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.

Fig. 1.

The propeptide and mature form of Mpl are secreted in the host cell cytosol upon a decrease in pH. J774 cells were infected with 10403S expressing wild-type (WT) Mpl or isogenic mutants expressing Mpl E350Q or no Mpl (Mpl⁻). Cells were pulse-labeled with [³⁵S]Met-Cys and chased in a nigericin-containing potassium buffer equilibrated to pH 7.3 or pH 6.5 (indicated above each lane). Secreted Mpl was immunoprecipitated from cleared host cell lysates. Numbers on right indicate molecular mass markers in kDa. Lanes: 1 and 2, 10403S; 3 and 4, HEL-583; 5 and 6, DP-L2343.

Fig. 2.

Anti-Flag antibodies allow for the differentiation of Mpl species. (A) Schematics of Mpl-Flag constructs used in this study and predicted patterns of reactivity with two different antibodies. Anti-Flag M1 recognizes an N-terminal Flag, while anti-Flag M2 recognizes any surface-accessible Flag. (B) Detection of Mpl from culture supernatants. Strains were grown in LB-MOPS-Glc, and the equivalent of 1.0 ml of supernatant at an OD₆₀₀ of 1 was resolved by SDS-PAGE. Mpl and Mpl-Flag constructs were detected with either anti-Mpl, anti-Flag M1, or anti-Flag M2 antibodies. Numbers on right indicate molecular mass markers in kDa. Positions of the Mpl zymogen, mature Mpl, and propeptide are indicated on left. Lanes: 1, 6, and 9, NF-L943; 2, HEL-587; 3, 7, and 10, HEL-981; 4, 8, and 11, HEL-1084; 5, HEL-469.

Fig. 3.

The Mpl zymogen is bacterium associated at physiological pH. Bacterium-associated Mpl was detected by fluorescence microscopy. Prior to fixing, infected HeLa cells were incubated in a nigericin-containing potassium buffer equilibrated to either pH 7.3 or pH 6.5. Host cell nuclei and bacteria were then stained with bisbenzimide (Hoechst 33258). Mpl-Flag was detected with anti-Flag M1 or anti-Flag M2 antibody followed by an FITC-conjugated secondary antibody. (a to h) NF-L943; (i to p) HEL-981; (q to x) HEL-1084.

Fig. 4.

PC-PLC does not influence the secretion of mature Mpl. J774 cells were infected with 10403S expressing PC-PLC and Mpl (WT) or isogenic mutants with no PC-PLC (PC-PLC⁻) or no Mpl (Mpl⁻). Cells were pulse-labeled with [³⁵S]Met-Cys and chased in a nigericin-containing potassium buffer at pH 7.3 or pH 6.5 (indicated above each lane). Secreted Mpl was immunoprecipitated from cleared host cell lysates. Numbers on right indicate molecular mass markers in kDa. Lanes: 1 and 2, 10403S; 3 and 4, DP-L1935; 5 and 6, DP-L2343.

Fig. 5.

Mpl directly mediates the maturation of PC-PLC in a pH-dependent manner. (A) Stained protein gel of purified pro-PC-PLC fractions following size exclusion chromatography. (B) Stained protein gel of purified Mpl-Flag_C-cat and Mpl E350Q-Flag_C-cat following immunochromatography. The asterisk indicates GroEL which copurified with Mpl-Flag_C-cat. (C) PC-PLC activation in vitro assay. Purified pro-PC-PLC and Mpl were mixed in a reaction buffer equilibrated to either pH 6.0, 6.5, or 7.3 (indicated above the lanes) and incubated at 37°C for 4 h. Following incubation, the proteins were resolved by SDS-PAGE. PC-PLC activity was detected by the egg yolk soft agar overlay assay. A band of opacity in the agar indicates phospholipid hydrolysis (upper panel). Following the activity assay, the gel was stained with Coomassie blue (lower panel). Numbers on right indicate molecular mass markers in kDa.

Fig. 6.

Schematic representation of the behavior of PC-PLC and Mpl when L. monocytogenes is located within a double membrane vacuole formed upon cell-to-cell spread. Pro-PC-PLC and the Mpl zymogen remain at the membrane-cell wall interface until a decrease in pH triggers Mpl autocatalysis and Mpl-mediated maturation of PC-PLC. The mature proteins are then secreted across the cell wall where PC-PLC can hydrolyze membrane phospholipids.