Novel Synergistic Probiotic Intervention: Transcriptomic and Metabolomic Analysis Reveals Ameliorative Effects on Immunity, Gut Barrier, and Metabolism of Mice during Salmonella typhimurium Infection.

Salmonella typhimurium (S. typhimurium), a prevalent cause of foodborne infection, induces significant changes in the host transcriptome and metabolome. The lack of therapeutics with minimal or no side effects prompts the scientific community to explore alternative therapies. This study investigates the therapeutic potential of a probiotic mixture comprising Lactobacillus acidophilus (L. acidophilus 1.3251) and Lactobacillus plantarum (L. plantarum 9513) against S. typhimurium, utilizing transcriptome and metabolomic analyses, a novel approach that has not been previously documented. Twenty-four SPF-BALB/c mice were divided into four groups: control negative group (CNG); positive control group (CPG); probiotic-supplemented non-challenged group (LAPG); and probiotic-supplemented Salmonella-challenged group (LAPST). An RNA-sequencing analysis of small intestinal (ileum) tissue revealed 2907 upregulated and 394 downregulated DEGs in the LAPST vs. CPG group. A functional analysis of DEGs highlighted their significantly altered gene ontology (GO) terms related to metabolism, gut integrity, cellular development, and immunity (p ≤ 0.05). The KEGG analysis showed that differentially expressed genes (DEGs) in the LAPST group were primarily involved in pathways related to gut integrity, immunity, and metabolism, such as MAPK, PI3K-Akt, AMPK, the tryptophan metabolism, the glycine, serine, and threonine metabolism, ECM-receptor interaction, and others. Additionally, the fecal metabolic analysis identified 1215 upregulated and 305 downregulated metabolites in the LAPST vs. CPG group, implying their involvement in KEGG pathways including bile secretion, propanoate metabolism, arginine and proline metabolism, amino acid biosynthesis, and protein digestion and absorption, which are vital for maintaining barrier integrity, immunity, and metabolism. In conclusion, these findings suggest that the administration of a probiotic mixture improves immunity, maintains gut homeostasis and barrier integrity, and enhances metabolism in Salmonella infection.

Humoral regulation of iron metabolism by extracellular vesicles drives antibacterial response.

Immediate restriction of iron initiated by the host is a critical process to protect against bacterial infections and has been described in the liver and spleen, but it remains unclear whether this response also entails a humoral mechanism that would enable systemic sequestering of iron upon infection. Here we show that upon bacterial invasion, host macrophages immediately release extracellular vesicles (EVs) that capture circulating iron-containing proteins. Mechanistically, in a sepsis model in female mice, Salmonella enterica subsp. enterica serovar Typhimurium induces endoplasmic reticulum stress in macrophages and activates inositol-requiring enzyme 1α signaling, triggering lysosomal dysfunction and thereby promoting the release of EVs, which bear multiple receptors required for iron uptake. By binding to circulating iron-containing proteins, these EVs prevent bacteria from iron acquisition, which inhibits their growth and ultimately protects against infection and related tissue damage. Our findings reveal a humoral mechanism that can promptly regulate systemic iron metabolism during bacterial infection.

Salmonella Infection Causes Hyperglycemia for Decreased GLP-1 Content by Enteroendocrine L Cells Pyroptosis in Pigs.

Inflammatory responses have been shown to induce hyperglycemia, yet the underlying mechanism is still largely unclear. GLP-1 is an important intestinal hormone for regulating glucose homeostasis; however, few studies have investigated the influence of digestive tract Salmonella infection on enteroendocrine L cell secretions. In this study, we established a model of Salmonella-infected piglets by oral gavage in order to analyze the effects of Salmonella infection on enteroendocrine L cell function. Furthermore, in vitro lipopolysaccharide (LPS) was administered to STC-1 cells to clarify its direct effect on GLP-1 secretion. The results showed that significantly increased blood glucose in the group of Salmonella-infected piglets was observed, and Salmonella infection decreased blood GLP-1 content. Then, ileal epithelium damage was observed by histological detection, and this was further verified by TUNEL staining. We identified activation of TLR signaling demonstrating up-regulated expressions of TLR4 and nuclear factor-kappa B (NF-ΚB). Furthermore, it was shown that Salmonella induced pyroptosis of enteroendocrine L cells and enhanced the secretion of IL-1ß through augmenting gene and protein expressions of NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a carboxyl-terminal CARD (ASC), Caspase 1, and gasdermin D (GSDMD). Meanwhile, in vitro LPS treatment induced the pyroptosis of STC-1 cells and reduced the secretion of GLP-1. Altogether, the results demonstrated that Salmonella infection can reduce secretion of GLP-1 by inducing pyroptosis of intestinal L cells, which may eventually result in hyperglycemia. The results provided evidence for the cause of hyperglycemia induced by inflammation and shed new light on glucose homeostasis regulation.

