Restricted translocation across the cell wall regulates secretion of the broad-range phospholipase C of Listeria monocytogenes.
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.
Snyder
Aleksandra
A
Department of Microbiology, University of Colorado Health Sciences Center, School of Medicine, Denver, Colorado 80262, USA.
Marquis
Hélène
H
D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002473:Cell Wall / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008089:Listeria monocytogenes / Q000201:enzymology* / Q000254:growth & development; D008264:Macrophages / Q000382:microbiology; D051379:Mice; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism*
D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins; D010738:Type C Phospholipases; C105598:phosphatidylcholine-specific phospholipase C