Investigating the role of MAB_1915 in intrinsic resistance to multiple drugs in Mycobacterium abscessus

Abstract

The increasing clinical significance of Mycobacterium abscessus is owed to its innate high-level, broad-spectrum resistance to antibiotics and therefore rapidly evolves as an important human pathogen. This warrants the identification of novel targets for aiding the discovery of new drugs or drug combinations to treat M. abscessus infections. This study is inspired by the drug-hypersensitive profile of a mutant M. abscessus (U14) with transposon insertion in MAB_1915. We validated the role of MAB_1915 in intrinsic drug resistance in M. abscessus by constructing a selectable marker-free in-frame deletion in MAB_1915 and complementing the mutant with the same or extended version of the gene and then followed by drug susceptibility testing. Judging by the putative function of MAB_1915, cell envelope permeability was studied by ethidium bromide accumulation assay and susceptibility testing against dyes and detergents. In this study, we established genetic evidence of the role of MAB_1915 in intrinsic resistance to rifampicin, rifabutin, linezolid, clarithromycin, vancomycin, and bedaquiline. Disruption of MAB_1915 has also been observed to cause a significant increase in cell envelope permeability in M. abscessus. Restoration of resistance is observed to depend on at least 27 base pairs upstream of the coding DNA sequence of MAB_1915. MAB_1915 could therefore be associated with cell envelope permeability, and hence its role in intrinsic resistance to multiple drugs in M. abscessus, which presents it as a novel target for future development of effective antimicrobials to overcome intrinsic drug resistance in M. abscessus.

Importance: This study reports the role of a putative fadD (MAB_1915) in innate resistance to multiple drugs by M. abscessus, hence identifying MAB_1915 as a valuable target and providing a baseline for further mechanistic studies and development of effective antimicrobials to check the high level of intrinsic resistance in this pathogen.

Keywords: MAB_1915; Mycobacterium abscessus; cell envelope permeability; fatty acid-CoA ligase; intrinsic drug resistance.

Fig 1

Tn insertion and construction of an in-frame disruption in MAB_1915. A Position of Tn insertion in U14, a Tn insertion mutant in which the Tn is seen to integrate into M. abscessus chromosome between “T” and “A” dinucleotides at positions 61 and 62 within MAB_1915, respectively. This study was inspired by the drug-sensitivity profile of this mutant. The 272-bp intergenic region between MAB_1915 and MAB_1914c is also shown. B Depiction of the construction of AES, in-frame disruption of MAB_1915 by allelic exchange and unmarking of the gene disruption mutant. C Verification of gene disruption by amplifying the junctions between the chromosome of M. abscessus and the inserted zeoR cassette. Lane M, 1-kb DNA ladder; lanes 1 and 2, Mab^Wt and ∆MAB_1915 mutant, respectively (primers a/c, 726 bp); lanes 2 and 4, Mab^Wt and ∆MAB_1915 mutant, respectively (primers d/b, 1154 bp). D Verification of knockout by amplifying (primers e/f) disrupted MAB_1915 in Mab^Wt, ∆MAB_1915 mutant, and unmarked ∆MAB_1915 mutant (Un1915). Lane M, 1-kb DNA ladder; lane 1, Mab^Wt (2672 bp); lane 2, ∆MAB_1915 mutant (2564 bp); lane 3, Un1915 (1964 bp).

Fig 2

Drug susceptibility testing of the strains by SPOTi. Mab^KO is susceptible to RIF, RFB, LZD, CLA, VAN, and BDQ by both broth microdilution and SPOTi. However, it is observed to have some levels of susceptibility to CLF and LEV only on agar plates. The shown CP strains appear to partially restore resistance to the drugs.

Fig 3

Presumed start codons (Psc) upstream of MAB_1915. Two alternative “GTG” start positions were presumed some 69 and 27 bp upstream of MAB_1915. In contrast to MAB_1915, the expression of either Psc1-MAB_1915 or Psc2-MAB_1915 in the knockout strain enables partial restoration of drug resistance.

Fig 4

Assessment of cell envelope permeability. A Accumulation of EtBr in the strains. B Susceptibility testing against dyes and detergents. Mab^KO accumulated more EtBr and was also observed to be relatively more sensitive to dyes and detergents in contrast to the CP strains, which perhaps results from an increase in the permeability of its cell envelope. ^****, P < 0.0001; ns, not significant.