Antimicrobial peptide AP2 ameliorates Salmonella Typhimurium infection by modulating gut microbiota

Antimicrobial peptide AP2 ameliorates Salmonella Typhimurium infection by modulating gut microbiota | 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 Antimicrobial peptide AP2 ameliorates Salmonella Typhimurium infection by modulating gut microbiota Lianglan Li, Aikun Fu, Qiufen Mo, Yi Wan, Yuanhao Zhou, Zihan Zeng, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3990205/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 Endogenous antimicrobial peptides/proteins contribute to reshape a healthy gut microbiota which play benefit roles in anti-inflammation and pathogen colonization resistance. Salmonella infection is one of the most frequently reported bacterial diseases worldwide. Manipulation of the gut microbiota through exogenous antimicrobial peptide may protects against Salmonella enterica colonization and improve clinical outcomes. In this study, results showed that oral administration of antimicrobial peptide AP2, an optimized version of native apidaecin IB (AP IB) had a protective effect against ST infections in mice indicated by alleviated ST-induced body weight loss and reduced the serum inflammatory cytokines. 16S rRNA-based analysis of microbiota from the cecum content showed that AP2 altered gut microbiota by significantly increasing the proportion of Bifidobacterium and decreasing Akkermansia at the genus level. Furthermore, the transplantation of fecal microbiota from AP2-treated donor mice, instead of control mice, significantly reduced caecal damage caused by ST. In conclusion, these findings hightlighted one of novel action mechanisms of exogenous antimicrobial peptide on ameliorating Salmonella Typhimurium infection by modulating gut microbiota. Apidaecin Salmonella Typhimurium inflammation gut microbiota fecal microbiota transplantation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Salmonellosis caused by Salmonella Typhimurium infection is one of the most frequently reported bacterial diseases in humans and animals[ 1 , 2 ], which is characterized by fever, acute intestinal inflammation, and diarrhea within 24h. After entering the intestinal lumen, this pathogen is able to overcome the intestinal barrier and generate gaps in the epithelium, then spread rapidly to the organs such as mesenteric lymph nodes (MLNs), spleen and liver [ 3 – 8 ]. Antibiotics are critical for the treatment of invasive ST infections [ 9 ]. However, the emergence of multidrug-resistant ST makes these infections more difficult to treat [ 10 – 12 ]. Meanwhile, antibiotic treatment resulted in a disruption of the gut microbiota, which were demonstrated to preclude ST infection by conferring colonization resistance, further increases susceptibility to ST infection[ 13 ]. For example, streptomycin treatment prior to ST ingestion led to an acute inflammation of the gastrointestinal tract, along with weight loss, diarrhea, and extra-intestinal infection (e.g. in the spleen and liver) [ 14 , 15 ]. In this respect, it is urgent to identify new antimicrobial agents to defense against ST. Antimicrobial peptides (AMPs) are regarded as promising candidates. AMPs are found in all higher organisms[ 16 ] and contribute to the first line of defense against microbial infections. They are gene-encoded and highly conserved innate immune effector molecules. AMPs can inhibit or kill a broad range of pathogens [ 17 ] by different mechanisms, such as lysis of the bacterial membrane or inhibition of certain targets on the surface of or within the bacteria[ 18 ], but rarely induce bacterial resistance [ 19 ]. At the same time, it was found that gut commensal microbes are resistant to high doses of AMPs [ 20 ], and in turn, AMPs reduced the susceptible to enteric pathogens colonization and inflammation by keeping gut microbial context balance[ 21 , 22 ]. Apidaecin is a type of proline-rich AMPs (PrAMPs) that is produced by bees and wasps in response to bacterial infections [ 23 , 24 ]. Among the three isoforms, apidaecin IB (AP IB) is clearly the most potent and highly active against Escherichia coli ( E. coli ) [ 25 , 26 ]. AP2 is a structurally modified version of native AP IB and contains arginine-valine-arginine (RVR) in positions one to three instead of glycine-asparagine-asparagine (GNN), respectively. Our previous research has demonstrated that AP2 has greater antibacterial activity against Gram-negative bacteria, and good stability under acidic, pepsin, and high temperature conditions. These features suggest that AP2 can be used in vivo to treat intestinal pathogenic infections. The aim of this study was to investigate whether AP2 was effective in the prevention of ST infections in animals. We found that the antimicrobial peptide AP2 had protective effect against ST infection by modulating the gut microbiota in mice. Materials and methods Ethics statement Male C57BL/6 mice aged 6–7 weeks were purchased from Slac Animal Inc. (Shanghai, China) and maintained at the Laboratory Animal Center of Zhejiang University. All animal experiments were conducted in accordance with experimental protocols approved by the Institutional Animal Care and Use Committee of Zhejiang University (ZJU20181068). All animal experiments were performed in strict accordance with university guidelines. Mice were fed and watered ad libitum. Preparation of peptides AP2 peptide (amino acid sequence: RVRRPVYIPQPRPPHPRL) and AP IB (amino acid sequence: GNNRPVYIPQPRPPHPRL) peptide were synthesized and purified by GL Biochem Ltd. (Shanghai, China). The peptide purity was determined to be higher than 95% by reverse-phase high-performance liquid chromatography (RP-HPLC). Bacterial strain and culture Salmonell a Typhimurium CMCC 50115 (ST) was obtained from the Institute of Preventive Veterinary Medicine, Zhejiang University (China). ST was inoculated in Luria-Bertani (LB) medium (10 g L-1 peptone, 5 g L-1 yeast extract, and 10 g L-1 NaCl) and incubated at 37℃ to the exponential phase with shaking at 250 r min-1. Antibacterial activity assay. The minimum inhibitory concentrations (MIC) of AP2 and antibiotics were determined by the micro-broth dilution method [ 27 ]. Briefly, ST was grown at 37℃ to an OD600 of 0.4 in LB medium and was diluted to 5×105 colony-forming units (CFUs) mL-1. A total of 180 µL cell suspension and 20 µL serial 2-fold dilutions of the peptide (final concentration ranging 320, 160, 80, 40, 20, 10, 5, and 2.5 µg mL-1) were added into each well, respectively. After incubation at 37℃ for 12–16 h, MICs were determined as the lowest peptide concentration at which no bacterial growth was observed. All tests were conducted in triplicate. Animals and experimental protocols. Mice were weighed and randomly divided into four groups (n = 12 per group) without significant difference in body weight: Control, AP2, ST and AP2 + ST groups, respectively. Before the inoculation with ST, mice in the AP2 and AP2 + ST groups were treated with AP2 (10 µg mL-1, 200 µL, 2µg /mouse) by gavage, once a day for 2 weeks, while the control and ST groups were given sterile ultrapure water (200 µL) instead. After 2 weeks, mice in the ST and AP2 + ST groups were fasted for 12 hours prior to inoculation via oral gavage with 4×108 CFUs ST in 200 µL PBS, as determined by plating, while mice from the control and AP2 groups were inoculated with 200 µL PBS and housed separately. Animals were sampled and evaluated at the 4th day post-inoculation (5 repeated experiments) since the weight loss in the ST-inoculated mice was around 20% of the initial body weight [ 28 ]. Histopathology Tissues samples of cacum and colon were harvested and fixed in 4% paraformaldehyde, dehydrated and processed into paraffin sections according to standard procedure. The paraffin sections were subjected with hematoxylin-eosin (H&E) staining at the histology core facilities at Zhejiang University. Images were captured by a Zeiss Imager-M2. Blinded examination by a GI pathologist at Zhejiang University was used to score the pathology of samples with previously published methods (49). Each section was evaluated for the submucosal edema, inflammatory infiltrate and epithelium (50). The pathological changes were scored from 0 to 4 according to the following scale: 0 = none, 1 = low, 2 = moderate, 3 = high, and 4 = extreme. The inflammation score for each mouse was calculated by adding the score for each parameter (21). Transmission electron microscopy (TEM) The morphology and histology of intercellular tight junctions were characterized by TEM. For TEM assessment, briefly, a 2-cm-long jejunum specimen was excised and fixed. Ultrathin sections were obtained and stained by uranyl acetate and lead citrate before examination on a Hitachi Model H-7650 TEM [ 29 ]. Analysis of sera parameters Blood was collected from the femoral artery and serum cytokines were analyzed using kits (ELISA kit; e-Bioscience, USA) following the manufacturer’s protocols. Cytokines in sera were expressed as pg mL-1. ST recovered in feces