pH-regulated activation and release of a bacteria-associated phospholipase C during intracellular infection by Listeria monocytogenes.
Listeria monocytogenes grows in the cytosol of mammalian cells and spreads from cell to cell without exiting the intracellular milieu. During cell-cell spread, bacteria become transiently entrapped in double-membrane vacuoles. Escape from these vacuoles is mediated in part by a bacterial phospholipase C (PC-PLC), whose activation requires cleavage of an N-terminal peptide. PC-PLC activation occurs in the acidified vacuolar environment. In this study, the pH-dependent mechanism of PC-PLC activation was investigated by manipulating the intracellular pH of the host. PC-PLC secreted into infected cells was immunoprecipitated, and both forms of the protein were identified by SDS-PAGE fluorography. PC-PLC activation occurred at pH 7.0 and lower, but not at pH 7.3. Total amounts of PC-PLC secreted into infected cells increased several-fold over controls within 5 min of a decrease in intracellular pH, and the active form of PC-PLC was the most abundant species detected. Bacterial release of active PC-PLC was dependent on Mpl, a bacterial metalloprotease that processes the proform (proPC-PLC), and did not require de novo protein synthesis. The amount of proPC-PLC released in response to a decrease in pH was the same in wild-type and Mpl-minus-infected cells. Immunofluorescence detection of PC-PLC in infected cells was performed. When fixed and permeabilized infected cells were treated with a bacterial cell wall hydrolase, over 97% of wild-type and Mpl-minus bacteria stained positively for PC-PLC, in contrast to less than 5% in untreated cells. These results indicate that intracellular bacteria carry pools of proPC-PLC. Upon cell-cell spread, a decrease in vacuolar pH triggers Mpl activation of proPC-PLC, resulting in bacterial release of active PC-PLC.
Marquis
H
H
Department of Microbiology, University of Colorado Health Sciences Center, School of Medicine Box B-175, 4200 E. 9th Avenue, Denver, CO 80262, USA. helene.marquis@uchsc.edu
Hager
E J
EJ
D000818:Animals; D002460:Cell Line; D010450:Endopeptidases / Q000494:pharmacology; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008089:Listeria monocytogenes / Q000201:enzymology* / Q000254:growth & development*; D008666:Metalloendopeptidases / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D011233:Precipitin Tests; D010738:Type C Phospholipases / Q000378:metabolism*; D014617:Vacuoles / Q000382:microbiology
D010738:Type C Phospholipases; D010450:Endopeptidases; D008666:Metalloendopeptidases; C021816:mutanolysin