pH-regulated activation and release of a bacteria-associated phospholipase C during intracellular infection by Listeria monocytogenes.

Abstract:

: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.

journal_name

Mol Microbiol

journal_title

Molecular microbiology

authors

Marquis H,Hager EJ

doi

10.1046/j.1365-2958.2000.01708.x

subject

Has Abstract

pub_date

2000-01-01 00:00:00

pages

289-98

issue

2

eissn

0950-382X

issn

1365-2958

pii

mmi1708

journal_volume

35

pub_type

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