As protozoan organisms adapted to parasitism, they became unable to synthesise the purine ring using the de novo pathway, relying exclusively on the uptake of these nutrients from their hosts, using high affinity transporters. To date, only genes encoding for members of the Equilibrative Nucleoside Transporter (ENT) family have been associated with purine or pyrimidine uptake in these organisms, and are believed to play a major role in nucleobase and nucleoside transport in protozoan parasites. Given the requirement of these organisms to transport purines with high efficiency, the ENT transporters became potential carriers for chemotherapeutic molecules. T. brucei, the causative agent of sleeping sickness, is the best studied protozoan parasite in terms of purine and pyrimidine transport functions, and although a dozen genes have been isolated and characterized as members of the ENT family, the susceptibility of these parasites against melamine-containing molecules, even after the genetic knockout of its target carrier, the P2 transporter, suggested the existence of as yet uncharacterised purine transport mechanisms in these cells. Using a combination of gene knockouts, we discovered that bloodstream forms of T. brucei possess two previously undescribed transporters: HXT1 and ADET1, with exclusive affinity for hypoxanthine or adenine, respectively. HXT1 was characterised as a medium-affinity hypoxanthine transporter, with a Km of 22 ± 1.7 μM and Vmax of 0.49 ± 0.06 pmol.(107 cells)-1s-1 for this nucleobase, whereas ADET1 is a high-affinity adenine transporter, showing a Km of 573 ± 62 nM and Vmax of 0.23 ± 0.06 pmol.(107 cells)-1s-1. Neither HXT1 nor ADET1 showed any affinity for any other natural purine or pyrimidine, and could not be completely inhibited by hypoxanthine or adenine analogues either. Given the unprecedented specificity for their substrates, and the fact that all T. brucei ENT transporters have previously been studied, it is likely that HXT1 and ADET1 are not part of the ENT family and might indicate the presence of other nucleoside transporter family in these parasites. Different from T. brucei, and despite being the aetiological agent of Chagas’ disease, no systematic characterisation of T. cruzi ENT transporters has been done. We therefore decided to use a genetically adapted T. brucei procyclic cell line (TbNBT-KO) with reduced (~86%) [3H]-Hypoxanthine uptake as a surrogate system to express and characterise T. cruzi transporters. We successfully expressed and characterized three out of four ENT genes from the T. cruzi Y strain and showed that its transporters have higher affinity for oxopurines than for aminopurines. TcrNBT1 was shown to be a very high-affinity hypoxanthine/guanine transporter (Km of 93.8 ± 4.7 nM for hypoxanthine and Ki of 121.9 ± 22.4 nM for guanine), with a Ki of 3.7 ± 0.5 μM for adenine. TcrNBT1 harboured lower affinity for purine nucleosides and poor affinity for pyrimidines. In contrast, TcrNT1 was found to be a high-affinity inosine/guanosine transporter with a Km of 1.0 ± 0.03 μM for inosine and Ki of 0.92 ± 0.2 μM for guanosine. Interestingly, TcrNT1 showed higher affinity for hypoxanthine (Ki = 23.9 ± 5.5 μM) than for adenosine (Ki = 38.9 ± 5.8 μM) and virtually no affinity for other purines or pyrimidines. Different from the other two, TcrNT2 turned out to be a high-affinity thymidine transporter (Km = 223.5 ± 7.1 nM), also displaying some affinity for uridine and cytidine (Ki values of 66 ± 6 and 728 ± 70 μM), but barely sensitive to inhibition by pyrimidine nucleobases or purines. The fourth ENT gene cloned from T. cruzi (TcrNB2) could not be characterised despite our efforts, but its genomic and structural similarity to Leishmania NT4 transporters indicate it might be a low-affinity purine nucleobase transporter. We further used TbNBT-KO to characterise a codon-adapted Plasmodium falciparum ENT1 transporter and, different from previous reports, found it to be a medium-affinity guanine/hypoxanthine transporter, with a Km of 11.22 ± 1.18 μM for guanine. The existence of high-affinity purine transport mechanisms in trypanosomatids also led us to test the potential of C7-substituted tubercidin analogues against African trypanosomes (T. brucei and Trypanosoma congolense). We verified that, although the size of the substituent is not a determinant for transport into the cell, analogues containing small substitutions yielded the highest trypanocidal effects against wildtype and drug-resistant T. brucei strains, as well as against wildtype T. congolense, with special regard to 3’-deoxyanalogues. We verified that the 3’-deoxytubercidin analogues are likely to exert their trypanocide effect by inhibiting the synthesis of nucleic acids, whereas their ribonucleoside counterparts probably act like tubercidin, by inhibiting the glycolytic pathway. Our findings contribute to the understanding of purine and pyrimidine transport mechanisms in trypanosomes, reports the potential existence of an as yet unreported nucleobase transporter family in trypanosomatids, and shows the potential of TbNBT-KO as a model cell line for the systematic characterisation of ENT genes from other protozoans. Moreover, we show the potential exploitation of the purine transport mechanisms as carriers of drugs against trypanosomatids.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:744204 |
| Date | January 2018 |
| Creators | Campagnaro, Gustavo Daniel |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/30603/ |
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