and MLNs The colonization of ST in the feces and MLNs were detected as previously described [ 5 ]. Briefly, stools were collected aseptically, weighed, and homogenized in PBS containing 0.1% Triton X-100. MLNs were dissected and minced through a 45-µm nylon mesh. Triton X-100 at a final concentration of 0.1% was added to the cell suspensions and incubated for 30 second. Serial dilutions were made and then coated on Salmonella -Shigella agar plates (Britania, Buenos Aires, Argentina). After 24 h, the CFUs were quantified by visual counting of micro-colonies and data were presented as mean ± SD of triplicate samples. Effect of AP2 on ST in intestine The mice (n = 4 per group) were pre-treated with streptomycin (20 mg per mouse)[ 30 ]. After one day, mice were inoculated with ST. 4 h later, mice in the AP2 + ST group was treated with AP2 (10 µg mL-1, 200 µL, 2µg /mouse) by gavage, while the ST group was given sterile ultrapure water (200 µL) instead. Finally, feces were collected every three hours to monitor the amount of ST. DNA extraction, V3-V4 16S rRNA gene amplification and microbiota community analysis DNA from the cecum content (collected at the 4th day post-inoculation) was extracted with the TIANamp Stool DNA Kit (TIANGEN BIOTECH CO., LTD, Beijing, China) according to the manufacturer’s instructions. PCR amplification and sequencing were performed by the G-BIO Inc. (Hangzhou, China). Bacterial DNA was amplified by a two-step PCR enrichment of the 16S rDNA (V3 and V4 regions) with forward primers containing the sequence 5’-CCTACGGGNGGCWGCAG-3’ and reverse primers containing the sequence 5’-GACTACHVGGGTATCTAATCC-3’. The processed pair-end reads were assembled using PandaSeq v2.8 with default parameter [ 31 ]. Chimeras were identified and removed using USEARCH 6.1 within QIIME. The QIIME script “add_qiime_labels.py” was used to combine the non-chimeric sequences from each sample into one file. OTU picking and taxonomic assignments were performed using the open-reference OTU picking workflow in Qiime with the Greengenes reference database [ 32 ]. OTUs with abundances below 0.005% of the total number of sequences were discarded [ 33 ]. Alpha diversity measurements, including Shannon, Chao1, observed species, and Good’s coverage, were calculated using the alpha_rarefaction.py script in QIIME. Weighted and unweighted unifrac distances [ 34 ] were calculated from the rarefied OTU table using the beta_diversity_through_plots.py script in QIIME. Principal component analysis (PCA) was conducted using the website METAGENassist. The linear discriminant analysis (LDA) effect size (LEfSe) method [ 35 ] was performed using the Galaxy online interface ( http://huttenhower.sph.harvard.edu/galaxy ). Fecal microbiota transplant (FMT) experiment Fecal microbiota was obtained from fresh stool samples of control (n = 4) or AP2 treated mice (n = 4). Fresh stool samples were pooled and diluted 20-fold and homogenized in sterile and pre-reduced 0.1 M potassium phosphate buffer (PBS, pH 7.2) containing 15% glycerol (v/v) to produce a 5% fecal suspension according to the previous study [ 36 ]. The homogenate was centrifuged at 100 g for 5 min at 4℃ and the resulting suspension was then pipetted into 5 mL sterile tubes and stored at -80℃ [ 37 ]. For FMT, the mice (n = 4 per group) were pre-treated with streptomycin (20 mg per mouse). Previously frozen pooled fecal samples from control or AP2-treated mice were thawed on ice and delivered via anorectal inoculation (200 µl) by using a catheter made from a round-tip silicone tube with a diameter of 1 mm. After inoculation, the mouse was held vertically with its head down for 1 min to prevent loss of the infusion. 4 h later, mice were inoculated with ST (Fig. 6 a). Finally, feces and MLNs were collected aseptically for further determination. Statistical analysis Data are expressed as means ± standard deviations (SD). A Student’s t-test and one-way analysis of variance (ANOVAs) with Tukey’s post hoc tests were performed and considered significant at P -value < 0.05. Results The MIC of AP2 in vitro The MIC of AP2 and AP IB against ST in vitro was first measured to explore their antibacterial activity. The results showed that the MIC of AP2 against Salmonella was 5 µg mL-1, which was better than that of AP IB with MIC of 10 µg mL-1 and kanamycin (Kan) and streptomycin (Strep) (Table 1 ). Table 1 The MIC of antimicrobial peptides. Strain AP2 (µg mL-1) AP IB (µg mL-1) Kan (µg mL-1) Strep (µg mL-1) ST 5 10 12.5 32.5 ST: Salmonella Typhimurium CMCC 50115. AP IB: apidaecin IB. Kan: kanamycin. Strep: streptomycin. AP2 attenuated the symptoms of ST infections in vivo To investigate whether AP2 has a protective effect against ST infection in vivo , C57BL/6 mice were treated with or without AP2 before ST infection. Compared with the control group, none of the animals in the AP2 group showed obvious weight loss (Fig. 1 ), whereas mice challenged with ST exhibited significant body weight loss at day 2 (ST group) and day 3 (both ST and AP2 + ST groups). However, the body weight loss of mice in AP2 + ST group markedly alleviated at day 3 and day 4 compared with ST group (Fig. 1 ). The results of the intestinal histopathology showed that ST infection induced an acute inflammation in the mucosa characterized by the swelling of the lamina propria (Fig. 2 a), inflammatory infiltration and desquamation (Fig. 2 b) and the shedding of microvilli (Fig. 2 c). Furthermore, the ST infection also induced intestinal mitochondria swelling (Fig. 2 c), which was significantly ameliorated by AP2 administration (Fig. 2 ). The level of serum pro-inflammatory cytokines reflect the intensity of inflammation. Mice in the control mice and AP2-treat mice) groups had similar low levels of serum inflammatory cytokines (IL-1β, and IFN-γ (Fig. 3 ). While ST inoculation resulted in a significant increase in the serum pro-inflammatory cytokines IL-1β and IFN-γ, which were decreased by AP2 pretreatment (Fig. 3 ). ST infection may result in bacterial translocation across the intestinal barrier, followed by migration to the spleen and liver [ 38 ]. Glutamic pyruvic transaminase (GPT) is mainly present in the cytoplasm of hepatic cell. When hepatocyte is injured, GPT will release into blood, and thus increasing the serum GPT activity[ 39 ]. Therefore, the level of this enzymes in serum could be used to assess the extent of damage[ 40 ]. Results showed that GTP activity were significantly increased in the ST group, which was markedly reduced (P < 0.05) in the AP2 + ST group (Fig. 3 ). These results suggested that AP2 administration ameliorated ST-induced liver damage. AP2 does not inhibit ST growth in the intestinal tract Since AP2 conferred protection against ST infection, next we want to verify whether AP2 could inhibit the growth of ST within intestinal tract directly. Since the microbiota confers colonization resistance to block Salmonella gut colonization [ 41 ], the streptomycin mouse model was used to remove gut microbial community and exclude its interference, and the amount of ST in feces was monitored every 3 h after treated with AP2 (Fig. 4a). Surprisingly, we found that AP2 treatment did not decrease the ST loads in the feces (Fig. 4b), which is inconsistent with the results obtained from in vitro experiments. Thus, the protective effect of AP2 against ST in vivo was not associated with its bactericidal effect directly. Several studies have demonstrated that the gut microbiota and its metabolites provide colonization resistance to ST infection [ 13 , 30 , 42 ]. But ST exploits inflammation to compete with this colonization resistance [ 43 – 45 ]. A previous study also found that AMPs beneficially affected the intestinal health by shaping the microbial ecology [ 46 ]. This led us to focus our further analyses on the gut microbiota composition. AP2 treatment modified the gut microbiota composition Since AP2 exhibited strong antibacterial capacity, which may influence the composition of the microbiome, a 16S rRNA-based analysis was used to determine the microbiota from the cecum content. Results showed that there were no significant differences in alpha diversity of the microbiota as reflected by Shannon, Chao1, Faith’s phylogenetic, and observed species indexes among control, AP2, ST and AP2 + ST groups (Supplemental Fig. S1 ). However, beta diversity assessment with weighted UniFrac distance revealed that the microbial community significantly different (ANOSIM, p < 0.05) between control and AP2 groups and between AP2 + ST and ST groups both at the phylum (Fig. 5a) and the genus (Fig. 5b) level. AP2 treatment did not significantly change the major microbial composition at the phylum level (Fig. 5c), but significantly increased the proportion of Actinobacteria , Alcaligenaceae, Allobaculum, Bifidobacteriales, Betaproteobacteria, Burkholderiales, Clostridiaceae, Lachnospiraxeae, Mogibacteriaceae and Sutterella , and decreased the proportion of Coprobacillus and Verrucomicrobia compared with control group. Meanwhile, the proportion of Alcaligenaceae, Betaproteobacteria, Burkholderiales , and Sutterella in the AP2 + ST mice were significantly higher than those in ST mice. Notably AP2 treatment also decreased the proportion of Verrucomicrobia independent of ST-inoculation. (Supplemental Fig. S2). The linear discriminant analysis effect size (LEfSe) [ 35 ] was used to identify specific OTUs that differed between the control and AP2 with or without ST inoculation. 