increase the resistance of S. typhimurium to polymyxin B. Thus, transcriptome analysis was used, which revealed that the deletion of CpxA can disrupt the structure and function of flagella and is related to various pathways, including cell motility. These proteins may become key targets for S.typhimurium to cope with the toxicity of polymyxin B. Laboratory tests revealed that the deletion of CpxA/ArcB can significantly reduce the growth rate, motility, and tolerance to hydrogen peroxide and acidic environments of the S.typhimurium SL1344 strain, while increasing its resistance to polymyxin B. However, the strain with a single deletion of CpxA did not show significant changes in most of these biological characteristics besides polymyxin B resistance. To further explore the specific molecular mechanisms by which the CpxAR two-component system affects these biological characteristics, particularly the resistance to polymyxin B, this study conducted transcriptome sequencing on the CpxA deletion strain, and performed a transcriptomic analysis for them under polymyxin B stress. For the selection of transcriptom samples, this study chose the CpxA single-gene deletion strain, which presented some changes in resistance to polymyxin B, for low-concentration polymyxin B treatment and transcriptom sequencing. This design is only a part of our in-depth study of the CpxAR two-component system in conjunction with ArcB for their resistance against polymyxin B. In subsequent research, we will further investigate how ArcB enhances the function of CpxA and CpxR in polymyxin B resistance. Through transcriptome sequencing, we identified 223 DEGs. GO and KEGG enrichment analyses revealed the functions of these genes in various domains, such as cell motility, flagellar assembly, and transmembrane transporter activity. To further clarify the pathways involved there, we conducted a PPI network via STRING. Then we filtered out a cluster of DEGs, which is closely related to flagellar assembly functions. On the basis of these findings, we preliminarily determined that the CpxA gene in S.typhimurium can alter the structure and function of flagella under polymyxin B stress. In the CpxA deletion strain, the expression of most genes involved in the flagellar assembly related signaling pathways was upregulated, which may be a key factor in the increased antibiotic resistance of the CpxA deletion strain. Declarations Conflict of Interest Statement The authors have no conflict of interest. Ethics Statement Not applicable. Funding Information This study was supported by the Natural Science Foundation of Hebei Province of China (Grant no. C2022407019). Author Contribution X.Z. data analysis and writing; Y.F. experimental design and guidance; C.X. system construction and project implementation; Z.G. assistance in molecular experiments; C.L. assistance in molecular experiments; Y.L. project supervision. All authors reviewed the manuscript. Acknowledgments Not applicable. Data Availability Transcriptomics data will be made public after the upload is completed (currently being submitted in GEO database). References Dai W, Zhang Y, Zhang J, Xue C, Yan J, Li X, Zheng X, Dong R, Bai J, Su Y, et al. Analysis of antibiotic-induced drug resistance of Salmonella enteritidis and its biofilm formation mechanism. Bioengineered. 2021;12(2):10254–63. Chen K, Gao Y, Li L, Zhang W, Li J, Zhou Z, He H, Chen Z, Liao M, Zhang J. Increased Drug Resistance and Biofilm Formation Ability in ST34-Type Salmonella Typhimurium Exhibiting Multicellular Behavior in China. Front Microbiol. 2022;13:876500. Han ML, Liu X, Velkov T, Lin YW, Zhu Y, Creek DJ, Barlow CK, Yu HH, Zhou Z, Zhang J, et al. Comparative Metabolomics Reveals Key Pathways Associated With the Synergistic Killing of Colistin and Sulbactam Combination Against Multidrug-Resistant Acinetobacter baumannii. Front Pharmacol. 2019;10:754. Rigatto MH, Falci DR, Zavascki AP. Clinical Use of Polymyxin B. Adv Exp Med Biol. 2019;1145:197–218. Zavascki AP, Goldani LZ, Li J, Nation RL. Polymyxin B for the treatment of multidrug-resistant pathogens: a critical review. J Antimicrob Chemother. 2007;60(6):1206–15. Jin T, Zhan X, Pang L, Peng B, Zhang X, Zhu W, Yang B, Xia X. CpxAR two-component system contributes to virulence properties of Cronobacter sakazakii. Food Microbiol. 2024;117:104393. Pasqua M, Coluccia M, Eguchi Y, Okajima T, Grossi M, Prosseda G, Utsumi R, Colonna B. Roles of Two-Component Signal Transduction Systems in Shigella Virulence. Biomolecules 2022, 12(9). Gahlot DK, Wai SN, Erickson DL, Francis MS. Cpx-signalling facilitates Hms-dependent biofilm formation by Yersinia pseudotuberculosis. NPJ biofilms microbiomes. 2022;8(1):13. Yao Q, Xie T, Fu Y, Wan J, Zhang W, Gao X, Huang J, Sun D, Zhang F, Bei W, et al. The CpxA/CpxR two-component system mediates regulation of Actinobacillus pleuropneumoniae cold growth. Front Microbiol. 2022;13:1079390. Li H, Liu F, Peng W, Yan K, Zhao H, Liu T, Cheng H, Chang P, Yuan F, Chen H, et al. The CpxA/CpxR Two-Component System Affects Biofilm Formation and Virulence in Actinobacillus pleuropneumoniae. Front Cell Infect Microbiol. 2018;8:72. Liu F, Yao Q, Huang J, Wan J, Xie T, Gao X, Sun D, Zhang F, Bei W, Lei L. The two-component system CpxA/CpxR is critical for full virulence in Actinobacillus pleuropneumoniae. Front Microbiol. 2022;13:1029426. Srinivasan VB, Vaidyanathan V, Mondal A, Rajamohan G. Role of the two component signal transduction system CpxAR in conferring cefepime and chloramphenicol resistance in Klebsiella pneumoniae NTUH-K2044. PLoS ONE. 2012;7(4):e33777. Lecuru M, Nicolas-Chanoine MH, Tanaka S, Montravers P, Armand-Lefevre L, Denamur E, Mammeri H. Emergence of Imipenem Resistance in a CpxA-H208P-Variant-Producing Proteus mirabilis Clinical Isolate. Microb drug Resist (Larchmont NY). 