By Hannah Lee & Faith St. Clair
Faculty mentor: Dr. Swati Agrawal
Kinetoplastids are a group of pathogenic, unicellular parasites that can cause fatal diseases in humans, such as Leishmaniasis and sleeping sickness, most prominent in tropical and developing parts of the world. Although some treatment methods are available for parasitic infections, they are often not optimal and can induce side effects. Leishmania tarentolae is a nonpathogenic Kinetoplastid that can be used as a model organism in the lab to study possible treatment methods. A major asset to the parasites’ survival is apoptosis which allows for adaptation to the environment as well as avoidance of immune detection by the host. Phosphoglycerate mutase family member 5 (PGAM5) is a metabolic enzyme that has been suspected to play a role in cell death as a pro-apoptotic gene in L. tarentolae. This dodecamer protein regulates mitochondrial homeostasis and is associated with the development of diverse diseases. A gene replacement strategy using the CRISPR-cas9 system followed by homology directed replacement at the PGAM5 locus was used. PF16, a flagellar motility factor in L. tarentolae that is crucial for promastigote motility, was used as a control in the experiment. Interference with the genes encoding for PF16 render the parasite immobile and increase drug susceptibility. Deletion of PF16 has been found to show significant phenotypic changes in the parasite. After verifying the sequences by sequencing, the PF16 and PGAM5 constructs will be transfected into L. tarentolae. Once a knockout clone is isolated, knockout parasites will be observed and compared to wild type cells to understand the role of PGAM5 in the apoptosis pathway. We hypothesize that the absence of PGAM5 will inhibit apoptosis in the parasite, reducing its rate of survival which could possibly create an alternative treatment method for parasitic infections.
