Lactiplantibacillus plantarum Postbiotics Suppress Salmonella Infection via Modulating Bacterial Pathogenicity, Autophagy and Inflammasome in Mice

Simple Summary Salmonella infection is an urgent problem in animal husbandry, which causes salmonellosis in livestock and poses threats to human health through contaminated livestock products. As an alternative to antibiotics, probiotics play an important role in maintaining animal health. However, some probiotics, such as lactic acid bacteria, have limitations in storage and hostile environments. In this context, exploring the bacterial components or metabolites of probiotics has gradually drawn more research interest, and these are termed “postbiotics”. This study investigated the antibacterial effect of Lactiplantibacillus plantarum (LP) postbiotics and compared the effects to the live bacteria on intestinal health and autoimmunity in mice challenged with Salmonella. The results showed LP culture supernatant directly inhibited Salmonella growth and pathogenicity. LP postbiotics (the heat-killed bacteria and metabolites) showed similar or even superior effects to the active probiotic against Salmonella in mice. Furthermore, we found that LP postbiotics alleviated Salmonella infection via modulating bacterial pathogenicity, autophagy and inflammatory response. These results provide a theoretical basis for the protection of LP postbiotics against Salmonella and help to further explore its role in animal husbandry. Abstract Our study aimed to explore the effects of postbiotics on protecting against Salmonella infection in mice and clarify the underlying mechanisms. Eighty 5-week-old C57BL/6 mice were gavaged daily with Lactiplantibacillus plantarum (LP)-derived postbiotics (heat-killed bacteria, LPBinactive; culture supernatant, LPC) or the active bacteria (LPBactive), and gavaged with Salmonella enterica Typhimurium (ST). The Turbidimetry test and agar diffusion assay indicated that LPC directly inhibited Salmonella growth. Real-time PCR and biofilm inhibition assay showed that LPC had a strong ability in suppressing Salmonella pathogenicity by reducing virulence genes (SopE, SopB, InvA, InvF, SipB, HilA, SipA and SopD2), pili genes (FilF, SefA, LpfA, FimF), flagellum genes (FlhD, FliC, FliD) and biofilm formation. LP postbiotics were more effective than LP on attenuating ST-induced intestinal damage in mice, as indicated by increasing villus/crypt ratio and increasing the expression levels of tight junction proteins (Occludin and Claudin-1). Elisa assay showed that LP postbiotics significantly reduced ST-induced inflammation by regulating the levels of inflammatory cytokines (the increased IL-4 and IL-10 and the decreased TNF-α) in serum and ileum (p < 0.05). Furthermore, LP postbiotics inhibited the activation of NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) inflammasome by decreasing the protein expression of NLRP3 and Caspase-1, and the gene expression of Caspase-1, IL-1β and IL-18. Meanwhile, both LPC and LPB observably activated autophagy under ST infection, as indicated by the up-regulated expression of LC3 and Beclin1 and the downregulated p62 level (p < 0.05). Finally, we found that LP postbiotics could trigger an AMP-activated protein kinase (AMPK) signaling pathway to induce autophagy. In summary, Lactiplantibacillus plantarum-derived postbiotics alleviated Salmonella infection via modulating bacterial pathogenicity, autophagy and NLRP3 inflammasome in mice. Our results confirmed the effectiveness of postbiotics agents in the control of Salmonella infection.

Simple Summary: Salmonella infection is an urgent problem in animal husbandry, which causes salmonellosis in livestock and poses threats to human health through contaminated livestock products.As an alternative to antibiotics, probiotics play an important role in maintaining animal health.However, some probiotics, such as lactic acid bacteria, have limitations in storage and hostile environments.In this context, exploring the bacterial components or metabolites of probiotics has gradually drawn more research interest, and these are termed "postbiotics".This study investigated the antibacterial effect of Lactiplantibacillus plantarum (LP) postbiotics and compared the effects to the live bacteria on intestinal health and autoimmunity in mice challenged with Salmonella.The results showed LP culture supernatant directly inhibited Salmonella growth and pathogenicity.LP postbiotics (the heat-killed bacteria and metabolites) showed similar or even superior effects to the active probiotic against Salmonella in mice.Furthermore, we found that LP postbiotics alleviated Salmonella infection via modulating bacterial pathogenicity, autophagy and inflammatory response.These results provide a theoretical basis for the protection of LP postbiotics against Salmonella and help to further explore its role in animal husbandry.
Abstract: Our study aimed to explore the effects of postbiotics on protecting against Salmonella infection in mice and clarify the underlying mechanisms.Eighty 5-week-old C57BL/6 mice were gavaged daily with Lactiplantibacillus plantarum (LP)-derived postbiotics (heat-killed bacteria, LPB inactive ; culture supernatant, LPC) or the active bacteria (LPB active ), and gavaged with Salmonella enterica Typhimurium (ST).The Turbidimetry test and agar diffusion assay indicated that LPC directly inhibited Salmonella growth.Real-time PCR and biofilm inhibition assay showed that LPC had a strong ability in suppressing Salmonella pathogenicity by reducing virulence genes (SopE, SopB, InvA, InvF, SipB, HilA, SipA and SopD2), pili genes (FilF, SefA, LpfA, FimF), flagellum genes (FlhD, FliC, FliD) and biofilm formation.LP postbiotics were more effective than LP on attenuating ST-induced intestinal damage in mice, as indicated by increasing villus/crypt ratio and increasing the expression levels of tight junction proteins (Occludin and Claudin-1).Elisa assay showed that LP postbiotics significantly reduced ST-induced inflammation by regulating the levels of inflammatory cytokines (the increased IL-4 and IL-10 and the decreased TNF-α) in serum and ileum (p < 0.05).Furthermore, LP postbiotics inhibited the activation of NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) inflammasome by decreasing the protein expression of NLRP3 and Caspase-1, and the gene expression of Caspase-1, IL-1β and IL-18.Meanwhile, both LPC and LPB observably activated autophagy under ST infection, as indicated by the up-regulated expression of LC3 and Beclin1 and the downregulated p62 level (p < 0.05).Finally, we found that LP postbiotics could trigger an AMP-activated protein kinase (AMPK) signaling pathway to induce autophagy.In summary, Lactiplantibacillus plantarum-derived postbiotics alleviated Salmonella infection via modulating bacterial

