is a gram-positive bacterial pathogen that multiplies in the cytosol of infected cells and spreads from cell-to-cell using an actin-based mechanism of motility (10
). Upon initial invasion of a host cell and during cell-to-cell spread, L. monocytogenes
becomes entrapped in vacuoles that it must escape to perpetuate its intracellular cycle. Among the factors contributing to bacterial escape from double-membrane vacuoles are PC-PLC and Mpl (31
). Both enzymes are made as proproteins, which undergo maturation by cleavage of an N-terminal propeptide. The propeptide of PC-PLC serves to inhibit enzymatic activity and to retard PC-PLC translocation across the bacterial cell wall (18
). Rapid maturation and translocation of PC-PLC across the cell wall depend on a decrease in pH and on Mpl activity. In this study, we sought to determine the function of the Mpl propeptide. Our results indicate that the propeptide retains Mpl bacterium associated and that the compartmentalization of Mpl is integral to its ability to process its bacterium-associated substrates during intracellular infection.
Propeptides of bacterial proteases are normally important for catalyzing the folding of their covalently bound catalytic domains and can often perform this function either in cis
or in trans
). In this study, we first investigated whether the propeptide of Mpl serves as a folding catalyst by testing the behavior of an L. monocytogenes
mutant lacking the sequence coding for the Mpl propeptide. Detection of Mpl from broth-grown bacteria by Western immunoblotting indicated that the propeptide and catalytic domain fold into a stable form when synthesized as independent monomers. Whether MplΔpro achieves native conformation was determined by assessing its enzymatic activity. The EYA activity assay demonstrated that active Mpl can be generated in the absence of the propeptide. Therefore, contrary to the large majority of studied proproteases, the propeptide of Mpl is dispensable for the production of active enzyme. This is not the first example of a bacterial metalloprotease whose propeptide is dispensable for activity, as a similar observation was made for the neutral protease of Bacillus stearothermophilus
). Additional observations led us to consider that perhaps the propeptide of Mpl does not function as a folding catalyst. First, MplΔpro is less active than wild-type Mpl in the EYA activity assay, and this defect cannot be rescued by providing the propeptide in trans
. Second, the propeptide appeared to be stable as it was detectable by Western immunoblotting after cleavage from the proenzyme and when provided in trans
. Together, these two observations suggested that the propeptide of Mpl does not interact with its associated protease in trans
, since according to the folding-catalyst model, it would be degraded by its protease partner once the native conformation is attained. Third, two destabilizing propeptide mutations, H75V and H95L, did not prevent the generation of an active mature form of Mpl. Alternatively, our observations could indicate that the propeptide of Mpl functions as a folding catalyst but that the free energy required for the transition from the intermediate to the native state is low, enabling the mature form to reach its native state in the absence of the propeptide although with slower kinetics. This hypothesis would explain the decreased activity of MplΔpro detected on EYA, but would not explain the propeptide stability or why it cannot complement this defect in trans
. Biochemical and biophysical analyses of purified Mpl will be required to solve whether the propeptide functions as a folding catalyst. However, our data clearly indicate that Mpl is an atypical thermolysin-like protease, as its propeptide is not required to generate active enzyme.
PC-PLC and Mpl are virulence factors that contribute to the intracellular life cycle of L. monocytogenes. Therefore, we assessed Mpl activity during intracellular infection. Mpl mediates the rapid maturation and translocation of bacterium-associated PC-PLC and cleaves membrane-anchored ActA in acid-pH vacuoles. These two functions were tested by manipulating the intracellular pH of infected cells to synchronize the reaction for all intracellular bacteria. Interestingly, the MplΔpro protein did not demonstrate activity toward either of its two substrates during intracellular infection. In addition, the two propeptide single-site mutants, Mpl(H75V) and Mpl(H95L), did not mediate the rapid maturation and translocation of PC-PLC across the bacterial cell wall during intracellular infection. ActA proteolysis by Mpl was not assessed for these two mutants. Together, these data indicated that the propeptide of Mpl is integral to the intracellular function of Mpl.
The behavior of PC-PLC during intracellular infection suggests that Mpl and PC-PLC interaction occurs prior to translocation across the bacterial cell wall, in the confined interface between cell wall and membrane. Perhaps the propeptide of Mpl serves a function similar to the propeptide of PC-PLC, to retard translocation of the protein across the bacterial cell wall. To test this possibility, the compartmentalization of Mpl during intracellular infection was determined. By immunofluorescence microscopy, we observed that wild-type Mpl, but not MplΔpro or Mpl(H75V), associates with intracellular bacteria. Additionally, using an immunoprecipitation assay, we found three times more (P
= 0.02) MplΔpro molecules than wild-type Mpl in the secreted fraction from intracellular bacteria. Mpl(H75V), whose propeptide appears to be misfolded and subject to degradation after autocatalysis, was also found in the secreted fraction of intracellular bacteria in a larger amount than wild-type Mpl (3.3-fold; P
= 0.02). Together, these data support the hypothesis that the propeptide of Mpl serves a function similar to the propeptide of PC-PLC, to retard translocation of the protein across the bacterial cell wall. Conceivably, the proform of Mpl is sequestered at the membrane-cell wall interface by interacting with cell envelope components, preventing efficient translocation of the protein across the cell wall. Alternatively, the Mpl zymogen may adopt a conformation that is incompatible with cell wall translocation. In either case, it appears that bacterial association is a prerequisite for Mpl to mediate PC-PLC maturation during intracellular infection, but that prerequisite is less stringent when bacteria are grown on agar plates. In fact, when a plcB
deletion mutant and an mpl
deletion mutant were streaked on EYA perpendicular to each other without touching, a small zone of opacity developed between the two strains (data not shown), indicating that Mpl-mediated maturation of PC-PLC can occur in the extracellular milieu. However, when L. monocytogenes
becomes entrapped in a vacuole, Mpl-mediated maturation of PC-PLC must occur very rapidly as the efficacy of bacterial escape from vacuoles is in part dependent on PC-PLC activity (31
). Therefore, the presence of PC-PLC and Mpl at the cell wall membrane interface may serve to increase the probabilities of interaction between these two proteins and consequently of bacterial escape from vacuoles.
In conclusion, this study revealed that the propeptide of Mpl serves to retain the bacterium-associated protease, and this function is integral to the ability of Mpl to mediate the rapid maturation and translocation of PC-PLC across the bacterial cell wall during intracellular infection. There are many similarities between Mpl and PC-PLC. Both enzymes are synthesized as a proprotein; both enzymes are found to be bacterium associated during intracellular infection; their individual propeptides serve to retard protein diffusion across the bacterial cell wall; neither propeptide contains a transmembrane domain or a cell wall anchoring motif; and, finally, maturation of PC-PLC is dependent on Mpl activity. Future studies will aim to determine the mechanism by which these propeptides interfere with protein translocation across the bacterial cell wall, a phenomenon that is critical to the pathogenesis of L. monocytogenes.
Results from this study also revealed that the propeptide of Mpl is not required for Mpl to fold into its native state, although there is evidence that the propeptide enhances the ability of Mpl to fold into an active form. Additional biochemical and biophysical studies will be required to determine if the propeptide of Mpl functions as a folding catalyst.