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J. Biol. Chem., Vol. 283, Issue 1, 155-165, January 4, 2008

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The Role of the Mitochondrial Glycine Cleavage Complex in the Metabolism and Virulence of the Protozoan Parasite Leishmania major^*

From the Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110

For the human pathogen Leishmania major, a key metabolic function is the synthesis of thymidylate, which requires 5,10-methylenetetrahydrofolate (5,10-CH₂-THF). 5,10-CH₂-THF can be synthesized from glycine by the mitochondrial glycine cleavage complex (GCC). Bioinformatic analysis revealed the four subunits of the GCC in the L. major genome, and the role of the GCC in parasite metabolism and virulence was assessed through studies of the P subunit (glycine decarboxylase (GCVP)). First, a tagged GCVP protein was expressed and localized to the parasite mitochondrion. Second, a gcvP^– mutant was generated and shown to lack significant GCC activity using an indirect in vivo assay after incorporation of label from [2-¹⁴C]glycine into DNA. The gcvP^– mutant grew poorly in the presence of excess glycine or minimal serine; these studies also established that L. major promastigotes require serine for optimal growth. Although gcvP^– promastigotes and amastigotes showed normal virulence in macrophage infections in vitro, both forms of the parasite showed substantially delayed replication and lesion pathology in infections of both genetically susceptible or resistant mice. These data suggest that, as the physiology of the infection site changes during the course of infection, so do the metabolic constraints on parasite replication. This conclusion has great significance to the interpretation of metabolic requirements for virulence. Last, these studies call attention in trypanosomatid protozoa to the key metabolic intermediate 5,10-CH₂-THF, situated at the junction of serine, glycine, and thymidylate metabolism. Notably, genome-based predictions suggest the related parasite Trypanosoma brucei is totally dependent on the GCC for 5,10-CH₂-THF synthesis.

Received for publication, September 25, 2007 , and in revised form, November 2, 2007.

^* This work was supported by National Institutes of Health Grant AI21903 (to S. M. B.) and European Molecular Biology Organization Long Term Fellowship ALTF 106-2005 (to T. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1–S3.

¹ To whom correspondence should be addressed: Dept. of Molecular Microbiology, Campus Box 8230, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis MO 63110. E-mail: beverley{at}borcim.wustl.edu.

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