2021;27(6):747–51. Hu M, Huang X, Xu X, Zhang Z, He S, Zhu J, Liu H, Shi X. Characterization of the Role of Two-Component Systems in Antibiotic Resistance Formation in Salmonella enterica Serovar Enteritidis. mSphere 2022, 7(6):e0038322. Huang H, Sun Y, Yuan L, Pan Y, Gao Y, Ma C, Hu G. Regulation of the Two-Component Regulator CpxR on Aminoglycosides and β-lactams Resistance in Salmonella enterica serovar Typhimurium. Front Microbiol. 2016;7:604. Hu WS, Chen HW, Zhang RY, Huang CY, Shen CF. The expression levels of outer membrane proteins STM1530 and OmpD, which are influenced by the CpxAR and BaeSR two-component systems, play important roles in the ceftriaxone resistance of Salmonella enterica serovar Typhimurium. Antimicrob Agents Chemother. 2011;55(8):3829–37. Szklarczyk D, Kirsch R, Koutrouli M, Nastou K, Mehryary F, Hachilif R, Gable AL, Fang T, Doncheva NT, Pyysalo S, et al. The STRING database in 2023: protein-protein association networks and functional enrichment analyses for any sequenced genome of interest. Nucleic Acids Res. 2023;51(D1):D638–46. Tables Tables 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files T1.xlsx T2.xlsx 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-5322344","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":372181731,"identity":"0cbb5ddd-d954-470b-b9dc-002aadb4beb8","order_by":0,"name":"Xiaoqing Zhang","email":"","orcid":"","institution":"Hebei Normal University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Xiaoqing","middleName":"","lastName":"Zhang","suffix":""},{"id":372181732,"identity":"e3c442ef-c720-4560-bc4c-d13f234e944d","order_by":1,"name":"Yupeng Fang","email":"","orcid":"","institution":"Hebei Normal University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yupeng","middleName":"","lastName":"Fang","suffix":""},{"id":372181733,"identity":"2ea72db5-6f8c-4bc5-a6e9-1d665dd3466b","order_by":2,"name":"Chengbin Xue","email":"","orcid":"","institution":"Hebei Normal University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Chengbin","middleName":"","lastName":"Xue","suffix":""},{"id":372181734,"identity":"18eb7135-3ce4-41bf-9eee-a668247240cb","order_by":3,"name":"Ziwei Guo","email":"","orcid":"","institution":"Hebei Normal University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Ziwei","middleName":"","lastName":"Guo","suffix":""},{"id":372181735,"identity":"b37a31d7-e369-4bc3-8b82-bf1eafa0d303","order_by":4,"name":"Cunlei Liu","email":"","orcid":"","institution":"Hebei Normal University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Cunlei","middleName":"","lastName":"Liu","suffix":""},{"id":372181736,"identity":"8a23fa10-273a-4c43-a8ff-574be551d619","order_by":5,"name":"Yongsheng Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYBACPmYQySNhx8beAxc0wKuFDaxFxiaZj+cMkJFgQIQWMGmTxjhPIodYLew8ZpI/cg4zs0m+PfiY98cfeQb25m0SDDV38DiMx0ya58xhPjbpvGRjngQDwwaeY2USDMee4dfC2AO0RTrHTHJGgkECg0SOmQRjw2G8WiR//jvM2CZ5xvwnWIv8G8JaJHh40hjbJHjMGD6AbeEhpIWt2JqHxyaZjSfHWOJDmrFhG09asUXCMdxa+PkPb7z5AxiV8u1nDD8k2MjJ87Mf3njjQw1uLUDAIoFqL4hIwKeBgYH5A375UTAKRsEoGPEAABLMQfXWsVPBAAAAAElFTkSuQmCC","orcid":"","institution":"Hebei Normal University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Yongsheng","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2024-10-24 03:23:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5322344/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5322344/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":68329727,"identity":"a7ccf612-3149-4af1-a3fb-d8d640e28952","added_by":"auto","created_at":"2024-11-06 06:51:54","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1285905,"visible":true,"origin":"","legend":"\u003cp\u003eBiological characteristics of the \u003cem\u003eCpxA\u003c/em\u003e and \u003cem\u003eCpxA/ArcB\u003c/em\u003e deficient strains A Growth curves of SL1344 and its gene deletion strains. B-C Bacterial motility detection and motility ring determination. D Biofilm formation ability assay. E Oxidative stress experiments with 10 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. F Hypertonic stress test with 2.5 M NaCl. G-H Acid stress experiments with LB broth at pH=5.5(G) and pH=4.5(H). I MIC determination of resistance to \u003cem\u003epolymyxin\u003c/em\u003e B. J Determination of resistance to\u003cem\u003e polymyxin\u003c/em\u003e B. WT: \u003cem\u003eS.typhimurium\u003c/em\u003e SL1344 wild type; KO1: \u003cem\u003eCpxA\u003c/em\u003e deletion strain SL1344Δ\u003cem\u003eCpxA\u003c/em\u003e; KO2: \u003cem\u003eCpxA\u003c/em\u003e and \u003cem\u003eArcB\u003c/em\u003e deletion strain SL1344\u003cem\u003eΔCpxAΔArcB\u003c/em\u003e; RS1:\u003cem\u003eCpxA\u003c/em\u003e knockout and complementation strain SL1344Δ\u003cem\u003eCpxA\u003c/em\u003e/pBR322-\u003cem\u003eCpxA\u003c/em\u003e; RS2: \u003cem\u003eCpxA\u003c/em\u003e and \u003cem\u003eArcB\u003c/em\u003e\u0026nbsp; knockout and complementation strain SL1344\u003cem\u003eΔCpxAΔArcB\u003c/em\u003e/ pBR322-\u003cem\u003eCpxA-ArcB\u003c/em\u003e. The results are shown as mean±SEM，n=3 independent experiments. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"F1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5322344/v1/00c65f22f1cdb03c45b278c0.jpg"},{"id":68329365,"identity":"303e9802-81db-417a-99be-42cbd7a048f5","added_by":"auto","created_at":"2024-11-06 06:43:54","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1272015,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptomic sequencing analysis of \u003cem\u003eCpxA\u003c/em\u003e-knockout strains\u003c/p\u003e\n\u003cp\u003eA Distribution of gene expression within each group. B Correlation coefficients for samples based on the FPKM values of all genes in each sample. C PCA was used to assess distribution of genes compared between groups. E GO enrichment analysis of DEGs. BP: Biological Processes, CC: Cellular Components, MF: Molecular Functions. F KEGG pathway enrichment analysis of the DEGs.