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Restricted Translocation across the Cell Wall Regulates Secretion of the Broad-Range Phospholipase C of Listeria monocytogenes

Department of Microbiology, University of Colorado Health Sciences Center, School of Medicine, Denver, Colorado 80262,¹ Department of Microbiology and Immunology, Cornell University, Ithaca, New York 14853²

Received 26 March 2003/ Accepted 30 July 2003

The virulence of Listeria monocytogenes is directly related to its ability to spread from cell to cell without leaving the intracellular milieu. During cell-to-cell spread, bacteria become temporarily confined to secondary vacuoles. Among the bacterial factors involved in escape from these vacuoles is a secreted broad-range phospholipase C (PC-PLC), the activation of which requires processing of an N-terminal prodomain. Mpl, a secreted metalloprotease of Listeria, is involved in the proteolytic activation of PC-PLC. We previously showed that, during intracellular growth, bacteria maintain a pool of PC-PLC that is not accessible to antibodies and that is rapidly released in its active form in response to a decrease in pH. pH-regulated release of active PC-PLC is Mpl dependent. To further characterize the mechanism regulating secretion of PC-PLC, the bacterial localization of PC-PLC and Mpl was investigated. Both proteins were detected in the bacterial supernatant and lysate with no apparent changes in molecular weight. Extraction of bacteria-associated PC-PLC and Mpl required cell wall hydrolysis, but there was no indication that either protein was covalently bound to the bacterial cell wall. Results from pulse-chase experiments performed with infected macrophages indicated that the rate of synthesis of PC-PLC exceeded the rate of translocation across the bacterial cell wall and confirmed that the pool of PC-PLC associated with bacteria was efficiently activated and secreted upon acidification of the host cell cytosol. These data suggest that bacterially associated PC-PLC and Mpl localize at the cell wall-membrane interface and that translocation of PC-PLC across the bacterial cell wall is rate limiting, resulting in the formation of a bacterially associated pool of PC-PLC that would readily be accessible for activation and release into nascent secondary vacuoles.

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