Listeria monocytogenes (Lm) are Gram-positive, facultative intracellular bacteria that cause two types of brain infections in humans: diffuse (meningitis/meningoencephalitis) and focal brainstem (rhombencephalitis). Meningitis occurs in the setting of immune compromise, likely due to the failure of the immune system to limit exponential growth of Lm in tissues such as spleen or liver. This leads to high titer bacteremia and either direct invasion of the blood-brain-barrier (BBB) or “stealth transport” of Lm associated with myeloid-derived cells that cross the BBB. In contrast, rhombencephalitis is an infection of a delayed nature that occurs in young, otherwise healthy immune competent people, which suggests that the infection is dependent on bacterially encoded neurovirulence factors. The central hypothesis of this proposal is that the virulence factors which define neurotropism during rhombencephalitis mediate critical early events in the intestines, rather than in the brain. Support for this hypothesis comes from our preliminary data showing that when the neurotropic Lm are injected intravenously (bypassing the gut phase of the infection), they do not disseminate to the brain. In addition, others showed that rhombencephalitis isolates did not have an increased ability to survive and replicate in bovine organotypic brain slices ex vivo relative to other Lm strains. Until recently, rhombencephalitis could be studied only in cows or sheep due to the lack of a small animal model of Lm brainstem infections. However, we recently showed that some clinical isolates of Lm (UKVDL9 and SD4000) could preferentially colonize the brainstems of mice late in the course of infection, without reaching high titer in the blood. Both of the neurotropic strains we reported belong to lineage III, the smallest and least characterized group of Lm isolates. The goal of this proposal is to define the factors that promote neurotropism by Lm UKVDL9 and SD4000 during the gut phase of listeriosis. We predict that neurotropic Lm could have an advantage in three different aspects of the intestinal infection; these form the three Specific Aims of the proposal. In Aim 1, we determine if the route used to cross the gut mucosa or inflammatory response induced promotes localization of neurotropic Lm strains in neural bundles in the intestinal submucosa or within the vagal crypt endings surrounding lymphoid cryptopatches in the gut. In Aim 2, we identify the novel Lm surface protein that mediates enhanced invasion of enteric glial cells, the cell type that surrounds nerves in the gut. In Aim 3, we test the hypothesis that a truncated actA allele found in neurotropic Lm promotes an altered type of intracellular movement that allows the bacteria to migrate along the entire length of an axon all the way to the brainstem. This work will move the listeriosis field forward by providing the first real insights into neurotropism during this life-threatening foodborne infection and could al...