Listeria factors important for cell-to-cell spread

Listeria monocytogenes is a facultative intracellular bacterial pathogen of humans and a variety of animal species. After entering a host cell, L. monocytogenes is initially surrounded by a membrane-bound vacuole. The bacterium promotes its escape from this vacuole, grows within the host cell cytosol and spreads from cell-to-cell via actin- based motility. The virulence of L. monocytogenes is directly related to its ability to spread from cell-to-cell without leaving the intracellular milieu. Many factors required for the intracellular growth and spread of L. monocytogenes have been identified and their role as virulence determinants has been studied primarily in a mouse model of infection. One of the best-characterized virulence factors is the pore-forming cytolysin listeriolysin O (LLO). In all mouse cell lines tested, LLO is absolutely required for vacuolar escape and hence intracellular growth and spread of the bacterium. However, the observation has been made that in the human-derived epithelial cell line Henle 407, L. monocytogenes can grow and spread in the absence of LLO. In this cell line, the phospholipase PC- PLC is sufficient to promote vacuolar escape in the absence of LLO. Therefore, by using a mouse model of infection, the role of PC-PLC in a human infection might have been underestimated. During the first year of the Erwin Schrödinger stipend, I have shown that PC-PLC can promote vacuolar lysis in other human-derived epithelial cell lines, namely HeLa and Hep-2. Therefore, it is not unique to the human- derived cell line Henle 407 that PC-PLC is able to promote. vacuolar lysis. Furthermore, I have shown that hyper- secretion of PC-PLC cannot relieve the LLO requirement for vacuolar escape in the mouse-derived cell line J774. In the second year, I plan to determine the temporal requirement of PC-PLC during an infection of human cells. Specifically, I plan to determine the requirement of PC-PLC for degradation of the secondary double membrane spreading vacuole in human-derived epithelial cell lines. Also described in this proposal is a fluorescent activated cell sorter (FACS)-based strategy to isolate novel L. monocytogenes factors required for intracellular spread. Transposon mutagenesis will be performed in a green fluorescent protein (GFP)-expressing L. monocytogenes strain and spreading mutants will be identified by a FACS-based screen of host cells that have been infected with the L. monocytogenes mutant library. Listeria monocytogenes is an attractive model system for studying the molecular determinants of intracellular pathogenesis. The identification of novel factors required for intracellular spread and a more detailed understanding of factors required for intracellular growth and spread of L. monocytogenes will further our understanding of general virulence mechanisms shared by diverse species of intracellular pathogens.