Membrane permeabilization by Listeria monocytogenes phosphatidylinositol-specific phospholipase C is independent of phospholipid hydrolysis and cooperative with listeriolysin O.

Abstract

We have examined potential cooperative interactions of Listeria monocytogenes phosphatidylinositol-specific phospholipase C (PI-PLC) and listeriolysin O (LLO), a pore-forming hemolysin, in a liposome lysis assay, Large unilamellar vesicles, approximate to 0.1 mu m in diameter, encapsulating the fluorescent probe calcein, were treated with PI-PLC or LLO at pH 6.0, and each was capable of causing dye release, With phosphatidylcholine/phosphatidylinositol/cholesterol liposomes at 0.1 mu M lipid, minimal release of dye was observed on addition of 80 pM LLO or 7 nhl PI-PLC, Addition of the two proteins together produced rapid dye release, Unexpectedly, essentially identical results were obtained with phosphatidylcholine/cholesterol liposomes, Thus, the effect of PI PLC did not depend on lipid hydrolysis, Both proteins also released inulin (M(r) 5200) from liposomes, Membrane permeabilization was not accompanied by membrane fusion, Very little dye release from phosphatidylcholine/phosphatidylinositol/cholesterol liposomes was seen with PI-PLC from Bacillus thuringiensis, and addition of this enzyme to LLO produced no additional dye release; however PI-PLC from L. monocytogenes cooperated with perfringolysin O from Clostridium perfringens, PI-PLC from L. monocytogenes and LLO bind to phosphatidylcholine/cholesterol liposomes, and the rate of binding of each protein was not influenced by the presence of the other, These data support a postulated accessory role for PI-PLC with LLO in lysing the primary phagosome of a macrophage.