Trypanosoma brucei genome-wide RNAi library screen of methotrexate.

The aim of this study was to identify and characterise mechanisms of resistance to antifolate drugs in African trypanosomes. A genome-wide RNAi library screen was undertaken in bloodstream form Trypanosoma brucei exposed to the antifolate methotrexate. RNAi knockdown in conjunction with drug susceptibly and folate transport studies were used to validate the function of the putative folate transporters. RNA interference Target sequencing (RIT-seq) experiments identified putative folate transporters, Tb927.8.3620, Tb927.8.3630 and Tb927.8.3650 (TbFT1-3), as major contributors to antifolate drug resistance. RNAi knockdown of TbFT1-3 substantially reduced folate transport into cells and reduced the cells susceptibly to the classical antifolate methotrexate. In conjunction with other data, this shows that TbFT1-3 mediate the transport of methotrexate in trypanosomes and TbFT1-3 loss-of-function is a mechanism of anti-folate drug resistance.