Degradation of pteridine reductase 1 (PTR1) enzyme during growth phase in the protozoan parasite Leishmania donovani

Kumar, Pranav; Sundar, Shyam; Singh, Neeloo

doi:10.1016/j.exppara.2006.12.008

Cited by 16 publications

(10 citation statements)

References 31 publications

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“…S3) refutes the predicted glycosomal location for PTR1, based on the PST1 consensus sequence ‐VHA at its C‐terminus (Opperdoes and Szikora, 2006). This is consistent with the failure to detect PTR1 by a proteomic analysis of purified T. brucei glycosomes (Vertommen et al ., 2008) and agrees with the cytosolic location of a GFP‐PTR1 chimera in L. donovani (Kumar et al ., 2007). Likewise, both trypanothione reductase and trypanothione peroxidase from T. brucei also contain putative glycosomal targeting sequences, yet the former is clearly cytosolic (Smith et al ., 1991) and the latter is located in the cytosol and mitochondria (Schlecker et al ., 2005).…”

Section: Discussionsupporting

confidence: 79%

Trypanosoma brucei pteridine reductase 1 is essential for survival in vitro and for virulence in mice

Sienkiewicz

Ong

Fairlamb³

2010

Molecular Microbiology

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Gene knockout and knockdown methods were used to examine essentiality of pteridine reductase (PTR1) in pterin metabolism in the African trypanosome. Attempts to generate PTR1 null mutants in bloodstream form Trypanosoma brucei proved unsuccessful; despite integration of drug selectable markers at the target locus, the gene for PTR1 was either retained at the same locus or elsewhere in the genome. However, RNA interference (RNAi) resulted in complete knockdown of endogenous protein after 48 h, followed by cell death after 4 days. This lethal phenotype was reversed by expression of enzymatically active Leishmania major PTR1 in RNAi lines (oeRNAi) or by addition of tetrahydrobiopterin to cultures. Loss of PTR1 was associated with gross morphological changes due to a defect in cytokinesis, resulting in cells with multiple nuclei and kinetoplasts, as well as multiple detached flagella. Electron microscopy also revealed increased numbers of glycosomes, while immunofluorescence microscopy showed increased and more diffuse staining for glycosomal matrix enzymes, indicative of mis-localisation to the cytosol. Mis-localisation was confirmed by digitonin fractionation experiments. RNAi cell lines were markedly less virulent than wild-type parasites in mice and virulence was restored in the oeRNAi line. Thus, PTR1 may be a drug target for human African trypanosomiasis.

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Section: Discussionsupporting

confidence: 79%

Trypanosoma brucei pteridine reductase 1 is essential for survival in vitro and for virulence in mice

Sienkiewicz

Ong

Fairlamb³

2010

Molecular Microbiology

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“…Strikingly, it was shown that mutants lacking PTR1, and thus deficient in tetrahydropterin (H 4 B), generated significantly more metacyclics in culture and were more virulent (50). Moreover, PTR1 mRNA and protein were shown to be stage regulated and decreased dramatically in stationary versus log phase promastigotes (51, 52), reinforcing the role of pteridine metabolism in metacyclogenesis. In the present study, two folate/biopterin transporters (LmjF.10.0380 and LmjF.06.0310) and one pteridine transporter (LmjF.06.1260) were significantly upregulated in NP and MP (Data Set S3, modules 5, 37, and 122; Data Set S1, NP versus PP).…”

Section: Resultsmentioning

confidence: 84%

“…Among the consistencies found, the pteridine transporters LmjF.06.1260 and its ortholog in L. infantum , LinJ.06.1320, were in each case significantly higher in sand fly metacyclics than in culture-derived forms. Pteridine transport and metabolism are crucial for Leishmania differentiation and virulence (51, 52). …”

Section: Resultsmentioning

confidence: 99%

The Transcriptome ofLeishmania majorDevelopmental Stages in Their Natural Sand Fly Vector

