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

David A Scott,Suzanne M Hickerson,Tim J Vickers,Stephen M Beverley

doi:10.1074/jbc.m708014200

David A Scott, Suzanne M Hickerson + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m708014200

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Abstract

For the human pathogen Leishmania major, a key metabolic function is the synthesis of thymidylate, which requires 5,10-methylenetetrahydrofolate (5,10-CH(2)-THF). 5,10-CH(2)-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-(14)C]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-CH(2)-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-CH(2)-THF synthesis.

Highlights

We focused on the GCVP protein, which mediates the first catalytic step of the glycine cleavage complex (GCC), decarboxylation of glycine, and generated mutants and relevant control lines to explore its role and that of the GCC in Leishmania metabolism
In other species the GCC is localized to the mitochondrion, and correspondingly, presumptive mitochondrial targeting pre-sequences were identified for all of the Leishmania GCC subunit orthologs
We devised an alternative indirect in vivo assay for GCC activity after incorporation of label from [2-14C]glycine through 5,10-CH2-THF into thymidylate and DNA (Table 1)

Summary

EXPERIMENTAL PROCEDURES

Culture Media, and Drug Testing—L. major strain Friedlin clone V1 (MHOM/IL/81/Friedlin) was cultured as promastigotes in M199 medium as described [25] with the addition of 1 ␮g mlϪ1 biotin and 2 ␮g mlϪ1 biopterin. The disrupted gene was excised by digestion with BamHI and transfected as described above into heterozygote clone 8, and clonal lines were isolated on plates containing 20 ␮g mlϪ1 each puromycin and hygromycin. GcvPϪ or WT L. major were transfected with pXG-GCVP, and clones were isolated from plates containing 10 or 12.5 ␮g mlϪ1 G418; the presence of the plasmid was confirmed by re-isolating it from Leishmania, transforming Escherichia coli and verifying the plasmid by restriction digestion. These lines are referred to as gcvPϪ/ϩGCVP (formally gcvP::PAC/gcvP::HYG [pXGGCVP]) or WT/ϩGCVP

RESULTS

41 Ϯ 13 8Ϯ2

21 Ϯ 2 20 Ϯ 1

DISCUSSION