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Antimicrobial peptide AP2 ameliorates Salmonella Typhimurium infection by modulating gut microbiota

BMC Microbiology volume 25, Article number: 64 (2025) Cite this article

Abstract

Background

Endogenous antimicrobial peptides and proteins are essential for shaping and maintaining a healthy gut microbiota, contributing to anti-inflammatory responses and resistance to pathogen colonization. Salmonella enterica subsp. enterica serovar Typhimurium (ST) infection is one of the most frequently reported bacterial diseases worldwide. Manipulation of the gut microbiota through exogenous antimicrobial peptides may protect against ST colonization and improve clinical outcomes.

Results

This study demonstrated that oral administration of the antimicrobial peptide AP2 (2 µg /mouse), an optimized version of native apidaecin IB (AP IB), provided protective effects against ST infection in mice. These effects were evidenced by reduced ST-induced body weight loss and lower levels of serum inflammatory cytokines. A 16 S rRNA-based analysis of the cecal microbiota revealed that AP2 significantly modulated the gut microbiota, increasing the relative abundance of Bifidobacterium while decreasing that of Akkermansia at the genus level. Furthermore, the transplantation of fecal microbiota from AP2-treated donor mice, rather than from Control mice, significantly reduced cecal damage caused by ST and decreased the concentration of ST by one order of magnitude after infection.

Conclusions

These findings reveal a novel mechanism by which exogenous antimicrobial peptides mitigate Salmonella Typhimurium infection through the modulation of gut microbiota.

Peer Review reports

Introduction

Salmonellosis, caused by Salmonella enterica subsp. enterica serovar Typhimurium (ST) infection, is one of the most frequently reported bacterial diseases in humans and animals [1, 2]. It is characterized by fever, acute intestinal inflammation, and diarrhea within 24 h. Upon entering the intestinal lumen, this pathogen is capable of evading the intestinal epithelial barrier, inducing epithelial cell disruption, and subsequently disseminating to systemic organs, including the mesenteric lymph nodes (MLNs), spleen, and liver [3,4,5,6,7,8]. Antibiotics are essential for the treatment of invasive ST infections [9]. However, the emergence of multidrug-resistant ST makes these infections more difficult to treat [10,11,12]. The gut microbiota provides colonization resistance to its host, which is the ability of the microbiota to preclude infection by enteric pathogens such as ST. Disturbance of the microbiota due to antibiotic use leads to increased susceptibility to ST infection [13, 14]. For example, treatment with streptomycin prior to ST ingestion induced acute gastrointestinal inflammation, accompanied by weight loss, diarrhea, and extraintestinal infection (e.g., in the spleen and liver) [15, 16]. In this respect, there is an urgent need to identify new antimicrobial agents to defend against ST. Antimicrobial peptides (AMPs) are considered promising candidates, as they are found in all multicellular organisms [17] and serve as key components of the first line of defense against microbial infections. These gene-encoded, highly conserved molecules play critical roles in innate immunity. AMPs can inhibit or eliminate a broad spectrum of pathogens [18] through various mechanisms, including bacterial membrane lysis and the inhibition of specific targets on the surface or within the bacterial cell [19]. Notably, they rarely induce bacterial resistance [20]. Furthermore, it has been observed that gut commensal microbes exhibit resistance to high doses of AMPs [21]. AMPs have evolved in most multicellular organisms primarily to protect local microenvironments rather than to function within the systemic circulation [17]. Beyond their direct antimicrobial activity, AMPs exhibit a range of additional properties, including immunomodulatory effects, barrier repair promotion, maintenance of intestinal microbial homeostasis, anti-tumor activity, and potential as vaccine adjuvants [22, 23]. In turn, AMPs help maintain gut microbial balance, thereby reducing susceptibility to enteric pathogen colonization and inflammation [24, 25].

Apidaecin is a type of proline-rich AMP (PrAMP) produced by bees and wasps in response to bacterial infections [26, 27]. Among the three isoforms, apidaecin IB (AP IB) is clearly the most potent and highly active against Escherichia coli (E. coli) [28, 29]. 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 revealed that AP2 exhibits enhanced antibacterial activity against some Gram-negative (G−) bacteria in vitro and demonstrates good stability under acidic conditions, as well as in the presence of pepsin and high temperatures. These features suggest that AP2 may be a promising candidate for the in vivo treatment of intestinal pathogenic infections. Disruption of intestinal microbial homeostasis significantly increases host susceptibility to ST infection. By preemptively reshaping the gut microbiota and modulating immune responses, we hypothesize that AP2 may enhance the host’s resilience to infection, thereby reducing the risk of disease progression. The aim of this study was to evaluate the efficacy of AP2 in preventing ST infections in animal models. Our results demonstrate that AP2 exerts a 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 greater than 95% by reverse-phase high-performance liquid chromatography (RP-HPLC).

