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© 2016 Leija et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited., The human pathogenic parasite Trypanosoma brucei possess both de novo and salvage routes for the biosynthesis of pyrimidine nucleotides. Consequently, they do not require salvageable pyrimidines for growth. Thymidine kinase (TK) catalyzes the formation of dTMP and dUMP and is one of several salvage enzymes that appear redundant to the de novo pathway. Surprisingly, we show through analysis of TK conditional null and RNAi cells that TK is essential for growth and for infectivity in a mouse model, and that a catalytically active enzyme is required for its function. Unlike humans, T. brucei and all other kinetoplastids lack dCMP deaminase (DCTD), which provides an alternative route to dUMP formation. Ectopic expression of human DCTD resulted in full rescue of the RNAi growth phenotype and allowed for selection of viable TK null cells. Metabolite profiling by LC-MS/MS revealed a buildup of deoxypyrimidine nucleosides in TK depleted cells. Knockout of cytidine deaminase (CDA), which converts deoxycytidine to deoxyuridine led to thymidine/deoxyuridine auxotrophy. These unexpected results suggested that T. brucei encodes an unidentified 5'-nucleotidase that converts deoxypyrimidine nucleotides to their corresponding nucleosides, leading to their dead-end buildup in TK depleted cells at the expense of dTTP pools. Bioinformatics analysis identified several potential candidate genes that could encode 5'-nucleotidase activity including an HD-domain protein that we show catalyzes dephosphorylation of deoxyribonucleotide 5'-monophosphates. We conclude that TK is essential for synthesis of thymine nucleotides regardless of whether the nucleoside precursors originate from the de novo pathway or through salvage. Reliance on TK in the absence of DCTD may be a shared vulnerability among trypanosomatids and may provide a unique opportunity to selectively target a diverse group of pathogenic single-celled eukaryotes with a single drug., This work was supported by National Institutes of Health (grants AI078962 and AI034432) to MAP (https://www.niaid.nih.gov) and (grant GM007062) to CL (https://www.nigms.nih. gov), the Welch Foundation (grant I-1257) to MAP and (grant I-1686) to JJK (http://www.welch1.org), and Fundac ̧ão para a Ciência e Tecnologia (FCT, Portugal) SFRH/BD/51286/2010 (http://www.fct.pt) to FRF.

Polyamines are essential for cell growth of eukaryotes including the etiologic agent of human African trypanosomiasis (HAT), Trypanosoma brucei. In trypanosomatids, a key enzyme in the polyamine biosynthetic pathway, S-adenosylmethionine decarboxylase (TbAdoMetDC) heterodimerizes with a unique catalytically-dead paralog called prozyme to form the active enzyme complex. In higher eukaryotes, polyamine metabolism is subject to tight feedback regulation by spermidine-dependent mechanisms that are absent in trypanosomatids. Instead, in T. brucei an alternative regulatory strategy based on TbAdoMetDC prozyme has evolved. We previously demonstrated that prozyme protein levels increase in response to loss of TbAdoMetDC activity. Herein, we show that prozyme levels are under translational control by monitoring incorporation of deuterated leucine into nascent prozyme protein. We furthermore identify pathway factors that regulate prozyme mRNA translation. We find evidence for a regulatory feedback mechanism in which TbAdoMetDC protein and decarboxylated AdoMet (dcAdoMet) act as suppressors of prozyme translation. In TbAdoMetDC null cells expressing the human AdoMetDC enzyme, prozyme levels are constitutively upregulated. Wild-type prozyme levels are restored by complementation with either TbAdoMetDC or an active site mutant, suggesting that TbAdoMetDC possesses an enzyme activity-independent function that inhibits prozyme translation. Depletion of dcAdoMet pools by three independent strategies: inhibition/knockdown of TbAdoMetDC, knockdown of AdoMet synthase, or methionine starvation, each cause prozyme upregulation, providing independent evidence that dcAdoMet functions as a metabolic signal for regulation of the polyamine pathway in T. brucei. These findings highlight a potential regulatory paradigm employing enzymes and pseudoenzymes that may have broad implications in biology., Author summary Trypanosoma brucei is a single-celled eukaryotic pathogen and the causative agent of human African trypanosomiasis (HAT). Polyamines are organic polycations that are essential for growth in T. brucei to facilitate protein translation and to maintain redox homeostasis. The pathway is the target of eflornithine, a current frontline therapy for treatment of HAT. Polyamine biosynthetic enzymes are regulated at multiple levels in mammals (e.g. transcription, translation and protein turnover), but in contrast, T. brucei lacks these mechanisms. Instead in T. brucei a central enzyme in polyamine metabolism called AdoMetDC must form a complex with a sister protein (termed a pseudoenzyme) to be active. Herein, we show that cellular levels of this sister protein we call prozyme are in turn feedback regulated by both AdoMetDC and by its reaction product in response to cell treatments that reduce pathway output. This regulatory paradigm highlights how pseudoenzymes can evolve to play an important role in metabolic pathway regulation and in organismal fitness.