Introduction
Probiotics have been broadly used in animals for numerous benefits [1].However, some probiotics might produce toxins and possess antimicrobial resistance genes, which can be a potential risk for animal health [2].Additionally, the shortcomings of probiotics, such as their intolerance to high temperatures, low pH and long-time storage, have also limited their application [3].Evidence has shown that the metabolites and bacterial components of probiotics remain highly physiologically active after treatment with high temperatures or gastrointestinal digestive juices [4,5].These components are termed "postbiotics", defined as "preparation of inanimate microorganisms and/or their components that confers a health benefit on the host" [6].Accumulating research has indicated that postbiotics have similar or even better effects than their active bacteria [7].Compared with live strains, postbiotics of Saccharomyces boulardii showed better efficacy in alleviating dextran sulfate sodium-induced colitis by modulating inflammation and intestinal microbiota in mice [8].The killed lactic acid bacteria exhibited better immunomodulatory activities than live probiotic in mice spleen cells [9].The heat-inactivated Lactobacillus gasseri increased immunomodulatory activity more than the live strains in macrophages [10].
Salmonella is one of the most important pathogens which has threats to food safety and animal and human health.It is able to cause intractable septicemia, anemia, bacteremia, meningitis and fatal dysentery [11].At present, the main treatment method for Salmonella is using antibiotics.However, the abuse of antibiotics can lead to bacterial resistance and other side effects.Recent studies have reported the anti-Salmonella effect of postbiotics.For example, Lactobacillus rhamnosus SQ511 culture supernatant directly inhibited Salmonella growth [12]; Lactobacillus postbiotics improved growth performance and enhanced immunity of broiler chickens after Salmonella infection [13].However, their mechanism of action remains to be investigated.
Activating the host inflammatory response is one of the main manifestations of Salmonella infection [14].Inflammasomes play a complex and important influence in activating and releasing inflammatory factors.Among them, NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) inflammasome has attracted widespread attention as a protein complex that can be activated by multiple stimuli and make a vital difference to the immune function regulation and inflammatory signaling [15].Pathogenassociated molecular patterns (PAMPs) trigger a signaling cascade reaction that causes cytosolic pattern-recognition receptors (PRRs) like NLRs to form the "the inflammasome" complexed with multimeric protein [16].Salmonella interaction with the pyrin structural domain triggers the recruitment of apoptosis-associated speck-like proteins containing a CARD (ASC) by NLRP3, which increases pro-Caspase-1 through CARD-CARD interactions and activates it to form Caspase-1 and ultimately, NLRP3 inflammatory vesicles.Then, activated Caspase-1 cleaves pro-IL-1β and pro-IL-18 into mature forms, ultimately leading to an inflammatory response [17].Restraining NLPR3 inflammasome activation is therefore crucial to suppress Salmonella-mediated inflammation.
Autophagy is closely related to the inhibition of inflammation.When NLRP3 is activated, ASCs are recognized by the adaptor protein p62, bound to autophagosome and eventually degraded.Peng et al. [18] confirmed the relation of autophagy and inflammasome.Therefore, triggering autophagy to inhibit NLRP3 inflammasome may be essential to control inflammatory responses during Salmonella infection.Numerous studies have shown that autophagy exerts a key role in the host resistance to Salmonella [19,20].Post-biotics have been shown to have a modulatory effect on autophagy.L. paracasei culture supernatants stimulated autophagy of Caco-2 cells [21]; postbiotics of L. fermentum alleviated acetaminophen-induced hepatic intoxication through activating autophagy in HepG2 cells [22].Therefore, the present research aimed to investigate the effectiveness of postbiotics from Lactiplantibacillus plantarum (formerly known as Lactobacillus plantarum) [23] on protecting mice against Salmonella infection and elucidate the underlying mechanisms modulating NLRP3 inflammasome and autophagy.

Bacteria Preparation
Lactiplantibacillus plantarum HJZW08 (CGMCC: No. 23777) was provided by Zhejiang Vegamax Biotechnology Co., Ltd.(Huzhou, China) and was cultivated in MRS broth at 37 • C for 24 h.The active bacteria (LPB active ) were rinsed in PBS and transferred into new MRS broth to eventually reach 1 × 10 9 cfu/mL, and the LPB inactive were then prepared by heating at 100 • C for 15 min.LP metabolites (LP culture supernatant, LPC) were obtained by centrifugation and sterile filtration.NaOH-LPC was obtained by adjusting the pH of LPC to 6.5 with 0.1 mol/L NaOH.Catalase-LPC was obtained by removing H 2 O 2 via incubation with catalase (1 mg/mL) at 25 • C for 1 h.Protease-LPC was obtained by removing bacteriocins with trypsin (200 mg/mL) and protease K (1 mg/mL) at 37 • C for 1 h.Heat-LPC was obtained at 100 • C for 15 min.Salmonella Typhimurium (ST) SL1344 was cultivated in LB broth overnight, then washed in PBS and diluted to 3 × 10 9 cfu/mL.