Streptozotocin-induced hyperglycemia alters the cecal metabolome and exacerbates antibiotic-induced dysbiosis.

It is well established in the microbiome field that antibiotic (ATB) use and metabolic disease both impact the structure and function of the gut microbiome. But how host and microbial metabolism interacts with ATB susceptibility to affect the resulting dysbiosis remains poorly understood. In a streptozotocin-induced model of hyperglycemia (HG), we use a combined metagenomic, metatranscriptomic, and metabolomic approach to profile changes in microbiome taxonomic composition, transcriptional activity, and metabolite abundance both pre- and post-ATB challenge. We find that HG impacts both microbiome structure and metabolism, ultimately increasing susceptibility to amoxicillin. HG exacerbates drug-induced dysbiosis and increases both phosphotransferase system activity and energy catabolism compared to controls. Finally, HG and ATB co-treatment increases pathogen susceptibility and reduces survival in a Salmonella enterica infection model. Our data demonstrate that induced HG is sufficient to modify the cecal metabolite pool, worsen the severity of ATB dysbiosis, and decrease colonization resistance.

Salmonella Typhimurium impairs glycolysis-mediated acidification of phagosomes to evade macrophage defense.

Regulation of cellular metabolism is now recognized as a crucial mechanism for the activation of innate and adaptive immune cells upon diverse extracellular stimuli. Macrophages, for instance, increase glycolysis upon stimulation with pathogen-associated molecular patterns (PAMPs). Conceivably, pathogens also counteract these metabolic changes for their own survival in the host. Despite this dynamic interplay in host-pathogen interactions, the role of immunometabolism in the context of intracellular bacterial infections is still unclear. Here, employing unbiased metabolomic and transcriptomic approaches, we investigated the role of metabolic adaptations of macrophages upon Salmonella enterica serovar Typhimurium (S. Typhimurium) infections. Importantly, our results suggest that S. Typhimurium abrogates glycolysis and its modulators such as insulin-signaling to impair macrophage defense. Mechanistically, glycolysis facilitates glycolytic enzyme aldolase A mediated v-ATPase assembly and the acidification of phagosomes which is critical for lysosomal degradation. Thus, impairment in the glycolytic machinery eventually leads to decreased bacterial clearance and antigen presentation in murine macrophages (BMDM). Collectively, our results highlight a vital molecular link between metabolic adaptation and phagosome maturation in macrophages, which is targeted by S. Typhimurium to evade cell-autonomous defense.

Sodium butyrate modulates chicken macrophage proteins essential for Salmonella Enteritidis invasion.