Trypanosoma brucei is the causative agent of African sleeping sickness. The polyamine biosynthetic pathway has the distinction of being the target of the only clinically proven anti-trypanosomal drug with a known mechanism of action. Polyamines are essential for cell growth, and their metabolism is extensively regulated. However, trypanosomatids appear to lack the regulatory control mechanisms described in other eukaryotic cells. In T. brucei, S-adenosylmethionine decarboxylase (AdoMetDC) and ornithine decarboxylase (ODC) are required for the synthesis of polyamines and also for the unique redox-cofactor trypanothione. Further, trypanosomatid AdoMetDC is activated by heterodimer formation with a catalytically dead homolog termed prozyme, found only in these species. To study polyamine regulation in T. brucei, we generated inducible AdoMetDC RNAi and prozyme conditional knockouts in the mammalian blood form stage. Depletion of either protein led to a reduction in spermidine and trypanothione and to parasite death, demonstrating that prozyme activation of AdoMetDC is essential. Under typical growth conditions, prozyme concentration is limiting in comparison to AdoMetDC. However, both prozyme and ODC protein levels were significantly increased relative to stable transcript levels by knockdown of AdoMetDC or its chemical inhibition. Changes in protein stability do not appear to account for the increased steady-state protein levels, as both enzymes are stable in the presence of cycloheximide. These observations suggest that prozyme and ODC are translationally regulated in response to perturbations in the pathway. In conclusion, we describe the first evidence for regulation of polyamine biosynthesis in T. brucei and we demonstrate that the unique regulatory subunit of AdoMetDC is a key component of this regulation. The data support ODC and AdoMetDC as the key control points in the pathway and the likely rate-limiting steps in polyamine biosynthesis., Author Summary Human African trypanosomiasis (HAT) is an important vector-borne pathogen. The World Health Organization estimates that more than 50 million people are at risk for the disease, which occurs focally, in remote regions, and periodically reaches epidemic levels. Untreated HAT is always fatal, and the available drugs compromise toxicity and emerging resistance. The only safe treatment for late-stage disease is an inhibitor of an essential metabolic pathway that is involved in the synthesis of small organic cations termed polyamines. In this paper, we use genetic approaches to demonstrate how the parasite regulates this essential metabolic pathway. By modulating the protein levels of a trypanosome-specific activator of polyamine biosynthesis, the parasite has developed a mechanism to regulate pathway output. We also demonstrate that this pathway activator is essential to parasite growth. Our data strengthen the genetic and chemical validation of a key enzyme in this pathway as a drug target in the parasite, and they provide new insight into parasite-specific approaches that could be used to design novel drugs against this deadly disease.

Malaria drug resistance contributes to up to a million annual deaths. Judicious deployment of new antimalarials and vaccines could benefit from an understanding of early molecular events that promote the evolution of parasites. Continuous in vitro challenge of Plasmodium falciparum parasites with a novel dihydroorotate dehydrogenase (DHODH) inhibitor reproducibly selected for resistant parasites. Genome-wide analysis of independently-derived resistant clones revealed a two-step strategy to evolutionary success. Some haploid blood-stage parasites first survive antimalarial pressure through fortuitous DNA duplications that always included the DHODH gene. Independently-selected parasites had different sized amplification units but they were always flanked by distant A/T tracks. Higher level amplification and resistance was attained using a second, more efficient and more accurate, mechanism for head-to-tail expansion of the founder unit. This second homology-based process could faithfully tune DNA copy numbers in either direction, always retaining the unique DNA amplification sequence from the original A/T-mediated duplication for that parasite line. Pseudo-polyploidy at relevant genomic loci sets the stage for gaining additional mutations at the locus of interest. Overall, we reveal a population-based genomic strategy for mutagenesis that operates in human stages of P. falciparum to efficiently yield resistance-causing genetic changes at the correct locus in a successful parasite. Importantly, these founding events arise with precision; no other new amplifications are seen in the resistant haploid blood stage parasite. This minimizes the need for meiotic genetic cleansing that can only occur in sexual stage development of the parasite in mosquitoes., Author Summary Malaria parasites kill up to a million people around the world every year. Emergence of resistance to drugs remains a key obstacle against elimination of malaria. In the laboratory, parasites can efficiently acquire resistance to experimental antimalarials by changing DNA at the target locus. This happens efficiently even for an antimalarial that the parasite has never encountered in a clinical setting. In this study, we formally demonstrate how parasites achieve this feat: first, individual parasites in a population of millions randomly amplify large regions of DNA between short sequence repeats of adenines (A) or thymines (T) that are peppered throughout the malaria parasite genome. The rare lucky parasite that amplifies DNA coding for the target of the antimalarial, along with dozens of its neighboring genes, gains an evolutionary advantage and survives. In a second step, to withstand increasing drug pressure and to achieve higher levels of resistance, each parasite line makes additional copies of this region. This second expansion does not rely on the random A/T-based DNA rearrangements but, instead, a more precise amplification mechanism that retains the unique signature of co-amplified genes created earlier in each parasite. Generation of multiple copies of the target genes in the parasite genome may be the beginning of other beneficial changes for the parasite, including the future acquisition of mutations.