Mechanisms regulating vimentin-dependent invasion of the brain by Listeria monocytogenes

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Project Summary Listeria monocytogenes (Lm) is an intracellular bacterial pathogen capable of invading numerous host cell types. Lm infections can lead to severe disease in humans and most often affects immunocompromised individuals, pregnant women, and the elderly. Of particular concern is the ability of Lm to invade the central nervous system (CNS), leading to life-threatening meningitis and encephalitis. The identity of factors necessary to facilitate Lm brain infection has remained unclear. We have recently shown that a Lm surface protein, InlF, is required for successful colonization of the brain in mice. Moreover, we have determined that InlF binds vimentin, a cytosolic intermediate filament protein also present on the surface of brain endothelial cells. We hypothesize that InlF-vimentin interaction is required for Lm passage across the blood-brain barrier (BBB) to establish an infection in the brain. The focus of this proposal is to elucidate how the InlF-vimentin interaction mediates invasion of host cells in vitro and Lm infection of the brain in vivo. In Aim I, in vitro infection assays will be used to determine how InlF interacts with vimentin to mediate invasion of host cells. Confocal fluorescence microscopy and gentamicin protection assays will be used to directly examine the ability of InlF- expressing Lm to interact with brain microvascular endothelial cells. Biochemical approaches will be used to further characterize InlF-vimentin protein binding and identify the regions of vimentin involved in the InlF- vimentin protein-protein interaction. The mechanisms that regulate cell surface exposure of vimentin are unknown. We hypothesize that cellular membrane repair pathways facilitate redistribution of cytosolic vimentin to the surface of host cells. Cell biological studies will be performed to determine the role of membrane repair pathways for the localization of vimentin to the host cell surface. Additionally, potential vimentin co-receptor candidates previously identified by mass spectrometry will be examined to determine their role in InlF-mediated invasion of brain endothelial cells. In Aim II, the contribution of the InlF-vimentin interaction to Lm infection in vivo will be determined by infection studies in normal and vimentin knockout mice. In vitro infection assays and fluorescence microscopy will be performed with primary brain cells cultured from normal and knockout mice to determine if InlF mediates invasion of distinct primary brain cell types (astrocytes and neurons) and determine the importance of vimentin and potential co-receptors for infection. Gentamicin protection assays with primary mouse endothelial cells will also be performed to determine if InlF is required for invasion of primary cells that constitute the BBB. Finally, infection of mice with cell-to-cell spread-defective ΔactA or ΔactA/ΔinlF-derived Lm strains will determine the in vivo contribution of InlF for colonization of the brain by direct invasion vs. cell-to- cell spread. The proposed studies will provide insights into the protein-protein interactions and cellular mechanisms facilitating Lm invasion of the brain and may identify novel targets for preventing infections of the brain by Lm and other microbial pathogens.