Host cell factors involved in the vacuolar biology of Listeria monocytogenes infection

Listeria monocytogenes is a facultative intracellular bacterial pathogen. Upon entry into host cells, L. monocytogenes escapes the entry vacuole and replicates within the cytosol. As an intracellular pathogen, L. monocytogenes interacts extensively with the host cells it infects. In this dissertation, we characterized host cell factors involved in L. monocytogenes intracellular infection. We describe a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify host factors required for L. monocytogenes infection. 305 dsRNAs targeting a wide range of host cell functions were identified that altered L. monocytogenes infection. Comparison to a similar screen with Mycobacterium fortuitum, a vacuolar pathogen, identified host factors that may play a general role in intracellular pathogenesis and factors that specifically affect access to the cytosol by L. monocytogenes. Most striking was the large number of vesicular trafficking pathway components identified. We confirmed that knockdown of selected gene products from a variety of functional categories identified in the Drosophila screen similarly altered L. monocytogenes infection in human cells. Additionally, we used a sub-genomic shRNA library to identify human vesicular trafficking factors that affect vacuolar escape efficiency of L. monocytogenes lacking a pore-forming cytolysin, listeriolysin O. We compared L. monocytogenes vacuolar biology in human cells to the vacuolar biology in Drosophila and murine cells. We provide evidence that the vacuolar maturation state of L. monocytogenes -containing vacuoles can determine whether or not L. monocytogenes is able to productively infect a host cell. We propose a model in which partially mature vacuoles with an acidic pH are ideal for L. monocytogenes vacuolar escape, while immature or highly mature degradative vacuoles are less permissive for vacuolar escape.