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Infection and Immunity, January 2007, p. 44-51, Vol. 75, No. 1
0019-9567/07/$08.00+0     doi:10.1128/IAI.01001-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Compartmentalization of the Broad-Range Phospholipase C Activity to the Spreading Vacuole Is Critical for Listeria monocytogenes Virulence

P. S. Marie Yeung, Yoojin Na, Amanda J. Kreuder, and Hélène Marquis^*

Department of Microbiology and Immunology, Cornell University, Ithaca, New York 14853-6401

Received 23 June 2006/ Returned for modification 21 August 2006/ Accepted 12 October 2006

Listeria monocytogenes is a bacterial pathogen that multiplies in the cytosol of host cells and spreads directly from cell to cell by using an actin-based mechanism of motility. The broad-range phospholipase C (PC-PLC) of L. monocytogenes contributes to bacterial escape from vacuoles formed upon cell-to-cell spread. PC-PLC is made as an inactive proenzyme whose activation requires cleavage of an N-terminal propeptide. During infection, PC-PLC is activated specifically in acidified vacuoles. To assess the importance of compartmentalizing PC-PLC activity during infection, we created a mutant that makes constitutively active PC-PLC (the plcBpro mutant). Results from intracellular growth and cell-to-cell spread assays showed that the plcBpro mutant was sensitive to gentamicin, suggesting that unregulated PC-PLC activity causes damage to host cell membranes. This was confirmed by the observation of a twofold increase in staining of live infected cells by a non-membrane-permeant DNA fluorescent dye. However, membrane damage was not sufficient to cause cell lysis and was dependent on bacterial cell-to-cell spread, suggesting that damage was localized to bacterium-containing filopodia. Using an in vivo competitive infection assay, we observed that the plcBpro mutant was outcompeted up to 200-fold by the wild-type strain in BALB/c mice. Virulence attenuation was greater when mice were infected orally than when they were infected intravenously, presumably because the plcBpro mutant was initially outcompeted in the intestines, reducing the number of mutant bacteria reaching the liver and spleen. Together, these results emphasize the importance for L. monocytogenes virulence of compartmentalizing the activity of PC-PLC during infection.

Published ahead of print on 23 October 2006.

Editor: V. J. DiRita

Present address: Biological Sciences Department, California Polytechnic State University, San Luis Obispo, CA 93407-0401.

Present address: Iowa State University, College of Veterinary Medicine, Ames, IA 50011.