title: Proteolytic pathways of activation and degradation of a bacterial phospholipase C during intracellular infection by Listeria monocytogenes.
abstract: Listeria monocytogenes is a facultative intracellular bacterial pathogen that spreads cell to cell without exposure to the extracellular environment. Bacterial cell-to-cell spread is mediated in part by two secreted bacterial phospholipases C (PLC), a broad spectrum PLC (PC-PLC) and a phosphatidylinositolspecific PLC (PI-PLC). PI-PLC is secreted in an active state, whereas PC-PLC is secreted as an inactive proenzyme (proPC-PLC) whose activation is mediated in vitro by an L. monocytogenes metalloprotease (Mpl). Analysis of PI-PLC, PC-PLC, and Mpl single and double mutants revealed that Mpl also plays a role in the spread of an infection, but suggested that proPC-PLC has an Mpl-independent activation pathway. Using biochemical and microscopic approaches, we describe three intracellular proteolytic pathways regulating PCPLC activity. Initially, proPC-PLC secreted in the cytosol of infected cells was rapidly degraded in a proteasome-dependent manner. Later during infection, PCPLC colocalized with bacteria in lysosome-associated membrane protein 1-positive vacuoles. Activation of proPC-PLC in vacuoles was mediated by Mpl and an Mpl-independent pathway, the latter being sensitive to inhibitors of cysteine proteases. Lastly, proPC-PLC activation by either pathway was sensitive to bafilomycin A1, a specific inhibitor of vacuolar ATPase, suggesting that activation was dependent on acidification of the vacuolar compartment. These results are consistent with a model in which proPC-PLC activation is compartment specific and controlled by a combination of bacterial and host factors.
authors.affiliation: Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: H
authors.initials: H
authors.lastname: Marquis
authors.forename: H
authors.initials: H
authors.lastname: Goldfine
authors.forename: D A
authors.initials: DA
authors.lastname: Portnoy
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D002460:Cell Line; D003600:Cytosol; D010450:Endopeptidases / Q000378:metabolism; D004789:Enzyme Activation; D008089:Listeria monocytogenes / Q000201:enzymology* / Q000472:pathogenicity; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D010713:Phosphatidylcholines / Q000378:metabolism; D013109:Sphingomyelins / Q000378:metabolism; D013347:Subcellular Fractions; D010738:Type C Phospholipases / Q000378:metabolism*; D014774:Virulence
chemical_list: D001426:Bacterial Proteins; D010713:Phosphatidylcholines; D013109:Sphingomyelins; D010738:Type C Phospholipases; D010450:Endopeptidases; D008666:Metalloendopeptidases; C080367:mpl protein, Listeria monocytogenes