Exploitation of host cell processes for bacterial cell‐to‐cell spread (99.4)

The ubiquitous food‐borne bacterium Listeria monocytogenes causes spontaneous abortion in pregnant women and meningitis in immune‐compromised hosts. Bacteria in the intestine directly invade intestinal epithelial cells, but systemic dissemination requires that the bacteria use actin‐based motility to move into macrophages that carry them through the bloodstream to distal sites, and then that they cross the endothelial barrier to invade protected tissues such as the placenta and meninges. In tissue culture experiments, we have used fluorescence microscopy and flow cytometry to confirm that L. monocytogenes can directly invade and spread through a monolayer of human umbilical vein endothelial cells (HUVEC). We performed a targeted siRNA screen to identify host cell factors that modulate bacterial invasion and spread. Knocking down focal adhesion components including integrin, paxillin, and vinculin specifically increased bacterial invasion, suggesting that focal adhesions may normally inhibit invasion. We have also demonstrated that L. monocytogenes in infected macrophages can be robustly transferred to uninfected HUVEC via direct heterotypic cell‐to‐cell spread. Using siRNA screening, we have identified a number of cytoskeletal factors, including Arp2, capping proteins, Rac, Diaph2, and ZO1, which, in recipient cells, appear to decrease heterotypic bacterial transfer, suggesting that the endothelial cortex and tight junctions between endothelial cells may influence macrophage‐to‐endothelial transfer of bacteria.