Journal: bioRxiv

Article Title: Drug degradation caused by mce3R mutations confers contezolid (MRX-I) resistance in Mycobacterium tuberculosis

doi: 10.1101/2022.07.25.501497

Figure Lengend Snippet: The genetic variants in contezolid-resistant M. tuberculosis . (A) Distribution of mce3R mutations associated with contezolid resistance. The full length of mce3R gene is 1221bp, encoding a putative protein of 406 amino acids. SNPs are showed in gray with nonsense mutations in bold, with numbers that correspond to the amino acid positions. Insertion and deletion mutations are shown in indigo and purple, respectively, with numbers indicating base positions. (B) Two contezolid-resistant strains harbored multiple mutations within the 339-446 base region of the mce3R gene. SNPs, insertions and deletions are indicated by red, blue and purple boxes, respectively. The Query and Sbjct sequences belong to contezolid-resistant strains and the wild-type H37Rv strain, respectively. The numbers indicate the base position of the mce3R gene. (C) Distribution of hitchhiking mutations identified in contezolid-resistant strains by WGS analysis, among 10 independent selection cultures indicated with different colors.

Article Snippet: For each ten independent log-phase cultures of laboratory reference strain H37Rv, 3 ml aliquots were concentrated and spread onto three 7H10-OADC plates containing 5 μg/ml contezolid (Shanghai MicuRx Pharmaceutical Co. Ltd.), corresponding to 5 times the minimum inhibitory concentration (MIC) of 1 μg/ml contezolid for H37Rv.

Techniques: Selection

Journal: bioRxiv

Article Title: Drug degradation caused by mce3R mutations confers contezolid (MRX-I) resistance in Mycobacterium tuberculosis

doi: 10.1101/2022.07.25.501497

Figure Lengend Snippet: MTB Rv1936-promoted contezolid degradation. (A) Alignment of the amino acid sequences of the MTB Rv1936 and the flavin-dependent oxidoreductase MelF in M. marinum . Highlighted amino acids represent identity between sequences. The numbers indicate the amino acid position of Rv1936 or MelF. (B) Chromatogram representation of the parent contezolid and its two principal degradation products MRX445-1 and MRX401detected by UPLC/triple TOF MS, with their corresponding m/z values indicated. The retention time and mass are confirmed as described before . (C) Comparative quantification of contezolid, MRX445-1 and MRX401 based on their chromatographic peak area in the WT H37Rv, in the three contezolid-resistant mce3R mutants (L266P, 755_756insC and 417_527del), in Δ mce3R complemented with a constitutive expression vector for mce3R (Δ mce3R O/E mce3R ) and in the WT H37Rv that constitutively expresses Rv1936 (H37Rv O/E Rv1936 ). All strains were treated with 10 μl contezolid for 15 hours, with the exception of the WT H37Rv being treated with 2 μl contezolid to avoid drug inhibition effect. (D) Schematic representation of oxidation of contezolid by Rv1936, and subsequent hydrolysis of its DHPO ring-expanding enol lactone, the oxidative intermediate product indicated in dotted box, into two principal DHPO ring-opened metabolites MRX445-1 and MRX401. Chemical groups indicating the key DHPO ring-opening process of contezolid degradation are highlighted in red. The structures of contezolid and MRX445-1 were confirmed with authentic molecules, and the proposed structure of MRX401 is consistent with available high-resolution mass spectrometry data and its biotransformation pathway.

Article Snippet: For each ten independent log-phase cultures of laboratory reference strain H37Rv, 3 ml aliquots were concentrated and spread onto three 7H10-OADC plates containing 5 μg/ml contezolid (Shanghai MicuRx Pharmaceutical Co. Ltd.), corresponding to 5 times the minimum inhibitory concentration (MIC) of 1 μg/ml contezolid for H37Rv.

Techniques: Expressing, Plasmid Preparation, Inhibition, Mass Spectrometry