IL-22-dependent responses and their role during Citrobacter rodentium infection.

The mouse pathogen Citrobacter rodentium is utilized as a model organism for studying infections caused by the human pathogens enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) and to elucidate mechanisms of mucosal immunity. In response to C. rodentium infection, innate lymphoid cells and T cells secrete interleukin (IL)-22, a cytokine that promotes mucosal barrier function. IL-22 plays a pivotal role in enabling mice to survive and recover from C. rodentium infection, although the exact mechanisms involved remain incompletely understood. Here, we investigated whether particular components of the host response downstream of IL-22 contribute to the cytokine's protective effects during C. rodentium infection. In line with previous research, mice lacking the IL-22 gene (Il22-/- mice) were highly susceptible to C. rodentium infection. To elucidate the role of specific antimicrobial proteins modulated by IL-22, we infected the following knockout mice: S100A9-/- (calprotectin), Lcn2-/- (lipocalin-2), Reg3b-/- (Reg3ß), Reg3g-/- (Reg3γ), and C3-/- (C3). All knockout mice tested displayed a considerable level of resistance to C. rodentium infection, and none phenocopied the lethality observed in Il22-/- mice. By investigating another arm of the IL-22 response, we observed that C. rodentium-infected Il22-/- mice exhibited an overall decrease in gene expression related to intestinal barrier integrity as well as significantly elevated colonic inflammation, gut permeability, and pathogen levels in the spleen. Taken together, these results indicate that host resistance to lethal C. rodentium infection may depend on multiple antimicrobial responses acting in concert, or that other IL-22-regulated processes, such as tissue repair and maintenance of epithelial integrity, play crucial roles in host defense to attaching and effacing pathogens.

Toll-like receptor and inflammasome signals converge to amplify the innate bactericidal capacity of T helper 1 cells.

T cell effector functions can be elicited by noncognate stimuli, but the mechanism and contribution of this pathway to the resolution of intracellular macrophage infections have not been defined. Here, we show that CD4(+) T helper 1 (Th1) cells could be rapidly stimulated by microbe-associated molecular patterns during active infection with Salmonella or Chlamydia. Further, maximal stimulation of Th1 cells by lipopolysaccharide (LPS) did not require T-cell-intrinsic expression of toll-like receptor 4 (TLR4), interleukin-1 receptor (IL-1R), or interferon-γ receptor (IFN-γR) but instead required IL-18R, IL-33R, and adaptor protein MyD88. Innate stimulation of Th1 cells also required host expression of TLR4 and inflammasome components that together increased serum concentrations of IL-18. Finally, the elimination of noncognate Th1 cell stimulation hindered the resolution of primary Salmonella infection. Thus, the in vivo bactericidal capacity of Th1 cells is regulated by the response to noncognate stimuli elicited by multiple innate immune receptors.

Microcins mediate competition among Enterobacteriaceae in the inflamed gut.

The Enterobacteriaceae are a family of Gram-negative bacteria that include commensal organisms as well as primary and opportunistic pathogens that are among the leading causes of morbidity and mortality worldwide. Although Enterobacteriaceae often comprise less than 1% of a healthy intestine's microbiota, some of these organisms can bloom in the inflamed gut; expansion of enterobacteria is a hallmark of microbial imbalance known as dysbiosis. Microcins are small secreted proteins that possess antimicrobial activity in vitro, but whose role in vivo has been unclear. Here we demonstrate that microcins enable the probiotic bacterium Escherichia coli Nissle 1917 (EcN) to limit the expansion of competing Enterobacteriaceae (including pathogens and pathobionts) during intestinal inflammation. Microcin-producing EcN limits the growth of competitors in the inflamed intestine, including commensal E. coli, adherent-invasive E. coli and the related pathogen Salmonella enterica. Moreover, only therapeutic administration of the wild-type, microcin-producing EcN to mice previously infected with S. enterica substantially reduced intestinal colonization by the pathogen. Our work provides the first evidence that microcins mediate inter- and intraspecies competition among the Enterobacteriaceae in the inflamed gut. Moreover, we show that microcins can act as narrow-spectrum therapeutics to inhibit enteric pathogens and reduce enterobacterial blooms.

