Authors

Discussion

Several members of the ADP-ribosylation factor (ARFs) family of the small GTPases are known to be essential for viability in T. brucei bloodstream form cells. However, the molecular interactions of these proteins have not been fully characterised in T. brucei and there is a high level of identity shared between T. brucei and human ARF protein sequences, thus impacting on their potential as drug targets. An alternative method of indirectly targeting of ARFs may be through their regulators, the guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). These regulating molecules are responsible for maintaining the active/inactive state ARFs and are highly divergent in T. brucei. The aim of this study is to identify ARF regulators in T. brucei and to investigate whether they are essential in the parasite using RNAi. Further studies will determine which ARFs are regulated by these proteins, towards development of an assay for inhibitor screening. Two TbGEFs and Two TbGAPs have been identified using bioinformatics and through RNAi studies it was determined that TbGEF3 is essential for bloodstream form T. brucei viability. Following RNAi knockdown, cells exhibited the Big Eye phenotype, suggesting a defect in endocytosis. Flow cytometry was used to demonstrate that RNAi of TbGEF3 has a cytotoxic effect on BSF T. brucei, thus indicating an essential role of this protein in parasite viability.