Thymidine kinase (TK) is essential for growth of bloodstream form (BSF) T. brucei

To evaluate the essentiality of TK, we attempted to generate a TK null cell line in T. brucei BSF single marker (SM) cells. T. brucei is diploid, therefore minimally two alleles are present for each gene. Allelic replacement via homologous recombination was achieved by transfecting parasites with a PCR product containing a resistance marker flanked by the TK 5’ and 3’ UTRs. Removal of the first allele was successful; however we were unable to replace the last allele after repeated attempts, suggesting essentiality. To further evaluate this hypothesis, a TK conditional null (TK c-null) cell line was generated. The single allele knockout cells (SKO) were transfected with a vector conferring tetracycline (Tet) regulated expression of N-terminally FLAG tagged T. brucei TK (FLAG-TbTK). Expression of FLAG-TbTK was induced by addition of Tet and the remaining TK allele was successfully removed generating the TK c-null cell line. PCR amplification of the TK locus confirmed replacement by the two selectable markers (S1 Fig). To determine the effects caused by the loss of TK expression on cell growth, Tet was removed from the medium, which led to rapid growth arrest and near total cell death by day 3 (Fig 2A). Coincident with this growth arrest, TK transcript (by qPCR) and the TK protein (western blot) were depleted within 24 h after Tet removal, confirming good regulatory control of the ectopic TK copy (Fig 2A and 2B). Parasites reemerged several days later, likely due to the loss of Tet regulation. This has been reported to be a common phenomenon in T. brucei, likely due to mutations resulting in the loss of Tet regulation, e.g. [9, 27–29]. The TK c-null cells grew normally in pyrimidine-free medium (containing dialyzed fetal bovine serum) in the presence of Tet confirming that T. brucei is not auxotrophic for pyrimidines (Fig 2A). Upon subsequent removal of Tet, TK depletion led to cell death with a similar time course to medium containing non-dialyzed (normal) serum.

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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.

https://doi.org/10.1371/journal.ppat.1006010.g002

TK depleted cells are unable to establish an infection in mice

In vivo studies were performed to determine if TK was essential to support T. brucei infection in mice. In parallel to T. brucei SM infected mice, two groups (n = 3) of TK c-null infected mice were given either doxycycline (Dox) treated water or water only. As expected mice infected with SM cells in either condition (+/- Dox) had detectable levels of parasitemia by 72 h post infection, with fatalities occurring in all mice in both groups by day 6 (Fig 2C and S2 Fig). Similarly, all TK c-null infected mice treated with Dox to maintain expression of the Tet-regulated TK copy eventually died within the timeframe of the study. One mouse in this group showed a delayed time before succumbing to parasitemia suggesting some variability in TK expression levels in the TK c-null cells. Mice infected with the TK c-null strain treated only with water remained healthy and had no detectable levels of parasitemia past 30 days. Thus we conclude that TK is essential for T. brucei virulence and infectivity in vivo.

TK RNAi-induced growth arrest is reversed by expression of TK from multiple species

The finding that TK is essential in T. brucei is puzzling as no clear mechanistic role for TK in parasite fitness is apparent. T. brucei requires TK, which is a pyrimidine salvage enzyme; yet there is no requirement for salvageable pyrimidines for growth. To gain further mechanistic insight into this conundrum, we sought to address three possible explanations for the essentiality of TK: 1) parasites require an active TK enzyme, but it makes a novel product; 2) the TK protein, but not its catalytic activity is needed in some regulatory capacity; 3) parasites require formation of dUMP/dTMP by TK to balance pathway flux even under conditions where all pyrimidine precursors originate from the de novo pathway.

To provide additional mechanistic insight, an inducible RNAi cell line targeting TK mRNA was created so that we could easily introduce various rescue plasmids to address our mechanistic hypotheses. A Tet-regulated vector capable of producing a hairpin transcript targeting the 3’UTR of TK was generated and transfected into the TK SKO cell line. Induction of TK RNAi by addition of Tet led to a significant growth defect, although the growth defect was not as severe as observed for TK c-null cells (Fig 3A and 3B). TK mRNA expression was reduced to 20–25% of wild-type control levels by RNAi targeting the TK transcript (Fig 3C). However, the reduction in TK transcript levels was less in comparison to that observed in the TK c-null cells, explaining why the effect on cell growth was less pronounced.

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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.

https://doi.org/10.1371/journal.ppat.1006010.g003

To shed light on whether a novel TK product was being formed we transfected parasites with plasmids encoding rescue proteins from three different sources: AU1-tagged T. brucei TK, FLAG-tagged human HsTK, and Herpes simplex TK (HsvTK). HsTK has been shown to have more stringent substrate specificity than the T. brucei enzyme [24], whereas the viral HsvTK possesses broader substrate specificity than the human enzyme [30]. Rescue protein expression was also under control of Tet promoter. Thus, addition of Tet to these cells induces simultaneous knockdown of endogenous TK and expression of the tagged rescue protein. We found that the growth phenotype was reversed by expression of TK from all three species: T. brucei (TbTK)(Fig 3A), human (HsTK) (Fig 3B) and viral TK (HsvTK) (S3 Fig). Expression of TbTK and HsTK was confirmed by western blot (Fig 3A and 3B) and knockdown of endogenous TK was monitored by qPCR (Fig 3C). Viral TK expression was confirmed by the observance of ganciclovir sensitivity that was less apparent in cells expressing T. brucei TK (S3 Fig). Ganciclovir is a subversive substrate of HsvTK leading to premature chain termination of newly synthesized DNA [31]. The ability of both HsTK and HsvTK to rescue the TK RNAi growth phenotype shows that T. brucei TK is unlikely to catalyze a novel reaction, as the required activity is present in enzymes from other species that are known to have a range of substrate specificities.

