title: Competitive Growth Enhances Conditional Growth Mutant Sensitivity to Antibiotics and Exposes a Two-Component System as an Emerging Antibacterial Target in Burkholderia cenocepacia.
abstract: Chemogenetic approaches to profile an antibiotic mode of action are based on detecting differential sensitivities of engineered bacterial strains in which the antibacterial target (usually encoded by an essential gene) or an associated process is regulated. We previously developed an essential-gene knockdown mutant library in the multidrug-resistant Burkholderia cenocepacia by transposon delivery of a rhamnose-inducible promoter. In this work, we used Illumina sequencing of multiplex-PCR-amplified transposon junctions to track individual mutants during pooled growth in the presence of antibiotics. We found that competition from nontarget mutants magnified the hypersensitivity of a clone underexpressing gyrB to novobiocin by 8-fold compared with hypersensitivity measured during clonal growth. Additional profiling of various antibiotics against a pilot library representing most categories of essential genes revealed a two-component system with unknown function, which, upon depletion of the response regulator, sensitized B. cenocepacia to novobiocin, ciprofloxacin, tetracycline, chloramphenicol, kanamycin, meropenem, and carbonyl cyanide 3-chlorophenylhydrazone, but not to colistin, hydrogen peroxide, and dimethyl sulfoxide. We named the gene cluster esaSR for enhanced sensitivity to antibiotics sensor and response regulator. Mutational analysis and efflux activity assays revealed that while esaS is not essential and is involved in antibiotic-induced efflux, esaR is an essential gene and regulates efflux independently of antibiotic-mediated induction. Furthermore, microscopic analysis of cells stained with propidium iodide provided evidence that depletion of EsaR has a profound effect on the integrity of cell membranes. In summary, we unraveled a previously uncharacterized two-component system that can be targeted to reduce antibiotic resistance in B. cenocepacia.
authors: Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada.
authors: April S
authors: AS
authors: Gislason
authors: Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada.
authors: Matthew
authors: M
authors: Choy
authors: Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada.
authors: Ruhi A M
authors: RA
authors: Bloodworth
authors: Beijing Institute of Radiation Medicine, State Key Laboratory of Proteomics, Cognitive and Mental Health Research Center, Beijing, China.
authors: Wubin
authors: W
authors: Qu
authors: Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada.
authors: Maria S
authors: MS
authors: Stietz
authors: Department of Mathematics and Statistics, University of Minnesota Duluth, Duluth, Minnesota, USA.
authors: Xuan
authors: X
authors: Li
authors: Beijing Institute of Radiation Medicine, State Key Laboratory of Proteomics, Cognitive and Mental Health Research Center, Beijing, China.
authors: Chenggang
authors: C
authors: Zhang
authors: Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada Silvia.Cardona@umanitoba.ca.|Department of Medical Microbiology and Infectious Disease, University of Manitoba, Winnipeg, Canada.
authors: Silvia T
authors: 0000-0002-0629-8886
authors: ST
authors: Cardona
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D057508:Burkholderia cenocepacia / Q000187:drug effects* / Q000235:genetics; D002701:Chloramphenicol / Q000494:pharmacology; D002939:Ciprofloxacin / Q000494:pharmacology; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics; D006835:Hydrazones / Q000494:pharmacology; D007612:Kanamycin / Q000494:pharmacology; D000077731:Meropenem; D008826:Microbial Sensitivity Tests; D009675:Novobiocin / Q000494:pharmacology; D013752:Tetracycline / Q000494:pharmacology; D013845:Thienamycins / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D006835:Hydrazones; D013845:Thienamycins; C070053:carbonyl 3-chlorophenylhydrazone; D009675:Novobiocin; D007612:Kanamycin; D002939:Ciprofloxacin; D002701:Chloramphenicol; D013752:Tetracycline; D000077731:Meropenem
keywords: Burkholderia; Gram-negative bacteria; Illumina; antibiotic profiling; antibiotic resistance; drug efflux; drug targets; essential genes; transposon mutant; two-component regulatory systems