Lasso peptide MccY alleviates non-typhoidal salmonellae-induced mouse gut inflammation via regulation of intestinal barrier function and gut microbiota

ABSTRACT Non-typhoidal salmonellae-induced zoonotic intestinal bleeding infections are a crucial threat to public health worldwide, and the development of new medications is required to prevent and treat non-typhoid Salmonella (NTS) infections. MccY, a class of lasso peptide, is effective in biological sterilization against Salmonella and Shigella. In this study, the therapeutic effect of MccY on S. Typhimurium-infected BALB/c mice has been discussed. specific pathogen free (SPF) mice were infected with the ST53 strain with bacterial loads of 106 and 103 CFU/mL, respectively, and then treated with 9.92 mg/kg BW MccY. Results revealed that MccY significantly reduced mouse mortality by 20%–40% and effectively alleviated weight loss and diarrhea. The integrity of the intestinal structure was protected, and the expression of TNF-α, IL-6, IL-10, and IL-18 was inhibited. Particularly, MccY exerted multiple effects on gut microbiota ecology, including direct elimination of Salmonella, showing promise in limiting infection to the gut level and thereby halting the transmission of NTS. MccY indirectly inhibited other opportunistic pathogens and partially restored the microbiota, promoting an increase in Bacteroidetes, Lactobacillus, Muribaculum, and Clostridium. Consequently, this work provides an understanding of the link between host infection and therapeutic application of MccY. IMPORTANCE Diseases caused by Enterobacteriaceae multidrug-resistant strains have become increasingly difficult to manage. It is necessary to verify the new antibacterial drug MccY effect on non-typhoid Salmonella infection in mice since it is regarded as a promising microcin. The results demonstrated that MccY has a potential therapeutic application value in the protection against Salmonella-induced intestinal damage and alleviating related intestinal dysbiosis and metabolic disorders. MccY could be a promising candidate as an antimicrobial or anti-inflammatory agent for treating infectious diseases.

T he expansion of enterobacteria is a hallmark of microbial imbalance known as "dysbiosis." Diseases caused by Enterobacteriaceae multidrug-resistant strains have become increasingly difficult to manage, including Escherichia coli, Salmonella.and Shigella (1).Salmonella is crucial foodborne pathogen that causes diarrheal disease, intestinal dysbiosis, host intestinal barrier damage, and mortality in young animals and humans (2).An estimated 800,000 to 3.7 million cases of non-typhoid Salmonella (NTS) infections occur in the United States each year (3).The frequent appearance of super bacteria and the breakthrough against the last line of defense, polymyxin, have also increased the risk of the global NTS infection crisis (4).Broad-spectrum antibiotics have the potential to disrupt the intestinal microbiota balance in treating enterobacterial gut infections because their antibacterial effects target both harmful and beneficial bacteria (5).Therefore, there is an urgent need for new antibacterial drugs that do not disrupt the balance of gut microbiota.
Microcins are a class of ribosomally assembled and posttranslationally modified peptides (RiPPs) produced under conditions when similar bacteria respond competi tively, and they provide a competitive advantage in microbial communities (6,7).Microcin-producing Enterobacteriaceae in human feces have been reported to contribute to gut microbial ecology.E. coli (8) (EcN), which produces microcins and limits the growth of competitors such as adherent invasive Salmonella in the inflamed intestine (8).However, EcN only reduces the Salmonella gut burden 17-fold and does not reduce inflammation (9).These results indicate that the strategy of microcin intervention to minimize the risk of Salmonella infection is feasible; more research is required to determine the optimal intervention and treatment strategies.Lasso peptides, one of the small molecules within microcins, have potential for application as next-generation drugs (10).The involvement of lasso peptide in the inflammatory competition among and within Enterobacteriaceae makes it a possible therapy for specifically inhibiting intestinal pathogens and limiting bacterial growth.Microcin J25 (MccJ25) administra tion to mice with prior enterostreptococcus infection resulted in reduced intestinal colonization by this pathogen, as well as limited growth of competing gut species, adherent invasive E. coli (11).MccJ25 protects against enterotoxigenic Escherichia coli (ETEC)-induced intestinal injury and inflammatory responses, suggesting the applica tion of lasso peptide as an excellent antimicrobial or antiinflammation agent against pathogen infections.
A gene cluster isolated from S.

