Pyrimidine salvage enzymes are essential for de novo biosynthesis of Deoxypyrimidine nucleotides in Trypanosoma brucei

© 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.info:eu-repo/semantics/publishedVersio

A dual regulatory circuit consisting of S-adenosylmethionine decarboxylase protein and its reaction product controls expression of the paralogous activator prozyme in Trypanosoma brucei.

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

GMP synthase is essential for viability and infectivity of Trypanosoma bruceidespite a redundant purine salvage pathway

© 2015 John Wiley & Sons Ltd.The causative agent of human African trypanosomiasis, Trypanosoma brucei, lacks de novo purine biosynthesis and depends on purine salvage from the host. The purine salvage pathway is redundant and contains two routes to guanosine-5'-monophosphate (GMP) formation: conversion from xanthosine-5'-monophosphate (XMP) by GMP synthase (GMPS) or direct salvage of guanine by hypoxanthine-guanine phosphoribosyltransferase (HGPRT). We show recombinant T. brucei GMPS efficiently catalyzes GMP formation. Genetic knockout of GMPS in bloodstream parasites led to depletion of guanine nucleotide pools and was lethal. Growth of gmps null cells was only rescued by supraphysiological guanine concentrations (100 μM) or by expression of an extrachromosomal copy of GMPS. Hypoxanthine was a competitive inhibitor of guanine rescue, consistent with a common uptake/metabolic conversion mechanism. In mice, gmps null parasites were unable to establish an infection demonstrating that GMPS is essential for virulence and that plasma guanine is insufficient to support parasite purine requirements. These data validate GMPS as a potential therapeutic target for treatment of human African trypanosomiasis. The ability to strategically inhibit key metabolic enzymes in the purine pathway unexpectedly bypasses its functional redundancy by exploiting both the nature of pathway flux and the limited nutrient environment of the parasite's extracellular niche.This work was supported by National Institutes of Health grants 1R01AI078962 (to KS and MAP), 2R37AI034432 (to MAP) and GM007062 (to CL); the Welch Foundation grant I-1257 (to MAP) and Fundação para a Ciência e Tecnologia (FCT, Portugal) SFRH/BD/51286/2010 to FRF.info:eu-repo/semantics/publishedVersio

Metabolomic analysis of TK c-null cells.

<p>A. Detected pyrimidine and purine bases, nucleosides and nucleotides for cells grown in HMI-19 medium supplemented with normal serum (NS). The ratios (fold change) of metabolite levels in the absence of Tet for 24h compared to cells grown with Tet are plotted. B. HPAEC analysis of nucleotide sugars ±Tet at 24 h. C. Quantitation of dTTP by enzymatic assay ± Tet at 24 and 48 h for cell grown in HMI-19 supplemented with NS. D. Fold change of detected pyrimidine and purine bases, nucleosides and nucleotides ± Tet at 24 h for cells grown in HMI-19 medium supplemented with dialyzed serum (DS). E. Fold change of TCA intermediates ±Tet at 24 h for cells grown in HMI-19 medium supplemented with DS. Metabolites shown for C and D are from the same experiment. Data for additional detected metabolites for the normal serum (A) and dialyzed serum (D and E) studies are presented in Supplemental Figures. All data were collected in biological triplicate and error bars represent the SEM calculated for the ±Tet ratio by Graph Pad Prism using the baseline-correction algorithm. For A, D and E, multiple T test analysis was performed in GraphPad Prism comparing the +Tet and -Tet conditions for each study. Statistical significance was determined without correction for multiple comparisons and without assuming a consistent standard deviation. For C, data were analyzed using one way ANOVA with Dunnett’s multiple comparison test. Metabolites that showed a significant difference between the conditions are marked * P<0.05, ** P<0.01, *** P<0.001. Abbreviations are common nomenclature or have been previously defined except for CP, carbamoyl phosphate, R5P, ribose 5’-phosphate, 7m-guanosine, 7-methyl guanosine, succinate/m-malonic acid, succinate/methyl-malonic acid.</p

Steady-state kinetic analysis of <i>T</i>. <i>brucei</i> HD domain 5’-nucleotidase.

<p>A. Metal ion dependence. dCMP (1 mM) was used as the substrate and metal concentrations are noted on the figure. B. Substrate preference. Substrate concentrations were 1 mM and these assays were run in the presence of 0.5 mM Co⁺². The < symbol on the graph indicates that the activity was below the level of detection. Data were collected in triplicate and error bars represent the SD of the mean.</p

Catalytically active TK is required to rescue the <i>TK</i> RNAi growth phenotype.

