Single-molecule nanopore sequencing reveals extreme target copy number heterogeneity in arylomycin-resistant mutants.

Autor:	Girgis HS; Department of Infectious Diseases, Genentech, South San Francisco, CA 94080., DuPai CD; Department of OMNI Bioinformatics, Genentech, South San Francisco, CA 94080., Lund J; Department of Proteomics, Lipidomics and Next Generation Sequencing, Genentech, South San Francisco, CA 94080., Reeder J; Department of OMNI Bioinformatics, Genentech, South San Francisco, CA 94080., Guillory J; Department of Proteomics, Lipidomics and Next Generation Sequencing, Genentech, South San Francisco, CA 94080., Durinck S; Department of Oncology Bioinformatics, Genentech, South San Francisco, CA 94080., Liang Y; Department of Proteomics, Lipidomics and Next Generation Sequencing, Genentech, South San Francisco, CA 94080., Kaminker J; Department of OMNI Bioinformatics, Genentech, South San Francisco, CA 94080., Smith PA; Department of Infectious Diseases, Genentech, South San Francisco, CA 94080; smith.peter@gene.com skippington.elizabeth@gene.com., Skippington E; Department of OMNI Bioinformatics, Genentech, South San Francisco, CA 94080; smith.peter@gene.com skippington.elizabeth@gene.com.
Jazyk:	angličtina
Zdroj:	Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2021 Jan 05; Vol. 118 (1).
DOI:	10.1073/pnas.2021958118
Abstrakt:	Tandem gene amplification is a frequent and dynamic source of antibiotic resistance in bacteria. Ongoing expansions and contractions of repeat arrays during population growth are expected to manifest as cell-to-cell differences in copy number (CN). As a result, a clonal bacterial culture could comprise subpopulations of cells with different levels of antibiotic sensitivity that result from variable gene dosage. Despite the high potential for misclassification of heterogenous cell populations as either antibiotic-susceptible or fully resistant in clinical settings, and the concomitant risk of inappropriate treatment, CN distribution among cells has defied analysis. Here, we use the MinION single-molecule nanopore sequencer to uncover CN heterogeneity in clonal populations of Escherichia coli and Acinetobacter baumannii grown from single cells isolated while selecting for resistance to an optimized arylomycin, a member of a recently discovered class of Gram-negative antibiotic. We found that gene amplification of the arylomycin target, bacterial type I signal peptidase LepB, is a mechanism of unstable arylomycin resistance and demonstrate in E. coli that amplification instability is independent of RecA. This instability drives the emergence of a nonuniform distribution of lepB CN among cells with a range of 1 to at least 50 copies of lepB identified in a single clonal population. In sum, this remarkable heterogeneity, and the evolutionary plasticity it fuels, illustrates how gene amplification can enable bacterial populations to respond rapidly to novel antibiotics. This study establishes a rationale for further nanopore-sequencing studies of heterogeneous cell populations to uncover CN variability at single-molecule resolution. Competing Interests: Competing interest statement: P.A.S. is listed as an inventor on patent WO2017084630. H.S.G., C.D.D., J.R., J.L., S.D., Y.L., J.K., P.A.S. and E.S. are employees of Genentech, a member of the Roche Group, and are shareholders in Roche. J.G. is employed by Oxford Nanopore Technologies Ltd. (Copyright © 2021 the Author(s). Published by PNAS.)
Databáze:	MEDLINE
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