17 discriminative features (LDA score > 2) whose relative abundances varied significantly between the control and AP2 groups was identified. Furthermore, 11 bacterial taxa, such as Actinobacteria , Bifidobacterium , Allobaculum , and Sutterella were enriched in the AP2 group, while 6 bacterial taxa were increased in the control group, such as Verrucomicrobia , Coprobacillus , and Akkermansia. Based on the LEfSe, 17 bacterial taxa were significantly more abundant in the AP2 + ST group (e.g. Prevotella, AF12, Dehalobacterium, Oscillospira, Coprobacillus, Sutterella, Bilophila , and Desulfovibrio ; p < 0.05), while only 9 taxa were overrepresented in the ST group (e.g. Verrucomicrobia , Clostridium , Coprococcus , and Akkermansia ; p < 0.05 ) (Fig. 5d). Collectively, these data suggested that the gut microbiota modified by AP2 may be associated with its protective effect against ST infection in mice. Fecal microbiota transplant (FMT) of AP2-treated mice influences the course of ST-induced caecal inflammation in mice To further verify the hypothesis above, we performed FMT experiment (Fig. 6 a). The mice that were pre-treated with streptomycin [ 30 ], received microbiotas from the control or AP2-treated mice through anorectal inoculation, respectively. 4 h later, mice were infected with ST by oral gavage to evaluate the effects of the AP2-treated microbiota on ST infection (Fig. 6 a). Results showed that AP2-treated microbiota could significantly decrease levels of ST in the stool and MLN (Fig. 6 b), and also decreased intestinal pathology (Fig. 7), providing evidence that this alerted microbial community induced by AP2 treatment was effective against ST infection. These findings suggest that the beneficial effect of AP2 treatment on the course of infection was transferable via FMT. Discussion Oral infection of mice with ST leads to a fatal systemic disease [ 13 ]. During Salmonella invasion, PAMPs (pathogen-associated molecular patterns) and DAMPs (Danger-associated molecular patterns) initiate innate immunity, leading to recruitment of neutrophils and the increase expression of pro-inflammatory cytokines, most notably interleukin (IL)-6, IL-1β, and IFN-γ. Although neutrophils are indispensable in Salmonella resistance, neutrophils infiltration can also cause detrimental mucosa wounding. Under these circumstances, Salmonella gains a growth advantage over the gut microbiota from inflammatory conditions [ 47 ]. This study showed that AP2 has protective effects against ST infection, indicated by the decreased body weight loss, the attenuated intestinal and systemic inflammation ,lower ST translocation in ST-inoculated mice which were in line with the anti-Salmonella effects of other AMPs [ 48 ]. Furthermore, in this study, gut microbiota involved in the underlying mechanism of P2 function. Enteric pathogens interact extensively with the gut microbiota [ 49 , 50 ]. Once inoculated, ST would compete with the microbiota by exploits inflammation, however, gut microbiota indispensable to protect mice from ST colonization in the gut [ 51 ]. The complex interactions between ST, inflammation and microbiota have been researched and reviewed by some researchers [ 52 – 55 ]. Therefore, the inflammatory condition and microbiota modified by AP2 may also be the cause to elucidate its protective effect against ST infection. It has been reported that and that the gut microbiota plays key role in maintenance of health and the development of the mucosal immune system [ 56 – 59 ]. In current study, AP2 had no effect on the main categories of gut microbiota, which is consistent with results of one previous study that gut microbes from all dominant phyla were resistant to high levels of inflammation-associated AMPs [ 20 ]. We found that AP2 significantly favors the proliferation of some G + beneficial microorganisms, e.g., Bifidobacterium , Allobaculum , and Clostridia . Certain bacteria belonging to Bifidobacterium and Allobaculum were able to produce short-chain fatty acids (SCFAs), which were detrimental to the growth of S. Typhimurium [ 60 – 62 ]. Butyrate and propionate can act as signal molecules to downregulate the expression of the Salmonella pathogenicity island (SPI)1-encoded type 3 secretion system (T3SS) invasion genes [ 63 ], which were crucial for this bacterium to invade intestinal epithelial cells [ 64 ]. Furthermore, Clostridia had been proven to inhibit the ST colonization in the gut [ 30 ]. In addition, Oscillospira , Bilophila , and Desulfovibrio instead of Clostridium and Akkermansia were enriched in the AP2 + ST group compared to the ST group. Oscillospira were also able to secrete SCFA such as butyrate [ 65 ]. Many strains of Proteobacteria including Bilophila and Desulfovibrio , can induce the secretion of IgA and regulate intestinal homeostasis [ 66 ]. Proteobacteria, such as e.g., Bilophila and Desulfovibrio which was increased in AP2 + ST group compared with ST group, produce hydrogen sulfide (H2S), which have various biological effects in the immune system [ 67 , 68 ]. H2S exhibits several anti-inflammatory effects such as reduction of edema formation and suppression of the release of pro-inflammatory cytokines (such as TNF-α and IFN-γ) [ 69 ] and suppressing the activation of NF-κB [ 70 ]. Moreover, the proportions of G- bacteria such as Akkermansia were significantly decreased in the AP2 and AP2 + ST groups compared with the control and ST group, respectively. Previous studies have reported that the relative abundance of Akkermansia positively correlated with intestinal inflammation in a murine chronic enteritis model, and Akkermansia significantly exacerbated ST-induced intestinal inflammation [ 71 ]. However, some studies and reviews indicate that Akkermansia may be related to anti-inflammatory and have potential anti-inflammatory properties [ 72 , 73 ]. The impact of Akk on the inflammation may be related to its relative abundance, but the specific mechanism is not very clear yet. Hence, it could be concluded that the gut microbiota altered by AP2, characterizing with more abundance of anti-inflammatory bacteria and less abundance of pro-inflammatory bacteria, inhibit ST invasion and translocation, which verified by the results of FMT. In line with this, the pathogen colonization was reduced in the infected animals that received AP2-treated feces, indicating that the AP2 can shape an adaptive microbiota, which can then inhibit the growth of ST. Conclusion The work presented here indicated that AP2 ameliorated ST infection by modulating the gut microbiota and which play a key role in the protection against pathogen infection. These findings reveal a previously unrecognized mechanism by which AMPs alleviate the bacterial infection and thus will provide additional strategies for the further pharmaceutical investigations. Declarations Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ethics approval and consent to participate Ethics approval All animal experiments were conducted in accordance with experimental protocols approved by the Institutional Animal Care and Use Committee of Zhejiang University (ZJU20181068). All animal experiments were performed in strict accordance with the guidelines. Funding This work was supported by Young Scientists Fund of the National Natural Science Foundation of China (Grant No. 82003436), National Nature Science Foundation of China (Grant No. 32372892), the ‘twelfth five-year-plan’ in National Support Program for Science and Technology for rural development in China (Grant No. 2011BAD26B02) and Natural Science Foundation of China (Grant No. 31472128). Author Contribution All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by LL and AF. The first draft of the manuscript was written by LL, AF and QM, YZ, ZZ, AS, XZ and YW had been involved in analyzing the data and revising the manuscript critically. AF, QM participated in the experimental design. Weiqin Li and Weifen Li provided funding support. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Acknowledgements This work was financially supported by Young Scientists Fund of the National Natural Science Foundation of China (Grant No. 82003436), National Nature Science Foundation of China (Grant No. 32372892), the ‘twelfth five-year-plan’ in National Support Program for Science and Technology for rural development in China (Grant No. 2011BAD26B02) and Natural Science Foundation of China (Grant No. 31472128). 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Akkermansia muciniphila and its role in regulating host functions. Microb Pathog, 106, 171–181. https://doi.org/10.1016/j.micpath.2016.02.005 . Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial.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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-3990205","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":275093877,"identity":"bdc479a4-3444-4dde-9918-947a73b8ca4a","order_by":0,"name":"Lianglan Li","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Lianglan","middleName":"","lastName":"Li","suffix":""},{"id":275093878,"identity":"c1394f53-739a-403b-9e13-33167e9f521e","order_by":1,"name":"Aikun Fu","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Aikun","middleName":"","lastName":"Fu","suffix":""},{"id":275093879,"identity":"354269f8-7b9c-42b7-b328-9157ab601407","order_by":2,"name":"Qiufen Mo","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Qiufen","middleName":"","lastName":"Mo","suffix":""},{"id":275093880,"identity":"a387801e-85bc-4b4e-9dc6-0a2ef8f941fa","order_by":3,"name":"Yi Wan","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Wan","suffix":""},{"id":275093881,"identity":"6049dfd9-9b79-4f49-b475-ce0f33fcb4b0","order_by":4,"name":"Yuanhao Zhou","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Yuanhao","middleName":"","lastName":"Zhou","suffix":""},{"id":275093882,"identity":"f0ab1c7e-1567-463b-9cab-e00c30cba1e6","order_by":5,"name":"Zihan Zeng","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Zihan","middleName":"","lastName":"Zeng","suffix":""},{"id":275093883,"identity":"ecea5172-6335-4d93-bd23-8f8e622634d7","order_by":6,"name":"Anshan Shan","email":"","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Anshan","middleName":"","lastName":"Shan","suffix":""},{"id":275093884,"identity":"88a916a2-62de-4c3a-b72e-eb7307a2aa70","order_by":7,"name":"Xiaoping Zhang","email":"","orcid":"","institution":"China National Bamboo Research Center","correspondingAuthor":false,"prefix":"","firstName":"Xiaoping","middleName":"","lastName":"Zhang","suffix":""},{"id":275093885,"identity":"6e1c41ab-e353-4df6-bb46-7861c5b6d1c4","order_by":8,"name":"Weiqin Li","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Weiqin","middleName":"","lastName":"Li","suffix":""},{"id":275093886,"identity":"96c2b4c1-1595-4c71-996d-ab80516e1dcb","order_by":9,"name":"Weifen Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArElEQVRIiWNgGAWjYDAC5sMNBz4wsIGYBkRqYUtsODiDZC3MPBAmkVoMjjE2Hrb5w5fYwN68TYKh5g5RWhoO5/AA7eI5VibBcOwZYS1m9xuBWiSAWiRyzCSA2onQArLFwgCoRf4NKVoYEkC28BCpxR6o5WDPATbjNp60YouEY0RokWxjPvzhx59jsv3shzfe+FBDhBYoOAaJzASiNTAw1JCgdhSMglEwCkYcAADQRDmAbVsD8AAAAABJRU5ErkJggg==","orcid":"","institution":"Zhejiang University","correspondingAuthor":true,"prefix":"","firstName":"Weifen","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-02-26 06:49:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3990205/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3990205/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51792306,"identity":"ff231418-8eb7-4d9d-8ee3-eebafd5ca0e4","added_by":"auto","created_at":"2024-02-29 06:08:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":664446,"visible":true,"origin":"","legend":"\u003cp\u003eOral administration of AP2 restored weight loss caused by ST infection.\u003c/p\u003e\n\u003cp\u003eData presents the means ± SDs (n = 12 in each group). *** represents p\u0026lt;0.001 compared with ST group.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3990205/v1/86f386db545b61f4325cd003.png"},{"id":51792320,"identity":"a11bb4f0-e832-483a-be15-440a1dfadfd9","added_by":"auto","created_at":"2024-02-29 06:08:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":15666986,"visible":true,"origin":"","legend":"\u003cp\u003eAP2 treatment restored physiologic morphology of intestine. Representative images and blinded histopathology scores of H\u0026amp;E-stained cecal (a) and colon (b) sections (Optical microscope, 100×magnification) among control, AP2, ST and AP2+ST groups. Intestinal histopathology was scored from five mice. (c) Analysis of the jejunal morphology (TEM, 20K×, n=3). ** represents p \u0026lt; 0.01, *** \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3990205/v1/1adc9346ef5b18a81119fb8c.png"},{"id":51792313,"identity":"6e8305fa-0f77-471e-b36c-7e09ddc3b224","added_by":"auto","created_at":"2024-02-29 06:08:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":467269,"visible":true,"origin":"","legend":"\u003cp\u003eAP2 administration suppresses inflammation caused by ST infection. Levels of IL-1β, IFN-γ, IL-12/p40, IL-10 and Glutamic pyruvic transaminase (GPT) in the serum were determined using ELISA kit. Each column represents average data from five mice * represents p\u0026lt;0.05, ** \u0026lt;0.01, ***\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3990205/v1/8e109a1f539ccff74f623580.png"},{"id":51792316,"identity":"068d7852-2132-4812-9e9c-f6ab4300a4bf","added_by":"auto","created_at":"2024-02-29 06:08:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1352528,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of ST burden in the feces of the AP-treated and untreated mice that pre-treated with streptomycin. (a) Mice were treated with the streptomycin and then infected with 4×108 CFU of ST at 24 h after streptomycin treatment. And then mice were treated without AP2. (b) ST burden in the faeces was enumerated by plating serial dilutions of faeces homogenates on Salmonella-Shigella agar plates at 3, 6 and 9 h after AP2 treatment.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3990205/v1/e3fd0ebf84b9f029f51de79e.png"},{"id":51792321,"identity":"991de0ea-cc89-4db3-b3b3-61d1bf59bc44","added_by":"auto","created_at":"2024-02-29 06:08:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2143930,"visible":true,"origin":"","legend":"\u003cp\u003eAP2 can modify the microbiota before and after ST infection. (a) Principal coordinates analysis plots on unweighted UniFrac distances of cecal samples. Overall fecal microbiome diversity was represented by the first two principal coordinates on principal coordinates analysis of unweighted UniFrac distances. Each point represents a single sample. (b) Combined distribution at the phylum level of control, AP2, ST and AP2+ST groups. (c) Difference of intestinal microbiota of control, AP2, ST and AP2+ST groups.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3990205/v1/334d8e3f8900085cb7147234.png"},{"id":51792311,"identity":"3457967c-8ca5-4ad0-89ea-ee45a015d61a","added_by":"auto","created_at":"2024-02-29 06:08:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2721446,"visible":true,"origin":"","legend":"\u003cp\u003eAP2-altered microbiota inhibited the proliferation and invasion of ST. (a) Experimental design of FMT treatment and inoculation with ST. (b) Transfer of the microbiota from AP2 mice reduces ST shedding in feces and MLN. ** represents p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-3990205/v1/bc894b4d0e5a0ab4b49c2bcb.png"},{"id":51792303,"identity":"a5251017-f850-43e8-a178-9acf729b3358","added_by":"auto","created_at":"2024-02-29 06:08:35","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":20917018,"visible":true,"origin":"","legend":"\u003cp\u003eMicrobiota altered by AP2 contributed to the protection effect of AP2 against ST infection. (a) Representative H\u0026amp;E-stained images of the cecum. Necrosis, erosion (red arrow) and marked neutrophilic infiltration (black arrow) were observed accompanied by atypical crypt microabscess after 12 h of ST-inoculation from control mice. Transfer of the microbiota from AP2 mice is sufficient to decrease ST-induced inflammation. (b) A detailed scoring of cecal samples at 12 h post infection. Each stacked column represents an individual mouse. (c) Blinded histopathology scores of cecal samples. The score of individual mice (circles or boxes) and the geometric mean for each group (bars) are indicated. ** represents p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-3990205/v1/836b2c4f05e4e07d3d284b38.png"},{"id":56886005,"identity":"800dd87c-1d8f-4a3a-a654-6a4afd98b3d1","added_by":"auto","created_at":"2024-05-21 18:46:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":40547917,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3990205/v1/f864af8b-59b2-4f3f-903b-ee4d37385fcb.pdf"},{"id":51792314,"identity":"366e35a2-e8f2-4549-a035-05e315bd5e95","added_by":"auto","created_at":"2024-02-29 06:08:38","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":66348013,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-3990205/v1/1d82606047275e04b525c79d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antimicrobial peptide AP2 ameliorates Salmonella Typhimurium infection by modulating gut microbiota","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSalmonellosis caused by Salmonella Typhimurium infection is one of the most frequently reported bacterial diseases in humans and animals[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], which is characterized by fever, acute intestinal inflammation, and diarrhea within 24h. After entering the intestinal lumen, this pathogen is able to overcome