Host-mediated sugar oxidation promotes post-antibiotic pathogen expansion.

Changes in the gut microbiota may underpin many human diseases, but the mechanisms that are responsible for altering microbial communities remain poorly understood. Antibiotic usage elevates the risk of contracting gastroenteritis caused by Salmonella enterica serovars, increases the duration for which patients shed the pathogen in their faeces, and may on occasion produce a bacteriologic and symptomatic relapse. These antibiotic-induced changes in the gut microbiota can be studied in mice, in which the disruption of a balanced microbial community by treatment with the antibiotic streptomycin leads to an expansion of S. enterica serovars in the large bowel. However, the mechanisms by which streptomycin treatment drives an expansion of S. enterica serovars are not fully resolved. Here we show that host-mediated oxidation of galactose and glucose promotes post-antibiotic expansion of S. enterica serovar Typhimurium (S. Typhimurium). By elevating expression of the gene encoding inducible nitric oxide synthase (iNOS) in the caecal mucosa, streptomycin treatment increased post-antibiotic availability of the oxidation products galactarate and glucarate in the murine caecum. S. Typhimurium used galactarate and glucarate within the gut lumen of streptomycin pre-treated mice, and genetic ablation of the respective catabolic pathways reduced S. Typhimurium competitiveness. Our results identify host-mediated oxidation of carbohydrates in the gut as a mechanism for post-antibiotic pathogen expansion.

CRTAM Shapes the Gut Microbiota and Enhances the Severity of Infection.

Gut lymphocytes and the microbiota establish a reciprocal relationship that impacts the host immune response. Class I-restricted T cell-associated molecule (CRTAM) is a cell adhesion molecule expressed by intraepithelial T cells and is required for their retention in the gut. In this study, we show that CRTAM expression affects gut microbiota composition under homeostatic conditions. Moreover, Crtam-/- mice infected with the intestinal pathogen Salmonella exhibit reduced Th17 responses, lower levels of inflammation, and reduced Salmonella burden, which is accompanied by expansion of other microbial taxa. Thus, CRTAM enhances susceptibility to Salmonella, likely by promoting the inflammatory response that promotes the pathogen's growth. We also found that the gut microbiota from wild-type mice, but not from Crtam-/- mice, induces CRTAM expression and Th17 responses in ex-germ-free mice during Salmonella infection. Our study demonstrates a reciprocal relationship between CRTAM expression and the gut microbiota, which ultimately impacts the host response to enteric pathogens.

Factors Required for Adhesion of Salmonella enterica Serovar Typhimurium to Corn Salad (Valerianella locusta).

Salmonella enterica is a foodborne pathogen often leading to gastroenteritis and is commonly acquired by consumption of contaminated food of animal origin. However, frequency of outbreaks linked to the consumption of fresh or minimally processed food of nonanimal origin is increasing. New infection routes of S. enterica by vegetables, fruits, nuts, and herbs have to be considered. This leads to special interest in S. enterica interactions with leafy products, e.g., salads, that are mainly consumed in a minimally processed form. The attachment of S. enterica to salad is a crucial step in contamination, but little is known about the bacterial factors required and mechanisms of adhesion. S. enterica possesses a complex set of adhesive structures whose functions are only partly understood. Potentially, S. enterica may deploy multiple adhesive strategies for adhering to various salad species and other vegetables. In this study, we systematically analyzed the contributions of the complete adhesiome, of lipopolysaccharide (LPS), and of flagellum-mediated motility of S. enterica serovar Typhimurium (STM) in adhesion to Valerianella locusta (corn salad). We deployed a reductionist, synthetic approach to identify factors involved in the surface binding of STM to leaves of corn salad, with particular regard to the expression of all known adhesive structures, using the Tet-on system. This work reveals the contribution of Saf fimbriae, type 1 secretion system-secreted BapA, an intact LPS, and flagellum-mediated motility of STM in adhesion to corn salad leaves.IMPORTANCE Transmission of gastrointestinal pathogens by contaminated fresh produce is of increasing relevance to human health. However, the mechanisms of contamination of, persistence on, and transmission by fresh produce are poorly understood. We investigated the contributions of the various adhesive structures of STM to the initial event in transmission, i.e., binding to the plant surface. A reductionist system was used that allowed experimentally controlled surface expression of individual adhesive structures and analyses of the contribution to binding to leave surfaces of corn salad under laboratory conditions. The model system allowed the determination of the relative contributions of fimbrial and nonfimbrial adhesins, the type 3 secretion systems, the O antigen of lipopolysaccharide, the flagella, and chemotaxis of STM to binding to corn salad leaves. Based on these data, future work could reveal the mechanism of binding and the relevance of interaction under agricultural conditions.

