Purification of Sphingosine Kinase A, a Potential Drug Target, in Leishmania Parasites

Leishmania parasites are eukaryotic pathogens responsible for a spectrum of diseases (leishmaniasis) infecting both humans and animals. Transmitted by sandflies, these parasites are endemic in many tropical and subtropical areas. Current treatments are plagued with low efficacy, high cost, and strong side effects. Without a safe vaccine, there is a substantial need to develop new drugs and discover new drug targets.

Sphingosine kinase (SK) is an enzyme that catalyzes the formation of sphingosine-1-phosphate (S1P) from sphingosine. In mammals, S1P is an important sphingolipid metabolite with regulatory roles in diverse cellular processes including cell survival, growth, and differentiation. We recently identified a type A SK (SKa) in Leishmania major and generated a SKa-knockout (ska¯) strain through targeted gene replacement. Ska¯ parasites exhibited severe growth defects in vitro and failed cause disease in animals. The predicted amino acid sequence of SKa differs substantially from those of mammalian SKs. The activity of SKa can be determined using highthroughput assays. Based on these findings, we hypothesize that SKa may be a potential drug target.  In this study, we overexpressed SKa in E. coli using an expression vector (pGEX-4T-2). Our goal is to isolate SKa via affinity purification and characterize its biochemical properties. Future studies will identify specific SKa inhibitors that can serve as treatments for leishmaniasis.