Strategies for the identification and cloning of genes encoding pyrimidine transporters of Leishmania and Trypanosoma species

Alzahrani, Khalid Jamaan H. (2017) Strategies for the identification and cloning of genes encoding pyrimidine transporters of Leishmania and Trypanosoma species. PhD thesis, University of Glasgow.

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Abstract

In recent years, it has become clear that pyrimidine metabolism is an excellent target for anti-protozoan drug development, with multiple enzymes genetically validated as essential. Pyrimidine nucleobase and nucleoside analogues with promising activity against Leishmania and Trypanosoma species can be readily identified. Well-known drugs like 5-fluorouracil and 5-fluoro-2’deoxyuridine are rapidly metabolised by the parasites and incorporated into RNA and metabolic intermediates such as 5F-2’dUMP and UDP-glucose. We thus see that pyrimidine salvage is a major vulnerability in protozoa, with non-redundant pathways sensitive to inhibition by small pyrimidine analogues. Currently, the main factor slowing down progress towards actual therapeutic exploitation of this weakness is the lack of information on the transport proteins for pyrimidines that need to efficiently internalise the pyrimidine analogues into the parasites. The De Koning laboratory cloned the first protozoan purine nucleobase transporter in 2003 and here we report strategies to similarly identify the pyrimidine transporter family. We characterized thymidine transport in L. major promastigotes in order to establish which transporter is responsible for its uptake. We found two separate thymidine transport activities represent LmajNT1.1 and LmajNT1.2, with Km values of 4.2 mM and 26.9 mM, respectively. In addition, to verify the reported L. donovani model of two NT1-like genes encoding uridine/adenosine transporters, and an NT2 gene encoding an inosine transporter, we cloned the L. major and L. mexicana ENT-family genes that are syntenic with the L. donovani nucleoside transporters, expressing each in T. brucei for individual characterisation. Consistent with the L. donovani reports, the NT1-like genes of either species mediated the adenosine-sensitive uptake of [3H]-uridine but not of [3H]-inosine. Conversely, the NT2-like genes mediated the uptake of [3H]-inosine but not [3H]-uridine. These results showed that the organisation of nucleoside transport is strictly conserved among Leishmania species - a conclusion that has important implications for the development of a nucleoside-based chemotherapy. Comparative sequencing of wild-type strains and 5-fluorouracil-resistant lines of T. b. brucei and L. mexicana using RNA-seq was performed in order to identify the candidate pyrimidine transporter genes downregulated in the resistant lines. We found 17 candidate pyrimidine transporter genes encoding for at least 3-TM domains that were down-regulated in both 5-fluorouracil-resistant lines. We overexpressed the most promising candidate genes in Tbb-5FURes and Lmex-5FURes cell lines, aiming to determine their contribution to the 5-FU uptake and resistance. Moreover, RNAi library screening for T. brucei for 5-FU and 6-AU resistance, with and without prior disruption of pyrimidine biosynthesis, was performed. Data generated by RIT-seq was compared to the RNA-seq results. Several candidate pyrimidine transporter genes were highlighted by the returned data. In conclusion, our results make several noteworthy contributions toward the eventual identification of the pyrimidine transporter gene and, by increasing our understanding of 5-FU toxicity in kinetoplastids, we gain insight into the complexities of pyrimidine metabolism in these parasites.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Trypanosomabrucei, Leishmania, pyrimidine transporters.
Subjects: Q Science > QR Microbiology
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Supervisor's Name: De Koning, Professor Harry
Date of Award: 2017
Embargo Date: 2 November 2020
Depositing User: Mr Khalid Jamaan Alzahrani
Unique ID: glathesis:2017-8574
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 08 Nov 2017 10:53
Last Modified: 15 Nov 2017 12:27
URI: http://theses.gla.ac.uk/id/eprint/8574
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