Bacterial Conversion of Folinic Acid Is Required for Antifolate Resistance

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.

Potentiation of Available Antibiotics by Targeting Resistance – An Emerging Trend in Tuberculosis Drug Development

“…A novel determinant identified from this screen was further characterized in a recent report (Ogwang et al, 2011). The M. smegmatis mutant presented in this report exhibits hypersusceptibility to several combinations of trimethoprim/sulfonamides tested (Ogwang et al, 2011). For example, its MIC to trimethoprim/sulfachloropyridazine is 64 fold lower than that of the parental M. smegmatis strain.…”

“…Using this non-bias screen, the genome-wide collection of antifolate resistance determinants in mycobacteria (mycobacterial antifolate resistome) was found to be composed of fifty resistance determinants (unpublished data). A novel determinant identified from this screen was further characterized in a recent report (Ogwang et al, 2011). The M. smegmatis mutant presented in this report exhibits hypersusceptibility to several combinations of trimethoprim/sulfonamides tested (Ogwang et al, 2011).…”

“…These mutants are systematically tested for increased antifolate susceptibility, followed by chemical complementation using folate derivatives of both the de novo synthesis and the one-carbon interconversion network. This chemogenomic profiling approach allows for identification of novel determinants previously unknown to function in mycobacterial intrinsic antifolate resistance (Ogwang et al, 2011). Using this non-bias screen, the genome-wide collection of antifolate resistance determinants in mycobacteria (mycobacterial antifolate resistome) was found to be composed of fifty resistance determinants (unpublished data).…”

“…Therefore, novel potentiation approaches targeting resistance mechanisms might be more effective in both potentiating available antifolates and preventing the emergence of resistant strains. A recent study aimed at targeting intrinsic antifolate resistance in mycobacteria might reveal valuable targets for such resistance-targeted potentiation approaches (Ogwang et al, 2011). To identify novel antifolate resistance determinants, a genetic screen was first employed using a saturated transposon-insertion library of M. smegmatis.…”

Metabolomics Research with Tandem Mass Spectrometry

“…Poly-glutamate folate parent m/z is 129 more for one additional glutamate but with the same product ion m/z as the one corresponding to monoglutamate folate. Profiling of the folate derivatives showed that mono-glutamate folates had no change compared to the wild type Mycobacterium smegmatis, but dramatically decreased poly-glutamate 5-formyltetrahydrofolate, which indicates the important physiological role of poly-glutamate 5-formyltetrahydrofolate in the folate metabolism (Ogwang et al, 2011). …”