Salmonella Enteritidis is an intracellular foodborne pathogen that has developed multiple mechanisms to alter poultry intestinal physiology and infect the gut. Short chain fatty acid butyrate is derived from microbiota metabolic activities, and it maintains gut homeostasis. There is limited understanding on the interaction between S. Enteritidis infection, butyrate, and host intestinal response. To fill this knowledge gap, chicken macrophages (also known as HTC cells) were infected with S. Enteritidis, treated with sodium butyrate, and proteomic analysis was performed. A growth curve assay was conducted to determine sub-inhibitory concentration (SIC, concentration that do not affect bacterial growth compared to control) of sodium butyrate against S. Enteritidis. HTC cells were infected with S. Enteritidis in the presence and absence of SIC of sodium butyrate. The proteins were extracted and analyzed by tandem mass spectrometry. Our results showed that the SIC was 45 mM. Notably, S. Enteritidis-infected HTC cells upregulated macrophage proteins involved in ATP synthesis through oxidative phosphorylation such as ATP synthase subunit alpha (ATP5A1), ATP synthase subunit d, mitochondrial (ATP5PD) and cellular apoptosis such as Cytochrome-c (CYC). Furthermore, sodium butyrate influenced S. Enteritidis-infected HTC cells by reducing the expression of macrophage proteins mediating actin cytoskeletal rearrangements such as WD repeat-containing protein-1 (WDR1), Alpha actinin-1 (ACTN1), Vinculin (VCL) and Protein disulfide isomerase (P4HB) and intracellular S. Enteritidis growth and replication such as V-type proton ATPase catalytic subunit A (ATPV1A). Interestingly, sodium butyrate increased the expression of infected HTC cell protein involving in bacterial killing such as Vimentin (VIM). In conclusion, sodium butyrate modulates the expression of HTC cell proteins essential for S. Enteritidis invasion.

AvrA Exerts Inhibition of NF-κB Pathway in Its Naïve Salmonella Serotype through Suppression of p-JNK and Beclin-1 Molecules.

Avian salmonellosis caused by Salmonella enterica serovar Enteritidis (S. Enteritidis) and Pullorum (S. Pullorum) remains a big threat to the poultry industry and public hygiene. AvrA is an effector involved in inhibiting inflammation. Compared to AvrA from S. Enteritidis (SE-AvrA), the AvrA from S. Pullorum (SP-AvrA) lacks ten amino acids at the C-terminal. In this study, we compared the anti-inflammatory response induced by SP-AvrA to that of SE-AvrA. Transient expression of SP-AvrA in epithelial cells resulted in significantly weaker inhibition of NF-κB pathway activation when treated with TNF-α compared to the inhibition by SE-AvrA. SP-AvrA expression in the S. Enteritidis resulted in weaker suppression of NF-κB pathway in infected HeLa cells compared to SE-AvrA expression in the cells, while SP-AvrA expressed in S. Pullorum C79-13 suppressed NF-κB activation in infected HeLa and Caco 2 BBE cells to a greater extent than did SE-AvrA because of the higher expression of SP-AvrA than SE-AvrA in S. Pullorum. Further analysis demonstrated that the inhibition of NF-κB pathway in Salmonella-infected cells corresponded to the downregulation of the p-JNK and Beclin-1 protein molecules. Our study reveals that AvrA modifies the anti-inflammatory response in a manner dependent on the Salmonella serotype through inhibition of NF-κB pathway.

YAP in epithelium senses gut barrier loss to deploy defenses against pathogens.

Pathogens commonly disrupt the intestinal epithelial barrier; however, how the epithelial immune system senses the loss of intestinal barrier as a danger signal to activate self-defense is unclear. Through an unbiased approach in the model nematode Caenorhabditis elegans, we found that the EGL-44/TEAD transcription factor and its transcriptional activator YAP-1/YAP (Yes-associated protein) were activated when the intestinal barrier was disrupted by infections with the pathogenic bacterium Pseudomonas aeruginosa PA14. Gene Ontology enrichment analysis of the genes containing the TEAD-binding sites revealed that "innate immune response" and "defense response to Gram-negative bacterium" were two top significantly overrepresented terms. Genetic inactivation of yap-1 and egl-44 significantly reduced the survival rate and promoted bacterial accumulation in worms after bacterial infections. Furthermore, we found that disturbance of the E-cadherin-based adherens junction triggered the nuclear translocation and activation of YAP-1/YAP in the gut of worms. Although YAP is a major downstream effector of the Hippo signaling, our study revealed that the activation of YAP-1/YAP was independent of the Hippo pathway during disruption of intestinal barrier. After screening 10 serine/threonine phosphatases, we identified that PP2A phosphatase was involved in the activation of YAP-1/YAP after intestinal barrier loss induced by bacterial infections. Additionally, our study demonstrated that the function of YAP was evolutionarily conserved in mice. Our study highlights how the intestinal epithelium recognizes the loss of the epithelial barrier as a danger signal to deploy defenses against pathogens, uncovering an immune surveillance program in the intestinal epithelium.

