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Infection and Immunity, February 2009, p. 756-763, Vol. 77, No. 2
0019-9567/09/$08.00+0     doi:10.1128/IAI.01236-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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

Jan M. Boitz,1,{dagger} Phillip A. Yates,1,{dagger} Chelsey Kline,2 Upasna Gaur,3 Mary E. Wilson,3 Buddy Ullman,1* and Sigrid C. Roberts1,2,{ddagger}

Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, Oregon 97239-3098,1 Pacific University School of Pharmacy, Hillsboro, Oregon 97123,2 Department of Microbiology, University of Iowa, Iowa City, Iowa 522423

Received 9 October 2008/ Returned for modification 13 November 2008/ Accepted 1 December 2008

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 ~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.

* Corresponding author. Mailing address: Oregon Health & Science University, Department of Biochemistry and Molecular Biology, 3181 SW Sam Jackson Park Road, Portland, OR 97239-3098. Phone: (503) 494-2546. Fax: (503) 494-8393. E-mail: ullmanb{at}ohsu.edu

{triangledown} Published ahead of print on 8 December 2008.

Editor: W. A. Petri, Jr.

{dagger} J.M.B. and P.A.Y. contributed equally to this publication.

{ddagger} Senior author.

Infection and Immunity, February 2009, p. 756-763, Vol. 77, No. 2
0019-9567/09/$08.00+0     doi:10.1128/IAI.01236-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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