\u003c/p\u003e","description":"","filename":"F2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5322344/v1/0059025fc6732db0cdaff7c3.jpg"},{"id":68329726,"identity":"6cf4c7d4-87b1-4698-9b9d-6ec87f4f347b","added_by":"auto","created_at":"2024-11-06 06:51:54","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2495900,"visible":true,"origin":"","legend":"\u003cp\u003ePPI network analysis and hub gene cluster identification A Heatmap plot of DEGs processed with clustering analysis. B PPI network of DEGs with the color representing the Log\u003csub\u003e2\u003c/sub\u003eFC and the node size representing the p-value (the larger the node is, the smaller the p-value). \u003cem\u003eCpxA\u003c/em\u003e is highlighted. SL1344\u003cem\u003eΔCpxA-PolB\u003c/em\u003e vs SL1344-\u003cem\u003ePolB\u003c/em\u003e. C The main hub gene cluster extracted from the PPI network. D GO-BP enrichment analysis performed by STRING. E DEGs participated in the flagellar assembly pathway (KEGG-stm02040).\u003c/p\u003e","description":"","filename":"F3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5322344/v1/9aa8c618d72d5945f843894f.jpg"},{"id":75004776,"identity":"497245ac-645d-46e3-8c28-d832428f1977","added_by":"auto","created_at":"2025-01-29 10:24:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5726763,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5322344/v1/0fb697dd-a1c3-4129-8401-7d313590f9c5.pdf"},{"id":68329362,"identity":"700c513c-21e9-47cb-b0d7-b7b8f3735e0c","added_by":"auto","created_at":"2024-11-06 06:43:54","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10586,"visible":true,"origin":"","legend":"","description":"","filename":"T1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5322344/v1/d02aa357898fb8855dd81b50.xlsx"},{"id":68329367,"identity":"ea9f2005-170f-41bf-bebd-eb17b6ca6ce0","added_by":"auto","created_at":"2024-11-06 06:43:55","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10324,"visible":true,"origin":"","legend":"","description":"","filename":"T2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5322344/v1/d7eb53a86cefb91fdc9cb333.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eThe Impact of the CpxA on Regulating Polymyxin B Resistance in Salmonella Typhimurium\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eSalmonella typhimurium\u003c/em\u003e and \u003cem\u003eSalmonella\u003c/em\u003e enteritidis are the most common \u003cem\u003eSalmonella enteritidis\u003c/em\u003e that can infect humans, posing a serious threat to public health[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Salmonellosis is a major challenge in animal husbandry, not only seriously affects the health and production performance of animals, but also causes significant losses in the economic development of the livestock industry[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. \u003cem\u003ePolymyxin\u003c/em\u003e is a naturally occurring antibiotics that have been shown to have antibacterial activity against \u003cem\u003eSalmonella\u003c/em\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. \u003cem\u003ePolymyxin\u003c/em\u003e B is a clinically available \u003cem\u003epolymyxin\u003c/em\u003e used to treat infections caused by multidrug or extensively drug-resistant gram-negative bacteria[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and has ultimately been considered the last-resort treatment for such infections[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, solving the problem of \u003cem\u003eSalmonella\u003c/em\u003e resistance to \u003cem\u003epolymyxin\u003c/em\u003e B is of great significance to world public health security.\u003c/p\u003e \u003cp\u003eCpxAR (\u003cem\u003eCpxA/CpxR\u003c/em\u003e) is a regulatory two-component system that can contribute to the stress response and virulence in various gram-negative pathogens[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In \u003cem\u003eC. sakazakii\u003c/em\u003e, CpxAR deletion strains showed weakened bacterial adhesion and invasion ability, lower levels of tight junction proteins disruption and reduced apoptosis and cytotoxicity in cells cultured in vitro, as well as attenuated virulence in newborn mice[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In \u003cem\u003eShigella\u003c/em\u003e, the invasive phenotype can be repressed at low pH, and this pH-dependent regulation also requires CpxAR[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In enteropathogenic \u003cem\u003eYersinia pseudotuberculosis\u003c/em\u003e, CpxAR could influence biofilm development through its impact on exopolysaccharide production[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In \u003cem\u003eActinobacillus pleuropneumoniae\u003c/em\u003e, CpxAR plays an important role in cold resistance by upregulating \u003cem\u003eCspC\u003c/em\u003e expression[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. CpxAR contributes to the biofilm formation ability of \u003cem\u003eA. pleuropneumoniae\u003c/em\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], CpxAR and \u003cem\u003eCpxD\u003c/em\u003e mutants exhibited non-capsulated and were strongly impaired in virulence for mice, which suggested that CpxAR is a key modulator of capsule export that facilitates \u003cem\u003eA. pleuropneumoniae\u003c/em\u003e survival in the host[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These findings suggest a key role for CpxAR in the development of bacterial virulence, as well as the response to various environment stress.