Animal Experimental Design
The mice experiments were conducted according to the Guide for the Care and Use of Laboratory Animals, and all procedures were approved by the Animal Care and Use Committee at Zhejiang Agriculture and Forestry University (Approval number: ZA-FUAC2022011).Male C57BL/6 mice of 5 weeks were purchased from SLAC Laboratory Animal Co., Ltd.(Shanghai, China).They were raised in a biosafety and comfortable environment.The environmental conditions were temperature at 21 ± 1 • C and humidity at 50~60%, with a 12 h light/dark cycle.They were supplied with the standard forage and filtered water and allowed to eat and drink freely.The eighty mice were divided into five groups (Control, ST, LPB active + ST, LPB inactive + ST and LPC + ST).After a seven-day acclimatization period, mice were severally given intragastric administration of 0.2 mL MRS broth, LPB active (1 × 10 9 cfu/mL), LPB inactive (1 × 10 9 cfu/mL) or LPC every day.Following a 15-day pretreatment period, the ST, LPB active + ST, LPB inactive + ST and LPC + ST groups were all gavaged by 8-gauge gavage needles with 0.1 mL of ST suspension (3 × 10 9 cfu/mL) in PBS, while the control group received equal volume of PBS.The animals were then sacrificed by ether anesthesia three days later.All mice samples were instantly frozen in liquid nitrogen and stored at −80 • C.

Agar Diffusion Assay
The plates were filled with LB solid medium with 5% agar as the bottom layer.Approximately 10 8 cfu/mL of ST cells were then added into in the LB agar (1% w/v), and then, the mixture was transferred as the upper layer.Then, sterile oxford cups were placed on the solidified agar, and 250 µL MRS, 2% LPC and GM (25 µg/mL), respectively, were transferred into each well.The anti-Salmonella activity was estimated by measuring the diameter of the inhibition zone.NaOH-LPC, Catalase-LPC, Protease-LPC and Heat-LPC groups were added to detect the antibacterial components of LPC by the above method.

Biofilm Inhibition Assay
ST was co-cultured with 2% LPC or GM (25 µg/mL) in 24-well plates for 12 and 24 h.Then, planktonic ST cells were removed by sucking out the supernatant and washing three times with PBS gently.Then, plates were stained with 0.1% (w/v) crystal violet at 23-25 • C for 30 min.Then, every well was given 3-4 rinses using distilled water and dried at room temperature for 15 min.After incubating with 95% (v/v) ethanol for 30 min, the absorbance was determined by an automated microplate reader (SynergyH1, BioTeK, Winooski, VT, USA) at 545 nm [24].The equation of biofilm inhibition is as follows: Biofilminhibitionrate(%) = OD Salmonella −OD testgroup OD Salmonella × 100.OD Salmonella : contains only the OD value of Salmonella; OD test group : LPC+ Salmonella or GM+ Salmonella OD value.

Intestinal Morphological Analysis
A small section of middle ileum was fixed with paraformaldehyde at 4%, buried in paraffin and sliced to 3 µm, and then stained using hematoxylin and eosin (H&E).Then, images were photographed through a Nikon microsystem (Nikon, Tokyo, Japan).The villus length and crypt depth were statistically calculated from 8 fields per slide and 8 slides in each group.

Inflammatory Cytokines Analysis
The blood samples were immediately collected after mice sacrifice and the serum was obtained by centrifuging at 4000× g for 10 min and stored at −80 • C for further study.The ileum tissues were homogenized with PBS, followed by a centrifugation (10,000× g, 4 • C) for 15 min to obtain the supernatant.The contents of IL-1β, TNF-α, IL-6, IL-4 and IL-10 were detected using ELISA commercial kits (Angle Gene ELISA kits, Nanjing, China).

Western Blot
Mice ileum samples were homogenized in NP-40 (Beyotime, Shanghai, China) with 1% PMSF and phosphatase inhibitors (Beyotime, Shanghai, China) on ice for half an hour.The BCA kit (Beyotime, Shanghai, China) was used to quantify protein concentration.After centrifuging and denaturating, 20 µg protein of each sample was added to the well and separated using 12% SDS polyacrylamide gels at 80 V for 30 min, then at 120 V for 1 h, and transferred onto polyvinylidene difluoride (PVDF) membranes of 0.22 µm or 0.45 µm pore size (Millipore, Carlsbad, CA, USA) at 260 mA for 1.5 h.Afterwards, the membranes were blocked by 5% (w/v) skimmed milk and cultivated with corresponding rabbit-derived primary antibody at 1:1000 overnight at 4 • C. Primary antibodies against LC3, p62, Beclin1, NLRP3, Caspase-1, Occludin, Claudin-1, AMPK, p-AMPK, TAK1, ULK1 and β-actin were purchased from Hua'an Technology (Hua'an, Hangzhou, China), and p-ULK1 was purchased from Cell Signaling Technology (Cell Signaling Technology, Danvers, MA, USA).After incubating with the secondary antibody HRP-labeled Goat Anti-Rabbit IgG (H+L) (Beyotime, Shanghai, China) at 1:1000 for 1 h, a Tanon 4600 series automatic chemiluminescence image analysis system (Tanon, Shanghai, China) was applied to detect the immunoreactive bands.Eventually, the ImageJ software v1.8.0.was used to determine the relative band density, and β-actin was used as the control.