Identification of IL-40, a Novel B Cell-Associated Cytokine.

We describe a novel B cell-associated cytokine, encoded by an uncharacterized gene (C17orf99; chromosome 17 open reading frame 99), that is expressed in bone marrow and fetal liver and whose expression is also induced in peripheral B cells upon activation. C17orf99 is only present in mammalian genomes, and it encodes a small (∼27-kDa) secreted protein unrelated to other cytokine families, suggesting a function in mammalian immune responses. Accordingly, C17orf99 expression is induced in the mammary gland upon the onset of lactation, and a C17orf99-/- mouse exhibits reduced levels of IgA in the serum, gut, feces, and lactating mammary gland. C17orf99-/- mice have smaller and fewer Peyer's patches and lower numbers of IgA-secreting cells. The microbiome of C17orf99-/- mice exhibits altered composition, likely a consequence of the reduced levels of IgA in the gut. Although naive B cells can express C17orf99 upon activation, their production increases following culture with various cytokines, including IL-4 and TGF-ß1, suggesting that differentiation can result in the expansion of C17orf99-producing B cells during some immune responses. Taken together, these observations indicate that C17orf99 encodes a novel B cell-associated cytokine, which we have called IL-40, that plays an important role in humoral immune responses and may also play a role in B cell development. Importantly, IL-40 is also expressed by human activated B cells and by several human B cell lymphomas. The latter observations suggest that it may play a role in the pathogenesis of certain human diseases.

Exploiting host immunity: the Salmonella paradigm.

Pathogens have evolved clever strategies to evade and in some cases exploit the attacks of an activated immune system. Salmonella enterica is one such pathogen, exploiting multiple aspects of host defense to promote its replication in the host. Here we review recent findings on the mechanisms by which Salmonella establishes systemic and chronic infection, including strategies involving manipulation of innate immune signaling and inflammatory forms of cell death, as well as immune evasion by establishing residency in M2 macrophages. We also examine recent evidence showing that the oxidative environment and the high levels of antimicrobial proteins produced in response to localized Salmonella gastrointestinal infection enable the pathogen to successfully outcompete the resident gut microbiota.

A novel CsrA titration mechanism regulates fimbrial gene expression in Salmonella typhimurium.

A hierarchical control of fimbrial gene expression limits laboratory grown cultures of Salmonella enterica serovar typhimurium (S. typhimurium) to the production of type I fimbriae encoded by the fimAICDHF operon. Here we show that an unlikely culprit, namely the 5'-untranslated region (5'-UTR) of a messenger (m)RNA, coordinated the regulation. Binding of CsrA to the 5'-UTR of the pefACDEF transcript was required for expression of plasmid-encoded fimbriae. The 5'-UTR of the fimAICDHF transcript cooperated with two small untranslated RNAs, termed CsrB and CsrC, in antagonizing the activity of the RNA binding protein CsrA. Through this post-transcriptional mechanism, the 5'-UTR of the fimAICDHF transcript prevented production of PefA, the major structural subunit of plasmid-encoded fimbriae. This regulatory mechanism limits the costly expression of plasmid-encoded fimbriae to host environments in a mouse model. Collectively, our data suggest that the 5'-UTR of an mRNA coordinates a hierarchical control of fimbrial gene expression in S. typhimurium.

Direct visualization of endogenous Salmonella-specific B cells reveals a marked delay in clonal expansion and germinal center development.

