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Two distinct pathways for thymidylate (dTMP) synthesis …

The thymine-containing precursor of DNA is dTMP, which is synthesized after conversion of dUDP to dUMP

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dTMP biosynthetic process | SGD

In order to prove that the dTMP concentration in S. aureus affects the phenotypic appearance and intracellular persistence of S. aureus, this study was aimed at (i) correlating the effects of metabolic pathways that lead to altered dTMP concentrations with the associated phenotype and (ii) elucidating the impact of perturbed dTMP synthesis due to SXT exposure on the intracellular persistence of S. aureus in a CF cell line.

English: Synthesis of dTMP from dUMP, using 5,10-Methylenetetrahydrofolate

In view of the impact of SXT on the phenotypic appearance of S. aureus, an intracellular persistence assay was performed. Monolayers of IB3-1 epithelial CF cells were incubated with S. aureus SH1000 at an MOI of 50:1 in cell culture flasks for 3.5 h to enable intracellular uptake. Cell culture medium supplemented with lysostaphin was then added to inhibit extracellular growth of bacteria. In order to assess the impact of dTMP synthesis inhibitors on intracellular persistence, experiments were performed with and without SXT added to the cell culture medium. The numbers of intracellular bacteria were determined after 40 min and 24, 48, and 72 h of incubation as described in Materials and Methods. After 40 min of incubation approximately 10 bacteria per epithelial cell were observed (Fig. ). The average number of intracellular S. aureus SH1000 cells was found to decrease steadily over the 72-h time period to 0.03 bacterium per epithelial cell when cells were not exposed to SXT. S. aureus SH1000 exposed to SXT, however, showed significantly greater intracellular persistence after 48 and 72 h of incubation than unexposed S. aureus SH1000. Similar significantly greater intracellular persistence in IB3-1 cells after 72 h was found for S. aureus SCV S67 compared to the isogenic NCV revertant S. aureus S114 (data not shown). To verify the intracellular location of S. aureus, laser scanning microscopy was performed. After 3.5 h of incubation with S. aureus SH1000 and elimination of extracellular bacteria by lysostaphin exposure and washing, IB3-1 epithelial CF cells were stained with different markers as described in Materials and Methods. Staining with propidium iodide, which accumulates in dead bacteria, and SYTO9, which marks living bacteria, revealed that more than 99.9% of the bacteria were alive within F-actin-stained IB3-1 cells (data not shown). In addition, S. aureus-specific immunofluorescence staining confirmed the intracellular location of S. aureus (Fig. ).

Gene Ontology Term: dTMP biosynthetic process

Inhibition assays were used to analyze the metabolic pathways that are associated with the phenotypic appearance of thymidine-dependent S. aureus SCVs. The influence of 5-fluorouracil, acting mainly as an inhibitor of thymidylate synthase (), on the phenotype of S. aureus is shown in Fig. . S. aureus SH1000, an NCV, exhibited a zone of growth reduction around the disk impregnated with 5-fluorouracil on sheep blood agar (Fig. ). A thin bacterial lawn of small colonies grew around the disk, indicating that the strain utilized thymidine provided by the sheep blood agar to establish an alternative pathway for dTMP synthesis. In contrast, when S. aureus SH1000 was cultivated on Mueller-Hinton agar that did not contain thymidine, a clear-cut zone of inhibition was observed around the impregnated disk (Fig. ). The thymidine dependence of thymidylate synthase-inhibited S. aureus SH1000, in turn, was proven by growth of S. aureus SH1000 around a disk impregnated with thymidine within the zone of inhibition of 5-fluorouracil on Mueller-Hinton agar (Fig. ). S. aureus T1172 did not grow on Mueller-Hinton agar because this strain is a thymidine-dependent SCV. The growth on thymidine-providing sheep blood agar of S. aureus T1172 was not affected by 5-fluorouracil (Fig. ), because thymidylate synthase has no function in this strain ().

