Phosphoinositide-specific phospholipase C (PI-PLC) isozymes play a central role in receptor-mediated signal transduction in mammalian cells, cleaving the membrane lipid phosphatidylinositol 4,5-biphosphate to generate two second messengers, inositol 1,4,5-triphosphate and diacylglycerol. PI-PLCs are a large family of enzymes, and are also produced by parasitic protozoa including Trypanosoma brucei and the malaria-causing Plasmodium falciparum, by Drosophila, yeast, and plants. The smallest PI-PLCs are phosphatidylinositol-specific, i.e. only unphosphorylated PI is cleaved. They are secreted by the Gram plus bacteria Bacillus cereus and the nearly identical B. thuringiensis, by Staphylococcus aureus, which causes toxic-shock syndrome and related illnesses in humans, and by Listeria monocytogenes, responsible for bacterial infections in pregnant women, newborn children, and immunocompromised adults. The bacterial PI-PLCs are implicated virulence factors in these diseases. Bacterial PI-PLCs share with T. brucei PI-PLC the ability to cleave glycosylphosphatidylinositol (GPI) anchors, and are widely used to release mammalian GPI-anchored proteins. This laboratory has previously cloned, overexpressed, and solved the crystal structure of the B. cereus PI-PLC in informal collaboration with others in the Institute, and, in collaboration with Dr. John F. W. Keana, initiated the synthesis of inhibitors and substrate analogs. We now plan to complete mapping the active site region of B. cereus PI-PLC, to use this enzyme as a model system for the catalytic domain of mammalian PI-PLCs, and to examine related bacterial PI-PLCs. Specifically, we plan to complete the identification of residues of B. cereus PI-PLC important in substrate binding and catalysis through a combination of site-directed mutagenesis, crystallography, and enzyme kinetics. New fluorgenic substrate analogs will be synthesized in collaboration with Dr. Keana, to provide a sensitive continuous assay for the kinetic studies. Inhibitors, already available through this collaboration, will be used to identify which residues interact with the phosphoinositol, glycerol, and lipid groups of the substrate and to determine the effect on the pKa of catalytically critical residues by NMR. To exploit the fact that B. cereus PI-PLC closely resembles the catalytic domain of mammalian PI-PLC delta 1, the bacterial isozyme will be used as a model system, substituting critical mammalian segments into the enzyme to alter its substrate specificity. Finally, the new substrate analogs and inhibitors will be used in a comparative study of the PI-PLC virulence indicators of S. aureus and L. monocytogenes.
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