Parasite G6PD is a promising target for drug development against Leishmania

Abstract text

The enzyme glucose-6-phoshate dehydrogenase (G6PD) of the pentose phosphate pathway is required for redox regulation. As G6PD is found amongst numerous parasites causing neglected tropical diseases (NTD), including Leishmania (L.), identifying a drug to target G6PD in various Leishmania strains is a very promising strategy.

In order to genetically validate Leishmania G6PD as a potential drug target, we employed CRISPR/Cas9 to knockout this gene in both cutaneous and visceral Leishmaniasis (CL and VL) causing strains. Knock out of both alleles appeared to be lethal, viable parasites were only obtained while expressing Leishmania G6PD from a rescue plasmid. Loss of this plasmid resulted in parasite death, pointing to an essential role of G6PD for parasite survival. To further evaluate the role of G6PD in Leishmania, we established a CRISPR/Cas9/DiCre inducible knockout system. In this system parasites loose G6PD gene expression as well as G6PD enzymatic activity, over a time period of seve days resulting in both L. donovani and L. major full G6PD knockout strains. Interestingly wild type L. donovani contained a much higher level of G6PD activity as compared to wild type L. major parasites. By flow cytometry analysis, we could show that G6PD knockout decreases the parasites" tolerance against reactive oxygen species in the promastigote life stage of both species. In contrast to the viable L. major G6PD deficient axenic amastigotes, the L. donovani G6PD induced knockout strain was unable to survive as axenic amastigotes. Moreover preliminary data suggest that the L .major G6PD knockout had does not survive inside human macrophages. In summary, our data genetically validate G6PD as a promising drug target in both CL and VL causing Leishmania, and support the strategy to develop drugs for targeting multiple parasites causing NTDs.