Catalytically active TK is required for rescue of TbTK RNAi cells

To confirm that TbTK’s essential function is dependent on catalytic activity, mutations in the active site of both TbTK and HsTK were created. We targeted two conserved residues (S4 Fig) with described roles in the TK catalytic mechanism: T. brucei E286, which is reported to function as a proton acceptor [32] and human K32 which is an essential ATP binding residue [33]. Rescue plasmids were constructed as described above with the mutant TKs under the control of the Tet promoter and transfected into the TK RNAi line. In contrast to the wild type enzymes, neither the TbTK E286A nor HsTK K32I active site mutants were able to reverse the RNAi induced growth phenotype (Fig 3D and 3E). These data demonstrate that TK catalytic activity is required for its role in T. brucei cell survival.

Deoxyuridine supplementation partially rescues the RNAi-induced growth phenotype

While our data clearly show that T. brucei is not a pyrimidine auxotroph, we exploited the fact that the TK RNAi line retains partial TK activity (the knockdown is only 75–80% effective by RNAi (Fig 3C)) to assess whether we could use pyrimidine rescue to determine which TK product was needed for T. brucei growth. We found that high concentrations (significantly above physiological levels) of deoxyuridine (dUrd) resulted in the partial rescue of the RNAi growth phenotype (Fig 4A and S5A Fig) (5 mM rescued but 1 mM did not). However, similar levels of uridine (Urd) (Fig 4B and S5B Fig) or thymidine (dThd)(Fig 4C and S5C Fig) did not restore growth and dThd (0.15–1.0 mM) was in fact growth inhibitory to the TK RNAi +Tet induced cells but not to cells that expressed TK (-Tet), suggesting some type of feedback regulation. In contrast, the addition of dUrd or uracil to TK c-null cells, which are >99% depleted of TbTK, were unable to circumvent lethality of the TK knockout showing that TK activity is required for dUrd rescue (Fig 4C and S6 Fig). These data confirm that TK plays an essential role in maintaining dUMP pools.

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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.

https://doi.org/10.1371/journal.ppat.1006010.g004

Human dCMP deaminase rescues TK-deficient cells

The ability of dUrd to partially reverse the RNAi growth phenotype highlights an interesting feature in T. brucei pyrimidine metabolism. In most mammals, it has been suggested that a significant portion of dTTP is derived from dUMP produced by dCMP deaminase (DCTD)[17, 18]. Trypanosomatids lack this enzyme, restricting the number of metabolic routes dedicated to dUMP formation. We hypothesized that due to the lack of DCTD, trypanosomatids require TK to supplement dUMP pools. To test this hypothesis a Tet-regulated vector encoding human DCTD (HsDCTD) was transfected into the TK RNAi cell line to drive simultaneous expression of HsDCTD and knockdown of endogenous TbTK. The expression of FLAG-tagged HsDCTD completely rescued the TK RNAi-induced growth phenotype (Fig 5A). Expression of FLAG-tagged HsDCTD was confirmed by western blot and qPCR analysis confirmed that TK mRNA expression was simultaneously reduced (Fig 5B). To further demonstrate that HsDCTD can functionally replace TK, a TK null cell line was created in the background of FLAG-tagged HsDCTD Tet-regulated expression plasmid. Both TK alleles were replaced by selectable markers through homologous recombination in the presence of Tet to maintain expression of HsDCTD. PCR amplification of the region flanking the TK 5’ and 3’ UTRs confirmed replacement of TK with the selectable markers (S1 Fig). PCR analysis also confirmed that the TK gene was no longer detectable in genomic DNA from the TK null cells. Removal of Tet from this cell line led to depletion of FLAG-tagged HsDCTD and resulted in a severe growth phenotype by day 2 after Tet removal (Fig 5C). Cell growth of the HsDCTD TK null line was less severely impacted than the TK c-null line expressing Tet-regulated TbTK from the rescue plasmid, perhaps reflecting a higher residual expression level of HsDCTD (Fig 5C). By day 5 after Tet withdrawal cells began growing again coincident with re-expression of HsDCTD, again suggestive of emergence of cells that have mutations leading to loss of Tet regulation (Fig 5C). Thus, the data support the hypothesis that TK is essential in T. brucei and that its role is to contribute to the formation of dUMP in the absence of DCTD.

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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.

https://doi.org/10.1371/journal.ppat.1006010.g005

Metabolomic analysis reveals an accumulation of nucleoside substrates upon TK depletion

Analysis of ~130 soluble metabolites from TK c-null cells was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine the impact of TK depletion on metabolite pools (Fig 6 and S7–S9 Figs and S1 Appendix). Extracts were collected from TK c-null cells grown in medium containing normal non-dialyzed serum at 24 h ± Tet. An early time point was selected so that metabolite pools would be less affected by non-specific changes resulting from cell death later in the time course. Significant changes in the measured metabolite levels were mostly confined to pyrimidine nucleosides: there was a 15-30-fold accumulation of the TK substrates dUrd and dThd, a 3-fold accumulation of dCtd, and a 70-fold increase in thymine levels (Fig 6A). In contrast, the pyrimidine nucleotides CMP and UMP were not significantly changed by TK depletion, suggesting that the de novo pathway was able to maintain the uridine nucleotide pools. In further support, HPLC analysis of UDP-sugars was performed (Fig 6B). Nucleotide sugars are formed from UTP, thus their measurement provides a read-out of effects on intracellular UTP concentrations. The relative abundance of UDP-GlcNAc, UDP-Galactose, and UDP-Glucose were not significantly changed by TK depletion, confirming that UTP pools are not linked to TK activity. Thymidine nucleotide pools were not detected by the LC-MS/MS analysis so instead we quantitated dTTP levels using an enzymatic assay and found that dTTP levels were reduced to 30% of control levels 48 h after Tet removal, confirming TK is essential for synthesis of dTTP (Fig 6C).

