Defining the Intracellular Growth Niche of Foodborne Listeria Monocytogenes

Ingestion of Listeria monocytogenes (Lm)-contaminated food results in human disease ranging in severity from mild, self-limiting gastroenteritis to life-threatening septicemia and meningoencephalitis. The specific factors that influence disease severity are not well understood, and our knowledge of the intestinal phase of listeriosis, in particular, is severely limited. We recently developed a mouse model of foodborne infection to study the interaction of Lm with intestinal innate immune cells. In preliminary studies, we found that the majority of Lm in the gut were extracellular. This was an unexpected result, because the intracellular growth and spread from cell-to-cell without encountering the extracellular milieu is generally regarded as the primary virulence strategy for these facultative intracellular bacteria. LplA1-deficient Lm that were unable to replicate intracellularly could readily invade the gut mucosa and establish infection in the underlying lamina propria, but did not persist as well as wildtype Lm, and by three days post-infection had a severe defect in spreading to the mesenteric lymph nodes (MLN). This suggests that intracellular growth is not required for the initial stages of intestinal infection, but replication in some as-yet-unidentified cell type in the gut becomes increasingly more important as the infection proceeds. Multicolor flow cytometry can discriminate ten different subsets of myeloid-derived phagocytes that are unique to the gut. Preliminary data provided here verified that intestinal tissue contained at least one cell type that could support intracellular growth of Lm, and ruled out Ly6Chi monocytes and all four subsets of conventional dendritic cells as the intracellular niche. The primary goal of this proposal is to identify the cell type(s) in the gut that support intracellular growth of Lm and to define the innate immune response of intestinal cell types that that interact primarily with either intracellular or extracellular Lm. We hypothesize that exponential growth inside a myeloid-derived phagocyte subset that localizes to the lamina propria of the colon is needed to increase the number of Lm above a critical threshold that allows for dissemination to the MLN. In Aim 1, five candidate intestinal myeloid cell types will be sort purified, infected directly ex vivo and assayed for both intracellular localization and replication. In Aim 2, we will define the initial response of all ten subsets of intestinal myeloid cells by measuring the early cytokines and other immune mediators produced in response to Lm. In Aim 3, flow cytometry will be used to identify Lm-associated cells in the lamina propria and submucosa of the ileum and colon as well as the MLN that drain each of these tissues (sMLN and cMLN) to track the fate of Lm that invade the gut mucosa over the course of infection in mice. These exploratory studies will fill a key knowledge gap in the field by defining the early events that occur in the gut during foodborne transmission of Lm.