title: Mutations in pepQ Confer Low-Level Resistance to Bedaquiline and Clofazimine in Mycobacterium tuberculosis.
abstract: The novel ATP synthase inhibitor bedaquiline recently received accelerated approval for treatment of multidrug-resistant tuberculosis and is currently being studied as a component of novel treatment-shortening regimens for drug-susceptible and multidrug-resistant tuberculosis. In a limited number of bedaquiline-treated patients reported to date, ≥4-fold upward shifts in bedaquiline MIC during treatment have been attributed to non-target-based mutations in Rv0678 that putatively increase bedaquiline efflux through the MmpS5-MmpL5 pump. These mutations also confer low-level clofazimine resistance, presumably by a similar mechanism. Here, we describe a new non-target-based determinant of low-level bedaquiline and clofazimine cross-resistance in Mycobacterium tuberculosis: loss-of-function mutations in pepQ (Rv2535c), which corresponds to a putative Xaa-Pro aminopeptidase. pepQ mutants were selected in mice by treatment with clinically relevant doses of bedaquiline, with or without clofazimine, and were shown to have bedaquiline and clofazimine MICs 4 times higher than those for the parental H37Rv strain. Coincubation with efflux inhibitors verapamil and reserpine lowered bedaquiline MICs against both mutant and parent strains to a level below the MIC against H37Rv in the absence of efflux pump inhibitors. However, quantitative PCR (qPCR) revealed no significant differences in expression of Rv0678, mmpS5, or mmpL5 between mutant and parent strains. Complementation of a pepQ mutant with the wild-type gene restored susceptibility, indicating that loss of PepQ function is sufficient for reduced susceptibility both in vitro and in mice. Although the mechanism by which mutations in pepQ confer bedaquiline and clofazimine cross-resistance remains unclear, these results may have clinical implications and warrant further evaluation of clinical isolates with reduced susceptibility to either drug for mutations in this gene.
authors.affiliation: Center for Tuberculosis Research, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Deepak
authors.initials: D
authors.lastname: Almeida
authors.affiliation: Department of Computer Science, Texas A&M University, College Station, Texas, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Ioerger
authors.affiliation: Center for Tuberculosis Research, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Sandeep
authors.initials: S
authors.lastname: Tyagi
authors.affiliation: Center for Tuberculosis Research, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Si-Yang
authors.initials: SY
authors.lastname: Li
authors.affiliation: Global Alliance for TB Drug Development, New York, New York, USA.
authors.forename: Khisimuzi
authors.initials: K
authors.lastname: Mdluli
authors.affiliation: Janssen Pharmaceutica, Beerse, Belgium.
authors.forename: Koen
authors.initials: K
authors.lastname: Andries
authors.affiliation: Center for Tuberculosis Research, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Jacques
authors.initials: J
authors.lastname: Grosset
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas.
authors.forename: Jim
authors.initials: J
authors.lastname: Sacchettini
authors.affiliation: Center for Tuberculosis Research, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA Department of International Health, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA enuermb@jhmi.edu.
authors.forename: Eric
authors.initials: E
authors.lastname: Nuermberger
mesh_terms: D000818:Animals; D000995:Antitubercular Agents / Q000627:therapeutic use*; D002991:Clofazimine / Q000627:therapeutic use*; D064687:Diarylquinolines / Q000627:therapeutic use*; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D008826:Microbial Sensitivity Tests; D009154:Mutation / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics; D018088:Tuberculosis, Multidrug-Resistant / Q000188:drug therapy / Q000378:metabolism
chemical_list: D000995:Antitubercular Agents; D064687:Diarylquinolines; C493870:bedaquiline; D002991:Clofazimine