Bacterial strain and culture

Salmonella 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 peptone, 5 g/L yeast extract, and 10 g/L NaCl) and incubated at 37 °C to the exponential phase with shaking at 250 r/min.

Antibacterial activity assay

The minimum inhibitory concentrations (MICs) of AP2 and antibiotics were determined by the micro-broth dilution method [30]. Briefly, ST was grown at 37 °C to an OD600 of 0.4 in LB medium (approximately 1 × 109 colony-forming units (CFUs)/mL) and was diluted to 5 × 105 CFUs/mL. A total of 180 µL cell suspension and 20 µL serial 2-fold dilutions of the peptide (final concentrations ranging from 320, 160, 80, 40, 20, 10, 5, and 2.5 µg/mL) were added into each well, respectively. After incubation at 37 °C for 12‒16 h, the 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 assigned to four groups (n = 12 per group) with no significant differences in initial body weight: Control, AP2, ST, and AP2 + ST groups. Before ST inoculation, the mice in the AP2 and AP2 + ST groups received AP2 (10 µg/mL, 200 µL, 2 µg/mouse) via oral gavage once daily for two weeks, while those in Control and ST groups were administered 200 µL of sterile ultrapure water as a placebo. After two weeks, the mice in ST and AP2 + ST groups were fasted for 12 h before inoculation to facilitate ST colonization, and then were inoculated via oral gavage with 4 × 10⁸ CFUs of ST in 200 µL PBS, as confirmed by plating. Mice in Control and AP2 groups were inoculated with 200 µL PBS and housed separately. Animals were sampled (serum, jejunum, colon, cecum, and cecal contents) and evaluated on the 4th day post-inoculation (5 repeated experiments) since the weight loss in the ST-inoculated mice was approximately 20% of the initial body weight [31]. The mice were exposed to isoflurane anesthesia in a gas-tight chamber prefilled with 2.5% isoflurane in 100% O2 and stayed in the chamber and then sacrificed were by cervical dislocation.

Histopathology

Tissue samples from cecum and colon were harvested and fixed in 4% paraformaldehyde, dehydrated and processed into paraffin sections according to standard procedures. 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 [32, 33]. Each section was evaluated for submucosal edema, inflammatory infiltration and epithelium. 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.

Transmission electron microscopy (TEM)

The morphology and histology of intercellular tight junctions were characterized by TEM. Briefly, a 2-cm-long jejunum sample was excised and fixed. Ultrathin sections were obtained and stained with uranyl acetate and lead citrate before examination via a Hitachi Model H-7650 TEM [34].

Analysis of serum parameters

Blood was collected from the femoral artery and serum cytokines (IL-1β, IFN-γ, IL-12 and IL-10) were analyzed using kits (ELISA kit; e-Bioscience, USA) following the manufacturer’s protocols. Cytokines in serum were expressed as pg/mL.

ST recovered in feces and MLNs

The colonization of ST in the feces and MLNs was quantified 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 45-µm nylon mesh. Triton X-100 at a final concentration of 0.1% was added to the cell suspensions and incubated for 30 s. 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

Mice (n = 4 per group) were pre-treated with streptomycin (20 mg/mouse) [35]. After one day, mice were inoculated with ST. Four hours later, mice in the AP2 + ST group were treated with AP2 (10 µg/mL, 200 µL, 2 µg/mouse) by gavage, while those in the ST group were given an equivalent volume of sterile ultrapure water (200 µL). Finally, feces were collected every three hours to monitor the amount of ST.

DNA extraction, V3-V4 16 S rRNA gene amplification and microbiota community analysis

DNA from the cecum contents (collected on 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 the default parameter [36]. 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. Operational unit (OTU) picking and taxonomic assignments were performed using the open-reference OTU picking workflow in QIIME with the Greengenes reference database [37]. OTUs with abundances less than 0.005% of the total number of sequences were discarded [38].

