Characterization of the folylpolyglutamate synthetase gene and polyglutamylation of folates in the protozoan parasite Leishmania

Fadili, Amal El

doi:10.1016/s0166-6851(02)00163-9

Cited by 26 publications

(22 citation statements)

References 38 publications

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“…The latter system is a convenient and rapid quantitative method to look at intracellular growth of parasites [ 20]. The firefly luciferase gene as part of the Leishmania expression vector pSPαHYGα [ 22] was introduced in L. tropica strains by electroporation.…”

Section: Resultsmentioning

confidence: 99%

“…Recombinant parasites were produced by transfection of the Leishmania isolates with the firefly luciferase–containing vector pSP1.2 LUC αHYGα [ 22] and were used to infect THP1 cells (ratio 20:1) stimulated with phorbol myristic acetate (PMA) as described [ 21]. Luciferase activity was measured after 5 d of incubation in the presence of Glucantime.…”

Section: Methodsmentioning

confidence: 99%

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Unresponsiveness to Glucantime Treatment in Iranian Cutaneous Leishmaniasis due to Drug-Resistant Leishmania tropica Parasites

et al. 2006

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BackgroundRecent circumstantial evidence suggests that an increasing number of Iranian patients with cutaneous leishmaniasis are unresponsive to meglumine antimoniate (Glucantime), the first line of treatment in Iran. This study was designed to determine whether the clinical responses (healing, or non-healing) were correlated with the susceptibility of Leishmania parasites to Glucantime. Methods and FindingsIn vitro susceptibility testing was first performed on 185 isolated parasites in the intracellular mouse peritoneal macrophage model. A strong correlation between the clinical outcome and the in vitro effective concentration 50% (EC 50) values was observed. Parasites derived from patients with non-healing lesions had EC 50 values at least 4-fold higher than parasites derived from lesions of healing patients. A selection of these strains was typed at the molecular level by pulsed-field gels and by sequencing the pteridine reductase 1 (PTR1) gene. These techniques indicated that 28 out of 31 selected strains were Leishmania tropica and that three were Leishmania major. The L. major isolates were part of a distinct pulsed-field group, and the L. tropica isolates could be classified in three related additional pulsed-field groups. For each pulsed-field karyotype, we selected sensitive and resistant parasites in which we transfected the firefly luciferase marker to assess further the in vitro susceptibility of field isolates in the monocyte cell line THP1. These determinations confirmed unequivocally that patients with non-healing lesions were infected with L. tropica parasites resistant to Glucantime. Additional characterization of the resistant isolates showed that resistance is stable and can be reversed by buthionine sulfoximine, an inhibitor of glutathione biosynthesis. ConclusionsTo the authors' knowledge, this is the first report of proven resistant parasites contributing to treatment failure for cutaneous leishmaniasis and shows that primary Glucantime-resistant L. tropica field isolates are now frequent in Iran.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Unresponsiveness to Glucantime Treatment in Iranian Cutaneous Leishmaniasis due to Drug-Resistant Leishmania tropica Parasites

et al. 2006

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“…All the constructs were checked by sequencing with internal primers (Table S1). Similarly, the genes pyridoxal kinase (LmjF30.1250) and α-adaptin like protein (LmjF07.0050) were cloned in pSP72αNEOα or pSP72αHYGα[31] at Xba I and Hind III sites.…”

Section: Methodsmentioning

confidence: 99%

Multiple Mutations in Heterogeneous Miltefosine-Resistant Leishmania major Population as Determined by Whole Genome Sequencing

