An Investigation of 5-Fluorouracil Resistance in Leishmania and Trypanosoma species
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Abstract
Leishmaniasis is a parasitic vector-borne disease caused by the Leishmania parasite, which resides in female sandflies. African sleeping sickness or African trypanosomiasis is also a parasitic disease but it is spread by the tsetse fly (Glossina species). Chagas disease or American trypanosomiasis is a tropical disease caused by Trypanosoma cruzi and spread by insects called kissing bugs, Triatominae. Drug resistance has been one of the most important obstacles to the treatment of leishmaniasis and trypanosomiasis. For example, there has been evidence of resistance to melarsoprol and pentamidine for gHAT, and eflornithine for late stage HAT particularly in the T. b. rhodesiense, and pentavalent antimonials for leishmaniasis. This has limited the treatment options for these diseases. This has limited the treatment options for these diseases. Recent evidence has shown that pyrimidine metabolism is an excellent anti-protozoan drug development target, with multiple enzymes that are genetically essential. Pyrimidine nucleobase and nucleoside analogues have shown promising activity against Leishmania and Trypanosoma spp. Drugs like 5-fluorouracil and 5-fluoro-2’deoxyuridine are rapidly metabolized by the parasites into metabolic intermediates such as 5F-UDP-glucose, 5F-2’dUMP, 5F-UDP-galactose and 5F-UDP-N-acetylglucosamine, and incorporated into RNA. Pyrimidine analogue 5-FU was found to be a good inhibitor of high-affinity uracil transporters in T. b. brucei (TbU1 and TbU3) and Leishmania (LmajU1 and LmexU1).
Although the transporters for therapeutically active nucleobase allopurinol and antiparasitic nucleoside analogues have been identified, the transporter for 5-FU is still unknown. However, following the exclusion method, it is concluded that the 5-FU transporter is not an ENT transporter in Trypanosoma and Leishmania spps as their ENT transporters have all been cloned and characterised. Hence, our main interest is identifying the transporter gene (family) of kinetoplastids for pyrimidine nucleobases, using the antimetabolite 5-FU as a probe. It is expected that the 5-FU transporter is not of a gene family that has been previously associated with that activity. Resistance to 5-FU was generated in both T. b. brucei s427-wild type BSF and L. mexicana promastigotes, producing clonal lines Tbb-5FURes and Lmex-5FURes, respectively. RNA-seq and RIT-seq analyses of 5-FU resistant cell lines have identified candidate genes for pyrimidine transporters, including genes annotated as cation transporters (Tbb-CAT1-4), fatty acid desaturase (Tbb-FAD and Lmex-FAD) and glucose transporters. Apart from some of the glucose transporters, none of these potential transport genes have been previously characterised in protozoa and as such they are of interest in their own right as well.
Using the Alamar blue assay, the sensitivity to 5-FU in a single knockout of Tbb-CAT1-4 genes in T. b. brucei s427 WT cells was determined, and found to have no significant difference. Also, the results showed that [3H]-uracil uptake in T. b. brucei s427 WT + Tbb-CAT1-4+/- was almost the same as in wild type cells. Further, according to our results, the overexpression of Lmex-FAD gene in Lmex-5FURes and Tbb-FAD gene in Tbb-5FURes did not cause increased sensitivity to 5-FU in vitro, and similarly, did not change the rate of transport of [3H]-uracil. Following a full knockout of glucose transporter genes, their sensitivity to 5-FU was determined, revealing a significantly reduced sensitivity of the LmexGT1-3 double knockout genes in L. mexicana to 5-FU, in comparison to the wild type cell lines. Our results also revealed that the Lmex-GT1-3 KO cells do not accumulate 5-FU and uracil. In the re-expression of single LmexGT in Lmex-GT1-3 KO cells, the sensitivity to 5-FU increased significantly, but not quite back to the level of wild-type cells. After the introduction