Leishmania donovani Ornithine Decarboxylase Is Indispensable for Parasite Survival in the Mammalian Host

被引:57
|
作者
Boitz, Jan M. [1 ]
Yates, Phillip A. [1 ]
Kline, Chelsey [2 ]
Gaur, Upasna [3 ]
Wilson, Mary E. [3 ]
Ullman, Buddy [1 ]
Roberts, Sigrid C. [1 ,2 ]
机构
[1] Oregon Hlth & Sci Univ, Dept Biochem & Mol Biol, Portland, OR 97239 USA
[2] Pacific Univ, Sch Pharm, Hillsboro, OR 97123 USA
[3] Univ Iowa, Dept Microbiol, Iowa City, IA 52242 USA
关键词
S-ADENOSYLMETHIONINE DECARBOXYLASE; HUMAN AFRICAN TRYPANOSOMIASIS; DL-ALPHA-DIFLUOROMETHYLORNITHINE; BRUCEI-RHODESIENSE INFECTIONS; GAMBIENSE SLEEPING SICKNESS; GENE DELETION MUTANTS; VISCERAL LEISHMANIASIS; METHYLGLYOXAL BIS(GUANYLHYDRAZONE); IRREVERSIBLE INHIBITION; EFLORNITHINE;
D O I
10.1128/IAI.01236-08
中图分类号
R392 [医学免疫学]; Q939.91 [免疫学];
学科分类号
100102 ;
摘要
Mutations within the polyamine biosynthetic pathway of Leishmania donovani, the etiological agent of visceral leishmaniasis, confer polyamine auxotrophy to the insect vector or promastigote form of the parasite. However, whether the infectious or amastigote form of the parasite requires an intact polyamine pathway has remained an open question. To address this issue, conditionally lethal Delta odc mutants lacking ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, were created by double targeted gene replacement within a virulent strain of L. donovani. ODC-deficient promastigotes and axenic amastigotes were auxotrophic for polyamines and capable of robust growth only when exogenous putrescine was supplied in the culture medium, confirming that polyamine biosynthesis is an essential nutritional pathway for L. donovani promastigotes. To assess whether the Delta odc lesion also affected the ability of amastigotes to sustain a robust infection, macrophage and mouse infectivity experiments were performed. Parasite loads in murine macrophages infected with each of two independent Delta odc knockout lines were decreased similar to 80% compared to their wild-type counterpart. Furthermore, alpha-difluoromethylornithine, a suicide inhibitor of ODC, inhibited growth of wild-type L. donovani amastigotes and effectively cured macrophages of parasites, thereby preventing host cell destruction. Strikingly, however, parasitemias of both Delta odc null mutants were reduced by 6 and 3 orders of magnitude, respectively, in livers and spleens of BALB/c mice. The compromised infectivity phenotypes of the Delta odc knockouts in both macrophages and mice were rescued by episomal complementation of the genetic lesion. These genetic and pharmacological studies strongly implicate ODC as an essential cellular determinant that is necessary for the viability and growth of both L. donovani promastigotes and amastigotes and intimate that pharmacological inhibition of ODC is a promising therapeutic paradigm for the treatment of visceral and perhaps other forms of leishmaniasis.
引用
收藏
页码:756 / 763
页数:8
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