A role for adenine nucleotides in the sensing mechanism to purine starvation in Leishmania donovani.

Purine salvage by Leishmania is an obligatory nutritional process that impacts both cell viability and growth. Previously, we have demonstrated that the removal of purines in culture provokes significant metabolic changes that enable Leishmania to survive prolonged periods of purine starvation. In order to understand how Leishmania sense and respond to changes in their purine environment, we have exploited several purine pathway mutants, some in which adenine and guanine nucleotide metabolism is uncoupled. While wild type parasites grow in any one of a variety of naturally occurring purines, the proliferation of these purine pathway mutants requires specific types or combinations of exogenous purines. By culturing purine pathway mutants in high levels of extracellular purines that are either permissive or non-permissive for growth and monitoring for previously defined markers of the adaptive response to purine starvation, we determined that adaptation arises from a surveillance of intracellular purine nucleotide pools rather than from a direct sensing of the extracellular purine content of the environment. Specifically, our data suggest that perturbation of intracellular adenine-containing nucleotide pools provides a crucial signal for inducing the metabolic changes necessary for the long-term survival of Leishmania in a purine-scarce environment.

IMP dehydrogenase deficiency in Leishmania donovani causes a restrictive growth phenotype in promastigotes but is not essential for infection in mice.

Leishmania cannot synthesize purines de novo and therefore must scavenge purines from its host for survival and growth. Biochemical and genomic analyses have indicated that Leishmania species express three potential routes for the synthesis of guanylate nucleotides: (1) a two-step pathway that converts IMP to GMP; (2) a three-step pathway that starts with the deamination of guanine to xanthine, followed by phosphoribosylation to XMP and then conversion to GMP; or (3) direct guanine phosphoribosylation by HGPRT. To determine the role of the first of these pathways to guanylate nucleotide synthesis, an L. donovani line deficient in IMP dehydrogenase (IMPDH), the first step in the IMP to GMP pathway, was constructed by targeted gene replacement. The Δimpdh lesion triggered a highly restrictive growth phenotype in promastigotes in culture but did not impact parasitemias in mice. The dispensability of IMPDH in vivo is the first definitive demonstration that intracellular L. donovani amastigotes have access to a sufficient pool of guanine, xanthine, or guanylate precursors from the host.

Characterization of amplicons that suppress the conditional lethal growth phenotype of a Leishmania donovani mutant lacking normal purine salvage mechanisms.

A conditionally lethal mutant of Leishmania donovani that lacks both hypoxanthine-guanine phosphoribosyltransferase and xanthine phosphoribosyltransferase exhibits a strikingly restricted growth phenotype, can only survive as the promastigote under pharmacological constraints, and is profoundly compromised in its ability to infect macrophages and mice. Interestingly, the conditionally lethal growth phenotype displayed by these mutant parasites can be suppressed in vitro by selection of strains that have markedly amplified the adenine phosphoribosyltransferase gene on extrachromosomal elements that are unique to these suppressor strains. Employing pulsed field gel electrophoresis, we have now determined that the amplicons in two of these suppressor lines are linear molecules by: (1) their pulse time-dependent mobility; (2) the failure of γ-irradiation to generate new discrete bands; (3) their susceptibility to λ exonuclease digestion; and (4) the presence of telomeric sequences. Pulsed field gel electrophoresis also shows these amplicons to be approximately 200-275kb in size. However, quantitative polymerase chain reaction and Southern blot analyses demonstrated that the amplification units are ∼40kb in length, implying that the formation of these amplicons involved additional chromosomal rearrangements or oligomerization.

A rapid, efficient and economical method for generating leishmanial gene targeting constructs.

Targeted gene replacement is a powerful tool in Leishmania genetics that can be time-consuming to implement. One tedious aspect that delays progress is the multi-step construction of gene targeting vectors. To accelerate this process, we developed a streamlined method that allows the assembly of a complete targeting vector from all its constituent parts in a single-step multi-fragment ligation. The individual components to be assembled are flanked by sites for the restriction endonuclease SfiI that generates non-identical, non-palindromic three base 3'-overhangs designed to allow annealing and ligation of the parts only in the proper order. The method was optimized by generating constructs for targeting the Leishmania donovani inosine monophosphate dehydrogenase gene (LdIMPDH) encoding six different drug resistance markers, and was found to be rapid and efficient. These constructs were successfully employed to generate heterozygous LdIMPDH gene replacement mutants. This method is adaptable for generating targeting vectors for a variety of species.