Regulation Of The Metalloprotease Of Listeria Monocytogenes During Intracellular Infection

Abstract

Listeria monocytogenes is a Gram-positive, facultative intracellular bacterial pathogen that multiplies in the cytosol of host cells and relies on an actin-based mechanism of motility to spread from cell-to-cell without exiting the intracellular milieu. Virulence of L. monocytogenes is related to its ability to escape from vacuoles formed upon initial entry into a host cell and during cell-to-cell spread. One factor contributing to escape from vacuoles is a broad-range phospholipase C (PC-PLC). PC-PLC is translocated across the bacterial membrane as an inactive proenzyme and accumulates in the periplasmic space. PC-PLC maturation is dependent upon a decrease in pH and on the metalloprotease of L. monocytogenes (Mpl). Mpl, a thermolysin-like protease, is produced as a zymogen and matures via intramolecular autocatalysis. In this study, we wished to investigate the regulation of Mpl during intracellular infection. Our results show that at physiological pH, the zymogen remains bacterium-associated, presumably in the periplasm. Mpl produced in absence of its propeptide is aberrantly secreted suggesting that the propeptide regulates compartmentalization. Upon a decrease in pH, the bacterium-associated zymogen undergoes autocatalysis and mediates the proteolytic activation of PC-PLC. Mature Mpl and PC-PLC are then rapidly secreted across the cell wall. Two amino acids in the catalytic domain of Mpl, H226 and H241, influence mature Mpl secretion and its ability to process PC-PLC. The compartmentalization of the zymogen is also influenced by PrsA2, a peptidyl prolyl cis-trans isomerase that is anchored by an acyl chain to the trans side of the membrane. In absence of PrsA2, the proforms of Mpl and PC-PLC are aberrantly secreted across the cell wall and no longer undergo maturation upon a decrease in pH. Taken together, these results indicate that PrsA2 and the propeptide of Mpl serve to maintain the association of Mpl with bacteria and that this compartmentalization is required for Mpl to undergo autocatalysis at acidic pH and to mediate the proteolytic activation of PC-PLC.