Adaptive responses to purine starvation in Leishmania donovani

Abstract

SummaryStarvation 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 purinestarved 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.

Genetic Dissection of Pyrimidine Biosynthesis and Salvage in Leishmania donovani

“…LdBob is derived from the 1S2D strain (33,34), which had been acclimated for growth as axenic amastigotes (32,35). Promastigotes were cultured at 26°C in pH 7.4 DME-L medium that was supplemented with 10% FBS and 100 M hypoxanthine (36). Parasite lines that were auxotrophic for pyrimidines were routinely maintained in medium supplemented with 250 M uridine or, when necessary to bypass a concomitant deficiency in pyrimidine salvage, 2 mM orotate.…”

The Leishmania donovani UMP Synthase Is Essential for Promastigote Viability and Has an Unusual Tetrameric Structure That Exhibits Substrate-controlled Oligomerization

“…The pBB-CmR-ccdB, pCR-HYG, and pCR-PHLEO plasmids used in the construction of the UMPS gene targeting vectors have been described (23). Polyclonal antisera against the purified LdUMPS protein were raised in rabbits as described by Carter and colleagues (24). Guinea pig antibodies against the L. donovani inosine monophosphate dehydrogenase (IMPDH) protein were furnished by Dr. Armando Jardim (McGill University, Montreal, Canada), and the production of this antiserum has been reported (25).…”

“…A, genomic DNA prepared from wild-type, LdUMPS/umps, and ⌬umps parasites was digested with PvuII and hybridized to a 1.37-kb probe from the LdUMPS ORF and B, to a 0.73-kb probe from the 5Ј-flanking region of LdUMPS. C, a Western blot of wild-type, LdUMPS/umps, and ⌬umps cell lysates fractionated on an SDS-polyacrylamide gel and probed with monospecific antisera raised against the purified LdUMPS protein and monoclonal antibody directed against mouse ␣-tubulin (24). Lanes 1-3 in all panels refer to DNA or protein obtained from wild-type, LdUMPS/umps, and ⌬umps cells, respectively.…”

Nutrient Transport and Pathogenesis in Selected Parasitic Protozoa

“…In each study, purine restriction resulted in a pronounced upregulation (45-to 85-fold) of purine transport activities that resulted from an increase in the rate of transporter mRNA translation but did not significantly alter the abundance of the mRNA itself. An increase in the level of several purine salvage enzymes was also detected in parasites starved for purines (24). These results imply that Leishmania parasites sense purines and respond to decreasing levels of these essential nutrients by upregulating translation of mRNAs encoding purine transporters and salvage enzymes.…”

“…Two recent studies have examined expression of purine transporters in L. donovani and L. major parasites that have been restricted for uptake of essential purines, either by eliminating purines from the growth medium (24) or by restricting purine uptake in single or double null mutants for purine transporters (76). In each study, purine restriction resulted in a pronounced upregulation (45-to 85-fold) of purine transport activities that resulted from an increase in the rate of transporter mRNA translation but did not significantly alter the abundance of the mRNA itself.…”