\u003c/p\u003e \u003cp\u003eThe antibiotic resistance caused by CpxAR deficiency also received wide attention. In \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, the CpxAR deficient strain was more susceptible to \u003cem\u003eβ-lactams\u003c/em\u003e and \u003cem\u003echloramphenicol\u003c/em\u003e than the wild-type strain, which reveals its direct involvement in conferring antimicrobial resistance against clinically significant antibiotics for the very first time[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In a \u003cem\u003eProteus mirabilis\u003c/em\u003e clinical isolate, alterations in \u003cem\u003eCpxA\u003c/em\u003e might confer \u003cem\u003eimipenem\u003c/em\u003e and \u003cem\u003eamikacin\u003c/em\u003e resistance without significant defects in the growth rate[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The antibiotic resistance formation in \u003cem\u003eSalmonella enterica\u003c/em\u003e serovar Enteritidis could also be impaired by CpxAR, for the development of multidrug resistance through the regulation of cell membrane permeability and efflux pump activity[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. For example, \u003cem\u003eCpxR\u003c/em\u003e contributes to the resistance of \u003cem\u003eS.enterica\u003c/em\u003e serovar Typhimurium to \u003cem\u003eaminoglycosides\u003c/em\u003e and \u003cem\u003eβ-lactams\u003c/em\u003e by influencing the expression level of multidrug resistance-related genes including \u003cem\u003eAcrB\u003c/em\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In \u003cem\u003eSalmonella enterica\u003c/em\u003e serovar Typhimurium, the \u003cem\u003eCpxA/CpxR\u003c/em\u003e gene deletion mutant also presented decreased \u003cem\u003eceftriaxone\u003c/em\u003e resistance[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, the regulatory mechanism of antibiotic resistance regulated by CpxAR in a variety of gram-negative bacteria is still unclear, and its influence on \u003cem\u003epolymyxin\u003c/em\u003e B resistance is still not well understood.\u003c/p\u003e \u003cp\u003eIn this research, we offered a new perspective for understanding the function of the CpxAR two-component system in response to \u003cem\u003epolymyxin\u003c/em\u003e B through the knockout of \u003cem\u003eCpxA\u003c/em\u003e in \u003cem\u003eS. enterica\u003c/em\u003e serovar Typhimurium.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial strains and plasmids\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eS. enterica\u003c/em\u003e serovar Typhimurium strainand plasmids, including pKD46, pKD4, pCP20 and pBR322 used for gene modification, were maintained in our laboratory. All strains including SL1344, SL1344 Δ\u003cem\u003eCpxA\u003c/em\u003e, SL1344 Δ\u003cem\u003eCpxA\u003c/em\u003e/ pBR322-\u003cem\u003eCpxA\u003c/em\u003e, SL1344 Δ\u003cem\u003eCpxA\u003c/em\u003e Δ\u003cem\u003eArcB\u003c/em\u003e, SL1344 Δ\u003cem\u003eCpxA\u003c/em\u003e Δ\u003cem\u003eArcB\u003c/em\u003e/ pBR322-\u003cem\u003eCpxA-ArcB\u003c/em\u003e were validated via PCR or gene sequencing.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGrowth curve detection\u003c/h3\u003e\n\u003cp\u003eThe strains cultured for 12 h were inoculated into LB broth liquid medium at a ratio of 1:100, then cultured at 37 ℃ and 180 rpm. Their absorbance (OD\u003csub\u003e600\u003c/sub\u003e) was tested every two hours.\u003c/p\u003e\n\u003ch3\u003eBacterial motility determination\u003c/h3\u003e\n\u003cp\u003e2 mL of the corresponding bacterial suspension with OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.0 was washed with ice-cold PBS and resuspended with 1mL PBS. Then 2\u0026micro;L of the resuspended bacteria were absorbed and dropped on the center of LB agar semisolid medium, then cultured at 37℃ for 12h. Colony diameters were measured and recorded.\u003c/p\u003e\n\u003ch3\u003eBiofilm formation ability assay\u003c/h3\u003e\n\u003cp\u003e200 \u0026micro;L of the corresponding bacterial solution with OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.2 was taken and cultured in a 96-well plate at 37 ℃ for 36 h. Then fixed with methanol for 15 min, washed with PBS, dyed with 0.5% crystal violet for 30 min, and washed three times with PBS. Then 200 \u0026micro;L of ethanol was added to dissolve the crystal violet for 30 min, and the absorbance (OD\u003csub\u003e570\u003c/sub\u003e) of each sample was determined.\u003c/p\u003e\n\u003ch3\u003eResistance to environmental stress\u003c/h3\u003e\n\u003cp\u003eThe strains cultured overnight were inoculated into the corresponding LB broth at a ratio of 1:100. For the acid stress experiments, medium with pH\u0026thinsp;=\u0026thinsp;5.5/4.5 was used. For the hypertonic stress test, 2.5 M NaCl was added to the medium. For the oxidative stress experiments, 10 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was added to the medium. After being culture at 37℃, 180 rpm for 3h and dilute at a ratio of 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e, 100 \u0026micro;L was added to an LB agar plate. Then the bacteria were cultured overnight, and make bacterium colony computation. The relative survival rate of each sample was calculated by comparing the colony count results of each sample with those of the corresponding strain cultured under normal conditions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eResistance to\u003c/b\u003e \u003cb\u003epolymyxin\u003c/b\u003e \u003cb\u003eB\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe bacterial suspension, which was precultured with the corresponding concentration of \u003cem\u003epolymyxin\u003c/em\u003e B, was diluted to 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e CFU/mL with Mueller‒Hinton (MH) broth. The mixture was added to a 96-well plate, and the antibiotic concentration was adjusted to the corresponding value. The plate was incubated at 37\u0026deg;C for 16\u0026ndash;18 h, and the MICs of each strain were observed and recorded. After serial dilution, the suspension was spread on \u003cem\u003epolymyxin\u003c/em\u003e LB agar plates, and the control group was spread on LB agar plates. Plates were incubated overnight at 37\u0026deg;C and record with photographs.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAntibiotic Susceptibility Test\u003c/h2\u003e \u003cp\u003eAfter culture for 12 h, the bacterial strains were washed with PBS and adjusted to a McFarland standard turbidity of 0.5. The strains were spread on LB agar plates, and antimicrobial discs were applied. Plates were incubated at 37\u0026deg;C for 24 hours, and the diameters of the antimicrobial circles were measured.