Listeria monocytogenes is a food-borne, intracellular pathogen that causes gastroenteritis, abortions, or meningitis with a high mortality rate. Critical for Listeria virulence is its ability to replicate within mammalian cells and spread from one cell to another through an actin-dependent motility process that allows bacteria to remain within the protective environment of the host cytosol. Cell-cell spread of Listeria occurs in a variety of host cell types, including intestinal epithelial cells (enterocytes). Enterocytes are the first cell type infected by Listeria, and consequently play a key role in the onset of infection. Cell-cell spread is initiated by the bacterial surface protein ActA, which stimulates the formation of F-actin 'comet tails'that propel Listeria through the cytoplasm. Rocketing bacteria then contact the host cell plasma membrane and induce the formation of Listeria-containing protrusions that are engulfed by neighboring cells. ActA-mediated actin polymerization is well understood. However, little is known about the molecular mechanism of protrusion formation in enterocytes or other polarized epithelia. Specifically, it remains unclear whether bacterial proteins exist that function after ActA to directly control protrusion formation. Importantly, our preliminary data indicate that the secreted Listeria virulence protein InlC is needed for efficient protrusion formation and cell-cell spread in a polarized enterocyte cell line. InlC promotes bacterial protrusion generation by antagonizing the human adaptor protein Tuba. The precise mechanism by which InlC and Tuba control Listeria spreading remains unclear. As Tuba is a scaffolding protein, it likely affects spreading by interacting with one or more human ligands. The long-term goal of our work is to elucidate the molecular mechanism by which InlC promotes spreading of Listeria in polarized epithelial cells. The overall objective of this application is to identify human ligands of Tuba that control protrusion formation by Listeria. In addition, we aim to understand how these Tuba ligands are regulated by InlC. Our central hypothesis is that InlC controls protrusion formation by affecting Tuba and the activity and/or localization of one or more of Tuba's human ligands.
The Specific Aims i n this application, outlined below, are directed towards understanding how InlC, Tuba, and the ligands N-WASP, Cdc42, and ERM proteins control cell-cell spread.
AIM 1. Determine the role of the Tuba ligands N-WASP and Cdc42 in Listeria protrusion formation, and elucidate how these host proteins are controlled by InlC.
AIM 2. Determine if ERM proteins act together with Tuba to control bacterial spreading.
AIM 3. Assess the role of InlC/Tuba interaction in virulence. The above objectives will be addressed through a variety of approaches, including immunofluorescence microscopy, evaluation of ERM protein phosphorylation through Western blotting, RNA interference to investigate the function of Tuba and its ligands in spreading, and virulence studies using mice.
Listeria monocytogenes is a food-borne, intracellular bacterium that causes serious illnesses (listeriosis) leading to meningitis or abortion. Listeria evades the humoral immune system by spreading from one human cell to another, while remaining within the protective environment of the host cytosol. Epithelial cells lining the intestinal lumen (enterocytes) are the initial site of Listeria infection, and consequently play a critical role in the development of disease. The goal of this research is to identify bacterial and human components that mediate Listeria spreading in an enterocyte cell line. These bacterial and host proteins may be suitable as targets for drugs that limit Listeria infection.
