Listeria monocytogenes is a gram-positive bacterial pathogenthat multiplies in the cytosol of host cells and spreads directlyfrom cell to cell. During cell-to-cell spread, bacteria becometemporarily confined to secondary vacuoles. The broad-rangephospholipase C (PC-PLC) of L. monocytogenes contributes tobacterial escape from secondary vacuoles. PC-PLC requires cleavageof an N-terminal propeptide for activation, and Mpl, a metalloproteaseof Listeria, is involved in the proteolytic activation of PC-PLC.Previously, we showed that cell wall translocation of PC-PLCis inefficient, resulting in accumulation of PC-PLC at the membrane-cellwall interface. In infected cells, rapid cell wall translocationof PC-PLC is triggered by a decrease in pH and correlates withcleavage of the propeptide in an Mpl-dependent manner. To addressthe role of the propeptide and of Mpl in cell wall translocationof PC-PLC, we generated a cleavage site mutant and a propeptidedeletion mutant. The intracellular behavior of these mutantswas assessed in pulse-chase experiments. We observed efficienttranslocation of the proform of the PC-PLC cleavage site mutantin a manner that was pH sensitive and Mpl dependent. However,the propeptide deletion mutant was efficiently translocatedinto host cells independent of Mpl and pH. Overall, these resultssuggest that Mpl regulates PC-PLC translocation across the bacterialcell wall in a manner that is dependent on the presence of thepropeptide but independent of propeptide cleavage. In addition,similarly to Mpl-mediated cleavage of PC-PLC propeptide, Mpl-mediatedtranslocation of PC-PLC across the bacterial cell wall is pHsensitive.



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