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTranscriptome Sequencing\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eS.typhimurium\u003c/em\u003e and its \u003cem\u003eCpxA\u003c/em\u003e-delated strain were inoculated into LB broth containing 0.125 \u0026micro;g/ml \u003cem\u003epolymyxin\u003c/em\u003e B, and cultured overnight at 37\u0026deg;C, followed by dilution and subculture. After growing to the logarithmic phase, the bacteria were washed twice with PBS, collected, quickly frozen in liquid nitrogen, and then sent to Beijing Novogene Bioinformatics Technology Co., Ltd. for transcriptome sequencing and analysis.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThree biological replicates were set for each group (n\u0026thinsp;=\u0026thinsp;3) in all the experiments above. Statistical analysis was performed with GraphPad Prism 7. The results are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. The data were analyzed by Student\u0026rsquo;s t test. F test was used to compare the variances within each group, and the data without similar variance were adjusted by Welch\u0026rsquo;s correction. \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe purity of SL1344 kept in our laboratory was determined, which meets experimental standards without other bacterial contamination. On this basis, we constructed a \u003cem\u003eCpxA\u003c/em\u003e deletion strain SL1344∆\u003cem\u003eCpxA\u003c/em\u003e, and a \u003cem\u003eCpxA/ArcB\u003c/em\u003e deletion strain SL1344Δ\u003cem\u003eCpxA\u003c/em\u003eΔ\u003cem\u003eArcB\u003c/em\u003e, via the Red homologous recombination system. Concurrently, we also constructed their gene-complemented strains SL1344Δ\u003cem\u003eCpxA\u003c/em\u003e/pBR322-\u003cem\u003eCpxA\u003c/em\u003e and SL1344Δ\u003cem\u003eCpxA\u003c/em\u003eΔ\u003cem\u003eArcB\u003c/em\u003e/pBR322-\u003cem\u003eCpxA-ArcB\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003e1. Biological characteristics of the\u003c/b\u003e \u003cb\u003eCpxA\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eCpxA/ArcB\u003c/b\u003e \u003cb\u003edeficient strains\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe biological characteristics of these strains were then analyzed. Strains were cultured under the same conditions in LB broth, and growth curves were generated and plotted (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Results showed the deletion of \u003cem\u003eCpxA\u003c/em\u003e had no significant effect on the growth of SL1344, whereas combined deletion of \u003cem\u003eCpxA/ArcB\u003c/em\u003e inhibited its proliferation ability, indicating that \u003cem\u003eCpxA /ArcB\u003c/em\u003e may have influence on the growth metabolism of \u003cem\u003eS.typhimurium\u003c/em\u003e. The bacterial motility identification experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-C) revealed that the deletion of \u003cem\u003eCpxA\u003c/em\u003e had no significant effect on the motility of SL1344, but the motility of the \u003cem\u003eCpxA/ArcB\u003c/em\u003e deletion strain was significantly reduced, indicating that the double deletion of \u003cem\u003eCpxA/ArcB\u003c/em\u003e could inhibit the motility of \u003cem\u003eSalmonella Typhimurium\u003c/em\u003e. Meanwhile, the results of the biofilm formation ability assay showed no difference among SL1344 and all the gene deletion strains used above (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), indicating that \u003cem\u003eCpxA\u003c/em\u003e and \u003cem\u003eArcB\u003c/em\u003e may not affect the biofilm formation ability of \u003cem\u003eSalmonella Typhimurium\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn the experiment assessing resistance to environmental stress, the deletion of \u003cem\u003eCpxA/ArcB\u003c/em\u003e resulted in a decrease in tolerance to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). In the hypertonic stress test, there were no significant differences between the SL1344 and gene deletion strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). When pH\u0026thinsp;=\u0026thinsp;5.5, both the \u003cem\u003eCpxA\u003c/em\u003e and \u003cem\u003eCpxA/ArcB\u003c/em\u003e deletion strains exhibited significantly decreased growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, the resistance to acidic environments at pH\u0026thinsp;=\u0026thinsp;4.5 was enhanced when \u003cem\u003eCpxA\u003c/em\u003e was deleted, while decreased in \u003cem\u003eCpxA/ArcB\u003c/em\u003e deletion strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003eTo determine the tolerance of these strains to \u003cem\u003epolymyxin\u003c/em\u003e B, the broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) of the strains. Compared with that of SL1344, the MIC of the CpxA deletion strain increased by 2-fold, whereas the MIC of the CpxA/ArcB deletion strain increased by 4-fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). Meanwhile, the agar spot test also showed a certain advantage in \u003cem\u003epolymyxin\u003c/em\u003e B resistance for both \u003cem\u003eCpxA\u003c/em\u003e and \u003cem\u003eCpxA/ArcB\u003c/em\u003e deletion strains compaired with the original strain SL1344. These results indicate that the deletion of \u003cem\u003eCpxA\u003c/em\u003e and \u003cem\u003eArcB\u003c/em\u003e increased the resistance of SL1344 to \u003cem\u003epolymyxin\u003c/em\u003e B.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFinally, we used the K-B disk diffusion method (Table\u0026nbsp;1) to test the antibiotic susceptibility of these strains. The results showed that the \u003cem\u003eCpxA\u003c/em\u003e deletion strain had increased susceptibility to \u003cem\u003eamikacin, netilmicin\u003c/em\u003e, and \u003cem\u003ecephalothin\u003c/em\u003e, with further increased susceptibility observed in the \u003cem\u003eCpxA/ArcB\u003c/em\u003e deletion strain. This indicates the complex role of the CpxAR two-component system and the \u003cem\u003eArcB\u003c/em\u003e gene in regulating \u003cem\u003eSalmonella\u003c/em\u003e's response to antibiotics.