The correlated expression of immune and energy metabolism related genes in the response to Salmonella enterica serovar Enteritidis inoculation in chicken.

BACKGROUND: Salmonella enterica serovar Enteritidis (SE) is one of the food-borne pathogenic bacteria, which affects poultry production and poses severe threat to human health. The correlation of immune system and metabolism in chicken after SE inoculation is important but not clear. In the current study, we identified the expression of immune and energy metabolism related genes using quantitative PCR to evaluate the correlation between immune system and energy metabolism against SE inoculation in Jining Bairi chicken. RESULTS: ATP5G1, ATP5G3 and ND2 were significantly up-regulated at 1 dpi (day post inoculation), and ATP5E, ATP5G1, ATP5G3 were significantly down-regulated at 7 dpi (P < 0.05). IL-8 and IL-1ß were significantly down-regulated at 1 dpi, IL-8 and IL-18 were significantly down-regulated at 3 dpi, IL-8 and BCL10 were significantly up-regulated at 7 dpi (P < 0.05). CONCLUSIONS: These findings indicate that the correlation between immune and energy metabolism related genes gradually change with time points post SE inoculation, from one homeostasis to an opposite homeostasis with 3 dpi as a turning point. These results will pave the foundation for the relationship between immune system and energy metabolism in the response to SE inoculation in chicken.

The HOIL-1L ligase modulates immune signalling and cell death via monoubiquitination of LUBAC.

The linear ubiquitin chain assembly complex (LUBAC), which consists of HOIP, SHARPIN and HOIL-1L, promotes NF-κB activation and protects against cell death by generating linear ubiquitin chains. LUBAC contains two RING-IBR-RING (RBR) ubiquitin ligases (E3), and the HOIP RBR is responsible for catalysing linear ubiquitination. We found that HOIL-1L RBR plays a crucial role in regulating LUBAC. HOIL-1L RBR conjugates monoubiquitin onto all LUBAC subunits, followed by HOIP-mediated conjugation of linear chains onto monoubiquitin, and these linear chains attenuate the functions of LUBAC. The introduction of E3-defective HOIL-1L mutants into cells augmented linear ubiquitination, which protected the cells against Salmonella infection and cured dermatitis caused by reduced LUBAC levels due to SHARPIN loss. Our results reveal a regulatory mode of E3 ligases in which the accessory E3 in LUBAC downregulates the main E3 by providing preferred substrates for autolinear ubiquitination. Thus, inhibition of HOIL-1L E3 represents a promising strategy for treating severe infections or immunodeficiency.

Epithelial transport in digestive diseases: mice, monolayers, and mechanisms.

The transport of electrolytes and fluid by the intestinal epithelium is critical in health to maintain appropriate levels of fluidity of the intestinal contents. The transport mechanisms that underlie this physiological process are also subject to derangement in various digestive disease states, such as diarrheal illnesses. This article summarizes the 2019 Hans Ussing Lecture of the Epithelial Transport Group of the American Physiological Society and discusses some pathways by which intestinal transport is dysregulated, particularly in the setting of infection with the diarrheal pathogen, Salmonella, and in patients treated with small-molecule inhibitors of the tyrosine kinase activity of the epidermal growth factor receptor (EGFr-TKI). The burdensome diarrhea in patients infected with Salmonella may be attributable to decreased expression of the chloride-bicarbonate exchanger downregulated in adenoma (DRA) that participates in electroneutral NaCl absorption. This outcome is possibly secondary to increased epithelial proliferation and/or decreased epithelial differentiation that occurs following infection. Conversely, the diarrheal side effects of cancer treatment with EGFr-TKI may be related to the known ability of EGFr-associated signaling to reduce calcium-dependent chloride secretion. Overall, the findings described may suggest targets for therapeutic intervention in a variety of diarrheal disease states.

NLRC4 inflammasome activation is NLRP3- and phosphorylation-independent during infection and does not protect from melanoma.