MccY has a high-intensity continuous bactericidal effect by inhibiting the activity of respiratory chain
MccY identification and quantification were performed and described in Fig. 1A and B 1C showed the antimicrobial test of MccY proved that the inhibitory concentration of S. Typhimurium is 0.05 µg/mL, that of S. Infants and S. Sonnei is 0.10 µg/mL, and that of S. Flexneri was >1.0 µg/mL (Table S3).The results shown in Fig. 1D through F (Table S4) were analyzed in comparison to the previous time point.Strains could be killed by MccY inhibiting the activity of respiratory chain complex enzyme V within 12 h.In the logarithmic growth phase (4-6 h), ST53 and ATCC25931 enzyme were inhibited starting at 4 h (P < 0.0001); the SE63 strain was inhibited starting at 6 h (P < 0.0001).The results in the stationary phase (6-12 h) indicated that MccY inhibited the respiratory chain complex enzyme activities and the activity of ST53 (7.8 U/mg prot) was higher than those of SE63 (10.2 U/mg prot) and ATCC25931 (9.2 U/mg prot).It was confirmed that MccY has a high persistent bactericidal effect on foodborne bacteria by inhibiting the activity of respiratory chain complex enzyme V.

Administration of MccY alleviated symptoms of infection and decreased the mortality rate and bacterial burden in infected mice
The therapeutic effect of MccY on infections caused by foodborne opportunistic pathogens in the animal and human is still unclear.The experimental process in mice for the treatment of MccY after two different doses of infection was designed in Fig. 2, and the experimental process was based on previous studies of mouse models (11).The comprehensive detection method was referred to previous study on lasso peptide in mice, and the reference therapy dosage for MccJ25 in mice was 9.1 mg/kg body weight (BW) (15)(16)(17).In Fig. 3, the ST53-HD mice showed bloody stools on day 5 and succum bed to the infection on day 8.In contrast, the MccY treatment group showed delayed mortality for 1 day and a 20% increase in survival rate compared with the non-treated group (Fig. 3A).Similarly, the ST53-LD group mice died within 5-8 days, whereas those in the MccY treatment had a 40% increase (Fig. 3B).The average BW of ST53-HD mice was obviously decreased to the control group and the ST53-HD-MccY group (P < 0.05; P < 0.05) (Fig. 3C).The MccY-treated mice exhibited higher average BW compared with the ST53-LD mice (P < 0.05) (Fig. 3D).The weight loss observed in the infection group compared with the MccY-treated group indicates the effectiveness of MccY in preventing weight loss.The daily activity index (DAI) score (Table S5) was evaluated every day based on previous research on lasso peptide applications (11,18).The overall assessment of DAI  scores among the groups was provided in Fig. 3E and FTable S6 and S7.Infected mice displayed elevated body temperatures, ranging from 2°C to 5°C higher, and showed signs of bloody excretions.However, MccY treatment did not result in visible blood in the stool.The DAI score of ST53-HD groups has higher pathological score to the uninfected mice group (P < 0.001).On day 10, the pathological severity of MccY-treated mice was more severe (P < 0.01) than that of the uninfected group, but the score was lower than that of the infected group (P < 0.05).Thus, MccY alleviated the acute symptoms in the early phase of Salmonella-related bacteremia.
To investigate the effect of MccY on the clearance rate of Salmonella, the fecal bacterial count was based on the previous study on MccJ25 (18).On 3 dpi (Fig. 3G and  H), a remarkable decrease in the levels of Salmonella was observed in the MccY-treated mice compared with the infection group.Mice inoculated with 10 6 CFU/mL maintained approximately 10 4 to 10 5 CFU/mg Salmonella, and MccY reduced to 10 3 CFU/mg feces, resulting in a sterilization rate close to 99%.Notably, the excretion status revealed a dramatic reduction in colonization load in the 10 3 CFU/mL infection group to 10-20 CFU/mg, with a sterilization rate of 99.8%.Remarkably, MccY effectively eliminated a significant amount of Salmonella from the intestinal tract.