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2. Transcriptomic sequencing analysis of\u003c/b\u003e \u003cb\u003eCpxA\u003c/b\u003e\u003cb\u003e-knockout strains\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBased on the understanding of the CpxAR system and \u003cem\u003eArcB\u003c/em\u003e in regulating the response of \u003cem\u003eSalmonella\u003c/em\u003e to antibiotics, we chose to use transcriptome sequencing analysis to explore the changes in molecular signaling pathways in SL1344 after the knockout of \u003cem\u003eCpxA\u003c/em\u003e under \u003cem\u003epolymyxin\u003c/em\u003e B stress. In this way, the role of \u003cem\u003eCpxA\u003c/em\u003e and its related pathways under \u003cem\u003epolymyxin\u003c/em\u003e B stress could be elucidated.\u003c/p\u003e \u003cp\u003eThe Illumina sequencing platform was used in our study to analyze the gene expression levels of \u003cem\u003eS.enterica\u003c/em\u003e serovar Typhimurium pretreated with \u003cem\u003epolymyxin\u003c/em\u003e B (SL1344-\u003cem\u003ePolB\u003c/em\u003e), and its \u003cem\u003eCpxA\u003c/em\u003e-knockout strain pretreated with \u003cem\u003epolymyxin\u003c/em\u003e B (SL1344\u003cem\u003eΔCpxA-PolB\u003c/em\u003e). The sequencing fragments obtained from the high-throughput sequencer were transformed into sequence data (reads) via CASAVA base recognition and filtered to obtain clean reads. The Q30 percentages of the clean data for all samples were higher than 95.47%, and the GC contents of the clean data for all samples ranged between 52.12% and 53.03% (Table\u0026nbsp;2). The filtered sequencing reads were subjected to genomic location analysis via Bowtie2 software. Approximately 93.32%-95.47% of the clean reads were successfully mapped to the reference \u003cem\u003eSalmonella\u003c/em\u003e genome, indicating that the reference genome was appropriately selected and that there was no contamination in the relevant experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUsing the featureCounts function in the Subread software, we filtered out reads with low alignment quality, not properly paired, or aligned to multiple regions of the genome. In total, 4717 compounds were obtained from the sequenced sequences, and 140 features with a constant or single value across samples were identified and removed. After the expression values (FPKM) of all the genes in each sample were calculated, we presented a boxplot (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) which revealed good uniformity in the gene expression distribution. To ensure the rational selection of biological replicates, we analyzed the correlation of gene expression levels between samples and created a heatmap, which revealed that all the R\u003csup\u003e2\u003c/sup\u003e values between biological replicates were greater than 0.8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), indicating good reproducibility of the biological samples. Principal component analysis (PCA) was then performed using Metaboanalysis, and the results showed that the data grouping was acceptable. The transcripts obtained above were subsequently used for subsequent experiments.\u003c/p\u003e \u003cp\u003eAfter gene expression quantification, we conducted a statistical analysis to identify genes whose expression levels significantly differed under various conditions. We used DESeq2 to normalize the raw readcounts, adjust for sequencing depth, and calculate \u003cem\u003ep\u003c/em\u003e-values. Differentially expressed genes (DEGs) were obtained by filtering for |log2(FoldChange)| \u0026gt; 1 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. 223 DEGs were found, of which 139 were upregulated and 84 were down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eSubsequently, we performed Gene Ontology (GO) enrichment analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) on the aforementioned DEGs. These DEGs were enriched in 287 GO terms, with Cellular Components (CC) mainly enriched in components related to movement of cell or subcellular level, and cilium or flagellum-dependent cell movement; Biological Processes (BP) were primarily enriched in components of membranes and intrinsic components; Molecular Functions (MF) were mainly enriched in oxidoreductase activity and antioxidant activity. Concurrently, we also conducted KEGG pathway enrichment analysis for these DEGs, which were enriched across 60 KEGG pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). The enrichment was primarily observed in pathways such as flagellar components, bacterial chemotaxis, arginine biosynthesis, ABC transporters, and microbial metabolism in diverse environments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3. PPI network analysis and hub gene cluster identification\u003c/h2\u003e \u003cp\u003eThese DEGs were further analyzed to find out the key pathways affected by the knockout of \u003cem\u003eCpxA\u003c/em\u003e in \u003cem\u003eS.typhimurium\u003c/em\u003e. The mainstream hierarchical clustering method was used to analyze gene expression values. After the rows of the expression data were normalized, the clustering results were plotted, which revealed distinct intergroup differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThen the DEGs were subsequently analyzed using the STRING[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] database. The protein-protein interaction (PPI) network was exported and processed via Cytoscape software to display their interactive relationships (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). A prominent cluster with all nodes closely related to each other was observed, which was also closely associated with \u003cem\u003eCpxA\u003c/em\u003e. Therefore, we used the MCODE plugin in Cytoscape to extract this cluster. We successfully obtained a cluster containing 22 nodes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). GO-BP enrichment analysis of these 22 nodes via the STRING online tool revealed that the functions of this cluster are focused on Bacterial-type flagellum organization and cell motility (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Additionally, KEGG enrichment analysis of these 22 nodes also showed significant enrichment in the flagellar assembly (stm02040) pathway: of the 38 nodes in the Flagellar assembly pathway, 21 were in this cluster (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eThis result demonstrates the significant impact of \u003cem\u003eCpxA\u003c/em\u003e dysfunction on the structure and function of flagella in \u003cem\u003eSalmonella typhimurium\u003c/em\u003e under \u003cem\u003epolymyxin\u003c/em\u003e B stress. These findings provide us important insights into the role of the CpxAR two-component system in the resistance of \u003cem\u003eS.typhimurium\u003c/em\u003e to polymyxin B.