Invasion and intracellular replication of Salmonella Typhimurium in epithelial cells

National Institute Of Allergy And Infectious Diseases

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Abstract

Salmonella enterica serovar Typhimurium (Salmonella) is a common cause of enterocolitis in humans. Transmission to humans is primarily via ingestion of contaminated food or water. Once ingested the bacterium survives the acidic environment of the stomach and colonizes the intestines, a process that is dependent on several virulence factors, particularly the Type III Secretion System-1 (T3SS1) and flagellar-based motility. Motility is required for the bacteria to reach the luminal surface of the intestinal epithelium where invasion of intestinal epithelial cells is mediated by the T3SS1, which translocates bacterial effector proteins across the apical plasma membrane and into the cytosol. Once within epithelial cells most of the Salmonella reside within a modified phagosome, known as the Salmonella-Containing vacuole, where they replicate moderately. However, a significant sub-population of intracellular bacteria escape into the cytosol where they replicate rapidly, inducing cell death and extrusion of the host cell. Extrusion effectively limits systemic spread of Salmonella instead expelling the bacteria back into the gut lumen. Work from our lab and others have shown that the cytosolic and vacuolar populations of intracellular Salmonella are transcriptionally distinct. Vacuolar bacteria express genes required for adaption to the intracellular environment, particularly those encoding a second T3SS (T3SS2) that is required for maturation of the SCV. In contrast, cytosolic bacteria continue to express genes encoding the structural components and effector proteins of T2SS1 as well as chemotaxis and flagellar proteins. Thus, when released back into the gut lumen, these cytosolic bacteria are effectively primed to invade epithelial cells and thus ultimately contribute to persistence and fecal shedding levels. As a fecal-oral pathogen Salmonella transmission is depending on fecal shedding with the highest shedders, so-called super-shedders, contributing disproportionally. Studying the basic biology of how Salmonella interacts with the intestinal epithelium is essential, not only to develop our understanding of clinical Salmonellosis, but also to develop ways of limiting food contamination and transmission humans. Here we are studying regulation of T3SS1, the role of motility in invasion of intestinal epithelial cells and how the invasion of intestinal epithelial cells contributes to persistence and shedding. 1) Flagellar mediated motility is essential for pathogenesis but how it is coordinated with T3SS1-mediated invasion is not well understood. We have identified two methyl-accepting chemotaxis proteins (MCPs), McpA and McpC, that are co-expressed with the T3SS1 and showed that McpC promotes invasion of intestinal epithelial cells by promoting smooth swimming. Mutants lacking either of these MCPs are outcompeted by wild type Salmonella Typhimurium in the lumen of the inflamed mouse intestine. Unlike McpC, McpA is a cytosolic receptor and contains a chemoreceptor zinc-binding (CZB) domain, a conserved motif that is found in a variety of bacterial signal transduction proteins, including chemoreceptors, and may be involved in sensing inflammation. We found that zinc-binding via the CZB domain is essential for the ability of McpA to complement an mcpA deletion mutant. Altogether this work is revealing how important the coordination of invasion and motility is during the complex process of gut colonization. 2) In humans, Salmonella Typhimurium causes inflammatory diarrhea characterized by neutrophil infiltration. The pro-inflammatory cytokine il-8 plays a critical role in the recruitment of neutrophils from the blood stream into the gut tissue and lumen. Since mice do not have il-8 we are using the well-characterized HeLa cell infection model to study how Salmonella Typhimurium induces il-8 expression and secretion in human epithelial cells. 3) Following T3SS1-mediated invasion of epithelial cells Salmonella Typhimurium can replicate within the Salmonella Containing Vacuole (SCV) and within the cytosol, bacterial growth rates and transcriptional status within these two intracellular niches are quite different. Oral infection of naturally resistant mice (e.g. 129X1/SvJ) with Salmonella Typhimurium strain SL1344 leads to chronic infection with a small percentage of animals shedding high levels of Salmonella in their feces (super-shedders). We are now using this model to investigate the factors involved in shedding and persistence and the role played by intestinal epithelial cells. Infection with two other well characterized lab strains of Salmonella (LT2 and 14028s) also results in chronic infection, with no clinical disease, however the levels of fecal shedding are quite different. In LT2-infected mice super-shedders are rare, whereas in 14028s-infected mice almost all mice become super shedders. We are now analyzing the response of the innate immune system to each of these strains. In other recently completed studies, we have found that pre-infection sub-clinical intestinal inflammation is predictive for super-shedder development in mice. 4) We are investigating the ability of synthetic antimicrobial peptides to inhibit invasion of epithelial cells by Salmonella at sublethal concentrations.