mRNA Relative Expression Analysis by Real-Time PCR Assay
The total RNA was extracted from the Salmonella and C57BL/6 mice ileum using RNAiso plus (Takara Bio Inc., Otsu, Japan), referring to the manufacturer's instructions.Prime Script TM RT reagent Kit with g DNA Eraser kit were used to reverse transcribe 1 µL RNA (1000 ng/µL) into cDNA.The reaction conditions were 42 • C for 2 min to remove the DNA, then 37 • C for 15 min and 85 • C for 5 s to synthesize cDNA (50 ng/µL).Real-time PCR was performed in a CFX real-time PCR system (Bio-Rad Laboratories, Hercules, CA, USA) using SYBR Premix Ex TaqII (Takara Bio Inc., Otsu, Japan) under the following conditions: 95 • C for 30 s for initial denaturation, followed by 40 cycles at 95 • C for 5 s and 60 • C for 30 s.
β-actin and 16sRNA were regarded as reference genes.mRNA relative expression levels were calculated using the 2 −∆∆CT method (∆CT = CT target gene − CT reference gene ; ∆∆CT = ∆CT treated sample − ∆CT control sample ) [25].The primers are as shown in Table 1.
Table 1.Primer sequences used in real-time quantitative PCR 1 .

Statistical Analysis
All values are presented as means ± standard error.GraphPad Prism 8.0 was used to draw figures.Differences were conducted by one-way ANOVA and Tukey's test (SPSS Inc., Chicago, IL, USA).p-values of < 0.05 were deemed to be statistically significant.

Effects of LPC on Salmonella Growth
As shown in Figure 1a, LPC significantly inhibited the growth of ST (p < 0.05) and the inhibition zone was 23 mm.Results in Figure 1b show that LPC at the concentrations of 2-9% inhibited the growth of Salmonella starting from co-cultured with 4 h, while 1% LPC showed no antibacterial effect.Moreover, when incubated with 7-9% LPC, the inhibitory effect was similar to that of GM.As displayed in Figure 1c, we further found that when LPC was neutralized by NaOH, the antibacterial ability was completely blocked, whereas when treated with protease, catalase or heat showed no obvious changes.These results manifested that the main antibacterial ingredient of LPC was organic acids.
inhibition zone was 23 mm.Results in Figure 1b show that LPC at the concentrations of 2-9% inhibited the growth of Salmonella starting from co-cultured with 4 h, while 1% LPC showed no antibacterial effect.Moreover, when incubated with 7-9% LPC, the inhibitory effect was similar to that of GM.As displayed in Figure 1c, we further found that when LPC was neutralized by NaOH, the antibacterial ability was completely blocked, whereas when treated with protease, catalase or heat showed no obvious changes.These results manifested that the main antibacterial ingredient of LPC was organic acids.

Effects of LPC on Salmonella Pathogenicity
Figure 2a shows that 2% LPC notably restrained the relative expression levels of SPI-1 virulence genes SopE, SopB, InvF, InvA, SipA, SipB and HilA (p < 0.05).Compared to ST, the mRNA level of SopD2 encoded by SPI-2 was markedly decreased by LPC treatment (p < 0.05).As shown in Figure 2b, 2% LPC significantly inhibited the expression of pilus assembly genes including FilF, SefA, LpfA and FimF, compared with ST (p < 0.05).Figure 2c indicates that 2% LPC significantly inhibited the expression of flagella genes FlhD, FliC and FliD, compared to ST (p < 0.05).As demonstrated in Figure 2d, the inhibition ratio of 2% LPC on ST biofilm formation reached 80% at 12 h and 24 h, and the inhibition effect was extremely notable (p < 0.05).

Effects of LP Postbiotics on Salmonella-Induced Intestinal Injury in Mice
Figure 3a indicates that all the LP pretreatments significantly decreased Salmonella colonization in the ileum (p < 0.05), and LPC exerted a better effect with a marked decrease than LPB active (p < 0.05).As shown in Figure 3b, ST infection destroyed the intestinal villus construction and significantly reduced the length of the ileal villi and the villius/crypt ratio (p < 0.05).However, LP postbiotics and the active probiotic significantly reversed the trend by decreasing the crypt depth and increasing the villus/crypt ratio (p < 0.05).LPB inactive significantly reduced crypt depth and increased the villus/crypt ratio compared to LPB active and LPC (p < 0.05).Figure 3c shows that the expression of Occludin and Claudin-1 was significantly reduced after ST infection (p < 0.05), while pretreatments with LP postbiotics as well as LPB active significantly reversed this trend (p < 0.05).The expression level of protein Occludin notably maintained a higher level in LPB inactive + ST and LPC + ST groups than that of LPB active (p < 0.05).
Animals 2023, 13, x FOR PEER REVIEW 7 of 16 and FliD, compared to ST (p < 0.05).As demonstrated in Figure 2d, the inhibition ratio of 2% LPC on ST biofilm formation reached 80% at 12 h and 24 h, and the inhibition effect was extremely notable (p < 0.05).

