Development of a multiplexed assay in kinetoplastid parasites to identify probes for glycolysis

Clemson University, Clemson SC

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Abstract

Project Summary/Abstract This Diversity Supplement to the award R01AI156382, "Development of a multiplexed assay in kinetoplastid parasites to identify probes for glycolysis” will support the training of Sabrina Pizarro, a Clemson University graduate student in the Department of Genetics and Biochemistry that is from the Catawba Indian Nation, an under-represented group in the sciences. The goal of this project is two-fold. First, the project will support Sabrina’s research effort to develop and validate new molecular tools for use in the African trypanosome, Trypanosoma brucei. Sabrina will generate procyclic form (PF) and bloodstream form (BF) T. brucei parasites that express a protein pH biosensor based on pHluorin2. pHluorin2 is a ratiometric pHluorin variant that displays a pH (acid) dependent excitation that can be readily monitored by flow cytometry and microscopy. PF and BF parasites expressing these sensors will be used to resolve cellular mechanisms of acidification or the subcellular organelle that houses glycolysis, the glycosome. The pH sensing cell lines will serve an additional purpose, playing a role in the work described in the parent proposal. The pHluorin2 lines will be used as a counterscreen, allowing us to discriminate small molecules that influence glycolytic enzyme activity by disrupting cellular or glycosomal pH from those inhibitors that more directly inhibit enzymes in the pathway. The second goal of the project is to support Sabrina’s professional development as she works to achieve her career goals. Through interactions with the PI and co-mentor, along with participation in a variety of professional development opportunities, we anticipate that Sabrina will be fully positioned to successfully pursue the next step in her career path.