MccY reduced the expression pro-inflammatory factors in intestinal mucosa
To explore the capacity of MccY to inhibit Salmonella-induced intestinal immunity, the levels of inflammatory cytokines were detected (Fig. 4).The serum of SIgA and IgM has no statistical changes, whereas the level of IgG in the ST53-HD-MccY group was increased (P < 0.05) relative to the control group (Fig. 4A  the different groups in the cytokine levels in the jejunum.However, IL-6 was appreciably increased in cecum in the ST53-HD-MccY group compared with the ST53-HD group (P < 0.01).The IL-10 in the cecum of infected mice was significantly decreased compared with the control group (P < 0.05), and further significant reduction was observed after MccY treatment.MccY treatment downregulated IL-18 in ST53-HD-challenged mice (P < 0.05); on the contrary, IL-6 was notably upregulated (P < 0.01).The changes in pro-inflammatory and anti-inflammatory factors in the colon were mainly exhibited in the ST53-LD-challenged mice.The expression of TNF-α (P < 0.05) and IL-10 (P < 0.05) decreased in the ST53-LD-MccY group compared with the ST53-LD-challenged group.
MccY is involved in the regulation of intestinal mucosal immunity by reducing the secretion of pro-inflammatory factors, thereby enhancing the resistance of mice to NTS infection.

MccY ameliorating intestinal villi shedding, dampening inflammation, and enhancing gut integrity
Hematoxylin eosin (H&E) staining of the gut lumen further confirmed the intestinal protection of MccY during infection, and the histopathology and morphometrical characteristics were calculated in Fig. 5.The analysis excluded the data of cecum due to limited visibility of its crypt.In general, the intestinal damage degree of ST53-HD mice was significantly higher than that of ST53-LD mice.The jejunum of mice in the ST53-HD group exhibited severe intestinal tissue injury, including structural damage, and altered the functionality of cellular components such as goblet cells, Paneth cells, and neuroendocrine cells.The number of ST53-LD mice villi had been relatively maintained, and the mucosal morphology and crypt structure of jejunum in the MccY-treated group were protected (Fig. 5A).Specifically, in comparison to the jejunum, the cecum experienced less damage and a reduced shedding rate of goblet epithelial cells (Fig. 5B).In the ST53-HD group, the cecal mucosa and lamina propria were significantly separated, accompanied by villi shedding.However, in the ST53-LD infection group, the cecum exhibited a semi-exfoliative state, resulting in a monolayer of columnar epithelial cells.MccY-treated mice exhibited a general improvement in gut integrity, with minimal sloughing and an ordered arrangement of epithelial cells.After 14 days of infection, mice in the ST53-HD group displayed marked colitis (Fig. 5C), with almost complete loss and atrophy of the colon villi.The ST53-HD-MccY group demonstrated complete loss of most villi, and the colon showed a single-layered columnar epithelium with a thin brush border and a small number of scattered inflammatory cells within the lamina propria.The mucosal damage in ST53-LD mice was less severe compared with ST53-HD mice, and MccY therapy in mice colon resulted in no injury and did not exacerbate villus damage.
Inflammatory morphological changes in the intestine segment are indicative of chronic pathological changes in the gastrointestinal tract, and the villi and crypts are critical indicators of digestive and absorptive functions.Among the NTS-infected mice, several crypt epithelial cells were necrotic and sloughed off.The jejunum villi of ST53-HD mice were sparser, more damaged, and shorter, resulting in a lower V/C ratio in comparison with the control group (P < 0.05) (Fig. 5D).A common histological feature observed in colitis is the architectural distortion of the intestinal epithelium, character ized by the shortening and reduced branching of the crypts (21).MccY treatment led to alterations in the depth of colon crypts and a significant reduction in the colon V/C ratio compared with the control group (Fig. 5E).Based on these results, MccY therapy was found to decrease intestinal damage and enhance intestinal integrity.

