Adenylosuccinate Synthetase and Adenylosuccinate Lyase Deficiencies Trigger Growth and Infectivity Deficits in Leishmania donovani

Boitz, Jan M.; Strasser, Richard; Yates, Phillip A.; Jardim, Armando; Ullman, Buddy

doi:10.1074/jbc.m112.431486

Cited by 38 publications

(54 citation statements)

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“…As expected, wild type, Δ aprt , and Δ aah promastigotes displayed robust growth in adenine, while Δ aah /Δ aprt promastigotes failed to proliferate (Table I). As shown previously, neither Δ adss nor Δ hgprt /Δ xprt promastigotes could grow in adenine [9,17], although when a Δ aah mutation was inserted into each of these genetic backgrounds, the cells grew robustly [10,17]. Furthermore, pharmacologic simulation of the AAH deficiency with dCF enabled Δ adss [17] and Δ hgprt /Δ xprt (Table 1) promastigotes to proliferate with adenine as the sole purine nutrient but prevented growth of Δ aprt promastigotes by blocking adenine conversion to hypoxanthine via AAH.…”

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confidence: 77%

“…Included in this nutritional analysis were previously isolated gene deletion mutants with known 6-aminopurine growth deficits. These mutants include L. donovani harboring genetic lesions in adenylosuccinate synthetase (ADSS), AAH and ADSS (Δ aah /Δ adss ), HGPRT and XPRT (Δ hgprt /Δ xprt ), and the triple null mutant Δ aah /Δ hgprt /Δ xprt (Table I) [9,10,17]. Nutritional assessments were performed in purine-deficient Dulbecco’s modified Eagle medium- Leishmania (DME-L) growth medium supplemented with 5% dialyzed fetal bovine serum to which either 100 μM adenine or adenosine was added.…”

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confidence: 99%

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Adenine and adenosine salvage in Leishmania donovani

Boitz

Ullman

2013

Molecular and Biochemical Parasitology

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6-aminopurine metabolism in Leishmania is unique among trypanosomatid pathogens since this genus expresses two distinct routes for adenine salvage: adenine phosphoribosyltransferase (APRT) and adenine deaminase (AAH). To evaluate the relative contributions of APRT and AAH, adenine salvage was evaluated in Δaprt, Δaah, and Δaprt/Δaah null mutants of L. donovani. The data confirm that AAH plays the dominant role in adenine metabolism in L. donovani, although either enzyme alone is sufficient for salvage. Adenosine salvage was also evaluated in a cohort of null mutants. Adenosine is also primarily converted to hypoxanthine, either intracellularly or extracellularly, but can also be phosphorylated to the nucleotide level by adenosine kinase when the predominant pathways are genetically or pharmacologically blocked. These data provide genetic verification for the relative contributions of 6-aminopurine metabolizing pathways in L. donovani and demonstrate that all of the pathways can function under appropriate conditions of genetic or pharmacologic perturbation.

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confidence: 77%

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confidence: 99%

Adenine and adenosine salvage in Leishmania donovani

Boitz

Ullman

2013

Molecular and Biochemical Parasitology

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“…GMPR was localized in L. donovani promastigotes by indirect immunofluorescence as described [21,22,25,26] using a 1:500 dilution of anti-LmGMPR antibody and a 1:10,000 dilution of secondary goat anti-rabbit Oregon Green-conjugated antibody (Thermo Fisher Scientific). Parasites were costained with guinea pig antibodies to the L. donovani IMPDH protein (1:200) and goat anti-guinea pig Rhodamine Red-conjugated (1:10,000) secondary antibody (Thermo Fisher Scientific) to stain glycosomes.…”

Section: Methodsmentioning

confidence: 99%

“…1) [5,7,17–21]. The importance of the nucleotide interconversion pathways in growth and infectivity was validated by the isolation and characterization of Δ adss and Δ asl and Δ impdh null mutants [22,23]. The Δ adss and Δ asl parasites exhibited growth phenotypes essentially indistinguishable from that of the previously characterized Δ hgprt /Δ xprt double knockout, whereas the Δ impdh cells could utilize guanine, guanosine, xanthine or xanthosine, all guanylate precursors, to fulfill their purine requirements [14,22,23].…”

