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



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