MccY promotes gut ecological balance by simultaneously reducing Salmo nella burden
Mice feces were detected at four time points (1 day, 3 days, 5 days, and 7 days) to observe the dynamic changes of microflora (Fig. 6; Table S9 and S10 ).The number of amplicon sequence variation (ASV) in the samples varied substantially according to the Venn diagram (Fig. 6A).Specifically, the number of operations of the classification unit (OTU) decreased in the order of control group > ST53 MccY group > ST53 group.The Venn diagram indicated that MccY groups showed a greater microbial abundance than the infected group.The stacked bar charts in Fig. 6B aim to characterize the composition of various species in the microbiome.A higher ratio indicates a larger proportion of that particular bacteria in the total microbial flora.The community composition of the bacterial species in the ST53-HD group and ST53-LD group diverged significantly at the genus level.The main bacteria in the HD infection group were Bacteroides spp.and those of the LD infection group were Lactobacillus spp.A great proportion of Salmonella was observed in the ST53-HD-infected group, while the relative abundance of fecal Salmonella in mice administered with MccY was decreased on day 5 (P < 0.001) and day 7 (P < 0.001) (Fig. 6C).As described in Fig. 6D, the abundance of Salmonella in the ST53-LD-MccY-infected group was reduced than that in the MccY group (day 3, P < 0.0001; day 5, P < 0.01, and day 7, P < 0.0001).The MccY groups elucidated a dramatic reduction in Salmonella content, which was consistent with the results of Salmonella clearance depicted in Fig. 3G and H. MccY was more efficient at reducing the abundance of Salmonella in low-dose infections compared with high-dose infections.
Figure 6E and F analyzed the major flora abundance of each group in an average of 7 days.In the ST53-HD-infected mouse, the abundance of microbes decreased in the infected group; Bacteroides remained dominant after recovery from MccY treatment (P < 0.001).The abundance of Bacteroides was 19.7-fold higher in the MccY-treated groups than in the infection groups, and Lactobacillus increased significantly (P < 0.001).Interestingly, Staphylococcus and Klebsiella were significantly reduced (Fig. 6B), and the proportion of Escherichia that can be involved in intestinal bacterial infections was also inhibited, decreasing from 12.353% to 0.683% (P < 0.001) (Fig. 6E).Lactobacillus (50.602%) was the dominant species in the ST53-LD group, followed by Bacteroides, Akkermansia, Escherichia, and Alistipes.In Fig. 6F, the averages abundance of Lactobacillus in the treatment group was significantly higher compared with other groups (P < 0.001; P < 0.0001), accounting for 49.648% of the total microbial abundance in ST53-LD-MccY mice.The abundance of Muribaculum, Bacteroides, and Akkermansia did not markedly changes.MccY exerts multiple positive effects on gut microbiota ecology, including direct elimination of Salmonella, indirect inhibition of other opportunistic pathogens, and partial restoration of the microbiota.

