Discussion

Most protozoa are capable of both salvaging performed pyrimidines and de novo pyrimidine biosynthesis. This study seeks to identify the gene (family) encoding the protozoan pyrimidine transporters using next-generation RNA-sequencing (RNA-seq). Resistance to 5-fluorouracil (5-FU) was generated in both T. b. brucei BSF s427- wild type and Leishmania mexicana promastigotes, yielding clonal lines Tbb-5FURes and Lmex-5FURes, respectively. RNA-seq was performed to identify and characterize the differentially expressed genes between the sensitive and resistant strains. Five folate/pteridine transporters genes (TbbPters) out of nine were down-regulated in Tbb-5FURes, and two folate/pteridine genes (LmexPter) out of eleven genes were down-regulated in Lmex-5FURes. Interestingly, overexpression of TbbPter1 (Tb927.1.2820) in Tbb-5FURes caused a 3-fold increase in 5-FU sensitivity (P< 0.0001). Overexpression of TbbPter1, TbbPter3, TbbPter4 and TbbPter5 in Tbb-5FURes revealed a statistically significant decrease (P<0.05) in sensitive to 6-azauracil, indicating a restored uracil salvage pathway and decreasing reliance on de novo synthesis. Characterization of LmexPters and generation of double gene deletions of TbbPters (dKO) is ongoing, and transport and sensitivity assays will be performed. Identification of pyrimidine transporter genes in protozoa will aid in the therapeutic targeting of pyrimidine metabolism of protozoa.