The Metalloprotease of Listeria monocytogenes Is Regulated by pH

Abstract

Listeria monocytogenes is an intracytosolic bacterial pathogen. Among the factors contributing to escape from vacuoles are a phosphatidylcholine phospholipase C (PC-PLC) and a metalloprotease (Mpl). Both enzymes are translocated across the bacterial membrane as inactive proproteins, whose propeptides serve in part to maintain them in association with the bacterium. We have shown that PC-PLC maturation is regulated by Mpl and pH and that Mpl maturation occurs by autocatalysis. In this study, we tested the hypothesis that Mpl activity is pH regulated. To synchronize the effect of pH on bacteria, the cytosolic pH of infected cells was manipulated immediately after radiolabeling de novo-synthesized bacterial proteins. Immunoprecipitation of secreted Mpl from host cell lysates revealed the presence of the propeptide and catalytic domain in samples treated at pH 6.5 but not at pH 7.3. The zymogen was present in small amounts under all conditions. Since proteases often remain associated with their respective propeptide following autocatalysis, we aimed at determining whether pH regulates autocatalysis or secretion of the processed enzyme. For this purpose, we used an Mpl construct that contains a Flag tag at the N terminus of its catalytic domain and antibodies that can distinguish N-terminal and non-N-terminal Flag. By fluorescence microscopy, we observed the Mpl zymogen associated with the bacterium at physiological pH but not following acidification. Mature Mpl was not detected in association with the bacterium at either pH. Using purified proteins, we determined that processing of the PC-PLC propeptide by mature Mpl is also pH sensitive. These results indicate that pH regulates the activity of Mpl on itself and on PC-PLC.Listeria monocytogenes is a Gram-positive, facultative intracellular bacterial pathogen. It is the causative agent of the food-borne disease listeriosis, which has a high mortality rate (37). L. monocytogenes is able to invade host cells and spread from cell to cell using host actin (35). To escape the vacuoles formed upon initial entry into a cell or cell-to-cell spread, L. monocytogenes relies on multiple virulence factors. These factors include listeriolysin O (LLO) (7, 35), a phosphatidylinositol-specific phospholipase C (4), and a broad-range phospholipase C known as PC-PLC (phosphatidylcholine phospholipase C) (32). PC-PLC is synthesized as an inactive proenzyme and translocates across the cell membrane, where it accumulates at the membrane-cell wall interface (21, 34). A decrease in pH and the metalloprotease of L. monocytogenes (Mpl) are required for PC-PLC maturation, which coincides with the rapid secretion of mature PC-PLC across the bacterial cell wall (21, 31).Mpl is a member of the thermolysin family of metalloproteases which contains a Zn 2ϩ ion in the active site (11). Mpl is produced as a zymogen with an N-terminal propeptide (22). Similar to PC-PLC, Mpl translocates across the bacterial membrane and accumulates at the membrane-cell wall interface (24,34). This compartmentalization...

Posttranslocation Chaperone PrsA2 Regulates the Maturation and Secretion of Listeria monocytogenes Proprotein Virulence Factors

“…This maturation process is followed by the rapid secretion of these two enzymes across the bacterial cell wall (12,28,40). We questioned the role of PrsA2 in regulating the compartmentalization of PC-PLC and Mpl.…”

“…Upon a decrease in pH, which is normally experienced when bacteria are in vacuoles, Mpl undergoes autocatalysis and mediates the maturation of PC-PLC (6,12). This maturation process is followed by the rapid secretion of these two enzymes across the bacterial cell wall (12,28,40).…”

“…Immunofluorescence staining of Mpl. Bacterium-associated Mpl-Flag N-cat (an Mpl construct that contains a Flag tag at the N terminus of its catalytic domain) was detected by immunofluorescence as described previously (12). Briefly, HeLa cells infected with L. monocytogenes were fixed and then treated with purified L. monocytogenes-specific phage endolysin Ply118.…”

“…Concomitant to maturation, both proteins are rapidly se-creted across the bacterial cell wall. The secretion of PC-PLC in response to pH is dependent on Mpl, but the secretion of Mpl is not dependent on PC-PLC (12,46).…”

“…During intracytosolic growth, these proteins translocate across the bacterial cytoplasmic membrane and accumulate at the membrane-cell wall interface (28,31,40). Upon cell-to-cell spread, bacteria in vacuoles experience a decrease in pH, which initiates Mpl autocatalysis, leading to the proteolytic maturation of PC-PLC by mature Mpl (6,12,27). Concomitant to maturation, both proteins are rapidly se-creted across the bacterial cell wall.…”