title: An investigation of 5-fluorouracil resistance in Leishmania and Trypanosoma species
Creators: name: ALfayez, Ibrahim Abdullah
Publisher: name: University of Glasgow
Year: 2021

description: 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 of the glucose transporter genes, all the three genes did appear to have a very similar ability of functioning with regards to the (regulation of) uptake of 5-FU and uracil, restoring uptake to ~50% of 5-FU and ~30% of uracil uptake of wild type, respectively. It was also discovered that 5-FU and uracil did not have any measurable effects on the transport of glucose by LmexGT1, LmexGT2 and LmexGT3, an indication that none of them inhibits the transport of 0.1 μM of [3H]-2-deoxy-D-glucose up to 2.5 mM and therefore, the GTs are not themselves transporting uracil. We successfully expressed and characterized the FurD transporter in the 5-FU resistant cell lines (Tbb-5FURes and Lmex-5FURes) in order to investigate whether the sensitivity to 5-FU in vitro resistant strains could be restored by the introduction of a confirmed uracil/5-FU transporter. This would allow a functional screening of potential transporter genes. However, we found that the EC50 values of 5-FU of the 5-FU resistant cell lines and the FurD-expressing cell lines were not significantly different, although the expression of FurD in Lmex-5FURes induced a very high level of [3H]-uracil/5-FU uptake, even much above the wild type activity. We also characterised the transport activity of FurD in Lmex-5FURes promastigotes, and found FurD to be a highly selective and high-affinity transporter for uracil with Km of 0.97 ± 0.17 μM. Interestingly, our results confirmed that the anticancer drug 5-FU was as good a substrate as uracil for FurD in Lmex-5FURes, with a Ki of 0.76 ± 0.25 μM. Using a targeted CRISPR-Cas9 gene knockout strategy, we show that deletion of the LmexNT1 locus in L. mexicana-Cas9 promastigotes completely abolished adenosine and thymidine uptake. Moreover, it became highly resistant to tubercidin (and its analogues) and to 5-fluoro-2’-deoxyuridine. We also tested the possibility of using L. mexicana-Cas9ΔNT1 promastigotes as a surrogate system for the expression of TcrNT2 and TcrNB2 transporters of T. cruzi. We found TcrNT2 to be a high-affinity thymidine transporter with a Km 0.156 ± 0.017 μM, while TcrNB2 could not be characterised despite our efforts. To conclude, the results obtained in this study provide significant contributions to the identification of the pyrimidine transporter genes. The results also increase our understanding of the cytotoxic activity of 5-FU in kinetoplastid parasites, gaining insight into the complex pyrimidine metabolism that occurs in these parasites. In addition, the study shows that pyrimidine transport mechanisms could potentially be exploited as drug carriers against kinetoplastid parasites.