title: 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.
authors.affiliation: Department of Microbiology and Immunology, VMC Rm C5-169, Cornell University, Ithaca, NY 14853-6401, USA.
authors.forename: Brian M
authors.initials: BM
authors.lastname: Forster
authors.forename: Alan Pavinski
authors.initials: AP
authors.lastname: Bitar
authors.forename: Emily R
authors.initials: ER
authors.lastname: Slepkov
authors.forename: Karthik J
authors.initials: KJ
authors.lastname: Kota
authors.forename: Holger
authors.initials: H
authors.lastname: Sondermann
authors.forename: Hélène
authors.initials: H
authors.lastname: Marquis
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D006863:Hydrogen-Ion Concentration; D047468:Immunoprecipitation; D008089:Listeria monocytogenes / Q000201:enzymology* / Q000235:genetics; D045726:Metalloproteases / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009154:Mutation; D016133:Polymerase Chain Reaction; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D010738:Type C Phospholipases; C105598:phosphatidylcholine-specific phospholipase C; D045726:Metalloproteases