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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.

https://doi.org/10.1371/journal.ppat.1006010.g006

To better understand the consequences of TK depletion in a pyrimidine-free medium the LC-MS/MS analysis was repeated for TK c-null cells grown in medium supplemented with dialyzed serum (Fig 6C and S7–S9 Figs and S1 Appendix). Similar to the results for cells grown in normal serum based medium, we observed a statistically significant buildup of TK substrates dUrd and dThd, and of the dUrd precursor dCyd, though the increases (3-6-fold) were less than observed for cells grown in normal serum based medium. We also observed changes in the levels of TCA intermediates including decreases in citrate and aconitate and an increase in α-ketoglutarate, and perturbations in other metabolites related to pyrimidine biosynthesis including decreased carbamoylphoshate (CP) and acetyl-ornithine (30 and 80%, respectively) and a 2-fold increase in homocysteine (Fig 6C and 6D). Not all of these latter changes reached statistical significance. Some modest effects were also observed on several purines: an increase in xanthine and/or hypoxanthine was observed in both medium conditions, which may suggest some type of cross-regulation between pyrimidine and purine biosynthetic pathways. Thymine was only observed in cells grown in medium supplemented with non-dialyzed serum, which could supply a source for thymine, confirming genomic analysis that suggests T. brucei does not encode thymine biosynthetic enzymes.

Deletion of cytidine deaminase induces pyrimidine auxotrophy

The finding that the deoxypyrimidine nucleoside pools buildup upon TK depletion for cells grown in pyrimidine-free (dialyzed serum based) medium suggests that an undiscovered biosynthetic route for formation of deoxypyrimidine nucleosides must be present in T. brucei. To investigate if deoxycytidine could be involved in formation of dUMP and dTMP we decided to characterize the effects of depleting cytidine deaminase (CDA) on parasite growth. Based on the current annotation of the pyrimidine biosynthetic pathway, CDA like TK should catalyze a redundant reaction, since the deoxynucleotide pools can be supplied from the de novo pathway. However, attempts to generate a CDA null were unsuccessful, and indeed the CDA null could only be obtained in the presence of 500 μM dThd (Fig 7 and S10 Fig). Upon removal of dThd, CDA null cells exhibited a severe growth defect (Fig 7A). Cells cultured for longer eventually died around day 7–10 after dThd removal. Additional pyrimidine rescue studies revealed that both dThd and dUrd were capable of rescuing growth (EC₅₀ of 6–20 μM), whereas uracil (up to 250 μM) was not (Fig 7C–7E). The concentrations of dThd and dUrd required for rescue are significantly above reported human blood levels (range of 0.2–0.6 μM; http://www.hmdb.ca).

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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.

https://doi.org/10.1371/journal.ppat.1006010.g007

CDA null cells were grown ± dThd for 12 h in media supplemented with dialyzed FBS and the metabolite pools were analyzed by LC-MS/MS as described above. Overall the effects of CDA depletion on cellular metabolism were very similar to the effects observed after depletion of TK, confirming a link between the roles of the two enzymes (S11 Fig, S12 Fig and S1 Appendix). We observed a significant buildup (~7-fold) in the CDA substrate, dCtd, for the CDA null cells grown in the absence of Thd that was accompanied by changes in TCA intermediates and other metabolites that serve as precursors for de novo pyrimidine biosynthesis. These changes include increases in α-ketoglutarate (11-fold), glutamate (7-fold) and homocysteine (2-fold), and decreases in carbamoyl phosphate (2-fold). We also observed decreased levels of several amino acids, of polyamines, particularly N-acetyl putrescine and decreased levels of several purine mono-phosphate nucleotides. Neither dUrd nor dThd were detected in CDA null cells in the presence or absence of added dThd, suggesting they were either not formed (dUrd) or rapidly metabolized (dThd). In the absence of CDA, the buildup of dCtd suggests that the pyrimidine deoxynucleotides have been sequestered into a dead-end product that would be expected to lead to depletion of key nucleotides and to cell death as was observed for the TK null cells. These data support a role for CDA in the interconversion of deoxycytidine, deoxyuridine and thymidine pools, which is needed to balance these pools in a cell like T. brucei that expresses TK but not DCTD.

T. brucei proteins with domains that can perform 5’-nucleotidase activity

In order to identify the potential enzyme(s) responsible for converting deoxypyrimidine nucleotides into their corresponding nucleosides we undertook a bioinformatics analysis of the T. brucei genome. 5'-nucleotidase activities that catalyze formation of deoxyuridine, deoxycytidine, or thymidine from their respective mono-phosphate nucleotides are ascribed to two EC numbers (EC3.1.3.5 and EC3.1.3.89). Inspection of domain types performing these activities revealed nine protein families defined by PFAM, with all having homologous structure representatives in the PDB (Table 1). The families further merge into five different homologous fold types; including three different α/β sandwich folds (phosphoglycerate mutase-like, SurE-like, and HAD domain-related), one α+β four-layer sandwich fold (metallo-dependent phosphatases), and one all-α fold (HD-domain). The phosphoglycerate mutase-like representatives are limited to mammalian acid phosphatases, prostate (ACPP) enzymes that convert extracellular AMP to adenosine (i.e. ecto 5' nucleotidase activity) [34]. An additional identified enzyme (NT5E) from the metallo-dependent phosphatase fold group exhibits a similar ecto 5'-nucleotidase activity [35]. Examples of this fold group in both eukaryotes and bacteria contain signal peptides and are extracellular. In contrast, the remaining SurE, HAD domain-related, and HD-domain representatives appear cytosolic.