CD4(+) T cells and B cells are both essential for acquired immunity to Salmonella infection. It is well established that Salmonella inhibit host CD4(+) T-cell responses, but a corresponding inhibitory effect on B cells is less well defined. Here, we utilize an Ag tetramer and pull-down enrichment strategy to directly visualize OVA-specific B cells in mice, as they respond to infection with Salmonella-OVA. Surprisingly, OVA-specific B-cell expansion and germinal center formation was not detected until bacteria were cleared from the host. Furthermore, Salmonella infection also actively inhibited both B- and T-cell responses to the same coinjected Ag but this did not require the presence of iNOS. The Salmonella Pathogenicity Island 2 (SPI2) locus has been shown to be responsible for inhibition of Salmonella-specific CD4(+) T-cell responses, and an examination of SPI2-deficient bacteria demonstrated a recovery in B-cell expansion in infected mice. Together, these data suggest that Salmonella can simultaneously inhibit host B- and T-cell responses using SPI2-dependent mechanisms.

Salmonella uses energy taxis to benefit from intestinal inflammation.

Chemotaxis enhances the fitness of Salmonella enterica serotype Typhimurium (S. Typhimurium) during colitis. However, the chemotaxis receptors conferring this fitness advantage and their cognate signals generated during inflammation remain unknown. Here we identify respiratory electron acceptors that are generated in the intestinal lumen as by-products of the host inflammatory response as in vivo signals for methyl-accepting chemotaxis proteins (MCPs). Three MCPs, including Trg, Tsr and Aer, enhanced the fitness of S. Typhimurium in a mouse colitis model. Aer mediated chemotaxis towards electron acceptors (energy taxis) in vitro and required tetrathionate respiration to confer a fitness advantage in vivo. Tsr mediated energy taxis towards nitrate but not towards tetrathionate in vitro and required nitrate respiration to confer a fitness advantage in vivo. These data suggest that the energy taxis receptors Tsr and Aer respond to distinct in vivo signals to confer a fitness advantage upon S. Typhimurium during inflammation by enabling this facultative anaerobic pathogen to seek out favorable spatial niches containing host-derived electron acceptors that boost its luminal growth.

How bile acids and the microbiota interact to shape host immunity.

Bile acids are increasingly appearing in the spotlight owing to their novel impacts on various host processes. Similarly, there is growing attention on members of the microbiota that are responsible for bile acid modifications. With recent advances in technology enabling the discovery and continued identification of microbially conjugated bile acids, the chemical complexity of the bile acid landscape in the body is increasing at a rapid pace. In this Review, we summarize our current understanding of how bile acids and the gut microbiota interact to modulate immune responses during homeostasis and disease, with a particular focus on the gut.

CCL28 modulates neutrophil responses during infection with mucosal pathogens.

The chemokine CCL28 is highly expressed in mucosal tissues, but its role during infection is not well understood. Here, we show that CCL28 promotes neutrophil accumulation in the gut of mice infected with Salmonella and in the lung of mice infected with Acinetobacter. Neutrophils isolated from the infected mucosa expressed the CCL28 receptors CCR3 and, to a lesser extent, CCR10, on their surface. The functional consequences of CCL28 deficiency varied between the two infections: Ccl28-/- mice were highly susceptible to Salmonella gut infection but highly resistant to otherwise lethal Acinetobacter lung infection. In vitro, unstimulated neutrophils harbored pre-formed intracellular CCR3 that was rapidly mobilized to the cell surface following phagocytosis or inflammatory stimuli. Moreover, CCL28 stimulation enhanced neutrophil antimicrobial activity, production of reactive oxygen species, and formation of extracellular traps, all processes largely dependent on CCR3. Consistent with the different outcomes in the two infection models, neutrophil stimulation with CCL28 boosted the killing of Salmonella but not Acinetobacter. CCL28 thus plays a critical role in the immune response to mucosal pathogens by increasing neutrophil accumulation and activation, which can enhance pathogen clearance but also exacerbate disease depending on the mucosal site and the infectious agent.

Salmonella bongori provides insights into the evolution of the Salmonellae.

