Fluorescence-Based Flow Sorting in Parallel with Transposon Insertion Site Sequencing Identifies Multidrug Efflux Systems in <named-content content-type="genus-species">Acinetobacter baumannii</named-content>

ABSTRACT Multidrug efflux pumps provide clinically significant levels of drug resistance in a number of Gram-negative hospital-acquired pathogens. These pathogens frequently carry dozens of genes encoding putative multidrug efflux pumps. However, it can be difficult to determine how many of these pu...

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Autores principales:	Karl A. Hassan, Amy K. Cain, TaoTao Huang, Qi Liu, Liam D. H. Elbourne, Christine J. Boinett, Anthony J. Brzoska, Liping Li, Martin Ostrowski, Nguyen Thi Khanh Nhu, Tran Do Hoang Nhu, Stephen Baker, Julian Parkhill, Ian T. Paulsen
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2016
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/009af4327ad6410bacf216272c40249a
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spelling	oai:doaj.org-article:009af4327ad6410bacf216272c40249a2021-11-15T15:50:16ZFluorescence-Based Flow Sorting in Parallel with Transposon Insertion Site Sequencing Identifies Multidrug Efflux Systems in <named-content content-type="genus-species">Acinetobacter baumannii</named-content>10.1128/mBio.01200-162150-7511https://doaj.org/article/009af4327ad6410bacf216272c40249a2016-11-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01200-16https://doaj.org/toc/2150-7511ABSTRACT Multidrug efflux pumps provide clinically significant levels of drug resistance in a number of Gram-negative hospital-acquired pathogens. These pathogens frequently carry dozens of genes encoding putative multidrug efflux pumps. However, it can be difficult to determine how many of these pumps actually mediate antimicrobial efflux, and it can be even more challenging to identify the regulatory proteins that control expression of these pumps. In this study, we developed an innovative high-throughput screening method, combining transposon insertion sequencing and cell sorting methods (TraDISort), to identify the genes encoding major multidrug efflux pumps, regulators, and other factors that may affect the permeation of antimicrobials, using the nosocomial pathogen Acinetobacter baumannii. A dense library of more than 100,000 unique transposon insertion mutants was treated with ethidium bromide, a common substrate of multidrug efflux pumps that is differentially fluorescent inside and outside the bacterial cytoplasm. Populations of cells displaying aberrant accumulations of ethidium were physically enriched using fluorescence-activated cell sorting, and the genomic locations of transposon insertions within these strains were determined using transposon-directed insertion sequencing. The relative abundance of mutants in the input pool compared to the selected mutant pools indicated that the AdeABC, AdeIJK, and AmvA efflux pumps are the major ethidium efflux systems in A. baumannii. Furthermore, the method identified a new transcriptional regulator that controls expression of amvA. In addition to the identification of efflux pumps and their regulators, TraDISort identified genes that are likely to control cell division, cell morphology, or aggregation in A. baumannii. IMPORTANCE Transposon-directed insertion sequencing (TraDIS) and related technologies have emerged as powerful methods to identify genes required for bacterial survival or competitive fitness under various selective conditions. We applied fluorescence-activated cell sorting (FACS) to physically enrich for phenotypes of interest within a mutant population prior to TraDIS. To our knowledge, this is the first time that a physical selection method has been applied in parallel with TraDIS rather than a fitness-induced selection. The results demonstrate the feasibility of this combined approach to generate significant results and highlight the major multidrug efflux pumps encoded in an important pathogen. This FACS-based approach, TraDISort, could have a range of future applications, including the characterization of efflux pump inhibitors, the identification of regulatory factors controlling gene or protein expression using fluorescent reporters, and the identification of genes involved in cell replication, morphology, and aggregation.Karl A. HassanAmy K. CainTaoTao HuangQi LiuLiam D. H. ElbourneChristine J. BoinettAnthony J. BrzoskaLiping LiMartin OstrowskiNguyen Thi Khanh NhuTran Do Hoang NhuStephen BakerJulian ParkhillIan T. PaulsenAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 7, Iss 5 (2016)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Karl A. Hassan Amy K. Cain TaoTao Huang Qi Liu Liam D. H. Elbourne Christine J. Boinett Anthony J. Brzoska Liping Li Martin Ostrowski Nguyen Thi Khanh Nhu Tran Do Hoang Nhu Stephen Baker Julian Parkhill Ian T. Paulsen Fluorescence-Based Flow Sorting in Parallel with Transposon Insertion Site Sequencing Identifies Multidrug Efflux Systems in <named-content content-type="genus-species">Acinetobacter baumannii</named-content>