the intestinal barrier and generate gaps in the epithelium, then spread rapidly to the organs such as mesenteric lymph nodes (MLNs), spleen and liver [\u003cspan additionalcitationids=\"CR4 CR5 CR6 CR7\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Antibiotics are critical for the treatment of invasive ST infections [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, the emergence of multidrug-resistant ST makes these infections more difficult to treat [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Meanwhile, antibiotic treatment resulted in a disruption of the gut microbiota, which were demonstrated to preclude ST infection by conferring colonization resistance, further increases susceptibility to ST infection[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. For example, streptomycin treatment prior to ST ingestion led to an acute inflammation of the gastrointestinal tract, along with weight loss, diarrhea, and extra-intestinal infection (e.g. in the spleen and liver) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In this respect, it is urgent to identify new antimicrobial agents to defense against ST. Antimicrobial peptides (AMPs) are regarded as promising candidates. AMPs are found in all higher organisms[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and contribute to the first line of defense against microbial infections. They are gene-encoded and highly conserved innate immune effector molecules. AMPs can inhibit or kill a broad range of pathogens [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] by different mechanisms, such as lysis of the bacterial membrane or inhibition of certain targets on the surface of or within the bacteria[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], but rarely induce bacterial resistance [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. At the same time, it was found that gut commensal microbes are resistant to high doses of AMPs [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and in turn, AMPs reduced the susceptible to enteric pathogens colonization and inflammation by keeping gut microbial context balance[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eApidaecin is a type of proline-rich AMPs (PrAMPs) that is produced by bees and wasps in response to bacterial infections [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Among the three isoforms, apidaecin IB (AP IB) is clearly the most potent and highly active against \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. AP2 is a structurally modified version of native AP IB and contains arginine-valine-arginine (RVR) in positions one to three instead of glycine-asparagine-asparagine (GNN), respectively. Our previous research has demonstrated that AP2 has greater antibacterial activity against Gram-negative bacteria, and good stability under acidic, pepsin, and high temperature conditions. These features suggest that AP2 can be used \u003cem\u003ein vivo\u003c/em\u003e to treat intestinal pathogenic infections. The aim of this study was to investigate whether AP2 was effective in the prevention of ST infections in animals. We found that the antimicrobial peptide AP2 had protective effect against ST infection by modulating the gut microbiota in mice.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003eMale C57BL/6 mice aged 6\u0026ndash;7 weeks were purchased from Slac Animal Inc. (Shanghai, China) and maintained at the Laboratory Animal Center of Zhejiang University. All animal experiments were conducted in accordance with experimental protocols approved by the Institutional Animal Care and Use Committee of Zhejiang University (ZJU20181068). All animal experiments were performed in strict accordance with university guidelines. Mice were fed and watered ad libitum.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of peptides\u003c/h2\u003e \u003cp\u003eAP2 peptide (amino acid sequence: RVRRPVYIPQPRPPHPRL) and AP IB (amino acid sequence: GNNRPVYIPQPRPPHPRL) peptide were synthesized and purified by GL Biochem Ltd. (Shanghai, China). The peptide purity was determined to be higher than 95% by reverse-phase high-performance liquid chromatography (RP-HPLC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eBacterial strain and culture\u003c/h2\u003e \u003cp\u003e \u003cem\u003eSalmonell\u003c/em\u003ea Typhimurium CMCC 50115 (ST) was obtained from the Institute of Preventive Veterinary Medicine, Zhejiang University (China). ST was inoculated in Luria-Bertani (LB) medium (10 g L-1 peptone, 5 g L-1 yeast extract, and 10 g L-1 NaCl) and incubated at 37℃ to the exponential phase with shaking at 250 r min-1.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAntibacterial activity assay.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe minimum inhibitory concentrations (MIC) of AP2 and antibiotics were determined by the micro-broth dilution method [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Briefly, ST was grown at 37℃ to an OD600 of 0.4 in LB medium and was diluted to 5\u0026times;105 colony-forming units (CFUs) mL-1. A total of 180 \u0026micro;L cell suspension and 20 \u0026micro;L serial 2-fold dilutions of the peptide (final concentration ranging 320, 160, 80, 40, 20, 10, 5, and 2.5 \u0026micro;g mL-1) were added into each well, respectively. After incubation at 37℃ for 12\u0026ndash;16 h, MICs were determined as the lowest peptide concentration at which no bacterial growth was observed. All tests were conducted in triplicate.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnimals and experimental protocols.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMice were weighed and randomly divided into four groups (n\u0026thinsp;=\u0026thinsp;12 per group) without significant difference in body weight: Control, AP2, ST and AP2\u0026thinsp;+\u0026thinsp;ST groups, respectively. Before the inoculation with ST, mice in the AP2 and AP2\u0026thinsp;+\u0026thinsp;ST groups were treated with AP2 (10 \u0026micro;g mL-1, 200 \u0026micro;L, 2\u0026micro;g /mouse) by gavage, once a day for 2 weeks, while the control and ST groups were given sterile ultrapure water (200 \u0026micro;L) instead. After 2 weeks, mice in the ST and AP2\u0026thinsp;+\u0026thinsp;ST groups were fasted for 12 hours prior to inoculation via oral gavage with 4\u0026times;108 CFUs ST in 200 \u0026micro;L PBS, as determined by plating, while mice from the control and AP2 groups were inoculated with 200 \u0026micro;L PBS and housed separately. Animals were sampled and evaluated at the 4th day post-inoculation (5 repeated experiments) since the weight loss in the ST-inoculated mice was around 20% of the initial body weight [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eHistopathology\u003c/h2\u003e \u003cp\u003eTissues samples of cacum and colon were harvested and fixed in 4% paraformaldehyde, dehydrated and processed into paraffin sections according to standard procedure. The paraffin sections were subjected with hematoxylin-eosin (H\u0026amp;E) staining at the histology core facilities at Zhejiang University. Images were captured by a Zeiss Imager-M2. Blinded examination by a GI pathologist at Zhejiang University was used to score the pathology of samples with previously published methods (49). Each section was evaluated for the submucosal edema, inflammatory infiltrate and epithelium (50). The pathological changes were scored from 0 to 4 according to the following scale: 0\u0026thinsp;=\u0026thinsp;none, 1\u0026thinsp;=\u0026thinsp;low, 2\u0026thinsp;=\u0026thinsp;moderate, 3\u0026thinsp;=\u0026thinsp;high, and 4\u0026thinsp;=\u0026thinsp;extreme. The inflammation score for each mouse was calculated by adding the score for each parameter (21).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eTransmission electron microscopy (TEM)\u003c/h2\u003e \u003cp\u003eThe morphology and histology of intercellular tight junctions were characterized by TEM. For TEM assessment, briefly, a 2-cm-long jejunum specimen was excised and fixed. Ultrathin sections were obtained and stained by uranyl acetate and lead citrate before examination on a Hitachi Model H-7650 TEM [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of sera parameters\u003c/h2\u003e \u003cp\u003eBlood was collected from the femoral artery and serum cytokines were analyzed using kits (ELISA kit; e-Bioscience, USA) following the manufacturer\u0026rsquo;s protocols. Cytokines in sera were expressed as pg mL-1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eST recovered in feces and MLNs\u003c/h2\u003e \u003cp\u003eThe colonization of ST in the feces and MLNs were detected as previously described [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Briefly, stools were collected aseptically, weighed, and homogenized in PBS containing 0.1% Triton X-100. MLNs were dissected and minced through a 45-\u0026micro;m nylon mesh. Triton X-100 at a final concentration of 0.1% was added to the cell suspensions and incubated for 30 second. Serial dilutions were made and then coated on \u003cem\u003eSalmonella\u003c/em\u003e-Shigella agar plates (Britania, Buenos Aires, Argentina). After 24 h, the CFUs were quantified by visual counting of micro-colonies and data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of triplicate samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eEffect of AP2 on ST in intestine\u003c/h2\u003e \u003cp\u003eThe mice (n\u0026thinsp;=\u0026thinsp;4 per group) were pre-treated with streptomycin (20 mg per mouse)[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. After one day, mice were inoculated with ST. 4 h later, mice in the AP2\u0026thinsp;+\u0026thinsp;ST group was treated with AP2 (10 \u0026micro;g mL-1, 200 \u0026micro;L, 2\u0026micro;g /mouse) by gavage, while the ST group was given sterile ultrapure water (200 \u0026micro;L) instead. Finally, feces were collected every three hours to monitor the amount of ST.