Biochemical and Genetic Analysis of Methylenetetrahydrofolate Reductase in Leishmania Metabolism and Virulence

Tim J Vickers,David A Scott,Rocío Díaz De La Garza,Giuseppe Orsomando,Song O Kang,Stephen M Beverley,Andrew D Hanson

doi:10.1074/jbc.m608387200

Tim J Vickers, David A Scott + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m608387200

Copy DOI

Abstract

Methylenetetrahydrofolate reductase (MTHFR; EC 1.5.1.20) is the sole enzyme responsible for generation of 5-methyltetrahydrofolate, which is required for methionine synthesis and provision of methyl groups via S-adenosylmethionine. Genome analysis showed that Leishmania species, unlike Trypanosoma brucei and Trypanosoma cruzi, contain genes encoding MTHFR and two distinct methionine synthases. Leishmania MTHFR differed from those in other eukaryotes by the absence of a C-terminal regulatory domain. L. major MTHFR was expressed in yeast and recombinant enzyme was produced in Escherichia coli. MTHFR was not inhibited by S-adenosylmethionine and, uniquely among folate-metabolizing enzymes, showed dual-cofactor specificity with NADH and NADPH under physiological conditions. MTHFR null mutants (mthfr(-)) lacked 5-methyltetrahydrofolate, the most abundant intracellular folate, and could not utilize exogenous homocysteine for growth. Under conditions of methionine limitation mthfr(-) mutant cells grew poorly, whereas their growth was normal in standard culture media. Neither in vitro MTHFR activity nor the growth of mthfr(-) mutants or MTHFR overexpressors were differentially affected by antifolates known to inhibit parasite growth via targets beyond dihydrofolate reductase and pteridine reductase 1. In a mouse model of infection mthfr(-) mutants showed good infectivity and virulence, indicating that sufficient methionine is available within the parasitophorous vacuole to meet the needs of the parasite.

Highlights

Approved vaccines are available against these pathogens and current chemotherapy relies on pentavalent antimony, which cannot be given orally, has significant toxicity, and is facing increasing resistance in the field [1, 2]
Current therapeutic antifolates are ineffective against Leishmania because dihydrofolate reductase (DHFR) inhibition is by-passed by pteridine reductase 1 (PTR1), which is relatively insensitive to classical antifolates (26 –28)
Identification of Leishmania Methylenetetrahydrofolate reductase (MTHFR)—L. major MTHFR (LmjF36.6390) was identified in searches of the Leishmania genome databases; orthologs were detected in Leishmania infantum (LinJ36.0220) and Leishmania braziliensis (LbrM35.6090)

Summary

EXPERIMENTAL PROCEDURES

Reagents—(6R,6S)-Tetrahydrofolate (THF) was obtained from Schircks Laboratories (Jona, Switzerland). [14C]Formaldehyde (55 mCi mmolϪ1) was from PerkinElmer Life Sciences. Expression of L. major MTHFR in Saccharomyces cerevisiae— The MTHFR coding sequence was amplified from the pGEMT.MTHFR template using primers 5Ј-TGCAGGATCCATGTCCAAGCTTATCAGT (forward) and 5Ј-TGCACTGCAGTCACTCCACTAGCGCGTT (reverse) This amplicon was digested with BamHI and PstI and cloned into the corresponding sites in pVT103-U [37]. Yeast Growth and Preparation of Extracts—Cultures of strain RRY3 expressing LmjMTHFR, human MTHFR, or the corresponding empty vector control were grown as described [12]. Expression and Purification of Recombinant LmjMTHFR— An expression construct of LmjMTHFR was kindly provided by the Structural Genomics of Parasitic Protozoa consortium (clone Lmaj006157AAA, B5503) This used the expression vector pAVA0421 [40] that adds an N-terminal hexahistidine tag to the recombinant protein. The NADH-5,10-CH2-THF oxidoreductase activity of MTHFR was determined essentially as described [41] in deoxygenated phosphate assay buffer containing 10 mM formaldehyde. Intracellular folate concentrations were calculated using an intracellular volume for L. major of 1.9 ␮l per 1 ϫ 108 cells [45]

RESULTS

90 Ϯ 10 87 Ϯ 5

50 Ϯ 40 70 Ϯ 30

DISCUSSION