MccY constrains Salmonella transmission route, resulting in decreased burden in liver tissue
Further analysis of bacterial communities was conducted to investigate the mecha nisms behind MccY supplementation enhancing host resistance against S. Typhimu rium.The abundance of 20 major genus bacteria of ST53-LD group mice liver on day 14 was depicted in Fig. 7; Table S8.Liver samples from the control group and ST53-LD-MccY group have similar relative abundances with the main flora structure, primarily comprising of Veillonella, Streptococcus, Ochrobactrum, and Capnocytophaga.And the infection group of Salmonella content is the highest, followed by Veillonella and Streptococcus.Specifically, MccY decreased the ratio of Salmonella from the gut to liver, as compared with that of the infection group; Salmonella abundance of the MccY-treated group were dramatically decreased (P < 0.0001) (Fig. 7B).The concentration of several other bacteria, including Prevotella, Lactobacillus, Bacteroides, and Lactococcus increased, but no statistically significant differences were observed.MccY shows promise in limiting NTS infection to the gut level and curbing its dissemination to other tissues, thereby halting the in vivo transmission of NTS at its origin.
Consequently, the results demonstrated that MccY has the potential for therapeu tic application in protecting against NTS Salmonella-induced intestinal damage and inflammation at different doses (10 6 and 10 3 CFU/mL).It also alleviates related intestinal dysbiosis and metabolic disorders (Fig. 8).This work provides into insights into the relationship between host and MccY, suggesting that MccY shows promise as a potential antimicrobial or anti-inflammatory agent for the treatment of pathogenic infections.

DISCUSSION
In this study, the inhibitory activity of MccY against Salmonella was evaluated to demonstrate MccY bactericidal persistence in vivo.Mitochondrial respiratory complex V is known as F1F0-ATP synthase, which utilizes the energy produced by the gradient of protons across the membrane (22,23).The sterilization data indicate that the efficacy of MccY persists, facilitating the targeted eradication of pathogens colonizing the digestive tract.In this work, mice challenged with S. Typhimurium from 1 dpi to 14 dpi displayed daily symptoms of inflammatory diarrhea and bloody diarrhea characteristic of bacillary dysentery.The results demonstrated that 9.92 mg/kg BW MccY prevented the onset of acute bowel disease in the early stage of infection, alleviated intestinal symptoms, and increased the survival rate by 20%-40%.The results in Fig. 4  The administration of MccY might contribute to higher levels of IL-6 in the cecum, particularly in cases of high-dose infection, through the expansion of intestinal epithelial cells.IL-6 maintains homeostasis by stimulating the proliferation and triggering an increase in neutrophil count, and IL-10 has been found to be negatively correlated with the occurrence and progression of gastrointestinal disease (24,25).Bacterial metabo lites regulate inflammatory body activation and IL-18 expression, ultimately influencing intestinal inflammation and barrier function (26).MccY maintain intestinal homeostasis by suppressing IL-10 and IL-18, mitigating damage to the mucous layer and inhibiting goblet cell maturation.MccY act as a mediator in intestinal inflammation by regulat ing TNF-α, IL-6, and IL-18, triggering an inflammatory response within the intestinal mucosa, leading to loss of integrity and increased permeability of the upper cortex.Villi are finger-like projections that protrude into the lumen of the intestine, increasing the surface area for nutrient absorption; crypts are formed by the intraepithelial lines surrounding the base of the villi (27).MccY contributed to adhesion of mucus and decreased epithelial adherence, inflammation, and desquamation, and the ratio of V/C decreased.MccY for therapeutic purposes did not exacerbate villus damage; it promoted gut integrity and mitigated overall intestinal damage yet.
MccY disrupted the early proliferation of Salmonella in the intestinal lumen, highlighting the targeted killing effect of MccY on enteric pathogens.The NTS strain remains dormant in the blood, waits for the right opportunity to replicate in the intestine, and becomes difficult to eliminate (28,29).Intracellular Salmonella bacteria can be absorbed into the blood through the intestine and spread to the other tissue, such as the liver (30,31).MccY has shown an inhibitory effect on the body, a small portion of Salmonella can enter the liver and replication, evading the effect of MccY.The similarity in the microflora distribution in the liver between the treatment group and the control group indicates the protective effect of MccY on mouse liver.MccY could confine NTS bacterial infection to the gut level and prevent its spread to other tissues, thereby preventing the transmission of Salmonella.
Microbial colonization is a major driver of changes in the gut metabolome (32,33).MccY has a beneficial effect on the colonization and recovery of dominant bacteria.The ST53-HD-MccY and ST53-LD-MccY groups were primarily composed of Bacteroides, Lactobacillus, Muribaculum, and Alistipes.Bacteroides, as new-generation probiotics, inhibit pathogenic bacterial colonization and alleviate intestinal inflammation (34,35).The abundance of Lactobacillus was notably increased, and previous research has reported that Lactobacillus reduces the susceptibility and severity of Salmonella infection, while Alistipes species produce short-chain fatty acids (SCFAs) and reduce intestinal inflammation (36,37).These probiotics utilize MccY to compete with related species and colonize the gut, replacing the niche of intestinal pathogens.There was a markedly decreased abundance of Staphylococcus and Klebsiella in the ST53-HD infection group treated with MccY (Fig. 6B).MccY could reduce the proportions of opportunistic bacterial pathogen.The mode of action of MccY and its expected minimal impact on the microbiome will avoid the drawbacks of chronic antibiotic treatments.This study provides the first description of how MccY achieves potent therapeutic outcomes against NTS infection.MccY promoted a favorable gut microbial ecology and immune homeosta sis.It effectively attenuates the elevated levels of Salmonella in the gut lumen and limits the expansion of opportunistic pathogen.This work demonstrated how microcin could be exploited to improve intestinal health and presents that the administration of MccY could be served as an alternative to antibiotic drugs.

