A non-catalytic histidine residue influences the function of the metalloprotease of Listeria monocytogenes.

Mpl, a thermolysin-like metalloprotease, and PC-PLC, a phospholipase C, are synthesized as proenzymes by the intracellular bacterial pathogen Listeria monocytogenes. During intracellular growth, L. monocytogenes is temporarily confined in a membrane-bound vacuole whose acidification leads to Mpl autolysis and Mpl-mediated cleavage of the PC-PLC N-terminal propeptide. Mpl maturation also leads to the secretion of both Mpl and PC-PLC across the bacterial cell wall. Previously, we identified negatively charged and uncharged amino acid residues within the N terminus of the PC-PLC propeptide that influence the ability of Mpl to mediate the maturation of PC-PLC, suggesting that these residues promote the interaction of the PC-PLC propeptide with Mpl. In the present study, we identified a non-catalytic histidine residue (H226) that influences Mpl secretion across the cell wall and its ability to process PC-PLC. Our results suggest that a positive charge at position 226 is required for Mpl functions other than autolysis. Based on the charge requirement at this position, we hypothesize that this residue contributes to the interaction of Mpl with the PC-PLC propeptide.

The metalloprotease of Listeria monocytogenes is regulated by pH.

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.

The propeptide of the metalloprotease of Listeria monocytogenes controls compartmentalization of the zymogen during intracellular infection.

Integral to the virulence of the intracellular bacterial pathogen Listeria monocytogenes is its metalloprotease (Mpl). Mpl regulates the activity and compartmentalization of the bacterial broad-range phospholipase C (PC-PLC). Mpl is secreted as a proprotein that undergoes intramolecular autocatalysis to release its catalytic domain. In related proteases, the propeptide serves as a folding catalyst and can act either in cis or in trans. Propeptides can also influence protein compartmentalization and intracellular trafficking or decrease folding kinetics. In this study, we aimed to determine the role of the Mpl propeptide by monitoring the behavior of Mpl synthesized in the absence of its propeptide (MplDeltapro) and of two Mpl single-site mutants with unstable propeptides: Mpl(H75V) and Mpl(H95L). We observed that all three Mpl mutants mediate PC-PLC activation when bacteria are grown on semisolid medium, but to a lesser extent than wild-type Mpl, indicating that, although not essential, the propeptide enhances the production of active Mpl. However, the mutant proteins were not functional in infected cells, as determined by monitoring PC-PLC maturation and compartmentalization. This defect could not be rescued by providing the propeptide in trans to the mplDeltapro mutant. We tested the compartmentalization of Mpl during intracellular infection and observed that the mutant Mpl species were aberrantly secreted in the cytosol of infected cells. These data indicated that the propeptide of Mpl serves to maintain bacterium-associated Mpl and that this localization is essential to the function of Mpl during intracellular infection.

The metalloprotease of Listeria monocytogenes is activated by intramolecular autocatalysis.

The metalloprotease (Mpl) of Listeria monocytogenes is a thermolysin-like protease that mediates the maturation of a broad-range phospholipase C, whose function contributes to the ability of this food-borne bacterial pathogen to survive intracellularly. Mpl is made as a proprotein that undergoes maturation by proteolytic cleavage of a large N-terminal prodomain. In this study, we identified the N terminus of mature Mpl and generated Mpl catalytic mutants to investigate the mechanism of Mpl maturation. We observed that Mpl activity was a prerequisite for maturation, suggesting a mechanism of autocatalysis. Furthermore, using a strain of L. monocytogenes expressing both the wild-type form and a catalytic mutant form of Mpl simultaneously, we determined that in vivo maturation of Mpl occurs exclusively by an intramolecular autocatalysis mechanism.

A mariner-based transposition system for Listeria monocytogenes.

In this study, we developed a new mariner-based transposition system for Listeria monocytogenes. The mariner-based system has a high rate of transposition and a low rate of plasmid retention, and transposition is very random, making it an ideal tool for high-throughput transposon mutagenesis in L. monocytogenes.