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.

The many thousands of people living with cystic fibrosis are at a greater risk of developing a chronic lung infection caused by Mycobacterium abscessus . Since M. abscessus is clinically resistant to most anti-TB drugs available, treatment options are limited to macrolides.

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 abscessus is 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 against M. abscessus with in vitro and in vivo activities and that the efficacy of epetraborole could be improved with the combination of the non-proteinogenic amino acid norvaline. Norvaline demonstrated activity against the M. abscessus epetraborole resistant mutants thus, limiting resistance to epetraborole in wild-type populations. Here we show M. abscessus mutants 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-IPMSA555V variant 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 that M. abscessus can 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 of M. abscessus and describe a unique mechanism of substrate-inhibitor competition.