Effects of LP Postbiotics on Salmonella-Induced Intestinal Injury in Mice
Figure 3a indicates that all the LP pretreatments significantly decreased Salmonella colonization in the ileum (p < 0.05), and LPC exerted a better effect with a marked decrease than LPBactive (p < 0.05).As shown in Figure 3b, ST infection destroyed the intestinal villus construction and significantly reduced the length of the ileal villi and the villius/crypt ratio (p < 0.05).However, LP postbiotics and the active probiotic significantly reversed the LPBinactive significantly reduced crypt depth and increased the villus/crypt ratio compared to LPBactive and LPC (p < 0.05).Figure 3c shows that the expression of Occludin and Claudin-1 was significantly reduced after ST infection (p < 0.05), while pretreatments with LP postbiotics as well as LPBactive significantly reversed this trend (p < 0.05).The expression level of protein Occludin notably maintained a higher level in LPBinactive + ST and LPC +ST groups than that of LPBactive (p < 0.05).

Effects of LP Postbiotics on the Levels of Inflammatory Cytokines in Mice under Salmonella Challenge
As shown in Figure 4a, ST challenge significantly increased the serum pro-inflammatory factors, IL-1β, IL-6 and TNF-α (p < 0.05), and significantly decreased the levels of antiinflammatory cytokines, IL-4 and IL-10 (p < 0.05).LP pretreatments significantly reversed this trend (p < 0.05).Interestingly, LPB inactive was superior to LPB active and LPC in inhibiting ST-induced inflammation, and LPC was superior to LPB active .Figure 4b demonstrates the results for ileal inflammatory factors, similar to the trend for inflammatory factors in serum.Both LP postbiotics and its live bacterial pretreatments significantly downregulated the contents of pro-inflammatory factors, IL-1β and IL-6, raised by ST infection (p < 0.05).LPB inactive was more effective than LPB active and LPC.

Effects of LP Postbiotics on the Levels of Inflammatory Cytokines in Mice under Salmonella Challenge
As shown in Figure 4a, ST challenge significantly increased the serum pro-inflammatory factors, IL-1β, IL-6 and TNF-α (p < 0.05), and significantly decreased the levels of antiinflammatory cytokines, IL-4 and IL-10 (p < 0.05).LP pretreatments significantly reversed this trend (p < 0.05).Interestingly, LPBinactive was superior to LPBactive and LPC in inhibiting ST-induced inflammation, and LPC was superior to LPBactive.Figure 4b demonstrates the results for ileal inflammatory factors, similar to the trend for inflammatory factors in serum.Both LP postbiotics and its live bacterial pretreatments significantly downregulated the contents of pro-inflammatory factors, IL-1β and IL-6, raised by ST infection (p < 0.05).LPBinactive was more effective than LPBactive and LPC.

Effects of LP Postbiotics on NLRP3 Inflammasome in Mice under Salmonella Challenge
As shown in Figure 5a, ST infection markedly improved the mRNA relative expression levels of inflammasome biomarkers including Caspase-1, IL-1β and IL-18 compared with the control group (p < 0.05).Nevertheless, LP postbiotics and its live bacteria significantly downregulated them (p < 0.05).Furthermore, Figure 5b shows that LP postbiotics and the live bacteria acted through depressing the assembly of NLRP3 inflammasome and suppressing the inflammation caused by ST infection.LPC was superior to LPB active and LPB inactive .LPB inactive and LPC significantly inhibited Caspase-1 expression (p < 0.05) compared to ST, with a better inhibitory effect than that of LP live bacteria.

Effects of LP Postbiotics on NLRP3 Inflammasome in Mice under Salmonella Challenge
As shown in Figure 5a, ST infection markedly improved the mRNA relative expression levels of inflammasome biomarkers including Caspase-1, IL-1β and IL-18 compared with the control group (p < 0.05).Nevertheless, LP postbiotics and its live bacteria significantly downregulated them (p < 0.05).Furthermore, Figure 5b shows that LP postbiotics and the live bacteria acted through depressing the assembly of NLRP3 inflammasome and suppressing the inflammation caused by ST infection.LPC was superior to LPBactive and LPBinactive.LPBinactive and LPC significantly inhibited Caspase-1 expression (p < 0.05) compared to ST, with a better inhibitory effect than that of LP live bacteria.

Effects of LP Postbiotics on Autophagy under Salmonella Challenge
The results in Figure 6a show that preliminary treatments of LP markedly increased the expression levels of Beclin1 and LC3-II, inhibited p62 expression compared to ST (p < 0.05) and activated cellular autophagy.In addition, LPBinactive and LPC were significantly more effective than LPBactive on LC3-II (p < 0.05).The results suggest that LP postbiotics pretreatments, particularly LPC, activated autophagy to counteract ST and was more effective than its active bacteria.As shown in Figure 6b, LP postbiotics and its live bacteria pretreatments significantly enhanced the expression levels of proteins such as TAK1, p-ULK1 (Ser757) and p-AMPK compared to the ST group (p < 0.05), and LPBinactive and LPC appeared to be stronger than LP active bacteria (Figure 6b).Thus, LP postbiotics promoted autophagy in mice through the AMPK/ULK1 signal pathway.

Effects of LP Postbiotics on Autophagy under Salmonella Challenge
The results in Figure 6a show that preliminary treatments of LP markedly increased the expression levels of Beclin1 and LC3-II, inhibited p62 expression compared to ST (p < 0.05) and activated cellular autophagy.In addition, LPB inactive and LPC were significantly more effective than LPB active on LC3-II (p < 0.05).The results suggest that LP postbiotics pretreatments, particularly LPC, activated autophagy to counteract ST and was more effective than its active bacteria.As shown in Figure 6b, LP postbiotics and its live bacteria pretreatments significantly enhanced the expression levels of proteins such as TAK1, p-ULK1 (Ser757) and p-AMPK compared to the ST group (p < 0.05), and LPB inactive and LPC appeared to be stronger than LP active bacteria (Figure 6b).Thus, LP postbiotics promoted autophagy in mice through the AMPK/ULK1 signal pathway.

