Systemic listeriosis is one of the most deadly foodborne bacterial diseases in North America, as evidencedby recent outbreaks caused by contaminated cantaloupes (2011; 21% mortality) and cold cuts (2008; 39%mortality). After Listeria monocytogenes (Lm) spread to the brain, it is difficult to eradicate the infection,and many patients die even while receiving aggressive antibiotic treatment. Two different types of braininfections are observed in humans: a diffuse meningitis/meningoencephalitis typically seen in the elderlyand other immune-compromised individuals; and a more focal infection localized in the brainstem(rhombencephalitis) that occurs in otherwise healthy adults. The central hypothesis of this proposal is thatmeningitis occurs via hematogenous spread, primarily due to a failure of the immune system to limitoverwhelming growth of Lm in peripheral tissues, while rhombencephalitis occurs when neurotropic strainsof Lm invade cranial nerves and spread to the brain by axonal migration. Progress in understanding braininfections has been limited by the lack of an appropriate small animal model to study the two proposedroutes of dissemination, and by the technical limitation that very few bacteria are needed to causesymptomatic disease in the brain. In Aim 1 of this proposal, bacterial colonization patterns in the brain(focal vs. diffuse) will be identified in mice given food contaminated with either neurotropic Lm (humanrhombencephalitis and sheep brain isolates) or non-neurotropic Lm (veterinary strains isolated from theliver). In Aim 2, an innovative approach will define the pathway used by neurovirulent Lm to disseminate tothe brain. Lm will be engineered to secrete Cre recombinase during intracellular growth, and then used toinfect mice with a ?floxed? GFP gene, allowing for permanent identification of all cells in the body thatharbored intracellular Lm during any point of the infection. A key strength of this proposal is theinterdisciplinary approach resulting from a collaboration between microbiologists in the D?Orazio lab withextensive expertise in manipulating Lm and studying host responses to infection, and neuroscientists inthe Gensel lab, who have the expertise to perform high resolution imaging of the nerves and brainstems ofmice. Working together, this group will advance the field by revealing how Lm disseminate to the brainfollowing natural foodborne transmission, and will develop a set of tools that may be used in future studiesto identify the features of neurotropic Lm strains that promote brain infection.