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Adaptive responses to purine starvation in Leishmania donovani.

ABSTRACT: Starvation of Leishmania donovani parasites for purines leads to a rapid amplification in purine nucleobase and nucleoside transport. Studies with nucleoside transport-deficient L. donovani indicate that this phenomenon is mediated by the nucleoside transporters LdNT1 and LdNT2, as well as by the purine nucleobase transporter LdNT3. The escalation in nucleoside transport cannot be ascribed to an increase in either LdNT1 or LdNT2 mRNA. However, Western analyses on parasites expressing epitope-tagged LdNT2 revealed a marked upregulation in transporter protein at the cell surface. Kinetic investigations of LdNT1 and LdNT2 activities from purine-replete and purine-starved cells indicated that both transporters exhibited significant increases in V(max) for their ligands under conditions of purine-depletion, although neither transporter displayed an altered affinity for its respective ligands. Concomitant with the increase in purine nucleoside and nucleobase transport, the purine salvage enzymes HGPRT, XPRT and APRT were also upregulated, suggesting that under conditions where purines are limiting, Leishmania parasites remodel their purine metabolic pathway to maximize salvage. Moreover, qRT-PCR analyses coupled with cycloheximide inhibition studies suggest that the underlying molecular mechanism for this augmentation in purine salvage occurs post-transcriptionally and is reliant on de novo protein synthesis.

SUBMITTER: Carter NS

PROVIDER: S-EPMC2964060 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Adaptive responses to purine starvation in Leishmania donovani.

Carter Nicola S NS Yates Phillip A PA Gessford Sarah K SK Galagan Sean R SR Landfear Scott M SM Ullman Buddy B

Molecular microbiology 20101001 1

Starvation of Leishmania donovani parasites for purines leads to a rapid amplification in purine nucleobase and nucleoside transport. Studies with nucleoside transport-deficient L. donovani indicate that this phenomenon is mediated by the nucleoside transporters LdNT1 and LdNT2, as well as by the purine nucleobase transporter LdNT3. The escalation in nucleoside transport cannot be ascribed to an increase in either LdNT1 or LdNT2 mRNA. However, Western analyses on parasites expressing epitope-tag ...[more]

PMID: 20923417

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Project description:The ability of Leishmania to survive in their insect or mammalian host is dependent upon an ability to sense and adapt to changes in the microenvironment. However, little is known about the molecular mechanisms underlying the parasite response to environmental changes, such as nutrient availability. To elucidate nutrient stress response pathways in Leishmania donovani, we have used purine starvation as the paradigm. The salvage of purines from the host milieu is obligatory for parasite replication; nevertheless, purine-starved parasites can persist in culture without supplementary purine for over 3 months, indicating that the response to purine starvation is robust and engenders parasite survival under conditions of extreme scarcity. To understand metabolic reprogramming during purine starvation we have employed global approaches. Whole proteome comparisons between purine-starved and purine-replete parasites over a 6-48 h span have revealed a temporal and coordinated response to purine starvation. Purine transporters and enzymes involved in acquisition at the cell surface are upregulated within a few hours of purine removal from the media, while other key purine salvage components are upregulated later in the time-course and more modestly.Â After 48 h, the proteome of purine-starved parasites is extensively remodeled and adaptations to purine stress appear tailored to deal with both purine deprivation and general stress. To probe the molecular mechanisms affecting proteome remodeling in response to purine starvation, comparative RNA-seq analyses, qRT-PCR, and luciferase reporter assays were performed on purine-starved versus purine-replete parasites.Â While the regulation of a minority of proteins tracked with changes at the mRNA level, for many regulated proteins it appears that proteome remodeling during purine stress occurs primarily via translational and post-translational mechanisms. One mRNA sample from each of Purine-Starved and Purine-Replete cells were analyzed using SL RNA-Seq methodology

Dataset Information

Adaptive responses to purine starvation in Leishmania donovani.

Publications

Adaptive responses to purine starvation in Leishmania donovani.

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