PC-PLC and Mpl are virulence factors of L. monocytogenes
that accumulate as inactive proenzymes at the bacterial membrane-cell wall interface during growth in host cells (34
). Upon cell-to-cell spread, bacteria in vacuoles experience a decrease in pH that triggers the rapid secretion of mature PC-PLC across the bacterial cell wall in an Mpl-dependent manner (21
). This mechanism of regulating PC-PLC activity is imperative to the virulence of L. monocytogenes
, as a propeptide-deletion mutant of PC-PLC is highly attenuated in vivo
). In the present study, we tested the hypothesis that Mpl maturation is the pH-limiting step for PC-PLC maturation. Previously, we determined that Mpl maturation occurs by intramolecular autocatalysis (3
). Herein, we observed that Mpl autocatalysis occurs upon a decrease in pH and correlates with the rapid secretion of mature Mpl and propeptide across the bacterial cell wall. Moreover, our data indicate that Mpl directly mediates the proteolytic maturation of PC-PLC and that this activity is also pH regulated.
The Mpl zymogen remains mostly bacterium associated at physiological pH while mature Mpl is found in host cells within minutes of a decrease in pH. This observation is in accordance with our previous result showing that when Mpl is synthesized in the absence of its propeptide, it is secreted more efficiently across the bacterial cell wall (24
). This phenomenon has also been observed for the serine protease subtilisin from Bacillus subtilis
. Subtilisin precursor remains bacterium associated while mature subtilisin is found in the supernatant (26
). Our study provides further evidence that propeptides serve as a means of controlling the compartmentalization of bacterial proproteins, similarly to what has been observed for several eukaryotic proproteins (6
There is evidence that following autocatalysis, the pro and catalytic domains of metalloproteases remain associated as an autoprocessed complex that is void of enzymatic activity because the C terminus of the propeptide remains in the active site. Degradation of the propeptide by its cognate catalytic domain is essential for full activation of the protease (8
). We would argue that this mechanism of regulation does not apply to Mpl for two reasons. First, the propeptide of Mpl appears to be stable as it is detected in amounts similar to those of the mature form under conditions that lead to Mpl-mediated maturation of PC-PLC. Measuring the levels of detected Mpl from our immunoprecipitation assays revealed a ratio of 1:1.6 ± 0.4 (n
= 17) for mature Mpl to propeptide, supporting our conclusion that the propeptide of Mpl is not degraded by its cognate catalytic domain (B. M. Forster and H. Marquis, unpublished data). Second, mature Mpl-FlagN-cat
was not detected in association with the bacterium when using an antibody that specifically recognizes a free N-terminal Flag. Together, the results support a mechanism by which pH-induced autocatalysis leads to full activation of Mpl.
Since Mpl autocatalysis is controlled by pH, we wished to determine if pH regulates Mpl activity on a nonself substrate. The obvious substrate would be PC-PLC. However, the evidence for the role of Mpl in mediating the maturation of PC-PLC is only circumstantial. To address whether Mpl directly activates PC-PLC, we performed an in vitro assay with purified proteins. The results indicated that Mpl directly mediates the proteolytic maturation of PC-PLC and that, moreover, this activity is pH regulated. Mature PC-PLC and PC-PLC activity were detected at pH 6.0, and to a lesser extent at pH 6.5, but not at pH 7.3 or when pro-PC-PLC was incubated alone or with the Mpl catalytic mutant. It is very unlikely that the heat shock protein GroEL, which contaminated the wild-type Mpl preparation, would have affected the results, as GroEL is a chaperone with no known proteolytic activity. Therefore, our data indicate that Mpl directly mediates the proteolytic maturation of PC-PLC and that Mpl activity on a nonself substrate is pH sensitive. Alternatively, pH may influence the conformation of mature Mpl and pro-PC-PLC, enabling an interaction between these two proteins.
There are precedents for the observation that pH regulates the activity of a protease. For example, the serine protease furin undergoes autocatalysis once it reaches the acidic trans-Golgi network (1
). The autocatalysis of furin is controlled by a histidine residue in its propeptide that acts as a pH sensor to acidic environments. Upon acidification, this histidine becomes protonated and disrupts a hydrophobic pocket allowing for autocatalysis (5
). Members of the cathepsin family of cysteine proteases undergo autocatalysis in acidified lysosomes. Under acidic conditions, the propeptide of cathepsin L loses its tertiary conformation, resulting in a loss of affinity for the catalytic domain (15
). Future studies will be focused on determining how pH controls the enzymatic activity of Mpl. Mpl activity may be controlled by amino acids acting as pH sensors. pH may also induce protein conformational changes in Mpl and/or in PC-PLC to allow Mpl and PC-PLC to interact.
In conclusion, our results indicate that pH regulates the enzymatic activity and compartmentalization of Mpl and support a model () in which the proforms of PC-PLC and Mpl accumulate at the membrane-cell wall interface when the bacteria are in an environment at physiological pH, such as in the host cell cytosol. Upon cell-to-cell spread, bacteria become confined to double membrane vacuoles that acidify, which is sensed by Mpl, leading to Mpl autocatalysis and proteolytic maturation of PC-PLC by mature Mpl. The mature proteins are rapidly secreted across the bacterial cell wall into the vacuolar environment where PC-PLC can begin hydrolyzing vacuolar membrane phospholipids, contributing to lysis of the vacuole. As for Mpl, perhaps it targets additional substrates after accessing the vacuolar environment.
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 (more ...)