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

ABSTRACT: 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 ¹³C α-carbon-²H 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.

SUBMITTER: Prosser GA

PROVIDER: S-EPMC3903091 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Structure of the Mycobacterium tuberculosis D-alanine:D-alanine ligase, a target of the antituberculosis drug D-cycloserine.

Project description:D-alanine:D-alanine ligase (EC 6.3.2.4; Ddl) catalyzes the ATP-driven ligation of two D-alanine (D-Ala) molecules to form the D-alanyl:D-alanine dipeptide. This molecule is a key building block in peptidoglycan biosynthesis, making Ddl an attractive target for drug development. D-Cycloserine (DCS), an analog of D-Ala and a prototype Ddl inhibitor, has shown promise for the treatment of tuberculosis. Here, we report the crystal structure of Mycobacterium tuberculosis Ddl at a resolution of 2.1 Å. This structure indicates that Ddl is a dimer and consists of three discrete domains; the ligand binding cavity is at the intersection of all three domains and conjoined by several loop regions. The M. tuberculosis apo Ddl structure shows a novel conformation that has not yet been observed in Ddl enzymes from other species. The nucleotide and D-alanine binding pockets are flexible, requiring significant structural rearrangement of the bordering regions for entry and binding of both ATP and D-Ala molecules. Solution affinity and kinetic studies showed that DCS interacts with Ddl in a manner similar to that observed for D-Ala. Each ligand binds to two binding sites that have significant differences in affinity, with the first binding site exhibiting high affinity. DCS inhibits the enzyme, with a 50% inhibitory concentration (IC(50)) of 0.37 mM under standard assay conditions, implicating a preferential and weak inhibition at the second, lower-affinity binding site. Moreover, DCS binding is tighter at higher ATP concentrations. The crystal structure illustrates potential drugable sites that may result in the development of more-effective Ddl inhibitors.

| S-EPMC3019625 | biostudies-literature

Metabolomics analysis identifies d-Alanine-d-Alanine ligase as the primary lethal target of d-Cycloserine in mycobacteria.

Project description:d-Cycloserine is an effective second line antibiotic used as a last resort to treat multi (MDR)- and extensively (XDR) drug resistant strains of Mycobacterium tuberculosis . d-Cycloserine interferes with the formation of peptidoglycan biosynthesis by competitive inhibition of alanine racemase (Alr) and d-alanine-d-alanine ligase (Ddl). Although the two enzymes are known to be inhibited, the in vivo lethal target is still unknown. Our NMR metabolomics work has revealed that Ddl is the primary target of DCS, as cell growth is inhibited when the production of d-alanyl-d-alanine is halted. It is shown that inhibition of Alr may contribute indirectly by lowering the levels of d-alanine, thus allowing DCS to outcompete d-alanine for Ddl binding. The NMR data also supports the possibility of a transamination reaction to produce d-alanine from pyruvate and glutamate, thereby bypassing Alr inhibition. Furthermore, the inhibition of peptidoglycan synthesis results in a cascading effect on cellular metabolism as there is a shift toward the catabolic routes to compensate for accumulation of peptidoglycan precursors.

| S-EPMC3975674 | biostudies-literature

Reinterpreting the mechanism of inhibition of Mycobacterium tuberculosis D-alanine:D-alanine ligase by D-cycloserine.

Project description:d-Cycloserine is a second-line drug approved for use in the treatment of patients infected with Mycobacterium tuberculosis, the etiologic agent of tuberculosis. The unique mechanism of action of d-cycloserine, compared with those of other clinically employed antimycobacterial agents, represents an untapped and exploitable resource for future rational drug design programs. Here, we show that d-cycloserine is a slow-onset inhibitor of MtDdl and that this behavior is specific to the M. tuberculosis enzyme orthologue. Furthermore, evidence is presented that indicates d-cycloserine binds exclusively to the C-terminal d-alanine binding site, even in the absence of bound d-alanine at the N-terminal binding site. Together, these results led us to propose a new model of d-alanine:d-alanine ligase inhibition by d-cycloserine and suggest new opportunities for rational drug design against an essential, clinically validated mycobacterial target.

| S-EPMC3944805 | biostudies-other

Role of Alanine Racemase Mutations in Mycobacterium tuberculosis d-Cycloserine Resistance.

