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David A. Revelli, Ph.D.

Vasil Lab


 

THESIS


Complete Title of Thesis:
"Purification and Characterization of the Nonhemolytic Phospholipase C of Pseudomonas aeruginosa"

Prepared under the direction of:  Michael Vasil, Ph.D.


SUMMARY

This work describes the purification and characterization of the nonhemolytic phospholipase C (PlcN) of the opportunistic pathogen, Pseudomonas aeruginosa. For these studies, PlcN was purified to near homogeneity from E. coli through the use of affinity chromatography in a one step purification using the Strep-Tag II affinity tag. The resulting recombinant enzyme activity was compared to the native enzyme activity to evaluate equivalency of the two enzymes. Various attributes of PlcN, using the recombinant enzyme, such as pH optima, temperature optima, cation effects, and cellular localization were determined. In vitro substrate specificity as well as substrate affinity was determined using multiple lipid substrates at varying substrate aggregation states. Finally, localization, cell cytotoxicity, and the ability of PlcN to hydrolyze phospholipids in the context of a cellular membrane were studied. The studies determined that the native and recombinant enzymes had equivalent activity. PlcN exhibited the same behavior as PlcH in the presence of various cations suggesting not only a shared mechanism of catalysis but also one different from other classes of phospholipases C. Optimal pH of PlcN was determined to be between 6 and 6.5, but PlcN exhibited activity over a broad range of pH values (pH 5-8.5). This suggests PlcN would be stable over a broad range of pH values and have optimal activity in environments, such as, intracellular endosomes. In substrate specificity studies, PlcN exhibited activity on phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE) but not on sphingomyelin (SM) or phosphatidylglycerol(PG). Mixed micelle experiments showed that the order of affinity for the substrates of PIcN is PE>PS>>PC. The preferred substrates of PlcN, PE and PS, are major constituents of the inner leaflet of the plasma membrane. This suggests that the outer leaflet has to be breached first before PlcN can access its preferred substrates. The cytotoxicity of PlcN was evaluated on a number of cell lines and it was concluded that PlcN is not cytotoxic, at least for those cell lines, suggesting that PlcN is not capable of breaching the outer leaflet of the membrane by itself. Further studies were performed to determine if PlcN was able to increase the leakage of cellular contents when its preferred substrates were present on the outer leaflet after a membrane symmetrizing event, such as, apoptosis. Under the conditions of the experiments, PlcN was unable to increase cytolysis or diacylglycerol (DAG) through hydrolysis of membrane phospholipids. Finally, PlcN was determined to associate with membranes and be secreted from P. aeruginosa in membrane vesicles that elaborate from the surface of the bacteria. This represents a method of secretion that may deliver PlcN directly to the surface of eukaryotic cells. While these studies still give little prediction as to the elusive role of PlcN in pathogenesis, they demonstrate that PlcN is a stable enzyme, capable of activity in numerous environments. They also suggest that, while PlcN is not cytotoxic, it may nave other roles in colonization or invasion. The purified PlcN will be an invaluable tool to help further understanding of the role of PlcN in pathogenesis.