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eDNA extraction, V3-V4 16S rRNA gene amplification and microbiota community analysis\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eDNA from the cecum content (collected at the 4th day post-inoculation) was extracted with the TIANamp Stool DNA Kit (TIANGEN BIOTECH CO., LTD, Beijing, China) according to the manufacturer\u0026rsquo;s instructions. PCR amplification and sequencing were performed by the G-BIO Inc. (Hangzhou, China). Bacterial DNA was amplified by a two-step PCR enrichment of the 16S rDNA (V3 and V4 regions) with forward primers containing the sequence 5\u0026rsquo;-CCTACGGGNGGCWGCAG-3\u0026rsquo; and reverse primers containing the sequence 5\u0026rsquo;-GACTACHVGGGTATCTAATCC-3\u0026rsquo;.\u003c/p\u003e \u003cp\u003eThe processed pair-end reads were assembled using PandaSeq v2.8 with default parameter [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Chimeras were identified and removed using USEARCH 6.1 within QIIME. The QIIME script \u0026ldquo;add_qiime_labels.py\u0026rdquo; was used to combine the non-chimeric sequences from each sample into one file. OTU picking and taxonomic assignments were performed using the open-reference OTU picking workflow in Qiime with the Greengenes reference database [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. OTUs with abundances below 0.005% of the total number of sequences were discarded [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlpha diversity measurements, including Shannon, Chao1, observed species, and Good\u0026rsquo;s coverage, were calculated using the alpha_rarefaction.py script in QIIME. Weighted and unweighted unifrac distances [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] were calculated from the rarefied OTU table using the beta_diversity_through_plots.py script in QIIME.\u003c/p\u003e \u003cp\u003ePrincipal component analysis (PCA) was conducted using the website METAGENassist. The linear discriminant analysis (LDA) effect size (LEfSe) method [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] was performed using the Galaxy online interface (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://huttenhower.sph.harvard.edu/galaxy\u003c/span\u003e\u003cspan address=\"http://huttenhower.sph.harvard.edu/galaxy\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFecal microbiota transplant (FMT) experiment\u003c/h2\u003e \u003cp\u003eFecal microbiota was obtained from fresh stool samples of control (n\u0026thinsp;=\u0026thinsp;4) or AP2 treated mice (n\u0026thinsp;=\u0026thinsp;4). Fresh stool samples were pooled and diluted 20-fold and homogenized in sterile and pre-reduced 0.1 M potassium phosphate buffer (PBS, pH 7.2) containing 15% glycerol (v/v) to produce a 5% fecal suspension according to the previous study [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The homogenate was centrifuged at 100 g for 5 min at 4℃ and the resulting suspension was then pipetted into 5 mL sterile tubes and stored at -80℃ [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor FMT, the mice (n\u0026thinsp;=\u0026thinsp;4 per group) were pre-treated with streptomycin (20 mg per mouse). Previously frozen pooled fecal samples from control or AP2-treated mice were thawed on ice and delivered via anorectal inoculation (200 \u0026micro;l) by using a catheter made from a round-tip silicone tube with a diameter of 1 mm. After inoculation, the mouse was held vertically with its head down for 1 min to prevent loss of the infusion. 4 h later, mice were inoculated with ST (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Finally, feces and MLNs were collected aseptically for further determination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations (SD). A Student\u0026rsquo;s t-test and one-way analysis of variance (ANOVAs) with Tukey\u0026rsquo;s post hoc tests were performed and considered significant at \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eThe MIC of AP2\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe MIC of AP2 and AP IB against ST \u003cem\u003ein vitro\u003c/em\u003e was first measured to explore their antibacterial activity. The results showed that the MIC of AP2 against Salmonella was 5 \u0026micro;g mL-1, which was better than that of AP IB with MIC of 10 \u0026micro;g mL-1 and kanamycin (Kan) and streptomycin (Strep) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe MIC of antimicrobial peptides.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAP2\u003c/p\u003e \u003cp\u003e(\u0026micro;g mL-1)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAP IB\u003c/p\u003e \u003cp\u003e(\u0026micro;g mL-1)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKan\u003c/p\u003e \u003cp\u003e(\u0026micro;g mL-1)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eStrep\u003c/p\u003e \u003cp\u003e(\u0026micro;g mL-1)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eST\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e32.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eST: \u003cem\u003eSalmonella\u003c/em\u003e Typhimurium CMCC 50115. AP IB: apidaecin IB. Kan: kanamycin. Strep: streptomycin.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAP2 attenuated the symptoms of ST infections\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate whether AP2 has a protective effect against ST infection \u003cem\u003ein vivo\u003c/em\u003e, C57BL/6 mice were treated with or without AP2 before ST infection. Compared with the control group, none of the animals in the AP2 group showed obvious weight loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), whereas mice challenged with ST exhibited significant body weight loss at day 2 (ST group) and day 3 (both ST and AP2\u0026thinsp;+\u0026thinsp;ST groups). However, the body weight loss of mice in AP2\u0026thinsp;+\u0026thinsp;ST group markedly alleviated at day 3 and day 4 compared with ST group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe results of the intestinal histopathology showed that ST infection induced an acute inflammation in the mucosa characterized by the swelling of the lamina propria (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), inflammatory infiltration and desquamation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) and the shedding of microvilli (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Furthermore, the ST infection also induced intestinal mitochondria swelling (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), which was significantly ameliorated by AP2 administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe level of serum pro-inflammatory cytokines reflect the intensity of inflammation. Mice in the control mice and AP2-treat mice) groups had similar low levels of serum inflammatory cytokines (IL-1β, and IFN-γ (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). While ST inoculation resulted in a significant increase in the serum pro-inflammatory cytokines IL-1β and IFN-γ, which were decreased by AP2 pretreatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eST infection may result in bacterial translocation across the intestinal barrier, followed by migration to the spleen and liver [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Glutamic pyruvic transaminase (GPT) is mainly present in the cytoplasm of hepatic cell. When hepatocyte is injured, GPT will release into blood, and thus increasing the serum GPT activity[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Therefore, the level of this enzymes in serum could be used to assess the extent of damage[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Results showed that GTP activity were significantly increased in the ST group, which was markedly reduced (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the AP2\u0026thinsp;+\u0026thinsp;ST group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These results suggested that AP2 administration ameliorated ST-induced liver damage.