Alpha diversity measurements, including Shannon, Chao1, observed species, and Good’s coverage, were calculated using the alpha_rarefaction.py script in QIIME. Weighted Unifrac distances [39] 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 [40] 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 a previous study [41]. 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℃ [42].

For FMT, the mice (n = 4 per group) were pre-treated with streptomycin (20 mg/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. Four hours later [43], mice were inoculated with ST. Finally, feces and MLNs were collected aseptically for further determination.

Statistical analysis

Data were expressed as means ± standard deviations (SD). Student’s t-test and one-way analysis of variance (ANOVA) with Tukey’s post hoc test were performed, and P values < 0.05 were considered significant.

Results

The MICs of AP2 in vitro

The MICs of AP2 and AP IB against ST in vitro was first measured to explore their antibacterial activity. AP2 exhibited a MIC of 5 µg/mL, outperforming AP IB (MIC: 10 µg/mL) as well as the conventional antibiotics kanamycin (Kan) and streptomycin (Strep) (Table 1).

Table 1 The MICs of antimicrobial peptides
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AP2 attenuated the symptoms of ST infections in vivo

To evaluate the protective effect of AP2 against ST infection in vivo, C57BL/6 mice were pre-treated with or without AP2 prior to ST challenge. Mice in AP2 group maintained stable body weight, while those in ST group experienced significant weight loss on day 2. By day 3, both ST and AP2 + ST groups showed weight loss. However, weight loss in AP2 + ST group was significantly alleviated on days 3 and 4 compared with that in ST group (Fig. 1).

Fig. 1
figure 1

Oral administration of AP2 restored weight loss caused by ST infection

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Histopathological analysis of the intestine revealed that ST infection induced acute mucosal inflammation, characterized by swelling of the lamina propria (Fig. 2a), inflammatory cell infiltration and epithelial desquamation (Fig. 2b), as well as microvilli shedding (Fig. 2c). Additionally, ST infection caused noticeable mitochondrial swelling in intestinal cells (Fig. 2c), which was significantly alleviated by AP2 treatment (Fig. 2).

Fig. 2
figure 2

AP2 treatment restored the physiologic morphology of intestine. Representative images and blinded histopathology scores of H&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 jejunal morphology (TEM, 20 K×, n = 3). ** represents p < 0.01, *** < 0.01

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Serum pro-inflammatory cytokine levels serve as indicators of inflammation severity. Control and AP2-treated mice exhibited similarly low levels of serum cytokines, including IL-1β and IFN-γ (Fig. 3). While ST inoculation led to a significant increase in the serum IL-1β and IFN-γ levels. Notably, AP2 pretreatment effectively reduced these elevated cytokine levels (Fig. 3).

Fig. 3
figure 3

AP2 administration suppresses inflammation caused by ST infection. The levels of IL-1β, IFN-γ, IL-12/p40, IL-10, Glutamic pyruvic transaminase (GPT) and Glutamic-oxaloacetic transaminase (GOT) in the serum were determined using ELISA kit. Each column represents average data from five mice * represents p < 0.05, ** <0.01, ***<0.001

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ST infection may lead to bacterial translocation across the intestinal barrier, followed by migration to the spleen and liver [33]. Glutamic pyruvic transaminase (GPT) is present mainly in the cytoplasm of hepatic cells. When hepatocytes are injured, GPT will release into blood, and thus increasing the serum GPT activity [44]. Thus, serum GPT levels can be used as a marker of liver damage [45]. The results showed a significant increase in GPT activity in the ST group, which was notably reduced (P < 0.05) in AP2 + ST group (Fig. 4). These findings suggest that AP2 administration alleviated ST-induced liver damage.

Data are presented the means ± SDs (n = 12 in each group). *** represents p < 0.001 compared with ST group.

AP2 does not inhibit ST growth in the intestinal tract

Given that AP2 provides protection against ST infection, we next sought to determine whether AP2 directly inhibits ST growth within the intestinal tract. Since the microbiota confers colonization resistance to block ST gut colonization [46], we used streptomycin to disrupt the gut microbiota, thereby facilitating ST colonization. Our in vitro experiments demonstrated that AP2 significantly inhibited ST growth at the three-hour mark (Fig. S1). Consequently, ST levels in feces were monitored every three hours following AP2 treatment (Fig. 4a). Surprisingly, we found that AP2 treatment did not decrease the ST load in the feces (Fig. 4b), which is inconsistent with the results observed in in vitro experiments. Thus, the protective effect of AP2 against ST in vivo does not appear to be directly related to its bactericidal activity.

