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Homologs of the Acinetobacter baumannii AceI transporter represent a new family of bacterial multidrug efflux systems.

ABSTRACT: UNLABELLED:Multidrug efflux systems are a major cause of resistance to antimicrobials in bacteria, including those pathogenic to humans, animals, and plants. These proteins are ubiquitous in these pathogens, and five families of bacterial multidrug efflux systems have been identified to date. By using transcriptomic and biochemical analyses, we recently identified the novel AceI (Acinetobacter chlorhexidine efflux) protein from Acinetobacter baumannii that conferred resistance to the biocide chlorhexidine, via an active efflux mechanism. Proteins homologous to AceI are encoded in the genomes of many other bacterial species and are particularly prominent within proteobacterial lineages. In this study, we expressed 23 homologs of AceI and examined their resistance and/or transport profiles. MIC analyses demonstrated that, like AceI, many of the homologs conferred resistance to chlorhexidine. Many of the AceI homologs conferred resistance to additional biocides, including benzalkonium, dequalinium, proflavine, and acriflavine. We conducted fluorimetric transport assays using the AceI homolog from Vibrio parahaemolyticus and confirmed that resistance to both proflavine and acriflavine was mediated by an active efflux mechanism. These results show that this group of AceI homologs represent a new family of bacterial multidrug efflux pumps, which we have designated the proteobacterial antimicrobial compound efflux (PACE) family of transport proteins. IMPORTANCE:Bacterial multidrug efflux pumps are an important class of resistance determinants that can be found in every bacterial genome sequenced to date. These transport proteins have important protective functions for the bacterial cell but are a significant problem in the clinical setting, since a single efflux system can mediate resistance to many structurally and mechanistically diverse antibiotics and biocides. In this study, we demonstrate that proteins related to the Acinetobacter baumannii AceI transporter are a new class of multidrug efflux systems which are very common in Proteobacteria: the proteobacterial antimicrobial compound efflux (PACE) family. This is the first new family of multidrug efflux pumps to be described in 15 years.

SUBMITTER: Hassan KA

PROVIDER: S-EPMC4337561 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Homologs of the Acinetobacter baumannii AceI transporter represent a new family of bacterial multidrug efflux systems.

Hassan Karl A KA Liu Qi Q Henderson Peter J F PJ Paulsen Ian T IT

mBio 20150210 1

<h4>Unlabelled</h4>Multidrug efflux systems are a major cause of resistance to antimicrobials in bacteria, including those pathogenic to humans, animals, and plants. These proteins are ubiquitous in these pathogens, and five families of bacterial multidrug efflux systems have been identified to date. By using transcriptomic and biochemical analyses, we recently identified the novel AceI (Acinetobacter chlorhexidine efflux) protein from Acinetobacter baumannii that conferred resistance to the bio ...[more]

PMID: 25670776

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Project description: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 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.

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Homologs of the Acinetobacter baumannii AceI transporter represent a new family of bacterial multidrug efflux systems.

Publications

Homologs of the Acinetobacter baumannii AceI transporter represent a new family of bacterial multidrug efflux systems.

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