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.
O'Neil
Heather S
HS
Department of Microbiology and Immunology, Cornell University, Ithaca, NY 14853-6401, USA.
Forster
Brian M
BM
Roberts
Kari L
KL
Chambers
Andrew J
AJ
Bitar
Alan Pavinski
AP
Marquis
Hélène
H
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D047468:Immunoprecipitation; D008089:Listeria monocytogenes / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D010446:Peptide Fragments / Q000235:genetics / Q000502:physiology*; D017354:Point Mutation; D055550:Protein Stability; D010738:Type C Phospholipases / Q000378:metabolism*
D001426:Bacterial Proteins; D010446:Peptide Fragments; D010738:Type C Phospholipases; C105598:phosphatidylcholine-specific phospholipase C; D008666:Metalloendopeptidases; C080367:mpl protein, Listeria monocytogenes