Listeria phospholipase PC-PLC in human cell infection

Listeria monocytogenes is a facultative intracellular pathogen and its virulence is directly related to its ability to spread from cell-to-cell without leaving the intracellular milieu. Many virulence factors required for this process have been identified and their roles in cell-to-cell spread have been studied with knockout mutants. One of the best-characterized virulence factors of Listeria is the pore-forming cytolysin listeriolysin O (LLO). LLO, is largely responsible for the destruction of the primary vacuole formed during bacterial uptake, allowing the bacterium access to the cell cytosol. Besides LLO two phospholipases, PI-PLC and PC-PLC interact with host membranes and promote respectively the escape from the primary vacuole and secondary vacuoles formed during cell-to-cell spread. In several other pathogenic bacteria, phospholipases have been implicated as virulence determinants. A better understanding of the role of these enzymes in an infection is of considerable importance for the future development of strategies to intervene host pathogen interactions. In my research, I will focus on the broad range phospholipase PC-PLC. In a mouse model system, PC-PLC is required for promoting the escape of the bacterium from the secondary vacuole. Interestingly and importantly, in human cell lines such as the epithelial cell line Henle 407 PC-PLC can functionally replace LLO and promote the escape of the bacterium from all vacuoles. This indicates that in a human infection PC-PLC might be of greater significance than suggested by mouse system studies. The initial goal of my research will be to determine whether the PC-PLC promoted escape from the primary vacuoles in human cells is a general phenomenon or if it is specific only for a few human cell lines. This will be done by utilizing other available human cell lines and examining the intracellular growth of different L. monocytogenes mutants. Furthermore, I will determine whether overexpression of PC-PLC can relieve the LLO requirement in mouse cell lines. I will address this question by examining an infection with a hypersecreting or an inducible PC-PLC L. monocytogenes strain. In the latter strain, expression of PC-PLC will be independent of other bacterial virulence factors and can be controlled throughout the intracellular life cycle of L. monocytogenes. By using this inducible strain, I will also be able to investigate the requirement of PC-PLC throughout the intracellular life cycle of L. monocytogenes in human cells. This will allow me to determine the exact role of PC-PLC during intracellular growth and cell-to-cell spread.