Discussion
Although numerous studies have shown the beneficial effects of probiotics, their potential disadvantages have gradually been recognized as research has deepened.Postbiotics are the inactivated bacteria and their metabolites of probiotics.Of these, the active components are mainly peptidoglycan and lipophosphate walls, and the metabolites are organic acids, hydrogen peroxide and bacteriocins [26,27].The inhibitory effect of postbiotics on pathogenic bacteria has been proved.For example, postbiotics of L. acidophilus and Enterococcus faecalis observably reduced the quantity of Clostridium perfringens in the chicken digestive tract [28].In this study, we found that postbiotics derived from Lactiplantibacillus plantarum significantly alleviated Salmonella infection by inhibiting bacterial pathogenicity and modulating autophagy and NLRP3 inflammasome in mice.
Our results showed that LP metabolites markedly inhibited ST growth.The growth inhibitory effect of LPC on ST began at a concentration of 2% and intensified with increasing concentration.High-concentration LPC had even the same effect as GM, which provided evidence for the study of metabolites as an alternative to antibiotics.Similarly, it was reported that the postbiotics of Lactiplantibacillus pentosus SLC13 had a good bacteriostatic effect on the Helicobacter pylori in a dose-dependent manner [29].Our study showed that the antibacterial substance produced by LPC was organic acids, which was in accordance with the outcomes of Russo et al. [30], and that the organic acids contained in Lactobacillus plantarum had a good suppressive effect on common foodborne pathogenic