Project description:A screening of more than 1,500 drug-resistant strains of Mycobacterium tuberculosis revealed evolutionary patterns characteristic of positive selection for three alanine racemase (Alr) mutations. We investigated these mutations using molecular modeling, in vitro MIC testing, as well as direct measurements of enzymatic activity, which demonstrated that these mutations likely confer resistance to d-cycloserine.

| S-EPMC5700341 | biostudies-literature

Roles of Mycobacterium smegmatis D-alanine:D-alanine ligase and D-alanine racemase in the mechanisms of action of and resistance to the peptidoglycan inhibitor D-cycloserine.

Project description:D-Cycloserine (DCS) targets the peptidoglycan biosynthetic enzymes D-alanine racemase (Alr) and D-alanine:D-alanine ligase (Ddl). Previously, we demonstrated that the overproduction of Alr in Mycobacterium smegmatis determines a DCS resistance phenotype. In this study, we investigated the roles of both Alr and Ddl in the mechanisms of action of and resistance to DCS in M. smegmatis. We found that the overexpression of either the M. smegmatis or the Mycobacterium tuberculosis ddl gene in M. smegmatis confers resistance to DCS, but at lower levels than the overexpression of the alr gene. Furthermore, a strain overexpressing both the alr and ddl genes displayed an eightfold-higher level of resistance. To test the hypothesis that inhibition of Alr by DCS decreases the intracellular pool of D-alanine, we determined the alanine pools in M. smegmatis wild-type and recombinant strains with or without DCS treatment. Alr-overproducing strain GPM14 cells not exposed to DCS displayed almost equimolar amounts of L- and D-alanine in the steady state. The wild-type strain and Ddl-overproducing strains contained a twofold excess of L- over D-alanine. In all strains, DCS treatment led to a significant accumulation of L-alanine and a concomitant decease of D-alanine, with approximately a 20-fold excess of L-alanine in the Ddl-overproducing strains. These data suggest that Ddl is not significantly inhibited by DCS at concentrations that inhibit Alr. This study is of significance for the identification of the lethal target(s) of DCS and the development of novel drugs targeting the D-alanine branch of mycobacterial peptidoglycan biosynthesis.

| S-EPMC149019 | biostudies-literature

Integron gene cassettes harboring novel variants of D-alanine-D-alanine ligase confer high-level resistance to D-cycloserine.

Project description:Antibiotic resistance poses an increasing threat to global health. To tackle this problem, the identification of principal reservoirs of antibiotic resistance genes (ARGs) plus an understanding of drivers for their evolutionary selection are important. During a PCR-based screen of ARGs associated with integrons in saliva-derived metagenomic DNA of healthy human volunteers, two novel variants of genes encoding a D-alanine-D-alanine ligase (ddl6 and ddl7) located within gene cassettes in the first position of a reverse integron were identified. Treponema denticola was identified as the likely host of the ddl cassettes. Both ddl6 and ddl7 conferred high level resistance to D-cycloserine when expressed in Escherichia coli with ddl7 conferring four-fold higher resistance to D-cycloserine compared to ddl6. A SNP was found to be responsible for this difference in resistance phenotype between the two ddl variants. Molecular dynamics simulations were used to explain the mechanism of this phenotypic change at the atomic scale. A hypothesis for the evolutionary selection of ddl containing integron gene cassettes is proposed, based on molecular docking of plant metabolites within the ATP and D-cycloserine binding pockets of Ddl.

| S-EPMC7691350 | biostudies-literature

Glutamate Racemase Is the Primary Target of ?-Chloro-d-Alanine in Mycobacterium tuberculosis.

Project description:The increasing global prevalence of drug resistance among many leading human pathogens necessitates both the development of antibiotics with novel mechanisms of action and a better understanding of the physiological activities of preexisting clinically effective drugs. Inhibition of peptidoglycan (PG) biosynthesis and cross-linking has traditionally enjoyed immense success as an antibiotic target in multiple bacterial pathogens, except in Mycobacterium tuberculosis, where it has so far been underexploited. d-Cycloserine, a clinically approved antituberculosis therapeutic, inhibits enzymes within the d-alanine subbranch of the PG-biosynthetic pathway and has been a focus in our laboratory for understanding peptidoglycan biosynthesis inhibition and for drug development in studies of M. tuberculosis During our studies on alternative inhibitors of the d-alanine pathway, we discovered that the canonical alanine racemase (Alr) inhibitor ?-chloro-d-alanine (BCDA) is a very poor inhibitor of recombinant M. tuberculosis Alr, despite having potent antituberculosis activity. Through a combination of enzymology, microbiology, metabolomics, and proteomics, we show here that BCDA does not inhibit the d-alanine pathway in intact cells, consistent with its poor in vitro activity, and that it is instead a mechanism-based inactivator of glutamate racemase (MurI), an upstream enzyme in the same early stage of PG biosynthesis. This is the first report to our knowledge of inhibition of MurI in M. tuberculosis and thus provides a valuable tool for studying this essential and enigmatic enzyme and a starting point for future MurI-targeted antibacterial development.

