Boitz Jan M, Yates Phillip A, Kline Chelsey, Gaur Upasna, Wilson Mary E, Ullman Buddy, Roberts Sigrid C
Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, Oregon 97239-3098, USA.
Infect Immun. 2009 Feb;77(2):756-63. doi: 10.1128/IAI.01236-08. Epub 2008 Dec 8.
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 Deltaodc 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 Deltaodc 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 Deltaodc knockout lines were decreased approximately 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 Deltaodc 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 Deltaodc 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.
内脏利什曼病的病原体杜氏利什曼原虫，其多胺生物合成途径中的突变会使昆虫载体或寄生虫的前鞭毛体形式产生多胺营养缺陷。然而，寄生虫的感染性或无鞭毛体形式是否需要完整的多胺途径仍是一个悬而未决的问题。为了解决这个问题，通过在杜氏利什曼原虫的一个强毒株内进行双靶点基因替换，创建了缺乏鸟氨酸脱羧酶（ODC）（多胺生物合成中的限速酶）的条件致死性Deltaodc突变体。ODC缺陷的前鞭毛体和体外无鞭毛体对多胺营养缺陷，并且只有在培养基中提供外源性腐胺时才能强劲生长，这证实了多胺生物合成是杜氏利什曼原虫前鞭毛体必不可少的营养途径。为了评估Deltaodc损伤是否也影响无鞭毛体维持强劲感染的能力，进行了巨噬细胞和小鼠感染性实验。与野生型对应物相比，感染两个独立的Deltaodc敲除系之一的小鼠巨噬细胞中的寄生虫载量降低了约80%。此外，ODC的自杀性抑制剂α-二氟甲基鸟氨酸抑制了野生型杜氏利什曼原虫无鞭毛体的生长，并有效清除了巨噬细胞中的寄生虫，从而防止宿主细胞破坏。然而，令人惊讶的是，两个Deltaodc缺失突变体的虫血症在BALB/c小鼠的肝脏和脾脏中分别降低了6个和3个数量级。通过遗传损伤的附加体互补挽救了Deltaodc敲除在巨噬细胞和小鼠中的受损感染性表型。这些遗传学和药理学研究强烈表明ODC是杜氏利什曼原虫前鞭毛体和无鞭毛体的生存能力和生长所必需的重要细胞决定因素，并暗示ODC的药理学抑制是治疗内脏利什曼病以及可能的其他形式利什曼病的一种有前景的治疗模式。