Discussion
Although numerous studies have shown the beneficial effects of probiotics, their potential disadvantages have gradually been recognized as research has deepened.Postbiotics are the inactivated bacteria and their metabolites of probiotics.Of these, the active components are mainly peptidoglycan and lipophosphate walls, and the metabolites are organic acids, hydrogen peroxide and bacteriocins [26,27].The inhibitory effect of postbiotics on pathogenic bacteria has been proved.For example, postbiotics of L. acidophilus and Enterococcus faecalis observably reduced the quantity of Clostridium perfringens in the chicken digestive tract [28].In this study, we found that postbiotics derived from Lactiplantibacillus plantarum significantly alleviated Salmonella infection by inhibiting bacterial pathogenicity and modulating autophagy and NLRP3 inflammasome in mice.
Our results showed that LP metabolites markedly inhibited ST growth.The growth inhibitory effect of LPC on ST began at a concentration of 2% and intensified with increasing concentration.High-concentration LPC had even the same effect as GM, which provided evidence for the study of metabolites as an alternative to antibiotics.Similarly, it was reported that the postbiotics of Lactiplantibacillus pentosus SLC13 had a good bacteriostatic effect on the Helicobacter pylori in a dose-dependent manner [29].Our study showed that the antibacterial substance produced by LPC was organic acids, which was in accordance with the outcomes of Russo et al. [30], and that the organic acids contained in Lactobacillus plantarum had a good suppressive effect on common foodborne pathogenic bacteria and spoilage bacteria.LPC was not sensitive to enzyme inhibitors (trypsin, proteinase K, catalase), indicating that the main antibacterial substances were not bacteriocin and hydrogen peroxide.Furthermore, we found that LPC had high thermal stability, which was identical to the results of Mekky et al. [31].In summary, the inhibitory substance in LPC was an organic acid, but the specific substance that exerted the inhibitory effect needs to be further studied.
LP metabolites exerted a strong capacity in suppressing Salmonella pathogenicity.The pathogenicity of Salmonella is driven by virulence factors, flagella, pili and biofilm, which not only help the bacteria to invade cells and colonize, but also protect the bacteria from the external environment [32].We found that LPC significantly decreased the mRNA expression of the virulence genes (SopE, SopB, InvA, InvF, SipB, HilA, SipA and SopD2).The virulence genes SopB, SopE, and HilA encoded by SPI-1 play key roles in Salmonella invasion of cells and cause inflammation.SPI-2 encodes SopD2, which contributes to its evasion of lysosomal degradation [33].These virulence genes play a decisive part in invading host cells and the pathogenicity of Salmonella.Consistent with our results, it has been reported that Lactococcus suppressed Salmonella virulence genes' expression levels [34].Flagella is the motor organ of Salmonella which enables it to move forward in a fluctuating environment and is also an important virulence factor mediating bacterial attachment and invasion [35].In the course of Salmonella infection, pili plays an important role in host recognition, colonization and biofilm formation, which is considered to be the main organelles mediating the interaction and adhesion between Salmonella and host intestinal epithelium [36].In this study, LPC markedly repressed the gene expression involving in flagella (FlhD, FliC, FliD) and pili (FilF, SefA, LpfA, FimF) indicating its ability.Shi et al. [37] found that the culture supernatant of Lactobacillus reuteri S5 resulted in a significant reduction in the expression of Salmonella virulence, motility and adhesion genes and had an inhibitory effect on biofilm formation.We further found that LPC could inhibit the formation of ST biofilm.After colonizing the host intestine through adhesion and invasion, Salmonella further forms a dense biofilm to enhance its pathogenicity [38].Bacteria in biofilms are generally well protected from environmental stress, antibiotics and the host immune system, making them extremely difficult to eradicate [39].The bacterial resistance to antibiotics is 1000 times higher than that of planktic bacteria, leading to longterm chronic infection [40].Previous studies have also shown inhibitory effects of probiotic culture supernatants on Salmonella biofilms [41,42].Therefore, the results showed that LPC reduced the gene expression of ST virulence, pili and flagella, and suppressed biofilm formation to inhibit the pathogenicity of Salmonella.
The mice experiment revealed that LP postbiotics showed a similar or even superior effect than the active probiotic in protecting against Salmonella infection.LP postbiotics, particularly the metabolites, significantly decreased ST colonization in mice ileum.After invading the host, Salmonella first invades the intestinal epithelial cells of the body, reaches the lamina propria of the intestinal wall and multiplies in them, causing inflammation at the same time [43].With the increase in the number of bacteria, damage and inflammation will be aggravated and transferred to other tissues.In line with our results, postbiotics of Bacillus subtilis and Bacillus licheniformis have been found to decrease the number of Salmonella in the gut and thus relieve damage [44].Salmonella infection can cause intestinal mucosal and villi damage.We found that ST infection significantly decreased the villus length and increased the crypt depth, while pretreatments with LP postbiotics and the active bacteria could reverse this trend.The increase in villi height can enlarge the absorption area of small intestine, which is conducive to absorbing more nutrients [45].Crypt depth reflects the cell formation rate, and the shallower crypt indicates increased cell maturation rate and secretion function [46].Thus, higher villus height to crypt depth ratio indicates an increased absorptive capacity and cell formation rate to maintain the intestinal health.Previous studies have shown that probiotics play a crucial part in miti-gating intestinal tissue damage [47,48], while there has been less research on postbiotics.Intestinal epithelial cells are made up of simple columnar intestinal epithelial cells whose gaps have many tight junction proteins (Occludin, Claudin, et al.), which are closely related to permeability [49].Previous studies have shown that Salmonella infection led to intestinal damage, increased permeability and downregulated the expression of Occludin and Claudin in mice [50].Consistently, we also found that ST infection dramatically reduced the expression of Occludin and Claudin-1, whereas LP postbiotics as well as the live probiotic could prevent the decrease, indicating their ability to enhance mucosal barrier.In summary, LP postbiotics showed a similar effect as the live probiotic in reducing ST colonization, alleviating villi damages, enhancing tight junction expression and ultimately, preventing Salmonella-induced gut injuries.
We found that LP postbiotics inhibited NLRP3 inflammasome to alleviate Salmonellainduced inflammation.Salmonella infection can activate inflammatory pathways by binding to TLRs on the cell surface to produce pro-inflammatory cytokines that cause an inflammatory response [51].Our results showed that LP postbiotics pretreatments significantly decreased the levels of pro-inflammatory cytokines, IL-1β, TNF-α and IL-6, and increased anti-inflammatory cytokines, IL-4 and IL-10, suggesting its role in anti-inflammation.Previous studies have shown that the anti-inflammatory effect of postbiotics has been confirmed.For example, metabolites of L. acidophilus and L. casei could resist inflammation by lowering TNF-α cytokine level and increasing IL-10 level [52].In addition, we found that the inactive bacteria showed stronger anti-inflammatory abilities than the live bacteria and the metabolites.Combined with previous studies on resisting inflammatory nature of lipoteichoic acid [53], we speculated that the anti-inflammatory component in postbiotics might be lipoteichoic acid.NLRP3 inflammasomes are typical inflammasomes that can be activated by Salmonella, and the expression of genes and proteins of its biomarkers could be inhibited by LP postbiotics.Consistent with our study, Huang and Lu et al. [54,55] found that postbiotics (active peptides and inactivated organisms) could inhibit the activation of inflammatory vesicles and attenuate Salmonella-induced intestinal inflammation.Therefore, our results suggest that LP, especially postbiotics, inhibited Salmonella-induced inflammatory responses and the activation of inflammasomes.
Our study demonstrates that LP postbiotics activated autophagy to protect against Salmonella infection.Autophagy is a significant innate immune recognition mechanism against pathogens.The autophagosome can recognize intracellular pathogens as a foreign entity, isolate them and then transport them to the lysosome for degradation and removal, which is known as xenophagy [56].During Salmonella infection, autophagy protects cells from bacterial invasion [57].In this work, we found that LP postbiotics significantly prevented ST-disrupted autophagy, as evidenced by the increased levels of LC3 and Beclin1 and the decreased p62.Similar with our results, recent studies have confirmed the activation of autophagy by probiotics upon pathogen infection.For example, Lactobacillus yohimbe prevented Salmonella-induced intestinal damage by regulating autophagy [58]; Exopolysaccharides from Bifidobacterium animalis ameliorated Escherichia coli-induced IPEC-J2 cell damage via inhibiting apoptosis and restoring autophagy [59].We further found that AMPK/ULK1 signaling played a pivotal role in LP postbiotics-induced autophagy.AMPK/ULK1 and its upstream molecule TGF-β-activated kinase 1 (TAK1) [60] have a vital impact on cell survival, apoptosis, as well as inflammatory responses, and it is also a classic signaling pathway to activate autophagy.When cells are hungry or stimulated, activated AMPK can directly promote the phosphorylation of ULK1-related sites and ultimately initiate autophagy [61].We found that LP postbiotics markedly upregulated the phosphorylation of AMPK and ULK1 and the expression of TAK1, suggesting their capacity of triggering autophagy.In line with our results, a growing number of studies have also revealed the ability of Lactobacillus plantarum to induce the AMPK signaling pathway [62].Furthermore, it was reported that activation of AMPK/ULK1 was an effective way to defend against Salmonella.For instance, Zhuang et al. [63] demonstrated that autophagy could be induced by activating AMPK and ULK1 to restrict intracellular S. typhimurium growth in vitro.Therefore, our study indicates that TAK1/AMPK/ULK1 plays a key role in LP postbiotic-activated autophagy to protect against Salmonella infection.

