Yanet Valdez

Toll-like receptor 2 plays a critical role in maintaining mucosal integrity during Citrobacter rodentium-induced colitis.

Inflammatory bowel diseases and infectious gastroenteritis likely occur when the integrity of intestinal barriers is disrupted allowing luminal bacterial products to cross into the intestinal mucosa, stimulating immune cells and triggering inflammation. While specific Toll‐like receptors (TLR) are involved in the generation of inflammatory responses against enteric bacteria, their contributions to the maintenance of intestinal mucosal integrity are less clear. These studies investigated the role of TLR2 in a model of murine colitis induced by the bacterial pathogen Citrobacter rodentium. C. rodentium supernatants specifically activated TLR2 in vitro while infected TLR2–/– mice suffered a lethal colitis coincident with colonic mucosal ulcerations, bleeding and increased cell death but not increased pathogen burden. TLR2–/– mice suffered impaired epithelial barrier function mediated via zonula occludens (ZO)‐1 in naïve mice and claudin‐3 in infected mice, suggesting this could underlie their susceptibility. TLR2 deficiency was also associated with impaired production of IL‐6 by bone marrow‐derived macrophages and infected colons cultured ex vivo. As IL‐6 has antiapoptotic and epithelial repair capabilities, its reduced expression could contribute to the impaired mucosal integrity. These studies report for the first time that TLR2 plays a critical role in maintaining intestinal mucosal integrity during infection by a bacterial pathogen.

Expression and Secretion of Salmonella Pathogenicity Island-2 Virulence Genes in Response to Acidification Exhibit Differential Requirements of a Functional Type III Secretion Apparatus and SsaL

Salmonella pathogenicity island (SPI)-2 is pivotal to the intracellular survival of Salmonella and for virulence in mammals. SPI-2 encodes virulence factors (called effectors) that are translocated into the host cell, a type III secretion apparatus and a two-component regulatory system that regulates intracellular expression of SPI-2. Salmonella SPI-2 secretion activity appears to be induced in response to acidification of the vacuole in which it replicates. Here we show that the expression of the SPI-2 proteins, SseB and SseD (filament and pore forming components of the secretion apparatus, respectively) in response to acidification requires an intact secretion system and SsaL, a Salmonella homologue of SepL, a regulator required for type III-dependent secretion of translocators but not effectors in attaching and effacing gastrointestinal pathogens. We show that the expression of SPI-2-encoded effectors is acid-regulated but can be uncoupled from the expression of filament and translocon components, thus showing a differential requirement of SsaL for expression. The secretion and translocation of SPI-2-encoded effectors requires SsaL, but SsaL is dispensable for the secretion of SPI-2 effectors encoded in other pathogenicity loci, suggesting a secretion regulation function for SsaL. Further, we demonstrate that the differential expression of adjacent genes within the sseA operon (sseD and sseE) occurs at the transcriptional level. These data indicate that a Salmonella SPI-2 activation state is achieved by an acidregulated response that requires SsaL. These data also suggest the existence of a previously unrecognized regulatory element within SPI-2 for the “effector operon” region downstream of sseD that might demarcate the expression of translocators and effectors.

Salmonella Pathogenicity Island 2 Is Expressed Prior to Penetrating the Intestine

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that causes disease in mice that resembles human typhoid. Typhoid pathogenesis consists of distinct phases in the intestine and a subsequent systemic phase in which bacteria replicate in macrophages of the liver and spleen. The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI-2) is a major virulence factor contributing to the systemic phase of typhoid pathogenesis. Understanding how pathogens regulate virulence mechanisms in response to the environment, including different host tissues, is key to our understanding of pathogenesis. A recombinase-based in vivo expression technology system was developed to assess SPI-2 expression during murine typhoid. SPI-2 expression was detectable at very early times in bacteria that were resident in the lumen of the ileum and was independent of active bacterial invasion of the epithelium. We also provide direct evidence for the regulation of SPI-2 by the Salmonella transcription factors ompR and ssrB in vivo. Together these results demonstrate that SPI-2 expression precedes penetration of the intestinal epithelium. This induction of expression precedes any documented SPI-2-dependent phases of typhoid and may be involved in preparing Salmonella to successfully resist the antimicrobial environment encountered within macrophages.

