title: Synthetic lethality reveals mechanisms of Mycobacterium tuberculosis resistance to β-lactams.
abstract: Most β-lactam antibiotics are ineffective against Mycobacterium tuberculosis due to the microbe's innate resistance. The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains has prompted interest to repurpose this class of drugs. To identify the genetic determinants of innate β-lactam resistance, we carried out a synthetic lethality screen on a transposon mutant library for susceptibility to imipenem, a carbapenem β-lactam antibiotic. Mutations in 74 unique genes demonstrated synthetic lethality. The majority of mutations were in genes associated with cell wall biosynthesis. A second quantitative real-time PCR (qPCR)-based synthetic lethality screen of randomly selected mutants confirmed the role of cell wall biosynthesis in β-lactam resistance. The global transcriptional response of the bacterium to β-lactams was investigated, and changes in levels of expression of cell wall biosynthetic genes were identified. Finally, we validated these screens in vivo using the MT1616 transposon mutant, which lacks a functional acyl-transferase gene. Mice infected with the mutant responded to β-lactam treatment with a 100-fold decrease in bacillary lung burden over 4 weeks, while the numbers of organisms in the lungs of mice infected with wild-type bacilli proliferated. These findings reveal a road map of genes required for β-lactam resistance and validate synthetic lethality screening as a promising tool for repurposing existing classes of licensed, safe, well-characterized antimicrobials against tuberculosis.
  
  
  OBJECTIVE
  The global emergence of multidrug-resistant and extensively drug-resistant M. tuberculosis strains has threatened public health worldwide, yet the pipeline of new tuberculosis drugs under development remains limited. One strategy to cope with the urgent need for new antituberculosis agents is to repurpose existing, approved antibiotics. The carbapenem class of β-lactam antibiotics has been proposed as one such class of drugs. Our study identifies molecular determinants of innate resistance to β-lactam drugs in M. tuberculosis, and we demonstrate that functional loss of one of these genes enables successful treatment of M. tuberculosis with β-lactams in the mouse model.
authors.affiliation: Center for Tuberculosis Research, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Shichun
authors.initials: S
authors.lastname: Lun
authors.affiliation: Center for Tuberculosis Research, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: David
authors.initials: D
authors.lastname: Miranda
authors.affiliation: Center for Tuberculosis Research, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Andre
authors.initials: A
authors.lastname: Kubler
authors.affiliation: Center for Tuberculosis Research, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Haidan
authors.initials: H
authors.lastname: Guo
authors.affiliation: Center for Tuberculosis Research, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Mariama C
authors.initials: MC
authors.lastname: Maiga
authors.affiliation: Center for Tuberculosis Research, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Winglee
authors.affiliation: Center for Tuberculosis Research, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Shaaretha
authors.initials: S
authors.lastname: Pelly
authors.affiliation: wbishai1@jhmi.edu.
authors.forename: William R
authors.initials: WR
authors.lastname: Bishai
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D015780:Carbapenems / Q000494:pharmacology; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D005260:Female; D015378:Imipenem / Q000494:pharmacology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics*; D012329:RNA, Bacterial / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D017423:Sequence Analysis, RNA
chemical_list: D000900:Anti-Bacterial Agents; D015780:Carbapenems; D012329:RNA, Bacterial; D015378:Imipenem