Genetic analysis of Listeria monocytogenes determinants of pathogenesis

Listeria monocytogenes is a gram-positive, facultative intracellular pathogen responsible for serious infections in immunocompromised individuals and pregnant women. Murine listeriosis has been used extensively as a model system for the study of cell-mediated immunity. However, until recently little was known about bacterial determinants of pathogenesis. We have constructed a highly useful derivative of Tn917 for random mutagenesis of the L. monocytogenes chromosome and, coupled with the isolation of small plaque mutants in fibroblast L2 cell monolayers, have identified L. monocytogenes genes necessary for intracellular growth and cell-to-cell spread in mammalian cells. Transposon insertions in one class of small plaque mutant identified a gene, plcA, which we show encoded a secreted phosphatidylinositol-specific phospholipase C (PI-PLC). A representative strain from this mutant class was 3 logs less virulent in a mouse model of infection, invaded tissue culture cells and grew in the cytoplasm normally, but was defective at polymerizing host actin and subsequently was defective for cell-to-cell spread. Complementation analyses suggested that a polar effect on expression of an adjacent gene, prfA, was responsible for the defect in cell-to-cell spread. A genetic approach was taken to prove the polar effect on prfA. We constructed a plcA internal deletion strain $(\Delta plcA)$ to test for a possible role of PI-PLC in pathogenesis. The $\Delta plcA$ strain formed wild-type size plaques but was defective for growth in primary macrophage cultures and was 2-4 fold less virulent in mice. Mixed infections revealed a striking growth defect of the $\Delta plcA$ strain in the liver but not spleen of mice. Electron microscopy of infected bone marrow-derived macrophages revealed that PI-PLC played a role in mediating bacterial escape from host cell phagosomes. Therefore, plcA has a dual role in L. monocytogenes pathogenesis. Its most critical role is to provide readthrough transcription of prfA which, in turn, encodes a positive transcription factor of other virulence genes, including determinants required for cell-to-cell spread. The other role of plcA is to encode a secreted PI-PLC which is necessary for efficient lysis of host phagosome membranes