THE BROAD-RANGE PHOSPHOLIPASE-C AND A METALLOPROTEASE MEDIATE LISTERIOLYSIN O-INDEPENDENT ESCAPE OF LISTERIA-MONOCYTOGENES FROM A PRIMARY VACUOLE IN HUMAN EPITHELIAL-CELLS

Intracellular growth of Listeria monocytogenes begins after lysis of the primary vacuole formed upon bacterial entry into a host cell. Listeriolysin O (LLO), a pore-forming hemolysin encoded by hly, is essential for vacuolar lysis in most cell types. However, in human epithelial cells, LLO(-) mutants are capable of growth, suggesting that gene products other than LLO are capable of mediating escape from a vacuole. In this study, we investigated the role of other bacterial gene products in lysis of the primary vacuole in the human epithelial cell line Henle 407. Double internal in-frame deletion mutants were constructed by introducing a mutated hly allele into strains harboring deletions in either of the phospholipase C (PLC)-encoding genes or a metalloprotease-encoding gene. Bacterial escape from the primary vacuole, intracellular growth, and cell-to-cell spread were evaluated in Henle 407 cells. The results indicated that, in the absence of LLO, the broad-range PLC and the metalloprotease were both required for lysis of the primary vacuole in Henle 407 cells. Although phosphatidylinositol-specific PLC was not required, the efficiency of escape was reduced in an LLO phosphatidylinositol-specific PLC double mutant. These observations suggest that the relative importance of LLO, the phospholipases, and the metalloprotease may vary in different cell types or in cells from different species. In addition, these studies provide insight into the mechanisms of action of virulence determinants involved in the lysis of vacuolar membranes.