The genus Salmonella contains two species, S. bongori and S. enterica. Compared to the well-studied S. enterica there is a marked lack of information regarding the genetic makeup and diversity of S. bongori. S. bongori has been found predominantly associated with cold-blooded animals, but it can infect humans. To define the phylogeny of this species, and compare it to S. enterica, we have sequenced 28 isolates representing most of the known diversity of S. bongori. This cross-species analysis allowed us to confidently differentiate ancestral functions from those acquired following speciation, which include both metabolic and virulence-associated capacities. We show that, although S. bongori inherited a basic set of Salmonella common virulence functions, it has subsequently elaborated on this in a different direction to S. enterica. It is an established feature of S. enterica evolution that the acquisition of the type III secretion systems (T3SS-1 and T3SS-2) has been followed by the sequential acquisition of genes encoding secreted targets, termed effectors proteins. We show that this is also true of S. bongori, which has acquired an array of novel effector proteins (sboA-L). All but two of these effectors have no significant S. enterica homologues and instead are highly similar to those found in enteropathogenic Escherichia coli (EPEC). Remarkably, SboH is found to be a chimeric effector protein, encoded by a fusion of the T3SS-1 effector gene sopA and a gene highly similar to the EPEC effector nleH from enteropathogenic E. coli. We demonstrate that representatives of these new effectors are translocated and that SboH, similarly to NleH, blocks intrinsic apoptotic pathways while being targeted to the mitochondria by the SopA part of the fusion. This work suggests that S. bongori has inherited the ancestral Salmonella virulence gene set, but has adapted by incorporating virulence determinants that resemble those employed by EPEC.

Murine Models of Salmonella Infection.

The pathogen Salmonella enterica encompasses a range of bacterial serovars that cause intestinal inflammation and systemic infections in humans. Mice are a widely used infection model due to their relative simplicity and versatility. Here, we provide standardized protocols for culturing the prolific zoonotic pathogen S. enterica serovar Typhimurium for intragastric inoculation of mice to model colitis or systemic dissemination, along with techniques for direct extraintestinal infection. Furthermore, we present procedures for quantifying pathogen burden and for characterizing the immune response by analyzing tissue pathology, inflammatory markers, and immune cells from intestinal tissues. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Murine colitis model utilizing oral streptomycin pretreatment and oral S. Typhimurium administration Basic Protocol 2: Intraperitoneal injection of S. Typhimurium for modeling extraintestinal infection Support Protocol 1: Preparation of S. Typhimurium inoculum Support Protocol 2: Preparation of mixed S. Typhimurium inoculum for competitive infection Basic Protocol 3: Assessment of S. Typhimurium burden Support Protocol 3: Preservation and pathological assessment of S. Typhimurium-infected tissues Support Protocol 4: Measurement of inflammatory marker expression in intestinal tissues by qPCR Support Protocol 5: Preparation of intestinal content for inflammatory marker quantification by ELISA Support Protocol 6: Immune cell isolation from Salmonella-infected intestinal tissues.

Siderophore Immunization Restricted Colonization of Adherent-Invasive Escherichia coli and Ameliorated Experimental Colitis.