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Table 1. 5'-nucleotidase PFAM domain representatives.

https://doi.org/10.1371/journal.ppat.1006010.t001

We found evidence for all of the 5'-nucleotidase homologous fold types in the T. brucei genome with the exception of the SurE fold class (S1 Table). We identified nine phosphoglycerate mutase-like sequences, 33 metallo-dependent phosphatase sequences, 16 HAD domain-related sequences, and one HD-domain sequence (S1 Table). The presence of numerous examples of potential 5'-nucleotidase domains, many of which are annotated as hypothetical proteins, suggests multiple possible proteins that T. brucei could use to form deoxyuridine, deoxycytidine, or thymidine de novo. However, three of the identified genes possess specific PFAM domains described as having 5’-nucleotidase activity (EC 3.1.3.5 or EC 3.1.3.89) and thus are the highest ranked candidates. One encodes a HD domain protein: hypothetical protein (Tb09.211.2190) and two encode HAD-like domains: a putative p-nitrophenylphosphatase (Tb927.8.7510) and a hypothetical protein (Tb09.211.1880). The T. brucei HD domain protein is a homolog of E. coli 5’-nucleotidase YfbR (S13 Fig) while the T. brucei HAD-domain proteins are related to enzymes shown to have 5’-nucleotidase activity in both yeast and E. coli. [36–38]. These enzymes have been reported to have broad substrate specificity functioning on all three pyrimidine deoxyribose monophosphates.

T. brucei HD domain protein encodes a metal dependent 5’-nucleotidase

To provide support for our hypothesis that T. brucei encodes a 5’-nucleotidase we cloned, expressed and purified the recombinant T. brucei HD domain homolog (Tb09.211.2190) of bacterial 5’-nucleotidase YfbR (S14 Fig). We found that the T. brucei YfbR-like HD protein showed a metal dependent 5’-nucleotidase activity (Fig 8A). Similar to the bacterial enzyme it was most active in the presence of Co⁺² (0.5 mM), but activities within 2-4-fold of levels observed for Co⁺² were also obtained using Mn⁺² (0.5 mM) and physiological levels of Mg⁺² (10 mM). No activity was observed in the presence of Zn⁺² or EDTA. The specific activity of the T. brucei HD domain 5’-nucleotidase was very similar to the reported activity of E. coli YfbR [38]. The T. brucei enzyme showed a broad substrate range functioning on both pyrimidine and purine deoxyribonucleoside and ribonucleoside 5’-monophosphates, though it was most active on the deoxypyrimidine nucleotides (dCMP, dUMP and dTMP)(Fig 8B). It showed no activity towards diphosphate nucleotides. The T. brucei enzyme was somewhat more promiscuous then E. coli YfbR, which was unable to catalyze hydrolysis of ribonucleoside 5-monophosphates [38]. Both T. cruzi and Leishmania encode homologs of the T. brucei HD-domain 5’-nucleotidase (S1 Table and S13 Fig) suggesting they both also will be able to convert 5’-deoxyribonucleotide monophosphates to their respective nucleosides.

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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.

https://doi.org/10.1371/journal.ppat.1006010.g008

Single-celled eukaryotic pathogens that encode TK but lack DCTD

To assess if TK essentiality was likely to extend to other pathogenic protozoa we utilized the KEGG pathway database to determine the distribution of TK and DCTD throughout eukaryotes (Fig 9). A striking disparity was observed within protists when compared to higher eukaryotes. The vast majority of higher eukaryotes possess both TK and DCTD, which may explain TK’s non-essential role in these organisms. In contrast, the kinetoplastids and a number of other protozoan human pathogens such as Giardia encode only TK, suggesting that TK may be essential in these organisms as well. We also note that several of the protists such as Entamoeba histolytica, which lack DCTD, instead encode dCTP deaminase, an enzyme found almost exclusively in bacteria. Similar to DCTD, the ability to deaminate dCTP to dUTP offers an alternative path from cytosine to thymine nucleotide pools and thus we would predict that TK would not be essential in these species. Interestingly, these organisms represent the only eukaryotic KEGG organisms that have dCTP deaminase.

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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.

https://doi.org/10.1371/journal.ppat.1006010.g009

Ethics statement

All animal care and experimental procedures were in accordance with the office of Laboratory Animal welfare (OLAW) guidelines provided by the National Institute of Health (NIH) USA (Assurance Number D16-00296) and the USDA (Registration Number 74-R-0072) as specified by the University of Texas Southwestern Medical Center Animal Care and Use Committee (IACUC) guidelines. Animal experiments were approved by the Ethical Review Committee at the University of Southwestern Medical Center and performed under the IACUC-2012-0021 protocol. All mice were housed in an animal facility with barrier SPF conditions.

Gene accession numbers

T. brucei gene sequences were obtained from TriTrypDB and the gene accession numbers are as follows: TK (Tb927.10.880), CDA (Tb927.9.3000), HD-domain 5’-nucleotidase (Tb09.211.2190), TERT (Tb927.11.10190). The HsTK1 (P04183) and HsDCTD (P32321) amino acid sequences were obtained from UniProt, and HsvTK was derived from Addgene plasmid #48356.

