Dissemination of Intracellular and Extracellular Listeria from the Gut

Foodborne transmission of the facultative intracellular bacterial pathogen Listeria monocytogenes (Lm) results in human disease that ranges in severity from mild gastroenteritis to life-threatening septicemia or meningoencephalitis. The host and bacterial factors that determine disease severity are not well defined, and our understanding of the intestinal phase of listeriosis, in particular, is severely limited. To address this knowledge gap, we developed a mouse model of foodborne Lm infection that closely mimics all phases of the human disease. In preliminary studies, we found that the majority of Lm in the gut were extracellular, and that Lm was not able to replicate in many of the mononuclear phagocyte subsets present in the intestinal lamina propria. This was an unexpected result, because intracellular growth and spread from cell-to-cell without encountering the extracellular milieu are generally regarded as the primary virulence strategies for Lm. The overall hypothesis of this proposal is that foodborne Lm can use one of three distinct pathways to disseminate from the intestinal lamina propria to the mesenteric lymph nodes (MLN): 1) Lm “hitch a ride” attached to migratory inflammatory Ly6Chi monocytes which they cannot efficiently invade; 2) Lm invade CD103+ conventional dendritic cells (cDC) which serve as a “Trojan horse” to deliver non-replicating, but intracellular bacteria to the MLN; and 3) extracellular Lm traffic free-flowing through lymphatic vessels to the subcapsular sinus of the MLN. We propose that these three pathways are likely to be redundant during in vivo infection, but may be influenced by the route used by Lm to invade the intestinal epithelium. In Aim 1, the relative roles of monocytes and cDC in transporting Lm will be assessed by using mice lacking specific chemokine receptors to block intestinal migration and treatment with cellular differentiation factors to increase output of these cell types. In Aim 2, whole mount confocal microscopy will be used to visualize Lm within lymphatic vessels in the gut following either ligated loop or foodborne infection. In Aim 3, we will identify the cell types associated with Lm as they first reach the the subcapsular sinus of the MLN to verify the relative proportion of bacteria that spread using each of the three pathways. These exploratory studies will advance the field by defining the mechanisms used by Lm to disseminate from the gut in susceptible individuals, and will provide key insights needed to develop therapeutic approaches to block systemic spread of this invasive pathogen.