Molecular genetic analysis of purine nucleobase transport in Leishmania major

doi:10.1111/j.1365-2958.2007.05730.x

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Wiley InterScience

Molecular Microbiology

Volume 64 Issue 5, Pages 1228 - 1243

Published Online: 22 Apr 2007

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Molecular genetic analysis of purine nucleobase transport in Leishmania major

Diana Ortiz, ¹ Marco A. Sanchez, ¹ Steven Pierce, ^1† Timo Herrmann, ^1‡ Nicola Kimblin, ² H. G. Archie Bouwer ³ and Scott M. Landfear ¹ *

¹ Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR 97239, USA.
² Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
³ Veterans Affairs Medical Center Immunology Research, Earle A. Chiles Research Institute, Portland, OR 97239, USA.

Correspondence to *E-mail landfear@ohsu.edu; Tel. (+1) 503 494 2426; Fax (+1) 503 494 6862.

Present addresses: ^†Department of Biological Sciences, Columbia University, New York, NY 10032, USA.

^‡ Medical and Natural Sciences Research Center, University of Tübingen, Tübingen 72074, Germany.

ABSTRACT

Leishmania major and all other parasitic protozoa are unable to synthesize purines de novo and are therefore reliant upon uptake of preformed purines from their hosts via nucleobase and nucleoside transporters. L. major expresses two nucleobase permeases, NT3 that is a high affinity transporter for purine nucleobases and NT4 that is a low affinity transporter for adenine. nt3^(–/–) null mutant promastigotes were unable to replicate in medium containing 10 μM hypoxanthine, guanine, or xanthine and replicated slowly in 10 μM adenine due to residual low affinity uptake of that purine. The NT3 transporter mediated the uptake of the anti-leishmanial drug allopurinol, and the nt3^(–/–) mutants were resistant to killing by this drug. Expression of the NT3 permease was profoundly downregulated at the protein but not the mRNA level in stationary phase compared with logarithmic phase promastigotes. The nt4^(–/–) null mutant was quantitatively impaired in survival within murine bone marrow-derived macrophages. Extensive efforts to generate an nt3^(–/–)/nt4^(–/–) dual null mutant were not successful, suggesting that one of the two nucleobase permeases must be retained for robust growth of the parasite. The phenotypes of these null mutants underscore the importance of purine nucleobase transporters in the Leishmania life cycle and pharmacology.