Abstract
Leishmania are auxotrophic for purines, and consequently purine acquisition from the host is a requisite nutritional function for the parasite. Both adenylosuccinate synthetase (ADSS) and adenylosuccinate lyase (ASL) have been identified as vital components of purine salvage in Leishmania donovani, and therefore Δadss and Δasl null mutants were constructed to test this hypothesis. Unlike wild type L. donovani, Δadss and Δasl parasites in culture exhibited a profoundly restricted growth phenotype in which the only permissive growth conditions were a 6-aminopurine source in the presence of 2'-deoxycoformycin, an inhibitor of adenine aminohydrolase activity. Although both knock-outs showed a diminished capacity to infect murine peritoneal macrophages, only the Δasl null mutant was profoundly incapacitated in its ability to infect mice. The enormous discrepancy in parasite loads observed in livers and spleens from mice infected with either Δadss or Δasl parasites can be explained by selective accumulation of adenylosuccinate in the Δasl knock-out and consequent starvation for guanylate nucleotides. Genetic complementation of a Δasl lesion in Escherichia coli implied that the L. donovani ASL could also recognize 5-aminoimidazole-(N-succinylocarboxamide) ribotide as a substrate, and purified recombinant ASL displayed an apparent Km of ∼24 μm for adenylosuccinate. Unlike many components of the purine salvage pathway of L. donovani, both ASL and ADSS are cytosolic enzymes. Overall, these data underscore the paramount importance of ASL to purine salvage by both life cycle stages of L. donovani and authenticate ASL as a potential drug target in Leishmania.
Highlights
Purine salvage in Leishmania is an essential nutritional function
The adenylosuccinate lyase (ASL) ORF was amplified by PCR from the ASL cosmid using primers that attached appropriate 5Ј- and 3Ј-restriction sites, subcloned into the pCR2.1TOPO vector, excised via an NdeI-EcoRV restriction digest, and ligated into the pLCNEO-EGFPCO vector to create pLCNEO-EFGPCO-ASL. pLCNEO-EFGPCO-ASL was transfected into wild type L. donovani promastigotes, the transfectants were selected in 20 M G418, and EFGP-tagged ASL protein was visualized as described above
Primary Structures of L. donovani adenylosuccinate synthetase (ADSS) and ASL—The L. donovani ADSS (ABS11225) and ASL (ABS11226) genes and their flanking sequences were isolated from purified cosmid clones that hybridized to the putative ADSS (LmjF.13.1190) and ASL (LmjF.04.0460) ORFs, respectively, from L. major [38]
Summary
Purine salvage in Leishmania is an essential nutritional function. Results: Null mutants deficient in either adenylosuccinate synthetase or adenylosuccinate lyase impact growth and infectivity phenotypes of Leishmania donovani. The ⌬hgprt/ ⌬xprt null mutant is highly compromised in its ability to establish a visceral infection in mice [21], implying a central role for HGPRT and XPRT in purine salvage by amastigotes as well These findings demonstrate, that virtually all host or extracellular purines are funneled into substrates of HGPRT or XPRT and strongly intimate the functional importance of the downstream nucleotide interconversion enzymes that distribute the enzymatic products of HGPRT and XPRT into adenylate and guanylate nucleotides. Recombinant ASL was purified and shown to be catalytically active, and the gene complemented asl-deficient Escherichia coli in a manner that suggested that it was a bifunctional enzyme, like its human counterpart, which could catalyze the cleavage of 5-aminoimidazole-(N-succinylocarboxamide) ribotide (SAICAR) to 5-aminoimidazole-4-carboxamide ribotide and fumarate [26] Both ADSS and ASL were localized to the parasite cytosol using immunocytochemistry and/or cell fractionation, implying a potentially intriguing portioning of the adenylate and guanylate branches of the L. donovani purine salvage pathway. This investigation is the first to validate a single component of the purine salvage pathway of Leishmania as a prospective antileishmanial drug target
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