Conclusions
In conclusion, LP postbiotics suppressed Salmonella infection via inhibiting bacterial pathogenicity and modulating autophagy and NLRP3 inflammasome in mice.Our findings confirm the antimicrobial activity of postbiotics and provide a strategy to prevent Salmonella infection in the livestock production.However, which components of LP postbiotics (the specific organic acids or bacterial components) exert the key effects warrants further investigation.

Figure 1 .
Figure 1.Effects of LPC on Salmonella Growth.(a) The inhibition effect of LPC on ST was detected via agar-well diffusion method; (b) inhibitory effect of different concentrations of LPC on ST; (c) the ST inhibitory components in LPC were determined via the method described in (a).Data were analyzed by one-way ANOVA and Tukey s test (n = 6).Different lowercase letters indicate statistical significance with p < 0.05.

Figure 1 .
Figure 1.Effects of LPC on Salmonella Growth.(a) The inhibition effect of LPC on ST was detected via agar-well diffusion method; (b) inhibitory effect of different concentrations of LPC on ST; (c) the ST inhibitory components in LPC were determined via the method described in (a).Data were analyzed by one-way ANOVA and Tukey's test (n = 6).Different lowercase letters indicate statistical significance with p < 0.05.

Figure 2 .
Figure 2. Effects of LPC on Salmonella Pathogenicity.(a-c) The relative mRNA expression levels of the virulence, pilli and flagellar related genes of ST; (d) the inhibition rates of ST biofilm.The concentration of LPC was 2%.Data analysis was conducted by one-way ANOVA and Tukey s test (n = 3).Different lowercase letters indicate statistical significance with p < 0.05.

Figure 2 .
Figure 2. Effects of LPC on Salmonella Pathogenicity.(a-c) The relative mRNA expression levels of the virulence, pilli and flagellar related genes of ST; (d) the inhibition rates of ST biofilm.The concentration of LPC was 2%.Data analysis was conducted by one-way ANOVA and Tukey's test (n = 3).Different lowercase letters indicate statistical significance with p < 0.05.

Figure 3 .
Figure 3. Effects of LP Postbiotics on Salmonella-Induced Intestinal Injury in Mice.(a) The amounts of ST in the ileum of mice; (b) effect of LPC on ileum villi, crypt and villus/crypt ratio in mice infected with ST; (c) effect of LPC on the tight junction proteins Occludin and Claudin-1 expression in mouse ileum.Data were carried out by one-way ANOVA and Tukey s test (n = 3).Different lowercase letters indicate statistical significance with p < 0.05 (original western blot figures in Figure S1).

Figure 3 .
Figure 3. Effects of LP Postbiotics on Salmonella-Induced Intestinal Injury in Mice.(a) The amounts of ST in the ileum of mice; (b) effect of LPC on ileum villi, crypt and villus/crypt ratio in mice infected with ST; (c) effect of LPC on the tight junction proteins Occludin and Claudin-1 expression in mouse ileum.Data were carried out by one-way ANOVA and Tukey's test (n = 3).Different lowercase letters indicate statistical significance with p < 0.05 (original western blot figures in Figure S1).

Figure 4 .
Figure 4. Effects of LP Postbiotics on the Levels of Inflammatory Cytokines in Mice under Salmonella Challenge.(a) The contents of inflammatory factors in mouse serum; (b) the levels of inflammatory cytokines in mouse ileum.Data analysis was conducted by one-way ANOVA and Tukey s test (n = 6).Different lowercase letters indicate statistical significance with p < 0.05.

Figure 4 .
Figure 4. Effects of LP Postbiotics on the Levels of Inflammatory Cytokines in Mice under Salmonella Challenge.(a) The contents of inflammatory factors in mouse serum; (b) the levels of inflammatory cytokines in mouse ileum.Data analysis was conducted by one-way ANOVA and Tukey's test (n = 6).Different lowercase letters indicate statistical significance with p < 0.05.

Figure 5 .
Figure 5. Effects of LP Postbiotics on NLRP3 Inflammasome in Mice under Salmonella Challenge.(a) The expression of inflammasome genes in mouse ileum; (b) the protein expression levels of NLRP3 and Caspase-1.Data were analyzed using one-way ANOVA and Tukey s test (n = 3).Different lowercase letters indicate statistical significance with p < 0.05 (original western blot figures in Figure S2).

Figure 5 .
Figure 5. Effects of LP Postbiotics on NLRP3 Inflammasome in Mice under Salmonella Challenge.(a) The expression of inflammasome genes in mouse ileum; (b) the protein expression levels of NLRP3 and Caspase-1.Data were analyzed using one-way ANOVA and Tukey's test (n = 3).Different lowercase letters indicate statistical significance with p < 0.05 (original western blot figures in FigureS2).

Figure 6 .
Figure 6.Effects of LP Postbiotics on Autophagy under Salmonella Challenge.(a) The protein expression levels of Beclin1, p62, LC3 and β-actin; (b) the protein expression of TAK1, p-ULK1, ULK1, p-AMPK, AMPK and β-actin.Data analysis was carried out by one-way ANOVA and Tukey s test (n = 3).Different lowercase letters indicate statistical significance with p < 0.05 (original western blot figures in Figures S3 and S4).