Fig. 4
figure 4

Assessment of ST load in the feces of the AP-treated and untreated mice that pre-treated with streptomycin. (a) Mice were treated with streptomycin and then infected with 4 × 108 CFU of ST at 24 h after streptomycin treatment. The mice were then treated without AP2. (b) ST load in the feces was enumerated by plating serial dilutions of feces homogenates on Salmonella-Shigella agar plates at 3, 6 and 9 h after AP2 treatment

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Several studies have shown that the gut microbiota and its metabolites provide colonization resistance against ST infection [13, 35, 47]. However, ST exploits inflammation to overcome this resistance [48,49,50]. A previous study also demonstrated that AMPs can beneficially influence intestinal health by modulating the microbial ecosystem [51]. This prompted us to focus our subsequent analyses on the composition of the gut microbiota.

AP2 treatment modified the gut microbiota composition

Since AP2 exhibited a strong antibacterial capacity, which may influence the microbiota composition, a 16 S rRNA-based analysis was used to determine the microbiota from the cecum content. The results revealed that there were no significant differences in alpha diversity of the microbiota as indicated by Shannon, Chao1, Faith’s phylogenetic, and observed species indexes among control, AP2, ST and AP2 + ST groups (Supplemental Fig. S2). However, beta diversity analysis using weighted UniFrac distance revealed significant differences in the microbial communities between Control and AP2 groups, as well as between the AP2 + ST and ST groups, both at the phylum (Fig. 5a) and genus (Fig. 5b) levels (ANOSIM, p < 0.05).

Fig. 5
figure 5

AP2 can modify the microbiota before and after ST infection. (a and b) Principal coordinate analysis plots of weighted UniFrac distances of cecal samples. Overall fecal microbiota diversity was represented by the first two principal coordinates on principal coordinates analysis of weighted UniFrac distances. Each point represents a single sample. a: Phylum-level, b: Genus-level (c) Combined distribution at the phylum level of Control, AP2, ST and AP2 + ST groups. (d) LEfSe analysis identified the microbes whose abundances significantly differed between Control and AP2 groups, ST and AP2 + ST groups

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AP2 treatment did not significantly change the major microbial composition at the phylum level but affected their relative abundances. (Fig. 5c). Compared with Control group, the relative abundance of Bacteroidetes decreased in ST group. AP2 treatment could increase the relative abundance of Bacteroidetes regardless of ST infection. In contrast, AP2 treatment reduced the relative abundance of both Firmicutes and Verrucomicrobia. Notably, the decrease was independent of ST inoculation. (Supplemental Fig. S3).

To further investigate the modulatory effect of AP2 on the microbiota, we used linear discriminant analysis effect size (LEfSe) [40] to identify specific OTUs that differed between Control and AP2 with or without ST inoculation. A total of 17 discriminative features (LDA score > 2) were identified, showing significant differences in relative abundance between Control and AP2 groups. At the phylum level, AP2 group exhibited a significant enrichment of Actinobacteria, while genera such as Bifidobacterium, Allobaculum, and Sutterella were notably more abundant in the AP2 group. Conversely, Control group showed higher relative abundances of phyla such as Verrucomicrobia, and genera like Bifidobacterium, Coprobacillus and Akkermansia. Further LEfSe analysis of ST and AP2 + ST groups identified 17 bacterial taxa that were significantly more abundant in the AP2 + ST group (p < 0.05), including several genera, such as Prevotella, Dehalobacterium, Oscillospira, Coprobacillus, Sutterella, Bilophila, and Desulfovibrio exhibiting higher relative abundances. At the species level, the abundance of Bacillus sp. AF12 also increased. 9 bacterial taxa were significantly overrepresented in ST group (p < 0.05), with notable changes at various taxonomic levels. At the phylum level, Verrucomicrobia was enriched, particularly the genus Akkermansia. The genera Clostridium and Coprococcus were also more abundant. 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 transplantation (FMT) of AP2-treated mice influences the course of ST-induced cecal inflammation in mice

To further verify the above hypothesis, we performed an FMT experiment (Fig. 6a). The mice pre-treated with streptomycin [35] received microbiota from either Control or AP2-treated mice via anorectal inoculation. 4 h later [43], mice were infected with ST by oral gavage to evaluate the effects of the AP2-treated microbiota on ST infection (Fig. 6a). The results showed that AP2-treated microbiota could significantly decrease levels of ST in both feces and MLNs (Fig. 6b) and also decreased intestinal pathology (Fig. 7). These findings provide evidence that the altered microbial community induced by AP2 treatment effectively protected against ST infection. These findings suggest that the beneficial effects of AP2 treatment on the infection course were transferable via FMT.

