Membrane permeabilization by Listeria monocytogenes phosphatidylinositol-specific phospholipase C is independent of phospholipid hydrolysis and cooperative with listeriolysin O.
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, approximately 0.1 micron 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 microM lipid, minimal release of dye was observed on addition of 80 pM LLO or 7 nM 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.
Goldfine
H
H
Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia 19104-6076, USA.
Knob
C
C
Alford
D
D
Bentz
J
J
D001426:Bacterial Proteins / Q000378:metabolism; D001427:Bacterial Toxins; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002463:Cell Membrane Permeability; D005452:Fluoresceins / Q000032:analysis; D006360:Heat-Shock Proteins / Q000378:metabolism* / Q000494:pharmacology; D006460:Hemolysin Proteins; D006868:Hydrolysis; D007202:Indicators and Reagents; D007700:Kinetics; D008081:Liposomes; D008089:Listeria monocytogenes / Q000378:metabolism*; D008563:Membrane Lipids / Q000378:metabolism*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010743:Phospholipids / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism* / Q000494:pharmacology; D013997:Time Factors
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D005452:Fluoresceins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D007202:Indicators and Reagents; D008081:Liposomes; D008563:Membrane Lipids; D010743:Phospholipids; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; C038705:hlyA protein, Listeria monocytogenes; C007740:fluorexon