Bacterial strain and culture conditions
Primers and plasmid used in this study were designed and listed in Table S1.The E. coli and Salmonella strains used in this study were from our laboratory and are presented in Table S2.Briefly, strains were inoculated on 100 mL of LB solid medium and incubated overnight, 1:1,000 dilution in fresh LB broth.The broth was incubated for 6 h with 200 rpm shaking at 37℃.The samples were diluted 1:1,000 for the overlay tests.

Preparation of microcin Y
The recombinant MccY plasmid was constructed in our laboratory (13).pYL01 was transferred into E. coli BL21 (DE3), then E. coli (YL02) was cultured in LB medium with 50 µg/mL kanamycin.The broth was induced with 0.5 mM IPTG and incubated for 24 h.The supernatant of the recombinant bacterial cell was collected by centrifugation.The purification and qualification of lasso peptide were performed using the QQQ-LC-MS approach, as described in our previous study.Inhibition assays on solid media were carried out with modifications to a previously published protocol with 0.005-1.0μg/mL MccY.All experiments were independently repeated three times.

Mitochondrial respiratory complex V activity assay
The mitochondrial respiratory complex V activity assay was conducted following the kit instructions.Bacteria in logarithmic phase were collected and diluted into 1 × 10 5 CFU/mL and then treated with 0.2 mg/mL MccY culture for 2-12 h, and the bacteria were collected for each 2 h.The bacterial pellet was retained, and an appropriate amount of lysis buffer was added to the suspended cells and the mitochondria were extracted.The activities of mitochondrial respiratory chain complexes V were detected using a colorimetric method.The activity values of the complexes were calculated as follows: Activity value = [(test sample OD value − background OD value) × system vol × test sample dilution multiple] / [test sample volume × X (millimole absorptivity) × reac tion time (min) × test sample protein concentration (mg prot/mL)] Prot is an abbreviation of protein.The administered group samples were prepared in triplicate.

Mouse experiments
All of the Specific Pathogen Free (SPF) mouse experiments were approved by the South China Agricultural University Institutional Animal Care and Use Committee, under ethics approval number 2021C084.Mice were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd.Six-to seven-week-old male and female SPF BALB/c mice were randomized and divided into six groups (control group, infected group, and treatment group, n = 10) and co-housed in groups of two to three under environmentally controlled conditions.Since the median lethal dose of ST53 is known to be LD 50 = 10 6 CFU/mL, two doses of infection were designed.The mice were infected with ST53 at two different doses: 10 6 and 10 3 CFU/mL.Before the infection, the mice were treated with 100 mg/kg BW streptomycin to rebuild their intestinal flora.One hour later, the animals were given 0.5% NaHCO 3 to neutralize the stomach acid.MccY diluted in sterile PBS at 9.92 mg/kg was performed starting from 1 dpi and continued every day during the experimental period.Survival was monitored for 15 days, and exposure to CO 2 is used to euthanize these mice.