T. brucei in vitro growth

Experiments were performed using T. brucei BSF SM cells genetically manipulated to express T7 RNA polymerase and the Tet repressor (TetR) [29]. Cells were grown in HMI-19 medium supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO₂. HMI-19 is a modified medium that we previously reported [9]. It was designed to contain more physiologically relevant purine and pyrimidine levels and it is supplemented with only 10 μM hypoxanthine and no added thymidine, except that present in FBS. To obtain completely pyrimidine-free conditions, normal FBS was replaced with dialyzed FBS in media where indicated. All cells were maintained in exponential growth (10⁵−10⁶ cells/mL). SM cells were maintained in G418 (2.5 μg/mL) to retain the T7 polymerase and TetR. TK and CDA RNAi and knockout lines were cultured in the appropriate antibiotic depending on the transfected plasmid at the following concentrations unless otherwise stated: 2.5 μg/mL G418 (Life Technologies), 2.5 μg/mL blasticidin (InvivoGen), 2.5 μg/mL phleomycin (InvivoGen), 1–2 μg/mL hygromycin (Sigma), 0.1 μg/mL puromycin (Sigma), and 1 μg/mL Tet (RPI). For all nucleoside supplementation experiments 100 mM stocks of sterile filtered deoxyuridine (Sigma), thymidine (Sigma), uridine (Sigma), and uracil (Sigma) were added to cultures at concentrations indicated. All c-null lines were supplemented with 1 μg/mL Tet daily to maintain steady expression of Tet-regulated proteins. For pyrimidine and Tet free conditions, cells were washed (3 x 20 mL) with the appropriate media prior to beginning the growth experiments. For evaluation of growth rates, cells were washed and replated in media containing no antibiotics at a density of 20,000 cells/mL and diluted over the course of the study to maintain exponential growth. Cell density was determined using a hemocytometer (Bright-Line) with a lower limit of detection of 10⁴cells/ml. Two technical replicates were averaged for each counted sample. Total cell numbers were calculated by multiplying cell density by the dilution factor and volume [48].

Transfection of T. brucei

For each transfection, parasites (10⁷) were suspended in Human T Cell Nucleofector Buffer (Lonzo)(100 μL) containing NotI linearized vector (5 μg) or purified PCR product (1 μg) as described [49]. All transfected DNA was confirmed by sequencing prior to transfection. Negative controls cells transfected with buffer only were prepared alongside samples to optimize selection conditions. Cells were transfected using protocol X-001 on the Amaxa Nucleofector (Lonza) and then transferred to media (25 mL) and allowed to recover 8 h prior to addition of selection antibiotics. Two dilutions (1:20 and 1:40) of culture, containing selection antibiotics, were plated in 24-well plates at 2 mL/well. Negative control plates were monitored throughout the experiment to ensure selection was achieved. After several days, wells containing a cell density of about 10⁶ cells/ml were selected for generation of clonal lines by limiting dilutions.

Generation of T. brucei TK conditional null cell lines

The T. brucei TK and human (Hs) DCTD expressing TK c-null cell lines were generated utilizing the fusion PCR method [49, 50]. Cloning primers are shown in S2 Table. The first TK allele was replaced by the HYG resistance gene by PCR fusion of TK 5’ and 3’ UTRs to HYG. The HYG resistance gene was derived from the pLew90 vector [29] (a gift from George A.M. Cross). UTRs were amplified from genomic DNA isolated from SM cells. To generate the TK single allele knockout (SKO) line the purified PCR product was transfected into SM cells and hygromycin resistant cells were selected in medium containing G418 and hygromycin. The TK SKO line was then used to generate the remaining cell lines. A Tet-regulated vector containing either FLAG-tagged TbTK or FLAG-tagged HsDCTD was cloned as follows. The T. brucei TK or the human DCTD genes were PCR amplified from T. brucei SM genomic DNA or from human cDNA synthesized from RNA extracted from a human breast adenocarcinoma cell line (MDA-MB-231), respectively. For both constructs, the forward direction PCR primer contained a flanking 5’ HindIII restriction site and an N-terminal FLAG tag; the reverse direction primer contained a flanking 3’ BamHI restriction site. The restriction digested PCR products were ligated into the pLew100v5-phleo vector (a gift from George A.M. Cross). The pLew100v5-phleo vector was linearized by the NotI restriction enzyme to facilitate integration into the rRNA spacer region. Linearized vector (5 μg) was transfected into the TK SKO cell line and selected for resistance to phleomycin. The resulting clones were screened to identify those with the tightest level of Tet regulation of the ectopically expressed protein. Finally, the remaining TK allele was replaced by a PAC resistance gene synthesized by GenScript. The PAC TK UTR fusion product was generated as described above and transfected into TK SKO cells expressing either T. brucei TK or HsDCTD grown in Tet containing medium for 2 days prior to transfection. TK c-null cells were selected and maintained in G418, phleomycin, hygromycin, puromycin, and Tet (added daily). PCR primers flanking the 5’ and 3’UTRs were used to confirm that the TK gene had been replaced by the selectable markers.

Generation of the TK RNAi T. brucei BSF cell line

RNAit (http://trypanofan.bioc.cam.ac.uk/software/RNAit.html) was used to identify a suitable 566 bp region located in the TK 3’UTR. The 3’UTR was targeted to allow compatibility with TbTK rescue plasmids (described below), that utilize instead the ALD 3’UTR. Genomic DNA isolated from SM cells was used as template for PCR amplification of the target region and TA cloned into the Gateway vector pCR8/GW/TOPO (Life Technologies). The Tet inducible stem loop was created by addition of Gateway LR Clonase to a reaction containing both the Gateway vector (100 ng) and pTrypRNAiGate vector (100 ng) [51]. TK SKO (hyg) cells were transfected with the vector and integration into the rRNA spacer region was selected using phleomycin. For studies of the effects of TK knockdown, Tet was added daily to induce formation of the hairpin leading to knockdown of TK mRNA. Cells were grown in the absence of other antibiotics for these studies.

