Authors
G D Campagnaro1; H P De Koning1; 1 Institute of Infection, Immunity & Inflammation, University of Glasgow, UK Discussion
Trypanosoma cruzi, the causative agent of Chagas’ disease, is a kinetoplastid parasite endemic to the American continent and infects around 6 to 7 million people. As a kinetoplastid, T. cruzi is unable to synthesise its own purines and relies on salvage from the host, but its transport mechanisms have not yet been characterised. In other protozoa, purine uptake is mediated by Equilibrative Nucleoside Transporters (ENTs). To investigate whether this is also the case in T. cruzi, its four ENT genes (provisionally named TcrNBT1, TcrNBT2, TcrNT1 and TcrNT2) were cloned and expressed in a Trypanosoma brucei procyclic cell line from which a cluster of three high-affinity nucleobase transporters was deleted (TbNBT-KO), and which displayed reduced (~86%) [3H]-Hypoxanthine uptake. TcrNBT1 was shown to be a very high affinity oxopurine nucleobase transporter with a Km of 93.8 ± 4.7 nM for hypoxanthine and a Kiof 121.9 ± 22.4 nM for guanine, while the Kifor adenine was determined as 3.7 ± 0.5 µM. TcrNBT1 harboured lower affinity for purine nucleosides and poor affinity for pyrimidines. In contrast, TcrNT1 was found to be a high-affinity guanosine/inosine transporter with a Kmof 1.0 ± 0.03 µM for inosine and a 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 TcrNB1 and TcrNT1, TcrNT2 turned out to be a high-affinity thymidine transporter (Km = 223.5 ± 7.1 nM), displaying some affinity for uridine and cytidine, (Ki values of 66 ± 6 and 728 ± 70 µM, respectively), but barely sensitive to inhibition by pyrimidine nucleobases or purines. At present, only TcrNB2 is still undergoing characterisation. We propose TbNBT-KO as system for rational characterisation of ENT genes, and established the kinetic parameters of some T. cruzi purine and pyrimidine transport systems. The results indicate that, surprisingly, this parasite has a preference for oxopurines over aminopurines. 