Disease activity index assessment
DAI assessment was performed by evaluating the physical signs and intestinal-related pathological indexes of mice before and during the experiments, as described previously (11,38).Symptoms include body weight, body temperature, stool consistency, and presence of blood in the stool.Each mouse was scored for changes in body characteris tics, and the scores for each group were added to obtain the average score.The DAI evaluation methods of three animal experiments were consistent.

Cytokine production measurement
ELISA kits (Proteintech, USA) were used to measure serum SIgA, IgG, and IgM production.Blood samples were centrifuged at 2,000 × g for 10 min after collection.Total tissue protein was extracted by homogenization, and the concentration was measured using the BCA Protein Assay Kit (Novagen, Denmark).Plates were coated with a 5 µg/mL solution of carbonate buffer and incubated at 4°C for 1 h.Plates were washed and incubated for 1 h with peroxidase conjugated anti-HRP secondary antibody (Proteintech, USA) diluted 1:20 (vol/vol) with diluent.TMB Substrate Reagent Set was added and incubated for 30 min in the dark, and finally, 2N sulfuric acid was added to stop the reaction.Results were measured by absorbance at 450 nm, and ratios were calculated by dividing by the respective values.All assays were performed in duplicate, and a standard curve was generated for each experiment.

Histological analyses
The gut lumen was removed from the mice and fixed in 40% paraformaldehyde, embedded in paraffin, and stained by hematoxylin eosin.Determination of the level of intestinal epithelial damage was based on the measurement of villus length and recess depth, followed by the use of software to calculate V/C.Changes in intestinal barrier integrity and inflammatory responses were assessed.The infection and pathology of mice after treatment were determined by assessing the presence of blood in the stool and degree of intestinal lesions after dissection I (9,11).

16s rRNA gene sequencing and analysis
The microbiota analysis pipeline and bioinformatics analysis were performed according to previous reports.A total of 240 mouse fecal samples and 30 liver samples were collected, and DNA was extracted.The bacterial genome V3-V4 region of 16s rRNA was amplified by PCR (39).Amplicons were sequenced on an Illumina MiSeq platform using the MiSeq Reagent Kit v2 (500 cycles) (Personal Biotechnology Co. Ltd., China).Differences found in negative extraction controls prior to analysis were removed, after which the inverse Simpson diversity index and OTU richness were calculated using the unweighted UniFrac algorithm.

Statistical analysis
GraphPad Prism 8 was used to calculate descriptive statistics, and all statistical compar isons of P-values were presented in the main and Additional files 1. One-way ANOVA analysis was used for comparisons of two and three or more groups.The results between each group were students t-tested to compare the significant differences between the groups.The data are presented as the mean ± SD.
Enterica has been demonstrated to encode microcin Y (MccY), a class I microcin that is 21 amino acids in length.Our previous study charac terized the biological function of MccY as well as the basic chemical structure of the peptide and its potential mechanism of entry into cells to kill bacteria.RNA polymer ase transcription inhibition using MccY against Enterobacteriaceae bacteria have been proved, and the bactericidal effect of MccY were observed at 0.05-0.8µg/mL in Shigella and Salmonella strains (12, 13).While the role of MccJ25 as a factor influencing the host physiological development is established, NTS strains are highly resistant to MccJ25 due to specific receptor FhuA differences (10, 14).MccY exhibited the antimicrobial potency and structural stability in addressing NTS strains, and MccY is poised to function as an antibiotic alternative with applications in diverse fields ranging from antimicrobial agents and animal feed addition to the treatment of NTS bacterial diseases.Evaluating the clinical application of MccY through in vitro trials presents apparent limitations, and the animal models are necessary to evaluate the impact of MccY on NTS-induced intestinal barrier protection.The research aimed to examine the impact of MccY on gut microbiota and intestinal barrier function by analyzing ecological shifts in murine intestinal physiology, systemic immunity, and microbiota.The study assessed the benefits and risks of in vivo MccY use to achieve a comprehensive understanding of MccY interactions in mice infected with NTS.