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003ePolymyxin\u003c/em\u003e B is ultimately considered as the last-resort treatment for infections caused by multidrug or extensively drug-resistant gram-negative bacteria. Solving the problem of \u003cem\u003eSalmonella\u003c/em\u003e resistance to \u003cem\u003epolymyxin\u003c/em\u003e B is of great significance to world public health security. The CpxAR two-component system has been proven to be associated with antibiotic resistance in various bacteria, and this study further demonstrated that the deletion of \u003cem\u003eCpxA/ArcB\u003c/em\u003e could increase the resistance of \u003cem\u003eS. typhimurium\u003c/em\u003e to \u003cem\u003epolymyxin\u003c/em\u003e B. Thus, transcriptome analysis was used, which revealed that the deletion of \u003cem\u003eCpxA\u003c/em\u003e can disrupt the structure and function of flagella and is related to various pathways, including cell motility. These proteins may become key targets for \u003cem\u003eS.typhimurium\u003c/em\u003e to cope with the toxicity of \u003cem\u003epolymyxin\u003c/em\u003e B.\u003c/p\u003e \u003cp\u003eLaboratory tests revealed that the deletion of \u003cem\u003eCpxA/ArcB\u003c/em\u003e can significantly reduce the growth rate, motility, and tolerance to hydrogen peroxide and acidic environments of the \u003cem\u003eS.typhimurium\u003c/em\u003e SL1344 strain, while increasing its resistance to \u003cem\u003epolymyxin\u003c/em\u003e B. However, the strain with a single deletion of \u003cem\u003eCpxA\u003c/em\u003e did not show significant changes in most of these biological characteristics besides \u003cem\u003epolymyxin\u003c/em\u003e B resistance. To further explore the specific molecular mechanisms by which the CpxAR two-component system affects these biological characteristics, particularly the resistance to \u003cem\u003epolymyxin\u003c/em\u003e B, this study conducted transcriptome sequencing on the \u003cem\u003eCpxA\u003c/em\u003e deletion strain, and performed a transcriptomic analysis for them under \u003cem\u003epolymyxin\u003c/em\u003e B stress. For the selection of transcriptom samples, this study chose the \u003cem\u003eCpxA\u003c/em\u003e single-gene deletion strain, which presented some changes in resistance to \u003cem\u003epolymyxin\u003c/em\u003e B, for low-concentration \u003cem\u003epolymyxin\u003c/em\u003e B treatment and transcriptom sequencing. This design is only a part of our in-depth study of the CpxAR two-component system in conjunction with \u003cem\u003eArcB\u003c/em\u003e for their resistance against \u003cem\u003epolymyxin\u003c/em\u003e B. In subsequent research, we will further investigate how \u003cem\u003eArcB\u003c/em\u003e enhances the function of \u003cem\u003eCpxA\u003c/em\u003e and \u003cem\u003eCpxR\u003c/em\u003e in \u003cem\u003epolymyxin\u003c/em\u003e B resistance.\u003c/p\u003e \u003cp\u003eThrough transcriptome sequencing, we identified 223 DEGs. GO and KEGG enrichment analyses revealed the functions of these genes in various domains, such as cell motility, flagellar assembly, and transmembrane transporter activity. To further clarify the pathways involved there, we conducted a PPI network via STRING. Then we filtered out a cluster of DEGs, which is closely related to flagellar assembly functions. On the basis of these findings, we preliminarily determined that the \u003cem\u003eCpxA\u003c/em\u003e gene in \u003cem\u003eS.typhimurium\u003c/em\u003e can alter the structure and function of flagella under \u003cem\u003epolymyxin\u003c/em\u003e B stress. In the \u003cem\u003eCpxA\u003c/em\u003e deletion strain, the expression of most genes involved in the flagellar assembly related signaling pathways was upregulated, which may be a key factor in the increased antibiotic resistance of the \u003cem\u003eCpxA\u003c/em\u003e deletion strain.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest Statement\u003c/h2\u003e \u003cp\u003eThe authors have no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthics Statement\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding Information\u003c/h2\u003e \u003cp\u003eThis study was supported by the Natural Science Foundation of Hebei Province of China (Grant no. C2022407019).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eX.Z. data analysis and writing; Y.F. experimental design and guidance; C.X. system construction and project implementation; Z.G. assistance in molecular experiments; C.L. assistance in molecular experiments; Y.L. project supervision. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eTranscriptomics data will be made public after the upload is completed (currently being submitted in GEO database).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDai W, Zhang Y, Zhang J, Xue C, Yan J, Li X, Zheng X, Dong R, Bai J, Su Y, et al. Analysis of antibiotic-induced drug resistance of Salmonella enteritidis and its biofilm formation mechanism. Bioengineered. 2021;12(2):10254\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen K, Gao Y, Li L, Zhang W, Li J, Zhou Z, He H, Chen Z, Liao M, Zhang J. Increased Drug Resistance and Biofilm Formation Ability in ST34-Type Salmonella Typhimurium Exhibiting Multicellular Behavior in China. Front Microbiol. 2022;13:876500.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan ML, Liu X, Velkov T, Lin YW, Zhu Y, Creek DJ, Barlow CK, Yu HH, Zhou Z, Zhang J, et al. Comparative Metabolomics Reveals Key Pathways Associated With the Synergistic Killing of Colistin and Sulbactam Combination Against Multidrug-Resistant Acinetobacter baumannii. Front Pharmacol. 