Inflammatory bowel diseases (IBD) are characterized by chronic inflammation of the gastrointestinal tract and profound alterations to the gut microbiome. Adherent-invasive Escherichia coli (AIEC) is a mucosa-associated pathobiont that colonizes the gut of patients with Crohn's disease, a form of IBD. Because AIEC exacerbates gut inflammation, strategies to reduce the AIEC bloom during colitis are highly desirable. To thrive in the inflamed gut, Enterobacteriaceae acquire the essential metal nutrient iron by producing and releasing siderophores. Here, we implemented an immunization-based strategy to target the siderophores enterobactin and its glucosylated derivative salmochelin to reduce the AIEC bloom in the inflamed gut. Using chemical (dextran sulfate sodium) and genetic (Il10-/- mice) IBD mouse models, we showed that immunization with enterobactin conjugated to the mucosal adjuvant cholera toxin subunit B potently elicited mucosal and serum antibodies against these siderophores. Siderophore-immunized mice exhibited lower AIEC gut colonization, diminished AIEC association with the gut mucosa, and reduced colitis severity. Moreover, Peyer's patches and the colonic lamina propria harbored enterobactin-specific B cells that could be identified by flow cytometry. The beneficial effect of siderophore immunization was primarily B cell-dependent because immunized muMT-/- mice, which lack mature B lymphocytes, were not protected during AIEC infection. Collectively, our study identified siderophores as a potential therapeutic target to reduce AIEC colonization and its association with the gut mucosa, which ultimately may reduce colitis exacerbation. Moreover, this work provides the foundation for developing monoclonal antibodies against siderophores, which could provide a narrow-spectrum strategy to target the AIEC bloom in Crohn's disease patients. IMPORTANCE Adherent-invasive Escherichia coli (AIEC) is abnormally prevalent in patients with ileal Crohn's disease and exacerbates intestinal inflammation, but treatment strategies that selectively target AIEC are unavailable. Iron is an essential micronutrient for most living organisms, and bacterial pathogens have evolved sophisticated strategies to capture iron from the host environment. AIEC produces siderophores, small, secreted molecules with a high affinity for iron. Here, we showed that immunization to elicit antibodies against siderophores promoted a reduction of the AIEC bloom, interfered with AIEC association with the mucosa, and mitigated colitis in experimental mouse models. We also established a flow cytometry-based approach to visualize and isolate siderophore-specific B cells, a prerequisite for engineering monoclonal antibodies against these molecules. Together, this work could lead to a more selective and antibiotic-sparing strategy to target AIEC in Crohn's disease patients.

Native mass spectrometry-based metabolomics identifies metal-binding compounds.

Although metals are essential for the molecular machineries of life, systematic methods for discovering metal-small molecule complexes from biological samples are limited. Here, we describe a two-step native electrospray ionization-mass spectrometry method, in which post-column pH adjustment and metal infusion are combined with ion identity molecular networking, a rule-based data analysis workflow. This method enabled the identification of metal-binding compounds in complex samples based on defined mass (m/z) offsets of ion species with the same chromatographic profiles. As this native electrospray metabolomics approach is suited to the use of any liquid chromatography-mass spectrometry system to explore the binding of any metal, this method has the potential to become an essential strategy for elucidating metal-binding molecules in biology.

Evolution of the chaperone/usher assembly pathway: fimbrial classification goes Greek.

Many Proteobacteria use the chaperone/usher pathway to assemble proteinaceous filaments on the bacterial surface. These filaments can curl into fimbrial or nonfimbrial surface structures (e.g., a capsule or spore coat). This article reviews the phylogeny of operons belonging to the chaperone/usher assembly class to explore the utility of establishing a scheme for subdividing them into clades of phylogenetically related gene clusters. Based on usher amino acid sequence comparisons, our analysis shows that the chaperone/usher assembly class is subdivided into six major phylogenetic clades, which we have termed alpha-, beta-, gamma-, kappa-, pi-, and sigma-fimbriae. Members of each clade share related operon structures and encode fimbrial subunits with similar protein domains. The proposed classification system offers a simple and convenient method for assigning newly discovered chaperone/usher systems to one of the six major phylogenetic groups.

The Vi capsular polysaccharide prevents complement receptor 3-mediated clearance of Salmonella enterica serotype Typhi.

Capsular polysaccharides are important virulence factors of invasive bacterial pathogens. Here we studied the role of the virulence (Vi) capsular polysaccharide of Salmonella enterica serotype Typhi (S. Typhi) in preventing innate immune recognition by complement. Comparison of capsulated S. Typhi with a noncapsulated mutant (ΔtviBCDE vexABCDE mutant) revealed that the Vi capsule interfered with complement component 3 (C3) deposition. Decreased complement fixation resulted in reduced bacterial binding to complement receptor 3 (CR3) on the surface of murine macrophages in vitro and decreased CR3-dependent clearance of Vi capsulated S. Typhi from the livers and spleens of mice. Opsonization of bacteria with immune serum prior to intraperitoneal infection increased clearance of capsulated S. Typhi from the liver. Our data suggest that the Vi capsule prevents CR3-dependent clearance, which can be overcome in part by a specific antibody response.