Chronic Enteric Salmonella Infection in Mice Leads to Severe and Persistent Intestinal Fibrosis

BACKGROUND & AIMS Intestinal fibrosis and stricture formation are serious complications of Crohns disease, often requiring surgical intervention. Unfortunately, the mechanisms underlying intestinal fibrosis development are poorly understood, in part because of the lack of relevant animal models. Here, we present a novel murine model of severe and persistent intestinal fibrosis caused by chronic bacterial-induced colitis. METHODS Mice were treated with streptomycin 24 hours prior to oral infection with Salmonella enterica serovar Typhimurium. Tissues were analyzed for bacterial colonization and inflammation, and fibrosis was assessed by Massons trichrome staining and collagen quantification. Expression of the profibrotic cytokines transforming growth factor-beta1, connective tissue growth factor and insulin-like growth factor-I was determined, and the cell types present in fibrotic tissues were assessed by immunohistochemistry. RESULTS Infection led to chronic Salmonella colonization of the cecum and colon followed by edema, mucosal ulcerations, and severe transmural inflammation. This pathology was accompanied by significantly elevated expression of transforming growth factor-beta1, connective tissue growth factor, and insulin-like growth factor-I along with extensive type I collagen deposition in the cecal mucosa, submucosa, and muscularis mucosa of infected mice. Fibrosis was evident by 7 days postinfection, peaking at day 21 and still present at day 70. The fibrotic regions were found to be rich in fibroblasts and myofibroblasts. CONCLUSIONS These data demonstrate that chronic Salmonella infection of the murine gastrointestinal tract leads to severe tissue fibrosis. Because this model is highly reproducible and easy to perform, it provides great potential for investigating both host and bacterial contributions to intestinal fibrosis.

Molecular Mechanisms of Salmonella Virulence and Host Resistance

Salmonella species can cause typhoid fever and gastroenteritis in humans and pose a global threat to human health. In order to establish a successful infection, Salmonella utilize a large number of genes encoding a variety of virulence factors. Different animal models of infection have been used to better understand the mechanisms underlying each disease including cattle, rodents, and nematodes. To date, a number of different bacterial virulence factors have been identified using such animal models, most of which are secreted by two type three secretion systems (T3SS) encoded within Salmonella pathogenicity islands (SPI) 1 and 2. These proteins alter various host cell pathways, facilitating the invasion of epithelial cells during infection, as well as the survival and replication of Salmonella inside phagocytic cells. On the other hand, host genetics and resistance also play a role in the susceptibility to Salmonella infection. The natural resistance-associated macrophage protein 1 (Nramp1), for example, is critical for host defense, since mice lacking Nramp1 fail to control bacterial replication and succumb to low doses of S. Typhimurium. In this chapter, we analyze the different pathogen and host factors that play a role in the dynamic interaction between Salmonella and its host and their impact on disease.

Commensal-pathogen interactions in the intestinal tract

The intestinal microbiota are pivotal in determining the developmental, metabolic and immunological status of the mammalian host. However, the intestinal tract may also accommodate pathogenic organisms, including helminth parasites which are highly prevalent in most tropical countries. Both microbes and helminths must evade or manipulate the host immune system to reside in the intestinal environment, yet whether they influence each other’s persistence in the host remains unknown. We now show that abundance of Lactobacillus bacteria correlates positively with infection with the mouse intestinal nematode parasite, Heligmosomoides polygyrus, as well as with heightened regulatory T cell (Treg) and Th17 responses. Moreover, H. polygyrus raises Lactobacillus species abundance in the duodenum of C57BL/6 mice, which are highly susceptible to H. polygyrus infection, but not in BALB/c mice, which are relatively resistant. Sequencing of samples at the bacterial gyrB locus identified the principal Lactobacillus species as L. taiwanensis, a previously characterized rodent commensal. Experimental administration of L. taiwanensis to BALB/c mice elevates regulatory T cell frequencies and results in greater helminth establishment, demonstrating a causal relationship in which commensal bacteria promote infection with an intestinal parasite and implicating a bacterially-induced expansion of Tregs as a mechanism of greater helminth susceptibility. The discovery of this tripartite interaction between host, bacteria and parasite has important implications for both antibiotic and anthelmintic use in endemic human populations.

