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J Biol Chem. 2011 Apr 29;286(17):15377-90. doi: 10.1074/jbc.M111.231076. Epub 2011 Mar 3.

Bacterial conversion of folinic acid is required for antifolate resistance.

Ogwang S, Nguyen HT, Sherman M, Bajaksouzian S, Jacobs MR, Boom WH, Zhang GF, Nguyen L.

Source

Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4960, USA.

Abstract

Antifolates, which are among the first antimicrobial agents invented, inhibit cell growth by creating an intracellular state of folate deficiency. Clinical resistance to antifolates has been mainly attributed to mutations that alter structure or expression of enzymes involved in de novo folate synthesis. We identified a Mycobacterium smegmatis mutant, named FUEL (which stands for folate utilization enzyme for leucovorin), that is hypersusceptible to antifolates. Chemical complementation indicated that FUEL is unable to metabolize folinic acid (also known as leucovorin or 5-formyltetrahydrofolate), whose metabolic function remains unknown. Targeted mutagenesis, genetic complementation, and biochemical studies showed that FUEL lacks 5,10-methenyltetrahydrofolate synthase (MTHFS; also called 5-formyltetrahydrofolate cyclo-ligase; EC 6.3.3.2) activity responsible for the only ATP-dependent, irreversible conversion of folinic acid to 5,10-methenyltetrahydrofolate. In trans expression of active MTHFS proteins from bacteria or human restored both antifolate resistance and folinic acid utilization to FUEL. Absence of MTHFS resulted in marked cellular accumulation of polyglutamylated species of folinic acid. Importantly, MTHFS also affected M. smegmatis utilization of monoglutamylated 5-methyltetrahydrofolate exogenously added to the medium. Likewise, Escherichia coli mutants lacking MTHFS became susceptible to antifolates. These results indicate that folinic acid conversion by MTHFS is required for bacterial intrinsic antifolate resistance and folate homeostatic control. This novel mechanism of antimicrobial antifolate resistance might be targeted to sensitize bacterial pathogens to classical antifolates.

PMID:: 21372133; [PubMed - indexed for MEDLINE]
PMCID:: PMC3083218

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FIGURE 1.

FUEL, a novel determinant of antifolate resistance. A, antifolate susceptibility of M. smegmatis strains measured by MIC determination using 2-fold serial dilution assays. Concentrations of antifolates shown on the y axis are those of trimethoprim and sulfonamides in 1:19 ratios with MICs plotted as logarithmic values. FUEL (red) was inhibited at antifolate concentrations that are 3–6 log₂ dilutions lower than those inhibiting wild-type M. smegmatis (dark blue). In trans expression of either the msmeg_5472 gene from M. smegmatis (green), the synthetic gene encoding human MTHFS (mthfs; purple), or the gene encoding M. tuberculosis MTHFS homolog (rv0992c; yellow) restored antifolate resistance to FUEL. Expression of an inactive allele of MTHFS (mthfs(D154A); blue) did not significantly change MICs, which were within one doubling dilution of MICs against FUEL. The data presented represent the least differences of six MIC assay replicates. B, growth of M. smegmatis strains in the absence of antifolates. The data presented are averages of biological triplicates with S.D. (error bars). C, susceptibility of FUEL to the sulfonamide drug SCP and chemical restoration of resistance by exogenous addition of pABA, PteGlu₁, or 5-CHO-H₄PteGlu₁ (all used at 0.3 mm). Exponentially growing cultures of wild-type M. smegmatis or FUEL (5 μl; A = 1) were spotted on solid NE plates supplemented with different combinations of chemicals as indicated. In contrast to pABA and PteGlu₁, 5-CHO-H₄PteGlu₁ failed to restore growth of FUEL on plates embedded with 10 μg ml⁻¹ SCP. Growth was recorded after 3 days of incubation at 37 °C. Similar results were obtained with six other sulfonamides. The data presented are representatives of at least five biological replicates. SDM, sulfadimethoxine; SDZ, sulfadiazine; SMP, sulfamethoxypyridazine; SMX, sulfamethoxazole; SMZ, sulfamethiazole; STZ, sulfathiazole.

Bacterial Conversion of Folinic Acid Is Required for Antifolate Resistance

J Biol Chem. 2011 April 29;286(17):15377-15390.

FIGURE 5.

Enzymatic activity of purified M. smegmatis MTHFS. A, the ATP-dependent conversion of 5-CHO-H₄PteGlu_n to 5,10-CH⁺-H₄PteGlu_n catalyzed by MTHFS enzymatic activity. Functional groups in red are those involved in the cycloligation reaction. Abs, characteristic absorbance (wavelength in nm). B, purification of His₆-tagged M. smegmatis MTHFS from E. coli. Samples from the total cell lysate (T), insoluble pellet (P), soluble fraction (S), wash eluent (W), and eluted fraction (E) were separated by SDS-PAGE followed by Coomassie Blue staining (upper panel). Purified His₆-tagged M. smegmatis MTHFS was eluted as a single band of ∼25 kDa (arrow). Molecular weight standards (MW) were loaded in the far right lane. Identity of the His₆-tagged protein was determined by Western blot using antibody against the His₆ peptide (lower panel). C, spectrophotometric demonstration of the MTHFS enzymatic activity of purified His₆-tagged M. smegmatis MTHFS_MS. The reaction (1-ml volume contained in a cuvette of 0.5-cm light path; 50 mm MES buffer (pH 6), 10 mm magnesium acetate, 1 mm DTT, 0.1 mm EDTA, 0.1 mm ATP, and 0.1 mm (6S)-5-CHO-H₄PteGlu₁ at 23 °C) was initiated by the addition of 0.865 milliunit of purified M. smegmatis MTHFS (2.5 μg). The blank was set as the same as the reaction without enzyme and only one-half the amount of (6S)-5-CHO-H₄PteGlu₁ (0.05 mm). Repetitive scans (30-min period, 2-min intervals) were recorded automatically. Shown are representative data of biological quadruplicates. Arrows indicate the directions of change in absorbance. D, formation of 5,10-CH⁺-H₄PteGlu₁ from 5-CHO-H₄PteGlu₁ by MTHFS proteins measured by spectrophotometric absorbance at 360 nm. E, LC-MS/MS detection of 5,10-CH⁺-H₄PteGlu₁ (m/z 456/412) formed in the reactions catalyzed by MTHFS proteins. The reaction without an MTHFS protein added (top) serves as the control. Int, intensity.

Bacterial Conversion of Folinic Acid Is Required for Antifolate Resistance

J Biol Chem. 2011 April 29;286(17):15377-15390.