<p>A-B. Growth analysis of TK RNAi cells (±Tet) expressing <i>Tb</i>TK or <i>Hs</i>TK under Tet control. Cell growth was monitored for the indicated days. Error bars represent SD for triplicate biological replicates. C. qPCR analysis of <i>Tb</i>TK mRNA levels in <i>TK</i> RNAi knockdown cells in the absence and presence of the <i>Hs</i>TK rescue plasmid 48 h after Tet addition. Error bars represent SEM for triplicate data. Data were normalized to TK levels in wild-type SM cells. D-E. Growth analysis of <i>TK</i> RNAi cells (±Tet) expressing active-site mutant TK enzymes, <i>Tb</i>TK E286A or <i>Hs</i>TK K32I under Tet control. Error bars represent SD for triplicate biological replicates. Insets show western blots of the AU1-<i>Tb</i>TK (A), FLAG-<i>Tb</i>TK (D) or FLAG-<i>Hs</i>TK (B,E) rescued RNAi lines comparing ±Tet for 48 h, though in panel E, <i>Hs</i>TK K32I was detected with a <i>Hs</i>TK antibody. <i>Tb</i>BiP was detected as a loading control.</p

<i>T</i>. <i>brucei</i> pyrimidine pathway.

<p>Green lines salvage routes, blue lines <i>de novo</i> pathway, black lines interconversion routes, and the red dotted line indicates a reaction that is not present in trypanosomatids. The numbers above each arrow represent the enzyme catalyzing the reaction (EC number): <b>1–6</b>: carbamoyl phosphate synthase (6.3.5.5), aspartate carbamoyl transferase (2.1.3.2), dihydroorotase (3.5.2.3), dihydroorotate dehydrogenase (1.3.98.1), orotate phosphoribosyltransferase (2.4.2.10), orotidine 5-phosphate decarboxylase (4.1.1.23); <b>7</b> UMP-CMP kinase (2.7.4.14); <b>8</b>: nucleoside diphosphatase (3.6.1.6); <b>9</b>: nucleoside diphosphate kinase (2.7.4.6); <b>10</b>: cytidine triphosphate synthase (6.3.4.2); <b>11</b>: ribonucleoside diphosphate reductase (1.17.4.1); <b>12</b>: thymidylate kinase (2.7.4.9); <b>13</b>: deoxyuridine triphosphatase (dUTPase) (3.6.1.23); <b>14</b>: dihydrofolate reductase-thymidylate synthase (2.1.1.45); <b>15</b>:cytidine deaminase (CDA) (3.5.4.5); <b>16</b>: thymidine kinase (TK)(2.7.1.21); <b>17</b>: uridine phosphorylase (2.4.2.3); <b>18</b>:uracil phosphoribosyltransferase (2.4.2.9); 19: HD-domain 5’-nucleotidase (3.1.3.89); <b>20</b>: UDP-glucose pyrophosphorylase (2.7.7.9); <b>21</b>: UTP N-acetyl-α-D-glucosamine-1-phosphate uridylyltransferase (2.7.7.23); <b>22</b>: UDP-glucose 4-epimerase (5.1.3.2). The pathway was constructed based on the annotation described in [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006010#ppat.1006010.ref010" target="_blank">10</a>] and modified to incorporate results from our studies. Additionally enzyme <b>7</b> was added based on the published report that one of seven encoded adenylate kinases (ADKG) was biochemically characterized and shown to be a UMP-CMP kinase [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006010#ppat.1006010.ref016" target="_blank">16</a>].</p

Venn diagram showing the distribution of TK, DCTD and dCTP deaminase in representative protists and higher eukaryotes.

<p>The overlapping regions represent organisms that possess both of the indicated genes.</p

TK is essential for <i>in vitro</i> growth and infectivity in mice.

<p>A. Growth analysis of <i>TK</i> c-null cells and wild-type SM cells ±Tet. Expression of ectopic FLAG-tagged <i>Tb</i>TK is under Tet control, thus removal of Tet leads to loss of <i>Tb</i>TK expression. Cells were grown in HMI-19 medium supplemented with either normal serum (NS) or dialyzed serum (DS). Cell growth was monitored for the indicated days. Error bars represent standard deviation (SD) for triplicate biological replicates. Inset shows western blot analysis of FLAG-tagged <i>Tb</i>TK expression ±Tet for 24h. <i>Tb</i>BiP was detected as a loading control. B. qPCR analysis comparing mRNA expression levels of <i>Tb</i>TK to the TERT control ±Tet for 24 h. Error bars represent standard error of the mean (SEM) for triplicate data. C. Survival analysis of wild-type SM and TK c-null infected mice (±Dox) 1–30 days post infection for three mice per group.</p

5'-nucleotidase PFAM domain representatives.

<p>5'-nucleotidase PFAM domain representatives.</p