The NAIP/NLRC4 inflammasome is a cytosolic sensor of bacteria that activates caspase-1 and initiates potent immune responses. Structural, biochemical, and genetic data demonstrate that NAIP proteins are receptors for bacterial ligands, while NLRC4 is a downstream adaptor that multimerizes with NAIPs to form an inflammasome. NLRC4 has also been proposed to suppress tumor growth, though the underlying mechanism is unknown. Further, NLRC4 is phosphorylated on serine 533, which was suggested to be critical for its function. In the absence of S533 phosphorylation, it was proposed that another inflammasome protein, NLRP3, can induce NLRC4 activation. We generated a new Nlrc4-deficient mouse line and mice with S533D phosphomimetic or S533A nonphosphorylatable NLRC4. Using these models in vivo and in vitro, we fail to observe a requirement for phosphorylation in NLRC4 inflammasome function. Furthermore, we find no role for NLRP3 in NLRC4 function, or for NLRC4 in a model of melanoma. These results clarify our understanding of the mechanism and biological functions of NAIP/NLRC4 activation.

Integrative analysis of gut microbiome and metabolites revealed novel mechanisms of intestinal Salmonella carriage in chicken.

Intestinal carriage of Salmonella Enteritidis (SE) in the chicken host serves as a reservoir for transmission of Salmonella to humans through the consumption of poultry products. The aim of the current study was to examine the three-way interaction that occurred between host metabolites, resident gut microbiota and Salmonella following inoculation of SE in two-week-old layer chicks. Our results revealed an overall alteration in gut microbiome and metabolites in association with SE infection. Enriched colonization by different microbial members throughout the course of experimental infection highlighted significant fluctuation in the intestinal microbial community in response to Salmonella infection. As changes in community membership occurred, there was also subsequent impact on differential regulation of interlinked predicted functional activities within the intestinal environment dictated by Salmonella-commensal interaction. Alteration in the overall microbial community following infection also has a ripple effect on the host regulation of cecum-associated metabolic networks. The findings showed that there was differential regulation in many of the metabolites in association with SE colonization in chickens. Perturbation in metabolic pathways related to arginine and proline metabolism as well as TCA cycle was most prominently detected. Taken together, the present findings provided a starting point in understanding the effect of intestinal Salmonella carriage on the microbiome and metabolome of developing young layer chicks.

Lactobacillus johnsonii L531 ameliorates enteritis via elimination of damaged mitochondria and suppression of SQSTM1-dependent mitophagy in a Salmonella infantis model of piglet diarrhea.

Newly weaned piglets challenged with Salmonella infantis were particularly susceptible, whereas oral preadministration of Lactobacillus johnsonii L531 alleviated enteritis and promoted intestinal secretory IgA production. Salmonella infantis-induced activation of NLRC4 and NLRP3 inflammasomes and (nuclear factor kappa B) NF-κB signaling in the small intestine was also inhibited by L. johnsonii L531 pretreatment, thus limiting inflammation. An IPEC-J2 cell model of S. infantis infection yielded similar results. Salmonella infantis infection also resulted in mitochondrial damage and impaired mitophagy in the ileum and IPEC-J2 cells, as demonstrated by immunofluorescence colocalization of mitochondria with microtubule-binding protein light chain 3 (LC3) and high expression of autophagy-related proteins PTEN-induced putative kinase 1 (PINK1), sequestosome 1 (SQSTM1/p62), optineurin (OPTN), and LC3 by Western blotting analysis. However, L. johnsonii L531 pretreatment reduced both the extent of mitochondrial damage and autophagy-related protein expression. Our findings suggest that the amelioration of S. infantis-associated enteritis by L. johnsonii L531 is associated with regulation of NLRC4 and NLRP3 inflammasomes and NF-κB signaling pathway activation and suppression of mitochondrial damage. Amelioration of impaired mitophagy by L. johnsonii L531 could involve eliminating damaged mitochondria and regulating S. infantis-induced activation of the NF-κB-SQSTM1mitophagy signaling pathway in host cells to prevent the further mitochondrial damage and S. infantis dissemination.

The Systemic Zinc Homeostasis Was Modulated in Broilers Challenged by Salmonella.