Inbar

Hughitt

Dillon

et al. 2017

mBio

148

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The life cycle of the Leishmania parasite in the sand fly vector involves differentiation into several distinctive forms, each thought to represent an adaptation to specific microenvironments in the midgut of the fly. Based on transcriptome sequencing (RNA-Seq) results, we describe the first high-resolution analysis of the transcriptome dynamics of four distinct stages of Leishmania major as they develop in a natural vector, Phlebotomus duboscqi. The early transformation from tissue amastigotes to procyclic promastigotes in the blood-fed midgut was accompanied by the greatest number of differentially expressed genes, including the downregulation of amastins, and upregulation of multiple cell surface proteins, sugar and amino acid transporters, and genes related to glucose metabolism and cell cycle progression. The global changes accompanying post-blood meal differentiation of procyclic promastigotes to the nectomonad and metacyclic stages were less extensive, though each displayed a unique signature. The transcriptome of nectomonads, which has not been studied previously, revealed changes consistent with cell cycle arrest and the upregulation of genes associated with starvation and stress, including autophagic pathways of protein recycling. Maturation to the infective, metacyclic stage was accompanied by changes suggesting preadaptation to the intracellular environment of the mammalian host, demonstrated by the amastigote-like profiles of surface proteins and metabolism-related genes. Finally, a direct comparison between sand fly-derived and culture-derived metacyclics revealed a reassuring similarity between the two forms, with the in vivo forms distinguished mainly by a stronger upregulation of transcripts associated with nutrient stress.

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“…Parasite culture Transgenic parasites overexpressing PTR1–green fluorescent protein (GFP chimaera, PTR1 tagged at the N-terminal with GFP) were cultured at 25°C in M-199 medium supplemented with 10% heat-inactivated FBS, 100 U penicillin, 100 μg/ml streptomycin and in the presence of 150 μg/ml geneticin sulphate (G418; Kumar et al 2007 ). These GFP-transfected parasites were used to determine the IC 50 of the compound by flow cytometric analysis as established by us (Singh and Dube 2004 ).…”

Section: Methodsmentioning

confidence: 99%

An orally effective dihydropyrimidone (DHPM) analogue induces apoptosis-like cell death in clinical isolates of Leishmania donovani overexpressing pteridine reductase 1

et al. 2009

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The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis. The enzyme pteridine reductase 1 (PTR1) of L. donovani acts as a metabolic bypass for drugs targeting dihydrofolate reductase (DHFR); therefore, for successful antifolate chemotherapy to be developed against Leishmania, it must target both enzyme activities. Leishmania cells overexpressing PTR1 tagged at the N-terminal with green fluorescent protein were established to screen for proprietary dihydropyrimidone (DHPM) derivatives of DHFR specificity synthesised in our laboratory. A cell-permeable molecule with impressive antileishmanial in vitro and in vivo oral activity was identified. Structure activity relationship based on homology model drawn on our recombinant enzyme established the highly selective inhibition of the enzyme by this analogue. It was seen that the leishmanicidal effect of this analogue is triggered by programmed cell death mediated by the loss of plasma membrane integrity as detected by binding of annexin V and propidium iodide (PI), loss of mitochondrial membrane potential culminating in cell cycle arrest at the sub-G0/G1 phase and oligonucleosomal DNA fragmentation. Hence, this DHPM analogue [(4-fluoro-phenyl)-6-methyl-2-thioxo-1, 2, 3, 4-tetrahydropyrimidine-5-carboxylic acid ethyl ester] is a potent antileishmanial agent that merits further pharmacological investigation.

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Degradation of pteridine reductase 1 (PTR1) enzyme during growth phase in the protozoan parasite Leishmania donovani

Cited by 16 publications

References 31 publications

Trypanosoma brucei pteridine reductase 1 is essential for survival in vitro and for virulence in mice

Trypanosoma brucei pteridine reductase 1 is essential for survival in vitro and for virulence in mice

The Transcriptome ofLeishmania majorDevelopmental Stages in Their Natural Sand Fly Vector

An orally effective dihydropyrimidone (DHPM) analogue induces apoptosis-like cell death in clinical isolates of Leishmania donovani overexpressing pteridine reductase 1

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