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Synthetic lethality reveals mechanisms of Mycobacterium tuberculosis resistance to ?-lactams.

ABSTRACT:

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Most ?-lactam antibiotics are ineffective against Mycobacterium tuberculosis due to the microbe's innate resistance. The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains has prompted interest to repurpose this class of drugs. To identify the genetic determinants of innate ?-lactam resistance, we carried out a synthetic lethality screen on a transposon mutant library for susceptibility to imipenem, a carbapenem ?-lactam antibiotic. Mutations in 74 unique genes demonstrated synthetic lethality. The majority of mutations were in genes associated with cell wall biosynthesis. A second quantitative real-time PCR (qPCR)-based synthetic lethality screen of randomly selected mutants confirmed the role of cell wall biosynthesis in ?-lactam resistance. The global transcriptional response of the bacterium to ?-lactams was investigated, and changes in levels of expression of cell wall biosynthetic genes were identified. Finally, we validated these screens in vivo using the MT1616 transposon mutant, which lacks a functional acyl-transferase gene. Mice infected with the mutant responded to ?-lactam treatment with a 100-fold decrease in bacillary lung burden over 4 weeks, while the numbers of organisms in the lungs of mice infected with wild-type bacilli proliferated. These findings reveal a road map of genes required for ?-lactam resistance and validate synthetic lethality screening as a promising tool for repurposing existing classes of licensed, safe, well-characterized antimicrobials against tuberculosis.

Importance

The global emergence of multidrug-resistant and extensively drug-resistant M. tuberculosis strains has threatened public health worldwide, yet the pipeline of new tuberculosis drugs under development remains limited. One strategy to cope with the urgent need for new antituberculosis agents is to repurpose existing, approved antibiotics. The carbapenem class of ?-lactam antibiotics has been proposed as one such class of drugs. Our study identifies molecular determinants of innate resistance to ?-lactam drugs in M. tuberculosis, and we demonstrate that functional loss of one of these genes enables successful treatment of M. tuberculosis with ?-lactams in the mouse model.

SUBMITTER: Lun S

PROVIDER: S-EPMC4172077 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Synthetic lethality reveals mechanisms of Mycobacterium tuberculosis resistance to β-lactams.

Lun Shichun S Miranda David D Kubler Andre A Guo Haidan H Maiga Mariama C MC Winglee Kathryn K Pelly Shaaretha S Bishai William R WR

mBio 20140916 5

<h4>Unlabelled</h4>Most β-lactam antibiotics are ineffective against Mycobacterium tuberculosis due to the microbe's innate resistance. The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains has prompted interest to repurpose this class of drugs. To identify the genetic determinants of innate β-lactam resistance, we carried out a synthetic lethality screen on a transposon mutant library for susceptibility to imipenem, a carbapenem β-lactam antibiotic. Mutations i ...[more]

PMID: 25227469

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Project description:We report GSK3011724A (DG167) as a binary inhibitor of β-ketoacyl-ACP synthase (KasA) in Mycobacterium tuberculosis Genetic and biochemical studies established KasA as the primary target. The X-ray crystal structure of the KasA-DG167 complex refined to 2.0-Å resolution revealed two interacting DG167 molecules occupying nonidentical sites in the substrate-binding channel of KasA. The binding affinities of KasA to DG167 and its analog, 5g, which binds only once in the substrate-binding channel, were determined, along with the KasA-5g X-ray crystal structure. DG167 strongly augmented the in vitro activity of isoniazid (INH), leading to synergistic lethality, and also synergized in an acute mouse model of M. tuberculosis infection. Synergistic lethality correlated with a unique transcriptional signature, including upregulation of oxidoreductases and downregulation of molecular chaperones. The lead structure-activity relationships (SAR), pharmacokinetic profile, and detailed interactions with the KasA protein that we describe may be applied to evolve a next-generation therapeutic strategy for tuberculosis (TB).IMPORTANCE Cell wall biosynthesis inhibitors have proven highly effective for treating tuberculosis (TB). We discovered and validated members of the indazole sulfonamide class of small molecules as inhibitors of Mycobacterium tuberculosis KasA-a key component for biosynthesis of the mycolic acid layer of the bacterium's cell wall and the same pathway as that inhibited by the first-line antitubercular drug isoniazid (INH). One lead compound, DG167, demonstrated synergistic lethality in combination with INH and a transcriptional pattern consistent with bactericidality and loss of persisters. Our results also detail a novel dual-binding mechanism for this compound as well as substantial structure-activity relationships (SAR) that may help in lead optimization activities. Together, these results suggest that KasA inhibition, specifically, that shown by the DG167 series, may be developed into a potent therapy that can synergize with existing antituberculars.