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Despite our best efforts to discover new antimicrobials, bacteria have evolved mechanisms to become resistant. Resistance to antimicrobials can be attributed to innate, inducible, and acquired mechanisms.Mycobacterium abscessusis one of the most antimicrobial resistant bacteria and is known to cause chronic pulmonary infections within the cystic fibrosis community. Previously, we identified epetraborole as an inhibitor againstM. abscessuswithin vitroandin vivoactivities and that the efficacy of epetraborole could be improved with the combination of the non-proteinogenic amino acid norvaline. Norvaline demonstrated activity against theM. abscessusepetraborole resistant mutants thus, limiting resistance to epetraborole in wild type populations. Here we showM. abscessusmutants with resistance to epetraborole can acquire resistance to norvaline in a leucyl-tRNA synthetase (LeuRS) editing-independent manner. After showing that the membrane hydrophobicity and efflux activity are not linked to norvaline resistance, whole-genome sequencing identified a mutation in the allosteric regulatory domain of α-isopropylmalate synthase (α-IPMS). We found that mutants with the α-IPMSA555Vvariant incorporated less norvaline in the proteome and produced more leucine than the parental strain. Furthermore, we found that leucine can rescue growth inhibition from norvaline challenge in the parental strain. Our results demonstrate thatM. abscessuscan modulate its metabolism through mutations in an allosteric regulatory site to upregulate the biosynthesis of the natural LeuRS substrate and outcompete norvaline. These findings emphasize the antimicrobial resistant nature ofM. abscessusand describe a unique mechanism of substrate-inhibitor competition.Significance StatementCystic fibrosis patients and individuals undergoing plastic surgery are at risk for acquiring chronic infections fromMycobacterium abscessus. Current antibiotics are not adequate and require increased drug discovery efforts to identify better treatments for these patients. The benzoxaborole, epetraborole has been shown by our group and others to be a promising candidate againstM. abscessusbut the emergence of resistance to epetraborole in a clinical trial for complicated urinary tract infections has hindered its development. Previously, we identified the combination of epetraborole and norvaline as a potential means to limit resistance against epetraborole. Our results here demonstrate thatM. abscessuscan acquire resistance to both epetraborole and norvaline. These results may help develop combination therapies to reduce the risk of resistance to benzoxaboroles and non-proteinogenic amino acids.

New antibiotics are urgently needed to counter the emergence of antimicrobial-resistant pathogenic bacteria. A major challenge in antibiotic drug discovery is to turn potent biochemical inhibitors of essential bacterial components into effective antimicrobials. This difficulty is underpinned by a lack of methods to investigate the physicochemical properties needed for candidate antibiotics to permeate the bacterial cell envelope and avoid clearance by the action of bacterial efflux pumps. To address these issues, here we used a target engagement assay to measure the equilibrium and kinetic binding parameters of antibiotics targeting dihydrofolate reductase (DHFR) in live bacteria. We also used this assay to identify novel DHFR ligands having antimicrobial activity. We validated this approach using the Gram-negative bacteria

Mycobacterium abscessus is the most common rapidly growing non-tuberculous mycobacteria to cause pulmonary disease in patients with impaired lung function such as cystic fibrosis. M. abscessus displays high intrinsic resistance to common antibiotics and inducible resistance to macrolides like clarithromycin. As such, M. abscessus is clinically resistant to the entire regimen of front-line M. tuberculosis drugs, and treatment with antibiotics that do inhibit M. abscessus in the lab results in cure rates of 50% or less. Here, we identified epetraborole (EPT) from the MMV pandemic response box as an inhibitor against the essential protein leucyl-tRNA synthetase (LeuRS) in M. abscessus. EPT protected zebrafish from lethal M. abscessus infection and did not induce self-resistance nor against clarithromycin. Contrary to most antimycobacterials, the whole-cell activity of EPT was greater against M. abscessus than M. tuberculosis, but crystallographic and equilibrium binding data showed that EPT binds LeuRSMabs and LeuRSMtb with similar residues and dissociation constants. Since EPT-resistant M. abscessus mutants lost LeuRS editing activity, these mutants became susceptible to misaminoacylation with leucine mimics like the non-proteinogenic amino acid norvaline. Proteomic analysis revealed that when M. abscessus LeuRS mutants were fed norvaline, leucine residues in proteins were replaced by norvaline, inducing the unfolded protein response with temporal changes in expression of GroEL chaperonins and Clp proteases. This supports our in vitro data that supplementation of media with norvaline reduced the emergence of EPT mutants in both M. abscessus and M. tuberculosis. Furthermore, the combination of EPT and norvaline had improved in vivo efficacy compared to EPT in a murine model of M. abscessus infection. Our results emphasize the effectiveness of EPT against the clinically relevant cystic fibrosis pathogen M. abscessus, and these findings also suggest norvaline adjunct therapy with EPT could be beneficial for M. abscessus and other mycobacterial infections like tuberculosis., Author summary Current antimycobacterial drugs are inadequate to handle the increasing number of non-tuberculous mycobacteria infections that eclipse tuberculosis infections in many developed countries. Of particular importance for cystic fibrosis patients, Mycobacterium abscessus is notoriously difficult to treat where patients spend extended time on antibiotics with cure rates comparable to extreme drug resistant M. tuberculosis. Here, we identified epetraborole (EPT) with in vitro and in vivo activities against M. abscessus. We showed that EPT targets the editing domain of the leucyl-tRNA synthetase (LeuRS) and that escape mutants lost LeuRS editing activity, making these mutants susceptible to misaminoacylation with leucine mimics. Most importantly, combination therapy of EPT and norvaline limited the rate of EPT resistance in both M. abscessus and M. tuberculosis, and this was the first study to demonstrate improved in vivo efficacy of EPT and norvaline compared to EPT in a murine model of M. abscessus pulmonary infection. The demonstration of norvaline adjunct therapy with EPT for M. abscessus infections is promising for cystic fibrosis patients and could translate to other mycobacterial infections, such as tuberculosis.

Isoniazid (INH) is a cornerstone of antitubercular therapy. Mycobacterium tuberculosis complex bacteria are the only mycobacteria sensitive to clinically relevant concentrations of INH. All other mycobacteria, including M. marinum and M. avium subsp. paratuberculosis are resistant. INH requires activation by bacterial KatG to inhibit mycobacterial growth. We tested the role of the differences between M. tuberculosis KatG and that of other mycobacteria in INH sensitivity. We cloned the M. bovis katG gene into M. marinum and M. avium subsp. paratuberculosis and measured the MIC of INH. We recombinantly expressed KatG of these mycobacteria and tested in vitro binding to, and activation of, INH. Introduction of katG from M. bovis into M. marinum and M. avium subsp. paratuberculosis rendered them 20 to 30 times more sensitive to INH. Analysis of different katG sequences across the genus found KatG evolution diverged from RNA polymerase-defined mycobacterial evolution. Biophysical and biochemical tests of M. bovis and nontuberculous mycobacteria (NTM) KatG proteins showed lower affinity to INH and substantially lower enzymatic capacity for the conversion of INH into the active form in NTM. The KatG proteins of M. marinum and M. avium subsp. paratuberculosis are substantially less effective in INH activation than that of M. tuberculosis, explaining the relative INH insensitivity of these microbes. These data indicate that the M. tuberculosis complex KatG is divergent from the KatG of NTM, with a reciprocal relationship between resistance to host defenses and INH resistance. Studies of bacteria where KatG is functionally active but does not activate INH may aid in understanding M. tuberculosis INH-resistance mechanisms, and suggest paths to overcome them.