Fig. 6
figure 6

AP2-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 load in feces and MLNs. ** represents p < 0.01

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Fig. 7
figure 7

Changes in the microbiota caused by AP2 contributed to the protective effect of AP2 against ST infection. (a) Representative H&E-stained images of the cecum. Necrosis, erosion (red arrow) and marked neutrophilic infiltration (black arrow) were observed accompanied by atypical crypt microabscesses after 12 h of ST-inoculation in Control mice. Transfer of the microbiota from AP2 mice is sufficient to decrease ST-induced inflammation. (b) Left: A detailed scoring of cecal samples at 12 h post infection. Each stacked column represents an individual mouse. Right: Blinded histopathology scores of cecal samples. The score of individual mice (circles or boxes) and the geometric means for each group (bars) are indicated. ** represents p < 0.01

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Discussion

Oral infection of mice with ST leads to fatal systemic disease [13]. During ST invasion, pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) activate the innate immune response, triggering the recruitment of neutrophils and increasing the expression of pro-inflammatory cytokines, most notably interleukin (IL)-6, IL-1β, and IFN-γ. Although neutrophils play a crucial role in resistance to ST infection, their infiltration can also contribute to damaging mucosal injury. Under these conditions, ST gains a growth advantage over the other gut microbiota due to the inflammatory environment [52]. This study demonstrated that AP2 exerts protective effects against ST infection, as indicated by decreased body weight loss, attenuated intestinal and systemic inflammation, and decreased ST translocation in ST-inoculated mice which were in line with the anti-ST effects of other AMPs [53]. Furthermore, this study highlights the involvement of gut microbiota in the underlying mechanism of AP2’s function. Enteric pathogens, including ST, interact extensively with the gut microbiota [54, 55]. Once inoculated, ST would compete with the microbiota by exploiting inflammation. However, the commensal gut microbiota is critical for maintaining host defense against ST colonization in the intestinal tract [56]. The complex interactions between ST, inflammation and gut microbiota have been extensively studied and reviewed [57,58,59,60]. Therefore, the inflammatory condition and microbiota modified by AP2 may help elucidate its protective effect against ST infection.

Gut microbiota plays a critical role in maintaining host health and in the development of the mucosal immune system [61,62,63,64]. In our research, AP2 had no significant effect on the major gut microbiota phyla. This finding is consistent with previous research indicating that gut microbes from dominant phyla exhibit resistance to high levels of inflammation-associated AMPs [21]. Although our previous experiments demonstrated that ST exhibits antimicrobial activity against certain G− pathogenic bacteria, such as ST, in this study, we found that AP2 treatment does not significantly inhibit the growth of G− bacteria in the gut. Also, we found that AP2 couldn’t directly inhibit the growth of ST in vivo. Notably, some G− anaerobic bacteria, such as Parabacteroides, Parasutterella, and Bacteroides, which have been reported to potentially assist the host by providing numerous health benefits [65,66,67], were not suppressed by AP2. AP2 exerts a nuanced modulatory effect on the gut microbiota rather than a broad-spectrum promotion of bacterial growth or direct inhibition of specific pathogens. We propose that AP2 subtly modulates the gut microbiota, shifting it toward a new steady state characterized by increased resilience against ST infections. We found that AP2 favors the proliferation of Bifidobacterium, Allobaculum, and Clostridia. Certain bacteria belonging to the genera Bifidobacterium and Allobaculum are capable of producing short-chain fatty acids (SCFAs), which suppress the growth of ST [68,69,70]. Butyrate and propionate function as signaling molecules that downregulate the expression of ST pathogenicity island (SPI)1-encoded type 3 secretion system (T3SS) invasion genes [71], which are crucial for ST to invade intestinal epithelial cells [72]. Additionally, Clostridia has been proven to inhibit ST colonization in the gut [35].

