Compartmentalization of the Broad-Range Phospholipase C Activity to the Spreading Vacuole Is Critical forListeria monocytogenesVirulence

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Abstract
Listeria monocytogenesis 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) ofL. monocytogenescontributes 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 (theplcBΔpro mutant). Results from intracellular growth and cell-to-cell spread assays showed that theplcBΔpro 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 theplcBΔpro 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 theplcBΔpro mutant was initially outcompeted in the intestines, reducing the number of mutant bacteria reaching the liver and spleen. Together, these results emphasize the importance forL. monocytogenesvirulence of compartmentalizing the activity of PC-PLC during infection.

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