Cloning of RNAi rescue constructs and TK mutants

The TbTK rescue construct under control of the Tet promoter was generated using the same approach described above for the c-null cell line except that an N-terminal AU1-tag was included instead of a FLAG-tag to allow detection of the expressed protein. The gene encoding the HsTK open reading frame was synthesized by GenScript and cloned into the pUC57 vector, which was used for subsequent PCR amplification to generate the FLAG-tagged HsTK rescue construct. HsvTK was amplified from the pHJ17 Hyg-TK-loxP vector (Addgene). To generate the TbTK E286A and HsTK K32I mutants, both wild-type genes were subcloned into the pCR2.1-TOPO TA vector (Invitrogen). Complimentary PCR primers containing the desired point mutation were synthesized. Phusion polymerase (NEB) was used to amplify the entire vector according the to the following parameters: initial denaturation at 95°C for 30 s followed by 18 cycles of denaturation at 95°C for 30 s, annealing at 68°C for 1 min, and amplification at 72°C for 5 min. Each reaction (50 μL) was treated with DpnI (NEB)(1 μL) overnight at 37°C followed by transformation into T10 cells and selection with ampicillin (100 μg/ml). Clones were sequenced using M13 primers. All constructs contained flanking 5’ HindIII and 3’ BamHI restriction sites that permitted ligation into the pLew100v5-bsd vector[49]. The vectors were linearized with NotI and transfected into TK RNAi cells as described above.

Generation of the T. brucei CDA null cell line

SM cells were transfected with the HYG resistance gene flanked by the CDA 5’ and 3’ UTRs, generated by fusion PCR as described above, to generate the SKO in medium containing G418 and hygromycin. For the remaining allele, a fusion PCR product containing the PAC resistance gene was transfected into the SKO cells. Null cells were selected in growth medium containing G418, hygromycin, puromycin, and Thd (500 μM). PCR primers flanking the CDA 5’ and 3’ UTRs were used to confirm replacement of the CDA alleles.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomics analysis

T. brucei TK expressing TK c-null cells were grown with or without Tet for 24 h and CDA null cells were grown with or without thymidine (0.5 mM) for 12 h. Cells (10⁸) were harvested by centrifugation (3500 RPM, 5 min) and then washed in cold PBS (50 mL). Washed pellet was resuspended in 1 mL pre-chilled (-80°C) 80% methanol and incubated on ice for 10 min. The cell extract was centrifuged (16,000 x g, 4°C, 20 min) to remove insoluble debris and 0.9 mL of supernatant was dried using a vacuum centrifuge. Samples were stored at -80°C prior to analysis. For pyrimidine-free studies, a starter culture was washed, as described above, and grown in pyrimidine-free medium for 48 h prior to the start of the experiment. Targeted metabolite profiling by LC-MS/MS was performed as previously described allowing for detection of ~ 130 standard metabolites [52]. While this method allowed for quantitation of many key nucleosides and bases, the deoxynucleotides were not profiled as they are not part of the trained set of the facility. In order to attempt to identify these metabolites we isolated a larger cell number (5×10⁸cells) and again used targeted LC-MS/MS for detection as described [53]. Levels of deoxynucleotides in wild-type control SM cells were barely detectable so null lines were not analyzed.

Thymidine triphosphate (dTTP) quantitation by enzymatic assay

Because we were unable to quantitate dTTP by LC-MS/MS approaches we employed a previously reported enzymatic assay that monitors Klenow DNA polymerase catalyzed incorporation of ³H-dATP into synthetic oligonucleotides in a reaction that is proportional to the amount of dNTP [54, 55]. Through use of a standard curve the targeted dNTP concentration in the sample was determined. The oligo template that was used for the assay was as reported [54]. To prepare cell extracts, TK c-null cells were grown with or without Tet for 24 or 48 h in standard HMI-19 media supplemented with normal FBS and were harvested by centrifugation (3500 RPM, 5 min) and washed once with PBS. The pellets were resuspended in 60% methanol (250 μL) and incubated at -20°C overnight. Cell extracts were placed in a boiling water bath for 5 min, centrifuged (16,000xg, 20 min, 4°C), and then the soluble fraction was dried by vacuum centrifugation. Dried extracts were dissolved in 100 μL sample buffer (40 mM Tris-HCl pH 7.4, 10 mM MgCl₂). Each reaction (100 μL) contained 40 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, 5 mM DTT, 0.25 μM oligonucleotide template, 1.5 μg RNase A, 0.25 μM ³H-dATP (ARC- 17.2 Ci/mmol), 0.3 units Klenow Fragment (NEB), and cell extract (10 μL) or dTTP standard. Reactions were incubated at 37°C for 1 hr before spotting (85 μL of reaction) onto DE81 paper disks (23 mm-GE Healthcare), which were then air dried. Disks were washed (3 x 10 min) with 25 mL 5% Na₂HPO₄, rinsed once with water (25 mL) and absolute ethanol (15 mL). Dried disks were placed in scintillation liquid and radioactivity was measured by scintillation counting. A standard curve was generated with 0–4 pmol dTTP (New England BioLabs).

