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pH‐regulated activation and release of a bacteria‐associated phospholipase C during intracellular infection by Listeria monocytogenes
Author(s) -
Marquis Hélène,
Hager Elizabeth J.
Publication year - 2000
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2000.01708.x
Subject(s) - intracellular , biology , listeria monocytogenes , vacuole , cytosol , bacteria , phagosome , phospholipase , phospholipase c , intracellular parasite , biochemistry , bacterial cell structure , microbiology and biotechnology , cell membrane , cell , enzyme , cytoplasm , genetics
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.