Perturbation of vacuolar maturation promotes listeriolysin O-independent vacuolar escape during Listeria monocytogenes infection of human cells

Abstract

Listeria monocytogenes is a bacterial pathogen that replicates within the cytosol of infected host cells. The ability to rapidly escape the phagocytic vacuole is essential for efficient intracellular replication. In the murine model of infection, the pore-forming cytolysin listeriolysin O (LLO) is absolutely required for vacuolar dissolution, as LLO-deficient (DLLO) mutants remain trapped within vacuoles. In contrast, in many human cell types DLLO L. monocytogenes are capable of vacuolar escape at moderate to high frequencies. To better characterize the mechanism of LLOindependent vacuolar escape in human cells, we conducted an RNA interference screen to identify vesicular trafficking factors that play a role in altering vacuolar escape efficiency of DLLO L. monocytogenes. RNA interference knockdown of 18 vesicular trafficking factors resulted in increased LLO-independent vacuolar escape. Our results suggest that knockdown of one factor, RABEP1 (rabaptin-5), decreased the maturation of vacuoles containing DLLO L. monocytogenes. Thus, we provide evidence that increased vacuolar escape of DLLO L. monocytogenes in human cells correlates with slower vacuolar maturation. We also determined that increased LLO-independent dissolution of vacuoles during RABEP1 knockdown required the bacterial broad-range phospholipase C (PC-PLC).We hypothesize that slowing the kinetics of vacuolar maturation generates an environment conducive for vacuolar escape mediated by the bacterial phospholipases. © 2009 Blackwell Publishing Ltd.