Abstract
Mycobacterium abscessus is an emerging human pathogen causing severe pulmonary infections and is refractory to standard antibiotherapy, yet few drug resistance mechanisms have been reported in this organism. Recently, mutations in MAB_4384 leading to up-regulation of the MmpS5/MmpL5 efflux pump were linked to increased resistance to thiacetazone derivatives. Herein, the DNA-binding activity of MAB_4384 was investigated by electrophoretic mobility shift assays using the palindromic sequence IRS5/L5 located upstream of mmpS5/mmpL5. Introduction of point mutations within IRS5/L5 identified the sequence requirements for optimal binding of the regulator. Moreover, formation of the protein/IRS5/L5 complex was severely impaired for MAB_4384 harboring D14N or F57L substitutions. IRS5/L5/lacZ reporter fusions in M. abscessus demonstrated increased β-galactosidase activity either in strains lacking a functional MAB_4384 or in cultures treated with the TAC analogs. In addition, X-ray crystallography confirmed a typical TetR homodimeric structure of MAB_4384 and unraveled a putative ligand binding site in which the analogs could be docked. Overall, these results support drug recognition of the MAB_4384 TetR regulator, alleviating its binding to IRS5/L5 and steering up-regulation of MmpS5/MmpL5. This study provides new mechanistic and structural details of TetR-dependent regulatory mechanisms of efflux pumps and drug resistance in mycobacteria.
Highlights
Mycobacterium abscessus is a rapid growing mycobacterium (RGM) that has recently become an important health problem (Mougari et al, 2016)
We recently showed that mutations in the MAB_4384 regulator were associated with the transcriptional induction of the divergently oriented adjacent genes coding for an MmpS5/MmpL5 efflux pump and accounting for high resistance levels toward various TAC analogs (Halloum et al, 2017)
A combination of genetic, biochemical and structural studies was used to demonstrate that MAB_4384 is part of the TetR family of regulators, which represses the transcriptional expression of the MmpS5/MmpL5 efflux pump
Summary
Mycobacterium abscessus is a rapid growing mycobacterium (RGM) that has recently become an important health problem (Mougari et al, 2016). Recent studies have started to unveil the basis of the multi-drug resistance characterizing M. abscessus, uncovering a wide diversity of mechanisms or regulatory networks. These involve, for example, the induction of the erm(41) encoded 23S rRNA methyltransferase and mutations in the 23S rRNA that lead to clarithromycin resistance (Nash et al, 2009), the presence of a broad spectrum β-lactamase that limits the use of imipenem (Dubée et al, 2015; Lefebvre et al, 2016, 2017) or the presence of eis, encoding an acetyltransferase that modifies aminoglycosides, induced by whiB7, which contributes to the intrinsic resistance to amikacin (Hurst-Hess et al, 2017; Rominski et al, 2017b). Other studies reported the role of the ADP-ribosyltransferase MAB_0591 as a major contributor to rifamycin resistance (Rominski et al, 2017a) whereas MAB_2385 was identified as an important determinant in innate resistance to streptomycin (Dal Molin et al, 2018)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