\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAP2 does not inhibit ST growth in the intestinal tract\u003c/h2\u003e \u003cp\u003eSince AP2 conferred protection against ST infection, next we want to verify whether AP2 could inhibit the growth of ST within intestinal tract directly. Since the microbiota confers colonization resistance to block \u003cem\u003eSalmonella\u003c/em\u003e gut colonization [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], the streptomycin mouse model was used to remove gut microbial community and exclude its interference, and the amount of ST in feces was monitored every 3 h after treated with AP2 (Fig.\u0026nbsp;4a). Surprisingly, we found that AP2 treatment did not decrease the ST loads in the feces (Fig.\u0026nbsp;4b), which is inconsistent with the results obtained from \u003cem\u003ein vitro\u003c/em\u003e experiments. Thus, the protective effect of AP2 against ST \u003cem\u003ein vivo\u003c/em\u003e was not associated with its bactericidal effect directly.\u003c/p\u003e \u003cp\u003e Several studies have demonstrated that the gut microbiota and its metabolites provide colonization resistance to ST infection [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. But ST exploits inflammation to compete with this colonization resistance [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. A previous study also found that AMPs beneficially affected the intestinal health by shaping the microbial ecology [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. This led us to focus our further analyses on the gut microbiota composition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAP2 treatment modified the gut microbiota composition\u003c/h2\u003e \u003cp\u003eSince AP2 exhibited strong antibacterial capacity, which may influence the composition of the microbiome, a 16S rRNA-based analysis was used to determine the microbiota from the cecum content. Results showed that there were no significant differences in alpha diversity of the microbiota as reflected by Shannon, Chao1, Faith\u0026rsquo;s phylogenetic, and observed species indexes among control, AP2, ST and AP2\u0026thinsp;+\u0026thinsp;ST groups (Supplemental Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). However, beta diversity assessment with weighted UniFrac distance revealed that the microbial community significantly different (ANOSIM, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between control and AP2 groups and between AP2\u0026thinsp;+\u0026thinsp;ST and ST groups both at the phylum (Fig.\u0026nbsp;5a) and the genus (Fig.\u0026nbsp;5b) level.\u003c/p\u003e \u003cp\u003eAP2 treatment did not significantly change the major microbial composition at the phylum level (Fig.\u0026nbsp;5c), but significantly increased the proportion of \u003cem\u003eActinobacteria\u003c/em\u003e, \u003cem\u003eAlcaligenaceae, Allobaculum, Bifidobacteriales, Betaproteobacteria, Burkholderiales, Clostridiaceae, Lachnospiraxeae, Mogibacteriaceae\u003c/em\u003e and \u003cem\u003eSutterella\u003c/em\u003e, and decreased the proportion of \u003cem\u003eCoprobacillus\u003c/em\u003e and \u003cem\u003eVerrucomicrobia\u003c/em\u003e compared with control group. Meanwhile, the proportion of \u003cem\u003eAlcaligenaceae, Betaproteobacteria, Burkholderiales\u003c/em\u003e, and \u003cem\u003eSutterella\u003c/em\u003e in the AP2\u0026thinsp;+\u0026thinsp;ST mice were significantly higher than those in ST mice. Notably AP2 treatment also decreased the proportion of \u003cem\u003eVerrucomicrobia\u003c/em\u003e independent of ST-inoculation. (Supplemental Fig. S2).\u003c/p\u003e \u003cp\u003e The linear discriminant analysis effect size (LEfSe) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] was used to identify specific OTUs that differed between the control and AP2 with or without ST inoculation. 17 discriminative features (LDA score\u0026thinsp;\u0026gt;\u0026thinsp;2) whose relative abundances varied significantly between the control and AP2 groups was identified. Furthermore, 11 bacterial taxa, such as \u003cem\u003eActinobacteria\u003c/em\u003e, \u003cem\u003eBifidobacterium\u003c/em\u003e, \u003cem\u003eAllobaculum\u003c/em\u003e, and \u003cem\u003eSutterella\u003c/em\u003e were enriched in the AP2 group, while 6 bacterial taxa were increased in the control group, such as \u003cem\u003eVerrucomicrobia\u003c/em\u003e, \u003cem\u003eCoprobacillus\u003c/em\u003e, and \u003cem\u003eAkkermansia.\u003c/em\u003e Based on the LEfSe, 17 bacterial taxa were significantly more abundant in the AP2\u0026thinsp;+\u0026thinsp;ST group (e.g. \u003cem\u003ePrevotella, AF12, Dehalobacterium, Oscillospira, Coprobacillus, Sutterella, Bilophila\u003c/em\u003e, and \u003cem\u003eDesulfovibrio\u003c/em\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while only 9 taxa were overrepresented in the ST group (e.g. \u003cem\u003eVerrucomicrobia\u003c/em\u003e, \u003cem\u003eClostridium\u003c/em\u003e, \u003cem\u003eCoprococcus\u003c/em\u003e, and \u003cem\u003eAkkermansia\u003c/em\u003e; \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) (Fig.\u0026nbsp;5d). Collectively, these data suggested that the gut microbiota modified by AP2 may be associated with its protective effect against ST infection in mice.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFecal microbiota transplant (FMT) of AP2-treated mice influences the course of ST-induced caecal inflammation in mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further verify the hypothesis above, we performed FMT experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The mice that were pre-treated with streptomycin [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], received microbiotas from the control or AP2-treated mice through anorectal inoculation, respectively. 4 h later, mice were infected with ST by oral gavage to evaluate the effects of the AP2-treated microbiota on ST infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Results showed that AP2-treated microbiota could significantly decrease levels of ST in the stool and MLN (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), and also decreased intestinal pathology (Fig.\u0026nbsp;7), providing evidence that this alerted microbial community induced by AP2 treatment was effective against ST infection. These findings suggest that the beneficial effect of AP2 treatment on the course of infection was transferable via FMT.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOral infection of mice with ST leads to a fatal systemic disease [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. During \u003cem\u003eSalmonella\u003c/em\u003e invasion, PAMPs (pathogen-associated molecular patterns) and DAMPs (Danger-associated molecular patterns) initiate innate immunity, leading to recruitment of neutrophils and the increase expression of pro-inflammatory cytokines, most notably interleukin (IL)-6, IL-1β, and IFN-γ. Although neutrophils are indispensable in \u003cem\u003eSalmonella\u003c/em\u003e resistance, neutrophils infiltration can also cause detrimental mucosa wounding. Under these circumstances, \u003cem\u003eSalmonella\u003c/em\u003e gains a growth advantage over the gut microbiota from inflammatory conditions [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. This study showed that AP2 has protective effects against ST infection, indicated by the decreased body weight loss, the attenuated intestinal and systemic inflammation ,lower ST translocation in ST-inoculated mice which were in line with the anti-Salmonella effects of other AMPs [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Furthermore, in this study, gut microbiota involved in the underlying mechanism of P2 function. Enteric pathogens interact extensively with the gut microbiota [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Once inoculated, ST would compete with the microbiota by exploits inflammation, however, gut microbiota indispensable to protect mice from ST colonization in the gut [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The complex interactions between ST, inflammation and microbiota have been researched and reviewed by some researchers [\u003cspan additionalcitationids=\"CR53 CR54\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Therefore, the inflammatory condition and microbiota modified by AP2 may also be the cause to elucidate its protective effect against ST infection.\u003c/p\u003e \u003cp\u003eIt has been reported that and that the gut microbiota plays key role in maintenance of health and the development of the mucosal immune system [\u003cspan additionalcitationids=\"CR57 CR58\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. In current study, AP2 had no effect on the main categories of gut microbiota, which is consistent with results of one previous study that gut microbes from all dominant phyla were resistant to high levels of inflammation-associated AMPs [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. We found that AP2 significantly favors the proliferation of some G\u0026thinsp;+\u0026thinsp;beneficial microorganisms, e.g., \u003cem\u003eBifidobacterium\u003c/em\u003e, \u003cem\u003eAllobaculum\u003c/em\u003e, and \u003cem\u003eClostridia\u003c/em\u003e. Certain bacteria belonging to \u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eAllobaculum\u003c/em\u003e were able to produce short-chain fatty acids (SCFAs), which were detrimental to the growth of \u003cem\u003eS.