| S-EPMC5038272 | biostudies-literature

The Biological Properties and Potential Interacting Proteins of d-Alanyl-d-alanine Ligase A from Mycobacterium tuberculosis.

Project description:(1) Background: d-alanine-d-alanine ligase (DdlA), an effective target for drug development to combat against Mycobacterium tuberculosis (Mtb), which threatens human health globally, supplies a substrate of d-alanyl-d-alanine for peptidoglycan crosslinking by catalyzing the dimerization of two d-alanines. To obtain a better understanding of DdlA profiles and develop a colorimetric assay for high-throughput inhibitor screening, we focused on explicating and characterizing Tb-DdlA. (2) Methods and Results: Rv2981c (ddlA) was expressed in Escherichia coli, and the purified Tb-DdlA was identified using (anti)-polyhistidine antibody followed by DdlA activity confirmation by measuring the released orthophosphate via colorimetric assay and the yielded d-alanyl-d-alanine through high performance thin layer chromatography (HP-TLC). The kinetic assays on Tb-DdlA indicated that Tb-DdlA exhibited a higher affinity to ATP (KmATP: 50.327 ± 4.652 μmol/L) than alanine (KmAla: 1.011 ± 0.094 mmol/L). A colorimetric assay for Tb-DdlA activity was developed for high-throughput screening of DdlA inhibitors in this study. In addition, we presented an analysis on Tb-DdlA interaction partners by pull-down assay and MS/MS. Eight putative interaction partners of Tb-DdlA were identified. (3) Conclusions: Our dataset provided a valuable resource for exploring Tb-DdlA biology, and developed an easy colorimetric assay for screening of Tb-DdlA inhibitors.

| S-EPMC6017538 | biostudies-literature

Whole genome sequencing of Mycobacterium tuberculosis strains resistant to D-cycloserine

Project description:11 Mycobacterium tuberculosis mutants resistant to D-cycloserine were isolated in the laboratory. Genomic DNA was isolated and whole genomes were sequenced to perform SNP calling and identify possible mutations associated with resistance.

2018-12-28 | E-MTAB-5935 | biostudies-arrayexpress

Overexpression of the D-alanine racemase gene confers resistance to D-cycloserine in Mycobacterium smegmatis.

Project description:D-Cycloserine is an effective second-line drug against Mycobacterium avium and Mycobacterium tuberculosis. To analyze the genetic determinants of D-cycloserine resistance in mycobacteria, a library of a resistant Mycobacterium smegmatis mutant was constructed. A resistant clone harboring a recombinant plasmid with a 3.1-kb insert that contained the glutamate decarboxylase (gadA) and D-alanine racemase (alrA) genes was identified. Subcloning experiments demonstrated that alrA was necessary and sufficient to confer a D-cycloserine resistance phenotype. The D-alanine racemase activities of wild-type and recombinant M. smegmatis strains were inhibited by D-cycloserine in a concentration-dependent manner. The D-cycloserine resistance phenotype in the recombinant clone was due to the overexpression of the wild-type alrA gene in a multicopy vector. Analysis of a spontaneous resistant mutant also demonstrated overproduction of wild-type AlrA enzyme. Nucleotide sequence analysis of the overproducing mutant revealed a single transversion (G-->T) at the alrA promoter, which resulted in elevated beta-galactosidase reporter gene expression. Furthermore, transformants of Mycobacterium intracellulare and Mycobacterium bovis BCG carrying the M. smegmatis wild-type alrA gene in a multicopy vector were resistant to D-cycloserine, suggesting that AlrA overproduction is a potential mechanism of D-cycloserine resistance in clinical isolates of M. tuberculosis and other pathogenic mycobacteria. In conclusion, these results show that one of the mechanisms of D-cycloserine resistance in M. smegmatis involves the overexpression of the alrA gene due to a promoter-up mutation.

| S-EPMC179361 | biostudies-other

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