Abstract
BackgroundAfrican trypanosomes are capable of both pyrimidine biosynthesis and salvage of preformed pyrimidines from the host, but it is unknown whether either process is essential to the parasite.Methodology/Principal FindingsPyrimidine requirements for growth were investigated using strictly pyrimidine-free media, with or without single added pyrimidine sources. Growth rates of wild-type bloodstream form Trypanosoma brucei brucei were unchanged in pyrimidine-free medium. The essentiality of the de novo pyrimidine biosynthesis pathway was studied by knocking out the PYR6-5 locus that produces a fusion product of orotate phosphoribosyltransferase (OPRT) and Orotidine Monophosphate Decarboxylase (OMPDCase). The pyrimidine auxotroph was dependent on a suitable extracellular pyrimidine source. Pyrimidine starvation was rapidly lethal and non-reversible, causing incomplete DNA content in new cells. The phenotype could be rescued by addition of uracil; supplementation with uridine, 2′deoxyuridine, and cytidine allowed a diminished growth rate and density. PYR6-5−/− trypanosomes were more sensitive to pyrimidine antimetabolites and displayed increased uracil transport rates and uridine phosphorylase activity. Pyrimidine auxotrophs were able to infect mice although the infection developed much more slowly than infection with the parental, prototrophic trypanosome line.Conclusions/SignificancePyrimidine salvage was not an essential function for bloodstream T. b. brucei. However, trypanosomes lacking de novo pyrimidine biosynthesis are completely dependent on an extracellular pyrimidine source, strongly preferring uracil, and display reduced infectivity. As T. brucei are able to salvage sufficient pyrimidines from the host environment, the pyrimidine biosynthesis pathway is not a viable drug target, although any interruption of pyrimidine supply was lethal.
Highlights
Human African Trypanosomiasis (HAT, or sleeping sickness) is caused by infection with the protozoan parasites Trypanosoma brucei gambiense and T. b. rhodesiense in West Africa and in East and Southern Africa, respectively
We have previously shown that in procyclic T. b. brucei pyrimidines are mainly taken up through the TbU1 uracil transporter [23,16] and recently completed a study of pyrimidine transport activities in bloodstream form T. b. brucei showing the presence of only one high affinity uracil transporter, TbU3, and almost no uptake of other pyrimidines at physiological levels [24]
The two pathways converge at UMP, the end-product of the 6-step biosynthesis pathway as well as the nexus for salvaged cytidine, uridine, 29dUrd, 29dCtd and uracil, through the actions of cytidine deaminase, uridine phosphorylase and uracil phosphoribosyltransferase (UPRT)
Summary
Human African Trypanosomiasis (HAT, or sleeping sickness) is caused by infection with the protozoan parasites Trypanosoma brucei gambiense and T. b. rhodesiense in West Africa and in East and Southern Africa, respectively. Human African Trypanosomiasis (HAT, or sleeping sickness) is caused by infection with the protozoan parasites Trypanosoma brucei gambiense and T. b. Brucei and the other non-human infective species T. vivax and T. congolense cause the veterinary condition African Animal Trypanosomiasis (AAT, or nagana) in livestock in much of sub-Saharan Africa. Both diseases continue to have profound health and economic implications in poor and isolated populations of the region. The quest for new drugs led to the study of nucleotide salvage and biosynthesis in protozoa, and initially focused on inhibitors of purine metabolism, as pathogenic protozoan parasites (but not free-living protists) have lost the de novo purine biosynthesis pathways [5,6]. African trypanosomes are capable of both pyrimidine biosynthesis and salvage of preformed pyrimidines from the host, but it is unknown whether either process is essential to the parasite
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