Section: Introductionmentioning

confidence: 99%

“…The importance of the nucleotide interconversion pathways in growth and infectivity was validated by the isolation and characterization of Δ adss and Δ asl and Δ impdh null mutants [22,23]. The Δ adss and Δ asl parasites exhibited growth phenotypes essentially indistinguishable from that of the previously characterized Δ hgprt /Δ xprt double knockout, whereas the Δ impdh cells could utilize guanine, guanosine, xanthine or xanthosine, all guanylate precursors, to fulfill their purine requirements [14,22,23]. All three null mutants exhibited reduced infectivity phenotypes in macrophages; however, only Δ asl parasites were severely incapacitated in their ability to establish a visceral infection in the mouse model, whereas parasite burdens in mice inoculated with Δ adss or Δ impdh cells were robust [22,23].…”

Section: Introductionmentioning

confidence: 99%

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GMP reductase and genetic uncoupling of adenylate and guanylate metabolism in Leishmania donovani parasites

Boitz

Jardim

Ullman

2016

Molecular and Biochemical Parasitology

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Purine acquisition is an essential nutritional process for Leishmania. Although purine salvage into adenylate nucleotides has been investigated in detail, little attention has been focused on the guanylate branch of the purine pathway. To characterize guanylate nucleotide metabolism in Leishmania and create a cell culture model in which the pathways for adenylate and guanylate nucleotide synthesis can be genetically uncoupled for functional studies in intact cells, we created and characterized null mutants of L. donovani that were deficient in either GMP reductase alone (Δgmpr) or in both GMP reductase and its paralog IMP dehydrogenase (Δgmpr/Δimpdh). Whereas wild type parasites were capable of utilizing virtually any purine nucleobase/nucleoside, the Δgmpr and Δgmpr/Δimpdh null lines exhibited highly restricted growth phenotypes. The Δgmpr single mutant could not grow in xanthine, guanine, or their corresponding nucleosides, while no purine on its own could support the growth of Δgmpr/Δimpdh cells. Permissive growth conditions for the Δgmpr/Δimpdh necessitated both xanthine, guanine, or the corresponding nucleosides, and additionally, a second purine that could serve as a source for adenylate nucleotide synthesis. Interestingly, GMPR, like its paralog IMPDH, is compartmentalized to the leishmanial glycosome, a process mediated by its COOH-terminal peroxisomal targeting signal. The restricted growth phenotypes displayed by the L. donovani Δgmpr and Δgmpr/Δimpdh null mutants confirms the importance of GMPR in the purine interconversion processes of this parasite.

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Leishmania donovani adenylosuccinate synthetase requires IMP for dimerization and organization of the active site

Mochi,

Jani,

Shah

et al. 2024

FEBS Letters

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Adenylosuccinate synthetase (AdSS), which catalyses the GTP‐dependent conversion of inosine monophosphate (IMP) and aspartic acid to succinyl‐AMP, plays a major role in purine biosynthesis. In some bacterial AdSS, it is implicated that IMP binding is important to organize the active site, but in certain plant AdSS, GTP performs this role. Here, we report that in Leishmania donovani AdSS, IMP binding favoured dimerization, induced greater conformational change and improved the protein stability more than GTP binding. IMP binding, which resulted in a network of hydrogen bonds, stabilized the conformation of active site loops and brought the switch loop to a closed conformation, which then facilitated GTP binding. Our results provide a basis for designing better inhibitors of leishmanial AdSS.

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Adenylosuccinate Synthetase and Adenylosuccinate Lyase Deficiencies Trigger Growth and Infectivity Deficits in Leishmania donovani

Cited by 38 publications

References 65 publications

Adenine and adenosine salvage in Leishmania donovani

Adenine and adenosine salvage in Leishmania donovani

GMP reductase and genetic uncoupling of adenylate and guanylate metabolism in Leishmania donovani parasites

Leishmania donovani adenylosuccinate synthetase requires IMP for dimerization and organization of the active site

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