Metabolomics Reveal D-Alanine:D-Alanine Ligase As the Target of D-Cycloserine in Mycobacterium tuberculosis

Stable isotope-mass spectrometry (MS)-based metabolomic profiling is a powerful technique for following changes in specific metabolite pool sizes and metabolic flux under various experimental conditions in a test organism or cell type. Here, we use a metabolomics approach to interrogate the mechanism of antibiotic action of D-cycloserine (DCS), a second line antibiotic used in the treatment of multidrug resistant Mycobacterium tuberculosis infections. We use doubly labeled C-13 alpha-carbon H-2 L-alanine to allow tracking of both alanine racemase and D-alanine:D-alanine ligase activity in M. tuberculosis challenged with DCS and reveal that D-alanine:D-alanine ligase is more strongly inhibited than alanine racemase at equivalent DCS concentrations. We also shed light on mechanisms surrounding D-Ala-mediated antagonism of DCS growth inhibition and provide evidence for a postantibiotic effect for this drug. Our results illustrate the potential of metabolomics in cellular drug-target engagement studies and consequently have broad implications in future drug development and target validation ventures.