With regard to auxotrophism for growth factors, two main groups of S. aureus SCVs are recovered from clinical specimens: (i) SCVs that are dependent on menadione or hemin, indicating deficiencies in electron transport; and (ii) SCVs that are dependent on thymidine, indicating deficiencies in dTMP synthesis. Construction of S. aureus hemB and menD mutants with perturbed electron transport chains has been demonstrated to result in SCV phenotypes (). However, the genetic alterations in S. aureus SCVs of clinical isolates that are auxotrophic for hemin and menadione have not been discovered yet. In contrast, the detection of detrimental mutations in the thyA gene, encoding thymidylate synthase, in clinical thymidine-dependent SCV isolates and the construction of a defined thyA knockout mutant provide direct evidence at the molecular level that defects in the thymidylate synthase cause thymidine dependence in S. aureus SCVs (). This enzyme catalyzes the methylation of dUMP to dTMP with concomitant conversion of methylene tetrahydrofolate to dihydrofolate. As the antimicrobial agent trimethoprim-sulfamethoxazole (SXT) interferes with the bacterial tetrahydrofolate pathway and thymidine-dependent SCVs apparently bypass the SXT-inhibited pathway by uptake of extracellular thymidine via a nucleoside transporter, these variants can resist SXT exposure (, ). Thymidine-dependent S. aureus SCVs have even been shown to be induced by SXT, an agent which is frequently used for long-term prophylaxis in CF patients (, , ). Taking these observations together, it is tempting to speculate that the amount of intracellular dTMP in S. aureus is the pivotal factor in generation of thymidine-dependent SCVs.

dTMP synthesis View GO Annotations in other species in AmiGO

The present data show that inhibition of the thymidylate synthase by 5-fluorouracil in an NCV gives rise to an SCV, thereby confirming the importance of thyA integrity and dTMP concentration for the S. aureus phenotype. Likewise, SXT, which inhibits the synthesis of methylene tetrahydrofolate, induces the emergence of SCVs since methylene tetrahydrofolate is a cosubstrate of the thymidylate synthase.

A little bit of searching on-line will show you that the metabolic pathways for the pyrimidines CTP, GTP and UTP are extensively covered! There is a thymidine triphosphate, but strictly speaking it is dTTP, deoxythymidine triphosphate. Thymidine triphosphate is required for the synthesis of DNA. dTTP is not made by the de novo pyrimidine synthesis pathway. e.g., in E.coli dTTP is made after UDP or CDP is converted to dUDP or dCDP (which can then be deaminated to dUTP) by action of the enzyme ribonucleoside diphosphate reductase (sometimes abbreviated as rNDPase). dUTP is converted to dUMP (by dUTPase), dUMP to dTMP (by thymidylate synthase), and dTMP is converted to TDP and dTTP by kinases for use in DNA synthesis. There are other ways (i.e., thymidine kinase salvage pathway for dTMP) for its recycling.

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De novo and salvage pathway synthesis of dTMP in the nucleus

Folic acid, also known as Vitamin B9 is important to several biological functions. The folate derivative, 5,10-methylene-tetrahydrofolate is essential for the synthesis of dTMP from dUMP and it is therefore crucial for DNA replication and cell division. Tetrahydrofolate is an essential substrate in the biosynthesis of amino acid, glycine. Drugs targeting folate biosynthesis pathway has long been prescribed as anti-malarial agents. The two essential precursors of folate biosynthesis are 4-aminobenzoate (a product of pathway) and GTP. Thymidylate cycle, a part of folate biosynthesis pathway (below) plays important role in the generation of amino acid glycine and dTMP. Dihydrofolate reductase enzyme replenishes tetrahydrofolate from dihydrofolate for the above mentioned biosynthetic processes. The dihydrofolate reductase and thymidylate synthase activities are catalysed by a bifunctional enzyme in both Plasmodium falciparum and Toxoplasma gondii. In addition to the de novo folate biosynthesis pathway, T. gondii can salvage folate from host. Massimine et al demonstrated the uptake of radio-labelled exogenous folic acid and revealed the presence of common folate transporter which has high affinity for folic acid. This transporter is suggested to be bidirectional and concentration-dependent. They also added that T. gondii and other apicomplexans encode folate transporters as there are putative transporters homologous to BT1 family proteins present in these Apicomplexa genomes [].

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