Abstract
Leishmania donovani cannot synthesize purines de novo and express a multiplicity of enzymes that enable them to salvage purines from their hosts. Previous efforts to generate an L. donovani strain deficient in both hypoxanthine-guanine phosphoribosyl-transferase (HGPRT) and xanthine phosphoribosyltransferase (XPRT) using gene replacement approaches were not successful, lending indirect support to the hypothesis that either HGPRT or XPRT is crucial for purine salvage by the parasite. We now report the genetic confirmation of this hypothesis through the construction of a conditional delta hgprt/delta xprt mutant strain that exhibits an absolute requirement for 2'-deoxycoformycin, an inhibitor of the leishmanial adenine aminohydrolase enzyme, and either adenine or adenosine as a source of purine. Unlike wild type parasites, the delta hgprt/delta xprt strain cannot proliferate indefinitely without 2'-deoxycoformycin or with hypoxanthine, guanine, xanthine, guanosine, inosine, or xanthosine as the sole purine nutrient. The delta hgprt/delta xprt mutant infects murine bone marrow-derived macrophages <5% as effectively as wild type parasites and cannot sustain an infection. These data establish genetically that either HGPRT or XPRT is absolutely essential for purine acquisition, parasite viability, and parasite infectivity of mouse macrophages, that all exogenous purines are funneled to hypoxanthine and/or xanthine by L. donovani, and that the purine sources within the macrophage to which the parasites have access are HGPRT or XPRT substrates.
Highlights
The institution of an effective parasite-specific therapeutic regimen for the treatment of leishmaniasis, or for that matter any parasitic disease, depends upon exploiting fundamental biochemical or metabolic differences between parasite and host
Because hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and xanthine phosphoribosyltransferase (XPRT) are colocalized within a 4359-bp EcoRI fragment in the L. donovani genome [5], the first copy of both genes was displaced with X63-NEO-⌬hgprt/⌬xprt, a construct containing the 5Ј-untranslated region (UTR) of HGPRT and the 3Ј-UTR of XPRT, to create the HGPRT/hgprt/XPRT/ xprt double heterozygote
The last round of transfection was performed in medium containing 20 M dCF and 100 M adenine, whereas the HGPRT/hgprt/XPRT/xprt and ⌬hgprt/XPRT/xprt progenitors of the double knock-out were isolated in medium lacking dCF and containing 100 M adenine as a purine source. 50 –100 drug-resistant colonies were obtained within days after the first two cycles of transfections, but surprisingly only two barely visible colonies were obtained after 4 weeks following the last round of transfection in the adenine-dCF medium
Summary
The institution of an effective parasite-specific therapeutic regimen for the treatment of leishmaniasis, or for that matter any parasitic disease, depends upon exploiting fundamental biochemical or metabolic differences between parasite and host. Implementing targeted gene replacement strategies, L. donovani promastigotes deficient in HGPRT, APRT, XPRT, and/or AK were created in almost every conceivable combination [5, 15, 16], it was not possible to create a ⌬hgprt/⌬xprt double mutant in any genetic background.
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