et al. 2012

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BackgroundMiltefosine (MF) is the first oral compound used in the chemotherapy against leishmaniasis. Since the mechanism of action of this drug and the targets of MF in Leishmania are unclear, we generated in a step-by-step manner Leishmania major promastigote mutants highly resistant to MF. Two of the mutants were submitted to a short-read whole genome sequencing for identifying potential genes associated with MF resistance.Methods/Principal FindingsAnalysis of the genome assemblies revealed several independent point mutations in a P-type ATPase involved in phospholipid translocation. Mutations in two other proteins—pyridoxal kinase and α-adaptin like protein—were also observed in independent mutants. The role of these proteins in the MF resistance was evaluated by gene transfection and gene disruption and both the P-type ATPase and pyridoxal kinase were implicated in MF susceptibility. The study also highlighted that resistance can be highly heterogeneous at the population level with individual clones derived from this population differing both in terms of genotypes but also susceptibility phenotypes.Conclusions/SignificanceWhole genome sequencing was used to pinpoint known and new resistance markers associated with MF resistance in the protozoan parasite Leishmania. The study also demonstrated the polyclonal nature of a resistant population with individual cells with varying susceptibilities and genotypes.

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“…Leishmania species possess a single FPGS gene, and FPGS over- or under-expression in L. tarentolae resulted in increased or decreased levels of folylpolyglutamates (with Glu 4–6 predominating). This caused corresponding changes in the parasites’ resistance to methotrexate, which is less efficiently polyglutamylated by FPGS [16–18]. Efforts to generate FPGS-homozygous replacements were unsuccessful, suggesting that this gene may be essential [16].…”

Section: Acquisition Of Folates and Pteridinesmentioning

confidence: 99%

“…This caused corresponding changes in the parasites’ resistance to methotrexate, which is less efficiently polyglutamylated by FPGS [16–18]. Efforts to generate FPGS-homozygous replacements were unsuccessful, suggesting that this gene may be essential [16]. De-glutamylating activities have also been reported, although their roles other than contributing to methotrexate resistance have not been explored [18].…”

Section: Acquisition Of Folates and Pteridinesmentioning

confidence: 99%

Folate metabolic pathways in Leishmania

Vickers

Beverley

2011

Essays in Biochemistry

107

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Trypanosomatid parasitic protozoans of the genus Leishmania are autotrophic for both folate and unconjugated pteridines. Leishmania salvage these metabolites from their mammalian hosts and insect vectors through multiple transporters. Within the parasite, folates are reduced by a bifunctional DHFR (dihydrofolate reductase)-TS (thymidylate synthase) and by a novel PTR1 (pteridine reductase 1), which reduces both folates and unconjugated pteridines. PTR1 can act as a metabolic bypass of DHFR inhibition, reducing the effectiveness of existing antifolate drugs. Leishmania possess a reduced set of folate-dependent metabolic reactions and can salvage many of the key products of folate metabolism from their hosts. For example, they lack purine synthesis, which normally requires 10-formyltetrahydrofolate, and instead rely on a network of purine salvage enzymes. Leishmania elaborate at least three pathways for the synthesis of the key metabolite 5,10-methylene-tetrahydrofolate, required for the synthesis of thymidylate, and for 10-formyltetrahydrofolate, whose presumptive function is for methionyl-tRNAMet formylation required for mitochondrial protein synthesis. Genetic studies have shown that the synthesis of methionine using 5-methyltetrahydrofolate is dispensable, as is the activity of the glycine cleavage complex, probably due to redundancy with serine hydroxymethyltransferase. Although not always essential, the loss of several folate metabolic enzymes results in attenuation or loss of virulence in animal models, and a null DHFR-TS mutant has been used to induce protective immunity. The folate metabolic pathway provides numerous opportunities for targeted chemotherapy, with strong potential for ‘repurposing’ of compounds developed originally for treatment of human cancers or other infectious agents.

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Characterization of the folylpolyglutamate synthetase gene and polyglutamylation of folates in the protozoan parasite Leishmania

Cited by 26 publications

References 38 publications

Unresponsiveness to Glucantime Treatment in Iranian Cutaneous Leishmaniasis due to Drug-Resistant Leishmania tropica Parasites

Unresponsiveness to Glucantime Treatment in Iranian Cutaneous Leishmaniasis due to Drug-Resistant Leishmania tropica Parasites

Multiple Mutations in Heterogeneous Miltefosine-Resistant Leishmania major Population as Determined by Whole Genome Sequencing

Folate metabolic pathways in Leishmania

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