Salmonella challenge leads to systemic responses that induce the hypozincaemia in mice, which is considered a vital strategy against Salmonella invasion. However, it is not yet known if this phenomenon occurs in broilers. To investigate the change in zinc homeostasis of broilers against Salmonella challenge, 1-day-old male broilers were fed with the basal diet for 7 days. Afterwards, broilers were orally inoculated with either 0 or 0.5 × 108 CFU Salmonella Typhimurium (ST). The serum and selected tissues of Salmonella-challenged and non-challenged broilers were collected at 1, 3 and 7 days post-challenge for zinc homeostasis analysis. Our results showed that Salmonella challenge results in hypozincaemia (serum zinc decrease and liver zinc increase) via modulating the systemic zinc homeostasis of broilers. A profound, zinc transporter-mediated zinc absorption and redistribution affecting zinc homeostasis provided a mechanistic explanation for this phenomenon. In addition, we found that the zinc importers Zip5, Zip10, Zip11, Zip12, Zip13 and Zip14 were mainly downregulated in Salmonella-challenged broilers to reduce zinc absorption in the duodenum, while the Zip14 mRNA expression was upregulated to redistribute zinc into the liver. Collectively, these findings reveal that broilers counteract Salmonella infection via modulating their systemic zinc homeostasis.

Host resistance factor SLC11A1 restricts Salmonella growth through magnesium deprivation.

The pleiotropic host resistance factor SLC11A1 (NRAMP1) defends against diverse intracellular pathogens in mammals by yet-unknown mechanisms. We compared Salmonella infection of coisogenic mice with different SLC11A1 alleles. SLC11A1 reduced Salmonella replication and triggered up-regulation of uptake systems for divalent metal cations but no other stress responses. SLC11A1 modestly diminished iron availability and acutely restricted Salmonella access to magnesium. Growth of Salmonella cells in the presence of SLC11A1 was highly heterogeneous and inversely correlated with expression of the crucial magnesium transporter gene mgtB We observed superimposable single-cell patterns in mice lacking SLC11A1 when we restricted Salmonella access to magnesium by impairing its uptake. Together, these findings identify deprivation of the main group metal magnesium as the main resistance mechanism of SLC11A1 against Salmonella.

Salmonella Coiled-Coil- and TIR-Containing TcpS Evades the Innate Immune System and Subdues Inflammation.

Toll-like receptors (TLRs) activate innate immunity via interactions between their Toll/interleukin-1 (IL-1) receptor (TIR) domain and downstream adaptor proteins. Here we report that Salmonella Enteritidis produces a secreted protein (TcpS) that contains both a TIR domain and a coiled-coil domain. TcpS blocks MyD88- and TRIF-mediated TLR signaling, inhibits inflammatory responses, and promotes bacterial survival. Early-stage immune evasion by TcpS results in severe tissue damage in the late stage of infection and contributes to Salmonella virulence. TcpS-derived peptides inhibit nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) activation and reduce lipopolysaccharide (LPS)-elicited systemic inflammation. Therapeutic peptide administration alleviates weight loss of mice infected with H1N1 influenza. Importantly, maximal TcpS-mediated TLR inhibition requires the critical TIR-TcpS residues Y191 and I284, as well as TcpS homodimerization via its N-terminal coiled-coil domain. Our study unveils a mechanism in which TcpS suppresses innate immunity via both its homodimerization and interaction with MyD88. TcpS is also a potential therapeutic agent for inflammation-associated diseases.

Inflammatory monocytes provide a niche for Salmonella expansion in the lumen of the inflamed intestine.

Salmonella exploit host-derived nitrate for growth in the lumen of the inflamed intestine. The generation of host-derived nitrate is dependent on Nos2, which encodes inducible nitric oxide synthase (iNOS), an enzyme that catalyzes nitric oxide (NO) production. However, the cellular sources of iNOS and, therefore, NO-derived nitrate used by Salmonella for growth in the lumen of the inflamed intestine remain unidentified. Here, we show that iNOS-producing inflammatory monocytes infiltrate ceca of mice infected with Salmonella. In addition, we show that inactivation of type-three secretion system (T3SS)-1 and T3SS-2 renders Salmonella unable to induce CC- chemokine receptor-2- and CC-chemokine ligand-2-dependent inflammatory monocyte recruitment. Furthermore, we show that the severity of the pathology of Salmonella- induced colitis as well as the nitrate-dependent growth of Salmonella in the lumen of the inflamed intestine are reduced in mice that lack Ccr2 and, therefore, inflammatory monocytes in the tissues. Thus, inflammatory monocytes provide a niche for Salmonella expansion in the lumen of the inflamed intestine.

