The Metalloprotease of<i>Listeria monocytogenes</i>Controls Cell Wall Translocation of the Broad-Range Phospholipase C

Yeung, P. S. Marie; Zagorski, Nicholas; Marquis, Hélène

doi:10.1128/jb.187.8.2601-2608.2005

Cited by 43 publications

(59 citation statements)

References 39 publications

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“…Previously, it was shown that the secretion of PC-PLC across the bacterial cell wall is dependent on both a decrease in pH and Mpl (39). Similarly, results from this study suggest that a decrease in pH leads to secretion of Mpl across the bacterial cell wall.…”

Section: Maturesupporting

confidence: 79%

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The Metalloprotease of Listeria monocytogenes Is Regulated by pH

et al. 2011

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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...

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Section: Maturesupporting

confidence: 79%

“…The hly deletion was verified by loss of hemolysis on blood agar plates (25). Deletion of plcB was verified by the loss of PC-PLC activity on egg yolk agar plates (39). Deletion of inlAB was confirmed by PCR using primers Marq41 (17) and Marq494 ( Table 2).…”

Section: Methodsmentioning

confidence: 99%

The Metalloprotease of Listeria monocytogenes Is Regulated by pH

et al. 2011

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“…The mutation was inserted into the genome of L. monocytogenes by allelic exchange. The phenotype of L. monocytogenes Mpl E350Q was identical to that of an isogenic mpl deletion mutant (30). L. monocytogenes Mpl E350Q was negative for PC-PLC activity on egg yolk agar ( Fig.…”

Section: Resultsmentioning

confidence: 77%

“…There is evidence that Mpl-mediated activation of PC-PLC occurs at the membrane cell wall interface before translocation of PC-PLC across the bacterial cell wall (30), indicating that Mpl maturation occurs before translocation across the cell wall. Although the mature form of Mpl is found predominantly in the bacterial supernatant, there is no evidence that it is active in this environment.…”

Section: Resultsmentioning

confidence: 99%

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The Metalloprotease ofListeria monocytogenesIs Activated by Intramolecular Autocatalysis

2008

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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.Listeria monocytogenes is a facultative intracellular bacterial pathogen that multiplies in the cytosol of host cells and uses an actin-based mechanism of motility to spread from cell to cell without exiting the intracellular milieu (26). Upon cell-to-cell spread, bacteria are temporarily located in double-membrane vacuoles from which they must exit to perpetuate the intracellular life cycle. Among the factors contributing to vacuolar lysis is a bacterial phospholipase C (PC-PLC) (24, 28). PC-PLC is made as a proenzyme whose activation requires cleavage of a 24-amino-acid propeptide. The metalloprotease of Listeria (Mpl) contributes to PC-PLC activation (21). Moreover, Mplmediated maturation of PC-PLC is regulated in a temporal and spatial manner during the intracellular life cycle of L. monocytogenes (15).Mpl possesses the HEXXH motif that is characteristic of the Zincins superfamily of metalloproteases. Within this family, Mpl is most closely related to thermolysin, which is the prototype member of the M4 family of metalloproteases (20). Thermolysin is made by Bacillus thermoproteolyticus, and all members of the thermolysin family originate from bacteria. The active site zinc ion of these enzymes is coordinated by a water molecule and three amino acid residues, including the two histidines present within the HEXXH motif and a glutamic acid located 20 residues downstream of this motif (2, 8). In addition, the glutamic acid residue located within the HEXXH motif and a histidine residue located 83 residues downstream of this motif interact with a water molecule at the active site and are required for catalysis (2, 3).Thermolysin and related metalloproteases are synthesized as preproenzymes. The prodomain, which accounts for ϳ40% of the proenzyme, serves as a chaperone and as an inhibitor of catalysis (17,23,29). Processing of the prodomain generates the mature active form of the protease. Autocatalysis has been suggested to be the mechanism of maturation of thermolysinlike proteases, as catalytic site mutants fail to generate mature proteases (10, 11, 13, 16). Furthermore, a mechanism of intramolecular autocatalysis was suggested for thermolysin itself, as purified active t...

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Listeria Monocytogenes

Balestrino

Cossart

2009

Intracellular Niches of Microbes

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The Metalloprotease ofListeria monocytogenesControls Cell Wall Translocation of the Broad-Range Phospholipase C

Cited by 43 publications

References 39 publications

The Metalloprotease of Listeria monocytogenes Is Regulated by pH

The Metalloprotease of Listeria monocytogenes Is Regulated by pH

The Metalloprotease ofListeria monocytogenesIs Activated by Intramolecular Autocatalysis

Listeria Monocytogenes

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