Nucleotide sugar analysis

T. brucei cells (2x10⁷) were pelleted by centrifugation (3500 RPM, 5 min), washed in cold PBS once and then resuspended in 1 mL pre-chilled methanol. Samples were freeze-thawed in liquid nitrogen, placed on ice for 10 min, and centrifuged (16,000 x g for 10 min at 4°C) to remove insoluble debris. A portion of the supernatant (0.8 mL) was vacuum dried and the residue resuspended in 40 mM sodium phosphate, pH 7.4. Samples were syringe filtered (Millex GV 0.22 μM–Millipore) to remove fine particles. High-Performance Anion Exchange Chromatography (HPAEC) analysis was performed as published [56] but with a modified elution gradient that was optimized for the separation of UDP-GalNAc, UDP-GlcNAc, UDP-Gal and UDP-Glc. Briefly, chromatography was performed on a Dionex ICS3000 HPAEC system with a CarboPac PA1 analytical column (4 mm Å~ 250 mm) and guard column (4 mm Å~ 50 mm). The method was performed with eluents 1 mM NaOH (E1) and 1 M NaOAc, 1 mM NaOH (E2) as follows: 0 min—20% E2, 10 min—45% E2, 25 min—45% E2, 35 min—45% E2, 40 min—100% E2, 50 min—100% E2, 55 min—20% E2, 65 min—20% E2. Equal amounts of sample (approximately 50 Mio cells) were analyzed within each experiment and the signal divided by cell count. Synthetic standards were acquired from Promega.

RNA and DNA purification

DNAzol (Molecular Research Center) was used to isolate genomic DNA from T. brucei cells. Typically, 5x10⁷ cells were collected for DNA extraction using guidelines recommended by the manufacturer. Total RNA was extracted from samples (3x10⁷cells) using TRIzol (Invitrogen), following the manufacturer’s protocol.

Quantitation of mRNA by qPCR

As described above, total RNA was isolated from samples and treated with DNaseI (Invitrogen) to eliminate gDNA contamination. A cDNA reverse transcription kit (Applied Biosystems) was used to synthesize cDNA for downstream analysis. Relative mRNA abundance was quantified using iTaq SYBR Green Supermix with ROX (Bio-Rad) utilizing a standard curve for each set of primers per experiment. For all experiments, TERT was used as a reference gene [57]. Data was collected on the CFX96 (Bio-Rad) and analysis was performed using the Pfaffl method [58].

Virulence studies in mice

Mice (C57BL/6J) were purchased from Wakeland Laboratory (UT Southwestern) and were group-housed in filter-top cages. The animal facility has standard laboratory conditions: 21 to 22°C ambient temperature and a 12 h light/12 h dark cycle. Chow and water were available ad libitum. Both doxycycline (Dox) water and water only (controls), were supplemented with 0.1% saccharin to ensure animals drank the Dox supplemented water. Mice from each group were introduced to the study drinking water 2 days prior to infection. Water bottles were protected from light and replaced every 2–3 days. Mice drank approximately 12.5 mL of water daily. Mice (8 weeks old, n = 6) (12 in total, 3 per study arm) were infected intraperitoneally with 10³ SM or TK c-null parasites ± Dox. Prior to the infection TK c-null parasites were propagated in +Tet conditions to ensure parasite viability at the start of the study. Mice were monitored for parasitemia starting three days post-infection by collecting 1 μL of blood from the tail in a 1:150 dilution of medium and counted using a hemocytometer as described [59]. Mice were monitored for 30 days post infection.

Western blot analysis

Cells (5x10⁷) were harvested by centrifugation (3500 RPM, 5 min), washed twice in cold PBS (10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 137 mM NaCl, 2.7 mM KCl, pH 7.4), and resuspended (50 uL) in trypanosome lysis buffer (50 mM Hepes, pH 8.0, 100 mM NaCl, 5 mM, β-mercaptoethanol, 2 mM PMSF, 1 mg/mL leupeptin, 2 mg/mL antipain, 10 mg/mL benzamidine, 1mg/mL pepstatin, 1 mg/mL chymostatin). Cells were lysed by 3 freeze/thaw cycles. Insoluble debris was pelleted by centrifugation (16,000 x g, 20 min, 4°C) and the soluble fraction was collected. The Bio-Rad Protein Assay reagent was used to determine protein concentration with bovine serum albumin (BSA) used to generate a standard curve. Total protein (20 μg) was resolved by 12% SDS-PAGE and transferred to a PVDF membrane using the Mini Trans-Blot Cell (BioRad). Membranes were blocked by 5% non-fat dry milk in Tris-buffered saline (TBS) (20 mM Tris (pH 7.6), 150 mM NaCl) for 1 h then incubated with a primary antibody in 5% milk and TBS-T (TBS + 0.1% Tween-20) overnight at 4°C. The following dilutions were used for each primary antibody: αFLAG 1:1000 (Rabbit polyclonal-Thermo Fisher), αAU1 1:1000 (Mouse monoclonal – Covance), αHsTK 1:500 (Mouse monoclonal—Thermo Fisher), αTbBiP 1:100,000 (Covance). For detection of the primary antibody, the membrane was incubated in a 1:10,000 dilution of a Protein A-HRP conjugate (Abcam) in TBS-T (5% milk) for 1 hour at room temperature. The membrane was washed 5 x 5min with TBS-T and incubated in SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) for 5 minutes. The membrane was visualized by the ImageQuant LAS 4000 (GE).

Data analysis

Graphs were generated in GraphPad Prism version 7.0a for Mac, GraphPad Software, San Diego California USA (www.graphpad.com), and statistical analysis was performed as indicated in the figure legends.