Std fimbriae-fucose interaction increases Salmonella-induced intestinal inflammation and prolongs colonization.

Expression of ABO and Lewis histo-blood group antigens by the gastrointestinal epithelium is governed by an α-1,2-fucosyltransferase enzyme encoded by the Fut2 gene. Alterations in mucin glycosylation have been associated with susceptibility to various bacterial and viral infections. Salmonella enterica serovar Typhimurium is a food-borne pathogen and a major cause of gastroenteritis. In order to determine the role of Fut2-dependent glycans in Salmonella-triggered intestinal inflammation, Fut2+/+ and Fut2-/- mice were orally infected with S. Typhimurium and bacterial colonization and intestinal inflammation were analyzed. Bacterial load in the intestine of Fut2-/- mice was significantly lower compared to Fut2+/+ mice. Analysis of histopathological changes revealed significantly lower levels of intestinal inflammation in Fut2-/- mice compared to Fut2+/+ mice and measurement of lipocalin-2 level in feces corroborated histopathological findings. Salmonella express fimbriae that assist in adherence of bacteria to host cells thereby facilitating their invasion. The std fimbrial operon of S. Typhimurium encodes the π-class Std fimbriae which bind terminal α(1,2)-fucose residues. An isogenic mutant of S. Typhimurium lacking Std fimbriae colonized Fut2+/+ and Fut2-/- mice to similar levels and resulted in similar intestinal inflammation. In vitro adhesion assays revealed that bacteria possessing Std fimbriae adhered significantly more to fucosylated cell lines or primary epithelial cells in comparison to cells lacking α(1,2)-fucose. Overall, these results indicate that Salmonella-triggered intestinal inflammation and colonization are dependent on Std-fucose interaction.

Persistent Salmonella enterica Serovar Typhimurium Infection Induces Protease Expression During Intestinal Fibrosis.

BACKGROUND: Intestinal fibrosis is a common and serious complication of Crohn's disease characterized by the accumulation of fibroblasts, deposition of extracellular matrix, and formation of scar tissue. Although many factors including cytokines and proteases contribute to the development of intestinal fibrosis, the initiating mechanisms and the complex interplay between these factors remain unclear. METHODS: Chronic infection of mice with Salmonella enterica serovar Typhimurium was used to induce intestinal fibrosis. A murine protease-specific CLIP-CHIP microarray analysis was employed to assess regulation of proteases and protease inhibitors. To confirm up- or downregulation during fibrosis, we performed quantitative real-time polymerase chain reaction (PCR) and immunohistochemical stainings in mouse tissue and tissue from patients with inflammatory bowel disease. In vitro infections were used to demonstrate a direct effect of bacterial infection in the regulation of proteases. RESULTS: Mice develop severe and persistent intestinal fibrosis upon chronic infection with Salmonella enterica serovar Typhimurium, mimicking the pathology of human disease. Microarray analyses revealed 56 up- and 40 downregulated proteases and protease inhibitors in fibrotic cecal tissue. Various matrix metalloproteases, serine proteases, cysteine proteases, and protease inhibitors were regulated in the fibrotic tissue, 22 of which were confirmed by quantitative real-time PCR. Proteases demonstrated site-specific staining patterns in intestinal fibrotic tissue from mice and in tissue from human inflammatory bowel disease patients. Finally, we show in vitro that Salmonella infection directly induces protease expression in macrophages and epithelial cells but not in fibroblasts. CONCLUSIONS: In summary, we show that chronic Salmonella infection regulates proteases and protease inhibitors during tissue fibrosis in vivo and in vitro, and therefore this model is well suited to investigating the role of proteases in intestinal fibrosis.