Nramp1 drives an accelerated inflammatory response during Salmonella-induced colitis in mice.

A recently developed model for enterocolitis in mice involves pre‐treatment with the antibiotic streptomycin prior to infection with Salmonella enterica serovar Typhimurium (S. Typhimurium). The contribution of Nramp1/Slc11a1 protein, a critical host defence mechanism against S. Typhimurium, to the development of inflammation in this model has not been studied. Here, we analysed the impact of Nramp1 expression on the early development of colitis using isogenic Nramp1+/+ and Nramp1−/− mice. We hypothesized that Nramp1 acts by rapidly inducing an inflammatory response in the gut mucosa creating an antibacterial environment and limiting spread of S. Typhimurium to systemic sites. We observed that Nramp1+/+ mice showed lower numbers of S. Typhimurium in the caecum compared with Nramp1−/− mice at all times analysed. Acute inflammation was much more pronounced in Nramp1+/+ mice 1 day after infection. The effect of Nramp1 on development of colitis was characterized by higher secretion of the pro‐inflammatory cytokines IFN‐γ, TNF‐α and MIP‐1α and a massive infiltration of neutrophils and macrophages, compared with Nramp1−/− animals. These data show that an early and rapid inflammatory response results in protection against pathological effects of S. Typhimurium infection in Nramp1+/+ mice.

Evasive maneuvers by secreted bacterial proteins to avoid innate immune responses.

To cause disease, bacterial pathogens must first breach physical barriers, such as the mucous membrane that lines organs, and then successfully replicate and disseminate while avoiding destruction by the immune system. Many bacterial pathogens accomplish this by secreting proteins into their host environment, which act to subvert or dampen the expanding immune response. Here, we discuss how bacterial pathogens use an arsenal of secreted virulence proteins to modify the outcome of innate immune activation by altering how the immune system recognizes microbial invaders.

Influence of the microbiota on vaccine effectiveness

Studies of the relationship between the microbiome and the development and function of the immune system are demonstrating novel concepts that could significantly alter the way we treat disease and promote wellness. Several diseases, including inflammatory bowel disease, allergy/asthma, and diabetes, are associated with changes in composition of the microbiome. Recent findings suggest novel complex mechanisms by which the microbiome impacts immune cell development and differentiation. A major implication of these findings is that the composition of microbiome may ultimately affect vaccine efficacy. We explore here the potential role of the microbiome in vaccine responses in the context of our growing understanding of the relationship between the gastrointestinal microbiota, resident immune cell populations, and systemic immunity.

SseK1 and SseK2 are novel translocated proteins of Salmonella enterica serovar Typhimurium

ABSTRACT Salmonella enterica is a gram-negative, facultative intracellular pathogen that causes disease symptoms ranging from gastroenteritis to typhoid fever. A key virulence strategy is the translocation of bacterial effector proteins into the host cell, mediated by the type III secretion systems (TTSSs) encoded in Salmonella pathogenicity island 1 (SPI-1) and SPI-2. In S. enterica serovar Typhimurium LT2, we identified the protein products of STM4157 and STM2137 as novel candidate secreted proteins by comparison to known secreted proteins from enterohemorrhagic Escherichia coli and Citrobacter rodentium. The STM4157 and STM2137 proteins, which we have designated SseK1 and SseK2, respectively, are 61% identical at the amino acid level and differ mainly in their N termini. Western analysis showed that in vitro accumulation and secretion of these proteins in serovar Typhimurium were affected by mutations in the two-component systems SsrA/B and PhoP/Q, which are key mediators of intracellular growth and survival. SPI-2 TTSS-dependent translocation of recombinant SseK1::Cya was evident at 9 h postinfection of epithelial cells, while translocation of SseK2::Cya was not detected until 21 h. Remarkably, the translocation signal for SseK1 was contained within the N-terminal 32 amino acids. Fractionation of infected epithelial cells revealed that following translocation SseK1 localizes to the host cytosol, which is unusual among the currently known Salmonella effectors. Phenotypic analysis of ΔsseK1, ΔsseK2, and ΔsseK1/ΔsseK2 mutants provided evidence for a role that was not critical during systemic infection. In summary, this work demonstrates that SseK1 and SseK2 are novel translocated proteins of serovar Typhimurium.