Apoptosis in Crithidia fasciculata

By Kaelynn Parker and Abigail Delapenha

Faculty mentor: Professor Swati Agrawal

Crithidia fasciculata belongs to a group of parasites called kinetoplastids that comprise many important human pathogens. Evidence of apoptosis has been found in these parasites with pathways that appear to be different than in mammalian cells. Therefore, careful characterization of these pathways can provide ways to manipulate parasite infection which could be used to create better treatments for these diseases. In this study, potential apoptosis genes conserved across all kinetoplastid parasites were identified using gene prediction programs in Tri-TrypDB and BLAST searches. Homologous genes were identified in C. fasciculata and a comprehensive q-PCR analysis showed differential upregulation upon induction of apoptosis. One of the genes significantly changed was Bax1 inhibitory gene (Bax1i), an inhibitor of the putative apoptosis promoting Bax1. In order to characterize this gene further we made gene modification constructs for tagging and gene deletion using the CRISPR-Cas-9 system. A homologous repair template was created for Bax1i using 500 bp homology arms and a drug resistance gene using a fusion PCR protocol. Constructs were made using both Puromycin and Blasticidin resistance genes. We have successfully created and optimized the fusion PCR protocol for generation of 3.5Kb drug repair cassettes. The same process was repeated for Phosphoglycerate mutase family member 5 (PGAM5). Drug selection trials using Puromycin found the optimum concentration of drug is 50 µg/mL. Blasticidin trials are still being performed. The optimization of the fusion PCR protocol and drug selection procedure, along with the identification of genes done in this project will be important for continuing work.