Fig 1.
Green lines salvage routes, blue lines de novo 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): 1–6: 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); 7 UMP-CMP kinase (2.7.4.14); 8: nucleoside diphosphatase (3.6.1.6); 9: nucleoside diphosphate kinase (2.7.4.6); 10: cytidine triphosphate synthase (6.3.4.2); 11: ribonucleoside diphosphate reductase (1.17.4.1); 12: thymidylate kinase (2.7.4.9); 13: deoxyuridine triphosphatase (dUTPase) (3.6.1.23); 14: dihydrofolate reductase-thymidylate synthase (2.1.1.45); 15:cytidine deaminase (CDA) (3.5.4.5); 16: thymidine kinase (TK)(2.7.1.21); 17: uridine phosphorylase (2.4.2.3); 18:uracil phosphoribosyltransferase (2.4.2.9); 19: HD-domain 5’-nucleotidase (3.1.3.89); 20: UDP-glucose pyrophosphorylase (2.7.7.9); 21: UTP N-acetyl-α-D-glucosamine-1-phosphate uridylyltransferase (2.7.7.23); 22: UDP-glucose 4-epimerase (5.1.3.2). The pathway was constructed based on the annotation described in [10] and modified to incorporate results from our studies. Additionally enzyme 7 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 [16].
Fig 2.
TK is essential for in vitro growth and infectivity in mice.
A. Growth analysis of TK c-null cells and wild-type SM cells ±Tet. Expression of ectopic FLAG-tagged TbTK is under Tet control, thus removal of Tet leads to loss of TbTK 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 TbTK expression ±Tet for 24h. TbBiP was detected as a loading control. B. qPCR analysis comparing mRNA expression levels of TbTK 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.
Fig 3.
Catalytically active TK is required to rescue the TK RNAi growth phenotype.
A-B. Growth analysis of TK RNAi cells (±Tet) expressing TbTK or HsTK under Tet control. Cell growth was monitored for the indicated days. Error bars represent SD for triplicate biological replicates. C. qPCR analysis of TbTK mRNA levels in TK RNAi knockdown cells in the absence and presence of the HsTK 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 TK RNAi cells (±Tet) expressing active-site mutant TK enzymes, TbTK E286A or HsTK K32I under Tet control. Error bars represent SD for triplicate biological replicates. Insets show western blots of the AU1-TbTK (A), FLAG-TbTK (D) or FLAG-HsTK (B,E) rescued RNAi lines comparing ±Tet for 48 h, though in panel E, HsTK K32I was detected with a HsTK antibody. TbBiP was detected as a loading control.
Fig 4.
Effects of nucleoside supplementation on growth in TK RNAi or c-null cells.
Growth analysis of TK RNAi cells supplemented with: A. dUrd (1 or 5 mM). B. Urd and dUrd (5 mM). C. dThd (0.5 or 1 mM) D. dUrd supplementation of TK c-null cells (±Tet). TK c-null cells express an ectopic copy of TbTK under Tet control. Error bars represent SD for triplicate biological replicates.
Fig 5.
HsDCTD rescues the growth defect in TbTK RNAi and TbTK null cell lines.
A. Growth curves for TK RNAi cells or TK RNAi cells containing a Tet-regulated expression plasmid for HsDCTD. Cell growth was monitored ±Tet for the indicated days. Error bars represent SD for triplicate biological replicates. Inset shows a Western blot of HsDCTD expression ± Tet at 48 h. B. qPCR analysis of TbTK mRNA expression in both the TK RNAi cell line and the TK RNAi HsDCTD rescue line (±Tet 48 h). Error bars represent SEM for triplicate data. Data were normalized to the -Tet control, which is in the background of the single allele TK knockout. C. Growth analysis of TK null cells expressing either FLAG-tagged TbTK (c-null) or FLAG-tagged HsDCTD under the control of the Tet promoter. Error bars represent SD for triplicate biological replicates. Inset shows western blot analysis of the HsDCTD TK null cells 2 days and 5 days after Tet withdraw.
Fig 6.
Metabolomic analysis of TK c-null cells.
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.
Fig 7.
Deletion of the T. brucei CDA gene induces pyrimidine auxotrophy.
A. Growth curves for CDA null cells grown in HMI-19 or HMI-19 media supplemented with 500 μM dThd. Cell growth was monitored for the indicated days. Error bars represent SD for triplicate biological replicates. B. qPCR analysis of CDA expression in wild-type SM and CDA null cells. Error bars represent SEM for triplicate data. C-E. Growth analysis of CDA null cells supplemented with dThd (C), dUrd (D) or uracil (E) over a range of concentrations 48 h post dThd withdrawl. Error bars represent the range for duplicate biological replicates. dThd and dUrd dose response curves were fitted to the Agonist vs response (three parameters) equation in GraphPad Prism (line represents the fit), to obtain ED50 for growth stimulation. ED50 = 20 μM (2.9–61) for dThd and 6.8 μM (3.8–13) for dUrd, where values in parenthesis represent the 95% confidence interval.
Table 1.
5'-nucleotidase PFAM domain representatives.
Fig 8.
Steady-state kinetic analysis of T. brucei HD domain 5’-nucleotidase.
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.
Fig 9.
Venn diagram showing the distribution of TK, DCTD and dCTP deaminase in representative protists and higher eukaryotes.
The overlapping regions represent organisms that possess both of the indicated genes.