9 research outputs found
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
<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
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<sup>+2</sup>. 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
5'-nucleotidase PFAM domain representatives.
<p>5'-nucleotidase PFAM domain representatives.</p
<i>Hs</i>DCTD rescues the growth defect in <i>Tb</i>TK RNAi and <i>Tb</i>TK null cell lines.
<p>A. Growth curves for <i>TK</i> RNAi cells or <i>TK</i> RNAi cells containing a Tet-regulated expression plasmid for <i>Hs</i>DCTD. Cell growth was monitored ±Tet for the indicated days. Error bars represent SD for triplicate biological replicates. Inset shows a Western blot of <i>Hs</i>DCTD expression ± Tet at 48 h. B. qPCR analysis of <i>Tb</i>TK mRNA expression in both the <i>TK</i> RNAi cell line and the <i>TK</i> RNAi <i>Hs</i>DCTD 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 <i>TK</i> null cells expressing either FLAG-tagged <i>Tb</i>TK (c-null) or FLAG-tagged <i>Hs</i>DCTD under the control of the Tet promoter. Error bars represent SD for triplicate biological replicates. Inset shows western blot analysis of the <i>Hs</i>DCTD <i>TK</i> null cells 2 days and 5 days after Tet withdraw.</p
Deletion of the <i>T</i>. <i>brucei CDA</i> gene induces pyrimidine auxotrophy.
<p>A. Growth curves for <i>CDA</i> 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 <i>CDA</i> null cells. Error bars represent SEM for triplicate data. C-E. Growth analysis of <i>CDA</i> 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 ED<sub>50</sub> for growth stimulation. ED<sub>50</sub> = 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.</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