description	ABSTRACT Multidrug efflux pumps provide clinically significant levels of drug resistance in a number of Gram-negative hospital-acquired pathogens. These pathogens frequently carry dozens of genes encoding putative multidrug efflux pumps. However, it can be difficult to determine how many of these pumps actually mediate antimicrobial efflux, and it can be even more challenging to identify the regulatory proteins that control expression of these pumps. In this study, we developed an innovative high-throughput screening method, combining transposon insertion sequencing and cell sorting methods (TraDISort), to identify the genes encoding major multidrug efflux pumps, regulators, and other factors that may affect the permeation of antimicrobials, using the nosocomial pathogen Acinetobacter baumannii. A dense library of more than 100,000 unique transposon insertion mutants was treated with ethidium bromide, a common substrate of multidrug efflux pumps that is differentially fluorescent inside and outside the bacterial cytoplasm. Populations of cells displaying aberrant accumulations of ethidium were physically enriched using fluorescence-activated cell sorting, and the genomic locations of transposon insertions within these strains were determined using transposon-directed insertion sequencing. The relative abundance of mutants in the input pool compared to the selected mutant pools indicated that the AdeABC, AdeIJK, and AmvA efflux pumps are the major ethidium efflux systems in A. baumannii. Furthermore, the method identified a new transcriptional regulator that controls expression of amvA. In addition to the identification of efflux pumps and their regulators, TraDISort identified genes that are likely to control cell division, cell morphology, or aggregation in A. baumannii. IMPORTANCE Transposon-directed insertion sequencing (TraDIS) and related technologies have emerged as powerful methods to identify genes required for bacterial survival or competitive fitness under various selective conditions. We applied fluorescence-activated cell sorting (FACS) to physically enrich for phenotypes of interest within a mutant population prior to TraDIS. To our knowledge, this is the first time that a physical selection method has been applied in parallel with TraDIS rather than a fitness-induced selection. The results demonstrate the feasibility of this combined approach to generate significant results and highlight the major multidrug efflux pumps encoded in an important pathogen. This FACS-based approach, TraDISort, could have a range of future applications, including the characterization of efflux pump inhibitors, the identification of regulatory factors controlling gene or protein expression using fluorescent reporters, and the identification of genes involved in cell replication, morphology, and aggregation.
format	article
author	Karl A. Hassan Amy K. Cain TaoTao Huang Qi Liu Liam D. H. Elbourne Christine J. Boinett Anthony J. Brzoska Liping Li Martin Ostrowski Nguyen Thi Khanh Nhu Tran Do Hoang Nhu Stephen Baker Julian Parkhill Ian T. Paulsen
author_facet	Karl A. Hassan Amy K. Cain TaoTao Huang Qi Liu Liam D. H. Elbourne Christine J. Boinett Anthony J. Brzoska Liping Li Martin Ostrowski Nguyen Thi Khanh Nhu Tran Do Hoang Nhu Stephen Baker Julian Parkhill Ian T. Paulsen
author_sort	Karl A. Hassan
title	Fluorescence-Based Flow Sorting in Parallel with Transposon Insertion Site Sequencing Identifies Multidrug Efflux Systems in <named-content content-type="genus-species">Acinetobacter baumannii</named-content>
title_short	Fluorescence-Based Flow Sorting in Parallel with Transposon Insertion Site Sequencing Identifies Multidrug Efflux Systems in <named-content content-type="genus-species">Acinetobacter baumannii</named-content>
title_full	Fluorescence-Based Flow Sorting in Parallel with Transposon Insertion Site Sequencing Identifies Multidrug Efflux Systems in <named-content content-type="genus-species">Acinetobacter baumannii</named-content>
title_fullStr	Fluorescence-Based Flow Sorting in Parallel with Transposon Insertion Site Sequencing Identifies Multidrug Efflux Systems in <named-content content-type="genus-species">Acinetobacter baumannii</named-content>
title_full_unstemmed	Fluorescence-Based Flow Sorting in Parallel with Transposon Insertion Site Sequencing Identifies Multidrug Efflux Systems in <named-content content-type="genus-species">Acinetobacter baumannii</named-content>
title_sort	fluorescence-based flow sorting in parallel with transposon insertion site sequencing identifies multidrug efflux systems in <named-content content-type="genus-species">acinetobacter baumannii</named-content>
publisher	American Society for Microbiology
publishDate	2016
url	https://doaj.org/article/009af4327ad6410bacf216272c40249a
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Fluorescence-Based Flow Sorting in Parallel with Transposon Insertion Site Sequencing Identifies Multidrug Efflux Systems in <named-content content-type="genus-species">Acinetobacter baumannii</named-content>

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