Listeria monocytogenes and Shigella flexneri are human intestinal pathogens that replicate in the cytosol of infected cells and spread directly from primary infected cells to adjacent cells. The dissemination process is a fundamental aspect of pathogenesis as spreading-defective bacterial mutants are essentially avirulent. The ability of L. monocytogenes and S. flexneri to spread from cell to cell is related to their ability to display actin- based motility in the cytosol of infected cells. These bacteria produce virulence factors that lead to the recruitment at their surface of an essential host cell actin nucleator, the ARP2/3 complex. This results in actin polymerization at one pole of the bacteria, which propels the rods throughout the cytosol. Seminal electron microscopy studies revealed that, when bacteria reach the cell cortex, they form plasma membrane extensions that project into the cytosol of adjacent cells. These protrusions are resolved in adjacent cells into double membrane vacuoles, from which the pathogens escape by producing virulence factors that disrupt the integrity of eukaryotic membranes. In contrast to our advanced understanding of the molecular mechanisms supporting cytosolic actin-based motility, the mechanisms supporting pathogen dissemination through membrane protrusion formation are unresolved. To address this gap in knowledge, we have developed innovative procedures for imaging intracellular pathogen dissemination and identified cellular and bacterial factors supporting bacterial pathogen spread from cell to cell. In previous work, we defined the cellular machinery and the bacterial factors specifically required for L. monocytogenes spread from cell to cell. Recently, we uncovered the notion that, although utilizing similar mechanisms of actin-based motility in the cytosol, L. monocytogenes and S. flexneri have evolved different strategies of cell-to-cell spread. Unlike L. monocytogenes, S. flexneri hijacks phosphoinositide (PI(3)P) signaling in protrusions in order to facilitate their resolution into vacuoles. Here, we propose to gain the first mechanistic insight into the bacterial and cellular mechanisms supporting S. flexneri PI(3)P-dependent dissemination (Aim1 and Aim2); and the role of PI(3)P-dependent dissemination in pathogenesis (Aim3).
Various intracellular pathogens have evolved the ability to manipulate host cell processes in order to disseminate from infected cells to adjacent cells. In this proposal, we present our plans to gain the first mechanistic insight into the bacterial and cellular factors supporting pathogen dissemination through cell-to- cell spread; and impact of the dissemination process on pathogenesis. The proposed approach will contribute to our general understanding of the mechanisms underlying microbial pathogenesis and may constitute the foundation for the rational design of preventive and therapeutic interventions.
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