Genomic analysis to identify possible nucleotidyltransferase enzymes

The KEGG (Kyoto Encyclopedia of Genes and Genomes[60, 61]) pyrimidine metabolic pathway highlights two enzymatic reactions (EC 3.1.3.5 and EC 3.1.3.89) that perform the 5'-(deoxy)nucleotidase activity required to produce pyrimidine deoxynucleosides in other organisms. We searched all reviewed UniProtKB [62] entries with these two described enzyme activities, identifying 561 genes with EC 3.1.3.5 activity and 70 genes with EC 3.1.3.89 activity. We sorted the identified genes according to their assigned PFAM domains [63], keeping nine representative sequences from each unique PFAM, which correspond to five different fold groups (Table 1). For stringent identification of T. brucei protein sequences with potential 5' nucleotidase activity, the representative sequences were used as queries to search the NCBI NR database using PSI-BLAST [64] (5 iterations, E-value cutoff 0.001), storing the resulting position-specific scoring matrix as a checkpoint file for re-initiating BLAST against a database of protein sequences from the T. brucei genome (E-value cutoff 1). Identified T. brucei protein sequences were assigned PFAM domains using batch CD search [65], keeping those sequences with PFAM domains described as possessing 5’-nucleotidase activity. To identify all potential T. brucei protein sequences with domains related to those described as having 5'-nucleotidase activity, we queried the T. brucei genome using RPS-BLAST (E-value cutoff 0.006) with a library of sequence profiles downloaded from the conserved domain database (CDD ID in S1 Table) corresponding to each described 5'-nucleotidase PFAM in Table 1. Identified sequences were crosschecked for the presence of the query domain using batch CD search [65] or HHPRED [66, 67], reporting the positive hits using the initial RPS-BLAST E-values in S1 Table. Additional methods details are provided in S1 Text.

Cloning, expression and enzymatic assay of T. brucei 5’-nucleotidase (HD-fold, YfbR-like)

The DNA sequence for the T. brucei 5’-nucleotidase (HD-fold, YfbR-like) was obtained from TriTryDB (Tb09.211.2190). The E. coli codon optimized gene was synthesized by GenScript. PCR was used to generate flanking BsaI and XbaI restriction sites that allowed for cloning into the pE-SUMO(KAN) vector (LifeSensors, Malvern, PA) and expression as a N-terminal His₆-SUMO fusion protein. The nucleotidase pE-SUMO vector was transformed into BL21 cells. Cells were cultured in 2L LB-KAN (50 μg/mL) media at 37°C until OD₆₀₀ 0.7, then cooled to 16°C and induced by 500 μM IPTG (Isopropyl β-D-1-thiogalactopyranoside) for 16 h. Cells were collected by centrifugation and suspended in buffer A (500 mM NaCl, 50 mM HEPES pH 7.5, 5 mM imidazole, 5% glycerol, 5 mM 2-mercaptoethanol) supplemented with 2 mM PMSF (phenylmethane sulfonyl fluoride) and a protease inhibitor cocktail (1 mg/mL leupeptin, 2 mg/mL antipain, 10 mg/mL benzamidine, 1 mg/mL pepstatin, 1 mg/mL chymostatin). Cells were lysed by cell disruptor and the cell debris removed by centrifugation. Supernatant was applied to a HisTrap HP column (GE Healthcare) and washed with buffer A. The His₆-SUMO nucleotidase fusion was eluted using a gradient of 5–45% buffer B (500 mM NaCl, 50 mM HEPES pH 7.5, 5% glycerol, 5 mM 2-mercaptoethanol, 500 mM imidazole). Fractions were analyzed by SDS-PAGE and those containing the T. brucei HD domain 5’-nucelotidase were pooled, concentrated (10 kDa MWCO Millipore) and dialyzed against buffer A. The His₆-Sumo tag was removed by overnight incubation at 4°C with His₆-tagged Ubiquitin-like-specific protease 1 (ULP1) as described [68]. After incubation the mixture was applied to a second HisTrap HP column and cleaved 5’-nucelotidase was collected in the flow-through, while His₆-tagged ULP-1 and impurities were retained on the resin. The flow through was concentrated and protein purity was assessed to be >98% by SDS-PAGE analysis (S14 Fig). This tagless protein was used to collect the data shown in Fig 8. To provide an alternative purification method, His₆-Sumo tagged 5’-nucleotidase eluted from the first Ni⁺² column was further purified by Gel Filtration chromatography on a SuperDex 200 Prep Grade column (GE Healthcare) using buffer A. The His₆-Sumo tagged 5’-nucleotidase showed similar activity (within 2-fold) of the untagged version. Protein concentrations were determined by Bradford Assay (Bio-Rad).

T. brucei 5’-nucleotidase activity was assessed using an endpoint assay for released inorganic phosphate (P_i) using Malachite Green as the detection reagent as described [38, 69]. Briefly, each reaction (160 μL) contained 50 mM HEPES pH 7.5, 0.5 mM CoCl₂, 1 mM substrate, and enzyme. The reactions were incubated for 10–30 min (to confirm linearity) at 37°C and then 50 uL was treated with 100 mM EDTA. Malachite green reagent (150 μL) was added to each quenched reaction and incubated at room temperature for 5 min prior to measurement at 650 nm. A range of substrate (0.1–1.0 mM), metal (0.5–10 mM) and enzyme (25–100 nM) concentrations were tested to confirm linear dependence on enzyme concentration and to confirm that the reaction rate versus substrate curve was a saturable process. The production of P_i was measured at 650 nm. Absorbance was converted to μmoles of Pi using a standard curve generated using phosphate standard (Cayman Chemical) ranging from 0–100 μM. For the substrate and metal ion specificity studies shown in Fig 8, assays were run with 100 nM enzyme for 20 min using a substrate concentration of 1 mM. Metal ion concentrations are indicated on the graph. All data were collected in triplicate.