Furthermore, Oscillospira, Bilophila, and Desulfovibrio were enriched in the AP2 + ST group compared with the ST group, whereas Clostridium and Akkermansia were not. Oscillospira has also been shown to produce SCFAs such as butyrate [73]. Proteobacteria, including Bilophila and Desulfovibrio, were enriched in AP2 + ST group compared with ST group. These bacteria are known to induce the secretion of IgA and regulate intestinal homeostasis [74]. They produce hydrogen sulfide (H2S), a compound with diverse biological effects on the immune system [75, 76]. H2S exhibits several anti-inflammatory effects, including the reduction of edema formation, suppression of the release of pro-inflammatory cytokines (such as TNF-α and IFN-γ) [77] and suppressing the activation of NF-κB [78]. Moreover, the proportions of certain G− bacteria such as Akkermansia, were significantly lower in the AP2 and AP2 + ST groups than in Control and ST groups, respectively. Previous studies have reported that the relative abundance of Akkermansia is positively correlated with intestinal inflammation in a murine chronic enteritis model, and Akkermansia significantly exacerbated ST-induced intestinal inflammation [79]. However, some studies and reviews suggest that Akkermansia may be related to anti-inflammatory activity and exhibit potential anti-inflammatory properties [80, 81]. The impact of Akkermansia on inflammation may be related to its relative abundance, although the specific mechanisms underlying this relationship remain unclear. In our study, a direct comparison between the effects of AP2 treatment and FMT treatment within the same experimental cohort was not performed. However, we compared each treatment group with its respective control group. Based on cecal histopathology, the inflammation score decreased by approximately 37.5% in the AP2-treated group compared to the untreated group. Similarly, in the AP2-FMT group compared to the Con-FMT group, the inflammation score decreased by approximately 36.3%. These findings suggest that AP2 treatment and AP2-FMT treatment exhibit comparable efficacy. Therefore, it can be concluded that the gut microbiota altered by AP2 serves to inhibit ST invasion and translocation, as corroborated by the FMT results. Consistent with these findings, AP2 modulates the gut microbiota to foster an adaptive microbial community that suppresses ST growth and mitigates disease severity.

Conclusion

The presented study demonstrated that AP2 ameliorated ST infection by modulating the gut microbiota, which plays a key role in protection against pathogen infection. These findings reveal a novel mechanism by which AMPs alleviate bacterial infection and offer valuable insights for future pharmaceutical investigations.

Data availability

The 16 S rRNA sequencing data in this study can be found in the Sequence Read Archive (SRA) of NCBI under BioProject accession PRJNA1139680, http://www.ncbi.nlm.nih.gov/bioproject/1139680.

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Acknowledgements

We are grateful to all the participants in this study. Especially thanks to Zihan Zeng, Anshan Shan, and Xiaoping Zhang for their guidance and contributions to this research. The authors are grateful to the Bio-ultrastructure analysis Lab. of Analysis center of Agrobiology and environmental sciences, Zhejiang Univ.

Funding

This work was supported by Young Scientists Fund of the National Natural Science Foundation of China (Grant No. 82003436), 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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Author notes

  1. Lianglan Li and Qiufen Mo contributed equally to this work.

Authors and Affiliations

  1. Department of Critical Care Medicine, Nanjing Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China

    Lianglan Li & Weiqin Li

  2. Key Laboratory of Animal Molecular Nutrition of Education of Ministry, College of Animal Sciences, Zhejiang University, Hangzhou, China

    Qiufen Mo, Yi Wan, Yuanhao Zhou & Weifen Li

  3. College of Food and Health, Zhejiang A&F University, Hangzhou, 311300, China

    Qiufen Mo

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  1. Lianglan Li
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Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by LLL and QFM. The first draft of the manuscript was written by LLL and QFM, YW, YHZ had been involved in analyzing the data and revising the manuscript critically. LLL and QFM participated in the experimental design. WQL and WFL provided funding support. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Weiqin Li or Weifen Li.

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Li, L., Mo, Q., Wan, Y. et al. Antimicrobial peptide AP2 ameliorates Salmonella Typhimurium infection by modulating gut microbiota. BMC Microbiol 25, 64 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12866-025-03776-0

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12866-025-03776-0

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BMC Microbiology

ISSN: 1471-2180

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