Abstract 1508 Parasitoid Wasp Venom Pyruvate Kinase: Expression, Purification, and Kinetic Characterization

Parasitoid wasp infection of the model organism Drosophila melanogaster allows the study of the fly's innate immune responses and the wasp's manipulation of fly metabolism and immune response. Parasitoid wasps such as Ganaspis hookeri are obligate parasitoids and inject their eggs into Drosophila early third instar larvae to complete their life cycle. Upon wasp infection, fly immune cells differentiate into lamellocytes that surround the egg leading to melanization and death of the wasp. In addition to its egg, the wasp injects venom proteins that hinder immunity and manipulate metabolism to the wasp's advantage. During normal development, larvae accumulate fat and glycogen during the third instar larva. However, upon wasp infection, glycolysis is stimulated in immune cells and gluconeogenesis, glycogenolysis and fatty acid mobilization are stimulated in the fat body. Of the 168 venom proteins in G. hookeri venom, eight are glycolytic enzymes. In fact, the collection of glycolytic enzymes is complete with the exception of hexokinase and phosphofructokinase. The overall goal of our work is to determine the effects of Ganaspis hookeri venom glycolytic enzymes on the metabolism of wasp-infected larvae. This study focuses on pyruvate kinase (PyK), the enzyme responsible for the final step of turning phosphoenol pyruvate into pyruvate with the generation of ATP. Specifically, our goal is to express pyruvate kinase in E. coli and use the purified protein to compare the wasp and fly kinetic parameters, KM and Kcat. To express PyK, E. coli was transformed with the pyruvate kinase cDNA in the pET23a vector which attaches a C-terminal hexahistidine tag. After induction, lysates were prepared and SDS-PAGE and immunoblotting were used to verify the production of the appropriate protein. Nickel column chromatography was used to purify the fusion protein. MALDI and LC-MS/MS analyses verified that the expected full-length proteins were purified. Comparing kinetic parameters of wasp venom PyK, non-venom wasp PyK, and Drosophila PyK will allow us to assess the potential effects wasp venom PyK during larval infection. This work was carried out as a course-based undergraduate research (CURE) project at Siena College, Oklahoma Christian University, and UNC Pembroke, and externally funded by NIH R25 IPERT grant 1R25GM130528-01A1 to PIs R. Kelly and C. Goller at North Carolina State University and Co-PI M. Santisteban at UNC Pembroke, and NIH R35GM133760 to NTM.