preparation of electrocompetent cells to achieve single&#45;copy gene expression in an efflux&#45;deficient bacterial strain

Expressão gênica de cópia única para caracterização de sistemas de efluxo multidrogas

Acinetobacter baumannii is a World Health Organization top priority pathogen due to its encompassing resistance to all classes of antibiotics1. It is an opportunistic pathogen mostly affecting hospitalized, injured, or immunocompromised people. A. baumannii largely evades antibiotics via efflux pumps, the most relevant being the Resistance-Nodulation-Division (RND) family of exporters2. Understanding how these efflux pumps work mechanistically will allow one to develop targeted therapeutic options.
One common way that cellular processes can be specifically distinguished is through genetic manipulation. However, the tools available for A. baumannii genetic studies are limited, and to further confound experimental design, clinical isolates often are resistant to the antibiotics routinely used for selection in genetic manipulations3. A second hurdle encountered when studying efflux pumps specifically is that they are strictly regulated-often by unknown factors-making it difficult to accurately isolate and attribute function to a single pump4. Seeing this need to expand the research toolbox, we developed a mini-Tn7-based, single-copy-insertion, inducible expression system that incorporates a Flp recombinase target (FRT) cassette, which allows for the removal of the selection marker5,6,7 (Figure 1). First created for Pseudomonas8,9,10, this elegant cloning and expression system was used to generate single-copy efflux pump complements into an RND efflux pump-deficient strain of A. baumannii (ATCC 17978::&#916;adeIJK,&#916;adeFGH,&#916;adeAB: hereafter referred to as A. baumannii AB258) that we generated11. Being able to study one efflux pump at a time and not overwhelm the bacterial cells with high-copy expression (as generally seen with plasmid-based expression systems), one can better learn about the critical, physiological aspects of each efflux pump with minimal interference and reduced complications.
This article describes how to use the mini-Tn7 system to complement a deleted gene of interest, RND efflux pump adeIJK, into the chromosome of A. baumannii AB258 through a series of uncomplicated steps performed over the course of 9 days7. The first set of steps re-introduces the deleted efflux pump genes cloned into the mini-Tn7-based insertion plasmid (Figure 2A) at the single attTn7 insertion site downstream of the well-conserved glmS gene (Figure 3A). This process is facilitated by a non-replicative helper plasmid (Figure 2B) that encodes for the transposase genes needed for Tn7-driven insertion. The second set of steps uses an excision plasmid (Figure 2C) for Flp recombinase-mediated removal of the gentamicin gene flanked by FRT sites (Figure 3B) to create an unmarked strain. Though this system is used to elucidate the essential roles and possible inhibitors of RND efflux pumps with respect to antibiotic resistance, it can be used to investigate any gene of interest.

Autor em destaque: Avançando na pesquisa de resistência a antibióticos usando uma cepa bacteriana deficiente em efluxo e um sistema de expressão gênica de cópia única

Caracterização de Sistemas de Efluxo Multidrogas em Acinetobacter baumannii Usando uma Cepa Bacteriana Deficiente em Efluxo e um Sistema de Expressão Gênica de Cópia Única

We study multi-drug resistance efflux systems in gram-negative bacteria. We are interested in learning about the natural functions of these pumps and how these pumps can be used as a potential drug target. Expression of multi-drug resistance efflux systems is tightly regulated, making it difficult to study their functions.Thus, an expression system that bypasses the regulatory system can be quite useful in the characterization of these pumps. This protocol allows for the expression of genes at levels usually seen in natural settings. Thus, the activity of the gene products mimics what is observed in nature.

Preparação de células eletrocompetentes para obter expressão gênica de cópia única em uma cepa bacteriana deficiente em efluxo 

Para começar, coloque todos os tubos de microfuga estéreis de 1,5 mililitro e cubetas de eletroporação estéreis no gelo. Em seguida, coloque a garrafa de água estéril, que foi armazenada a quatro graus Celsius, no gelo. Transfira 1,5 mililitros da cultura bacteriana durante a noite para um dos tubos de microfuga de 1,5 mililitro e centrífuga a 13.000 G por dois minutos para peletizar as células.Usando uma pipeta de um mililitro, remova todo o sobrenadante sem perturbar o pellet celular. Adicione mais 1,5 mililitros de cultura bacteriana no mesmo tubo de microfuga, centrifugar a 13.000 G por dois minutos e remover o sobrenadante. Adicione um mililitro de água gelada e estéril ao pellet de célula e ressuspenda com pipetagem suave até que o pellet não fique mais no fundo do tubo de microfuga.Centrifugar as células ressuspensas a 13.000 G por dois minutos e remover cuidadosamente o sobrenadante usando uma pipeta de um mililitro. Ressuspenda suavemente o pellet de célula final em 200 microlitros de água gelada e estéril e transfira 100 microlitros da suspensão final da célula para o segundo tubo de microfuge de 1,5 mililitro gelado.

Research

JoVE Journal

Immunology and Infection

Preparation of Electrocompetent Cells to Achieve Single&#45;Copy Gene Expression in an Efflux&#45;Deficient Bacterial Strain

Characterizing Multidrug Efflux Systems in Acinetobacter baumannii Using an Efflux&#45;Deficient Bacterial Strain and a Single&#45;Copy Gene Expression System

Acinetobacter baumannii is recognized as a challenging Gram-negative pathogen due to its widespread resistance to antibiotics. It is crucial to comprehend ...

Imunologia e Infecção

Electroporation Procedure for the Single&#45;Copy Gene Expression in an Efflux&#45;Deficient Bacterial Strain

Procedimento de eletroporação para a expressão gênica de cópia única em uma cepa bacteriana deficiente em efluxo