\u003c/em\u003e Typhimurium [\u003cspan additionalcitationids=\"CR61\" citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Butyrate and propionate can act as signal molecules to downregulate the expression of the \u003cem\u003eSalmonella\u003c/em\u003e pathogenicity island (SPI)1-encoded type 3 secretion system (T3SS) invasion genes [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], which were crucial for this bacterium to invade intestinal epithelial cells [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Furthermore, \u003cem\u003eClostridia\u003c/em\u003e had been proven to inhibit the ST colonization in the gut [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition, \u003cem\u003eOscillospira\u003c/em\u003e, \u003cem\u003eBilophila\u003c/em\u003e, and \u003cem\u003eDesulfovibrio\u003c/em\u003e instead of \u003cem\u003eClostridium\u003c/em\u003e and \u003cem\u003eAkkermansia\u003c/em\u003e were enriched in the AP2\u0026thinsp;+\u0026thinsp;ST group compared to the ST group. \u003cem\u003eOscillospira\u003c/em\u003e were also able to secrete SCFA such as butyrate [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Many strains of Proteobacteria including \u003cem\u003eBilophila\u003c/em\u003e and \u003cem\u003eDesulfovibrio\u003c/em\u003e, can induce the secretion of IgA and regulate intestinal homeostasis [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Proteobacteria, such as e.g., \u003cem\u003eBilophila\u003c/em\u003e and \u003cem\u003eDesulfovibrio\u003c/em\u003e which was increased in AP2\u0026thinsp;+\u0026thinsp;ST group compared with ST group, produce hydrogen sulfide (H2S), which have various biological effects in the immune system [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. H2S exhibits several anti-inflammatory effects such as reduction of edema formation and suppression of the release of pro-inflammatory cytokines (such as TNF-α and IFN-γ) [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e] and suppressing the activation of NF-κB [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Moreover, the proportions of G- bacteria such as \u003cem\u003eAkkermansia\u003c/em\u003e were significantly decreased in the AP2 and AP2\u0026thinsp;+\u0026thinsp;ST groups compared with the control and ST group, respectively. Previous studies have reported that the relative abundance of \u003cem\u003eAkkermansia\u003c/em\u003e positively correlated with intestinal inflammation in a murine chronic enteritis model, and \u003cem\u003eAkkermansia\u003c/em\u003e significantly exacerbated ST-induced intestinal inflammation [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. However, some studies and reviews indicate that \u003cem\u003eAkkermansia\u003c/em\u003e may be related to anti-inflammatory and have potential anti-inflammatory properties [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. The impact of Akk on the inflammation may be related to its relative abundance, but the specific mechanism is not very clear yet. Hence, it could be concluded that the gut microbiota altered by AP2, characterizing with more abundance of anti-inflammatory bacteria and less abundance of pro-inflammatory bacteria, inhibit ST invasion and translocation, which verified by the results of FMT.\u003c/p\u003e \u003cp\u003eIn line with this, the pathogen colonization was reduced in the infected animals that received AP2-treated feces, indicating that the AP2 can shape an adaptive microbiota, which can then inhibit the growth of ST.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe work presented here indicated that AP2 ameliorated ST infection by modulating the gut microbiota and which play a key role in the protection against pathogen infection. These findings reveal a previously unrecognized mechanism by which AMPs alleviate the bacterial infection and thus will provide additional strategies for the further pharmaceutical investigations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e \u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate Ethics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted in accordance with experimental protocols approved by the Institutional Animal Care and Use Committee of Zhejiang University (ZJU20181068). All animal experiments were performed in strict accordance with the guidelines.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by Young Scientists Fund of the National Natural Science Foundation of China (Grant No. 82003436), National Nature Science Foundation of China (Grant No. 32372892), the \u0026lsquo;twelfth five-year-plan\u0026rsquo; in National Support Program for Science and Technology for rural development in China (Grant No. 2011BAD26B02) and Natural Science Foundation of China (Grant No. 31472128).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by LL and AF. The first draft of the manuscript was written by LL, AF and QM, YZ, ZZ, AS, XZ and YW had been involved in analyzing the data and revising the manuscript critically. AF, QM participated in the experimental design. Weiqin Li and Weifen Li provided funding support. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was financially supported by Young Scientists Fund of the National Natural Science Foundation of China (Grant No. 82003436), National Nature Science Foundation of China (Grant No. 32372892), the \u0026lsquo;twelfth five-year-plan\u0026rsquo; in National Support Program for Science and Technology for rural development in China (Grant No. 2011BAD26B02) and Natural Science Foundation of China (Grant No. 31472128). We are grateful to all the participants in this study. The authors are grateful to the Bio-ultrastructure analysis Lab. of Analysis center of Agrobiology and environmental sciences, Zhejiang Univ.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDougan, G., John, V., Palmer, S., \u0026amp; Mastroeni, P. 2011. Immunity to salmonellosis. Immunological Reviews, 240(1), 196\u0026ndash;210. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1111/j.1600-065X.2010.00999.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1600-065X.2010.00999.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaiti, S., Patro, S., Purohit, S., Jain, S., Senapati, S., \u0026amp; Dey, N. 2014. Effective Control of Salmonella Infections by Employing Combinations of Recombinant Antimicrobial Human β-Defensins hBD-1 and hBD-2. 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Microb Pathog, 106, 171\u0026ndash;181. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.micpath.2016.02.005\u003c/span\u003e\u003cspan address=\"10.1016/j.micpath.2016.02.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"Apidaecin, Salmonella Typhimurium, inflammation, gut microbiota, fecal microbiota transplantation","lastPublishedDoi":"10.21203/rs.3.rs-3990205/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3990205/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEndogenous antimicrobial peptides/proteins contribute to reshape a healthy gut microbiota which play benefit roles in anti-inflammation and pathogen colonization resistance. Salmonella infection is one of the most frequently reported bacterial diseases worldwide. Manipulation of the gut microbiota through exogenous antimicrobial peptide may protects against Salmonella enterica colonization and improve clinical outcomes. In this study, results showed that oral administration of antimicrobial peptide AP2, an optimized version of native apidaecin IB (AP IB) had a protective effect against ST infections in mice indicated by alleviated ST-induced body weight loss and reduced the serum inflammatory cytokines. 16S rRNA-based analysis of microbiota from the cecum content showed that AP2 altered gut microbiota by significantly increasing the proportion of Bifidobacterium and decreasing Akkermansia at the genus level. Furthermore, the transplantation of fecal microbiota from AP2-treated donor mice, instead of control mice, significantly reduced caecal damage caused by ST. In conclusion, these findings hightlighted one of novel action mechanisms of exogenous antimicrobial peptide on ameliorating Salmonella Typhimurium infection by modulating gut microbiota.\u003c/p\u003e","manuscriptTitle":"Antimicrobial peptide AP2 ameliorates Salmonella Typhimurium infection by modulating gut microbiota","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-29 06:07:14","doi":"10.21203/rs.3.rs-3990205/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":"79de99d1-2727-4e37-ae37-16955e5dc61f","owner":[],"postedDate":"February 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-27T20:08:18+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-29 06:07:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3990205","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3990205","identity":"rs-3990205","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}