Discussion

It is currently believed that in bloodstream form (BF) T. brucei, the ATP/ADP carrier (TbAAC) transports cytosolic ATP generated from glycolysis into the mitochondrion, where it is hydrolyzed by FoF1-ATPase to maintain the essential mitochondrial (mt) membrane potential. Interestingly, BF parasites tolerate high doses of carboxyatractyloside (CATR), a specific inhibitor of AAC. Therefore, we explored the necessity of this transporter by generating BF TbAAC double knockout (DKO) cell lines, which display only a slight growth phenotype compared to their parental cells. While we further characterize these genetically modified trypansomes, we explored if the source of mt ATP doesn’t come from within the organelle by mt substrate phosphorylation, since a subunit of succinyl CoA synthetase (TbSCoAS) has already been established to be essential in BF parasites (Zhang X, Nucleic Acids Res (38) 2010). However, we again were able to generate TbSCoAS DKO cells that also demonstrate only a mild growth phenotype. We have determined that these cells have increased TbAAC expression and are now very sensitive to CATR. This suggests that these two proteins can compensate for each other in rich media, so we are characterizing the depletion of TbAAC in TbSCoAS DKO cell lines. Furthermore, it will be interesting to determine if the loss of fitness measured in culture for both DKO cell lines will be amplified in a mouse model.