FIG 1
FIG 1 Preparation and characterization of MccY.(A) MccY identification was detected by LC-MS-QQQ-TOF.(B) MccY quantification performed by HPLC.The x-axis represents the charge-to-mass ratio, and the y-axis represents the response value.(C) The antimicrobial test of MccY against different serotype Salmonella and Shigella strains.(D).Activity of respiratory chain enzyme V of bacteria.

FIG 2
FIG 2 Experimental design for MccY for 2 weeks.

FIG 3
FIG 3 Therapeutic of MccY administration during Salmonella infection.(A) Percentage of survival of mice subjected to ST53-HD infection.(B) Percentage of survival of mice subjected to ST53-LD infection.(C) Body weight changes in ST53-HD-infected mice.(D) Body weight changes in the ST53-LD infection groups.(E) DAI in the ST53-HD infection groups.(F) DAI in the ST53-LD infection groups.(G) Detection of bacterial excretion in mouse feces in the ST53-HD infection and treatment groups.(H) Detection of bacterial excretion in mouse feces in the ST53-LD infection and treatment groups.Data are presented as the mean ± SD.Significant difference values are indicated for each group.(t-test; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

FIG 4
FIG 4 Changes in the expression of immune cytokines in serum in different groups.(A) SIgA expression of mice serum.(B) IgM expression of mice serum.(C) IgG expression of mice serum.(D) Cytokine expression in the jejunum.(E) Cytokine expression in the cecum.(F) Cytokine expression in the colon.Data are presented as mean ± SD; each group indicates significant difference values (t-test; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

FIG 5
FIG 5 The effect of MccY on gut lumen morphology and inflammatory status in the ST53 infection BALB/c mice.Scale bar = 100 µm.(A) Images of the jejunum after H&E staining.(B) Images of the cecum lumen after H&E staining.(C) Images of the colon lumen after H&E staining.(D) Villous height and crypt depth ratio of the jejunum.(E) Villous height and crypt depth ratio of the colon.(Data were expressed as mean ± SD (n = 8), t-test; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 for MccY groups compared with control group).

FIG 6
FIG 6 Effect of MccY treatment on the modulation of gut microbiota structure in the feces of the ST53 infection groups.(A) Venn diagram of ASV/OTU sequence abundance in different groups.(B) The relative abundance of fecal of mice.The x-axis represents each group of mice, and the y-axis represents the relative content ratio of each bacterial community to the total bacterial community at the genus level.(C) The relative abundance of Salmonella in the mouse feces in the ST53-HD infection groups.(D) The abundance of gut microbiota at the genus level post ST53-HD infection.(E) The relative abundance of Salmonella in the mouse fecal in the ST53-LD infection groups.(F) The abundance of gut microbiota at the genus level post ST53-HD infection.Each column indicates a group, and each row represents the genus level.Comparing MccY with controls, the data are presented as the mean ± SD. (t-test; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

FIG 7 FIG 8
FIG 7 Effect of MccY on the modulation of liver microbiota structure of the ST53 infection groups.(A) The relative abundance of lives in the ST53 infection mice.(B) Salmonella composition in ST53 infection mouse livers.Each column indicates a group, and each row represents the genus level.Data are presented as the mean ± SD.Significantly different values are indicated for each group (t-test; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).
presented that MccY upregulate the IgM level, and IgG results indicate that MccY play a crucial role in the immunity during the middle and late stages of infection.