Loss of allosteric regulation in α-isopropylmalate synthase identified as an antimicrobial resistance mechanism

Abstract

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.

Challenging the gold standard: the limitations of molecular assays for detection ofMycobacterium tuberculosisheteroresistance

“…gDNA was extracted from RIF-R (RpoB S450L), INH-R (KatG AA428del), FQ-R (GyrA D94G) and CFZ/BDQ-R (Rv0678c S63R) monoresistant M. bovis BCG strains using the Van Sooligen protocol (previously described) 12 whereas gDNA was extracted from M. tb 20527 , M. tb 21 697 and BCG INH/FQ-R using the Qiagen UCP Pathogen Mini Kit with a modified mechanical lysis protocol as previously described. 13 Concentration (ng/uL) was measured using Quant-iT PicoGreen dsDNA assay per the manufacturer’s protocol (Life Technologies Corporation, Eugene, Oregon, USA). Samples were prepared in triplicate and one aliquot was sent to each of the three laboratories for analysis.…”