2019;10:754.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRigatto MH, Falci DR, Zavascki AP. Clinical Use of Polymyxin B. Adv Exp Med Biol. 2019;1145:197\u0026ndash;218.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZavascki AP, Goldani LZ, Li J, Nation RL. Polymyxin B for the treatment of multidrug-resistant pathogens: a critical review. J Antimicrob Chemother. 2007;60(6):1206\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJin T, Zhan X, Pang L, Peng B, Zhang X, Zhu W, Yang B, Xia X. CpxAR two-component system contributes to virulence properties of Cronobacter sakazakii. Food Microbiol. 2024;117:104393.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePasqua M, Coluccia M, Eguchi Y, Okajima T, Grossi M, Prosseda G, Utsumi R, Colonna B. Roles of Two-Component Signal Transduction Systems in Shigella Virulence. Biomolecules 2022, 12(9).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGahlot DK, Wai SN, Erickson DL, Francis MS. Cpx-signalling facilitates Hms-dependent biofilm formation by Yersinia pseudotuberculosis. NPJ biofilms microbiomes. 2022;8(1):13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYao Q, Xie T, Fu Y, Wan J, Zhang W, Gao X, Huang J, Sun D, Zhang F, Bei W, et al. The CpxA/CpxR two-component system mediates regulation of Actinobacillus pleuropneumoniae cold growth. Front Microbiol. 2022;13:1079390.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi H, Liu F, Peng W, Yan K, Zhao H, Liu T, Cheng H, Chang P, Yuan F, Chen H, et al. The CpxA/CpxR Two-Component System Affects Biofilm Formation and Virulence in Actinobacillus pleuropneumoniae. Front Cell Infect Microbiol. 2018;8:72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu F, Yao Q, Huang J, Wan J, Xie T, Gao X, Sun D, Zhang F, Bei W, Lei L. The two-component system CpxA/CpxR is critical for full virulence in Actinobacillus pleuropneumoniae. Front Microbiol. 2022;13:1029426.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSrinivasan VB, Vaidyanathan V, Mondal A, Rajamohan G. Role of the two component signal transduction system CpxAR in conferring cefepime and chloramphenicol resistance in Klebsiella pneumoniae NTUH-K2044. PLoS ONE. 2012;7(4):e33777.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLecuru M, Nicolas-Chanoine MH, Tanaka S, Montravers P, Armand-Lefevre L, Denamur E, Mammeri H. Emergence of Imipenem Resistance in a CpxA-H208P-Variant-Producing Proteus mirabilis Clinical Isolate. Microb drug Resist (Larchmont NY). 2021;27(6):747\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu M, Huang X, Xu X, Zhang Z, He S, Zhu J, Liu H, Shi X. Characterization of the Role of Two-Component Systems in Antibiotic Resistance Formation in Salmonella enterica Serovar Enteritidis. \u003cem\u003emSphere\u003c/em\u003e 2022, 7(6):e0038322.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang H, Sun Y, Yuan L, Pan Y, Gao Y, Ma C, Hu G. Regulation of the Two-Component Regulator CpxR on Aminoglycosides and β-lactams Resistance in Salmonella enterica serovar Typhimurium. Front Microbiol. 2016;7:604.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu WS, Chen HW, Zhang RY, Huang CY, Shen CF. The expression levels of outer membrane proteins STM1530 and OmpD, which are influenced by the CpxAR and BaeSR two-component systems, play important roles in the ceftriaxone resistance of Salmonella enterica serovar Typhimurium. Antimicrob Agents Chemother. 2011;55(8):3829\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSzklarczyk D, Kirsch R, Koutrouli M, Nastou K, Mehryary F, Hachilif R, Gable AL, Fang T, Doncheva NT, Pyysalo S, et al. The STRING database in 2023: protein-protein association networks and functional enrichment analyses for any sequenced genome of interest. Nucleic Acids Res. 2023;51(D1):D638\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"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},"keywords":"Salmonella typhimurium, CpxAR two-component system, Polymyxin resistance, CpxA, Flagellar assembly.","lastPublishedDoi":"10.21203/rs.3.rs-5322344/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5322344/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003ePolymyxin\u003c/em\u003e B is considered as a last-resort antibiotic for multidrug-resistant or extensively drug-resistant gram-negative bacterial infections. Addressing \u003cem\u003eSalmonella\u003c/em\u003e resistance to \u003cem\u003epolymyxin\u003c/em\u003e B is crucial for global public health. Our study revealed that \u003cem\u003eCpxA/ArcB\u003c/em\u003e knockout inhibits the motility, tolerance to acid stress and oxidative stress of \u003cem\u003eS.typhimurium\u003c/em\u003e. Most notably, the tolerance of \u003cem\u003eS.typhimurium\u003c/em\u003e to \u003cem\u003epolymyxin\u003c/em\u003e B could be increased by either \u003cem\u003eCpxA\u003c/em\u003e or \u003cem\u003eCpxA/ArcB\u003c/em\u003e deletion. In this study, transcriptomic detection and analysis were used to clarify the mechanisms by which \u003cem\u003eCpxA-\u003c/em\u003edeleted \u003cem\u003eS.typhimurium\u003c/em\u003e is involved in resistance to \u003cem\u003epolymyxin\u003c/em\u003e B stress, which may be related to processes such as increased assembly of bacterial flagella.\u003c/p\u003e","manuscriptTitle":"The Impact of the CpxA on Regulating Polymyxin B Resistance in Salmonella Typhimurium","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-06 06:43:50","doi":"10.21203/rs.3.rs-5322344/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":"ca5b055e-20b9-470a-bc4a-d4de16ee1184","owner":[],"postedDate":"November 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-29T10:23:30+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-06 06:43:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5322344","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5322344","identity":"rs-5322344","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}