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Comparative Study
. 2014 Dec;58(12):7250-7.
doi: 10.1128/AAC.03728-14. Epub 2014 Sep 22.

Properties of AdeABC and AdeIJK efflux systems of Acinetobacter baumannii compared with those of the AcrAB-TolC system of Escherichia coli

Affiliations
Comparative Study

Properties of AdeABC and AdeIJK efflux systems of Acinetobacter baumannii compared with those of the AcrAB-TolC system of Escherichia coli

Etsuko Sugawara et al. Antimicrob Agents Chemother. 2014 Dec.

Abstract

Acinetobacter baumannii contains RND-family efflux systems AdeABC and AdeIJK, which pump out a wide range of antimicrobial compounds, as judged from the MIC changes occurring upon deletion of the responsible genes. However, these studies may miss changes because of the high backgrounds generated by the remaining pumps and by β-lactamases, and it is unclear how the activities of these pumps compare quantitatively with those of the well-studied AcrAB-TolC system of Escherichia coli. We expressed adeABC and adeIJK of A. baumannii, as well as E. coli acrAB, in an E. coli host from which acrAB was deleted. The A. baumannii pumps were functional in E. coli, and the MIC changes that were observed largely confirmed the substrate range already reported, with important differences. Thus, the AdeABC system pumped out all β-lactams, an activity that was often missed in deletion studies. When the expression level of the pump genes was adjusted to a similar level for a comparison with AcrAB-TolC, we found that both A. baumannii efflux systems pumped out a wide range of compounds, but AdeABC was less effective than AcrAB-TolC in the extrusion of lipophilic β-lactams, novobiocin, and ethidium bromide, although it was more effective at tetracycline efflux. AdeIJK was remarkably more effective than a similar level of AcrAB-TolC in the efflux of β-lactams, novobiocin, and ethidium bromide, although it was less so in the efflux of erythromycin. These results thus allow us to compare these efflux systems on a quantitative basis, if we can assume that the heterologous systems are fully functional in the E. coli host.

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Figures

FIG 1
FIG 1
Expression levels of AdeB, AdeJ, and AcrB in the inner membrane fraction of AG100AΩ without induction. The inner membrane proteins (10 μg) in the sample buffer prepared as described in Materials and Methods were separated by SDS-PAGE with 8% (wt/vol) acrylamide in the running gel. Western immunoblots using antitetrahistidine antibody show the expression of RND pump proteins. Lane 1, AG100AΩ containing pKY9790; lane 2, AG100AΩ containing pKY-adeAB-6His; lane 3, AG100AΩ containing pKY-adeIJ-6His; lane 4, AG100AΩ containing pKY-acrAB-6His.
FIG 2
FIG 2
IPTG induces the expression of AdeB and AdeJ. The inner membrane proteins (10 μg) were prepared from AG100AΩ (ΔlacY) containing pKY9790-borne adeAB-6His and adeIJ-6His genes grown with the concentrations of IPTG specified below and were separated by SDS-PAGE. The expression levels of the AdeB and AdeJ proteins were determined by using immunoblotting with a monoclonal antibody against tetrahistidine, as described in Materials and Methods. Lane 1, AG100AΩ (ΔlacY) containing pKY-adeAB-6His without IPTG; lane 2, AG100AΩ (ΔlacY) containing pKY-adeAB-6His with 10 μM IPTG; lane 3, AG100AΩ (ΔlacY) containing pKY-adeAB-6His with 20 μM IPTG; lane 4, AG100AΩ (ΔlacY) containing pKY-adeIJ-6His without IPTG; lane 5, AG100AΩ (ΔlacY) containing pKY-adeIJ-6His with 5 μM IPTG; lane 6, AG100AΩ (ΔlacY) containing pKY-adeIJ-6His with 10 μM IPTG.
FIG 3
FIG 3
Adjustment of AcrB protein expression to a level comparable to the level of AdeB or AdeJ protein expression. (Lanes 1 to 3) In order to express similar levels of AdeB and AcrB proteins, AG100AΩ (ΔlacY) containing pKY-adeAB-6His was induced with 10 μM IPTG (lane 2) and the same strain containing pKY-acrAB-4His was grown without IPTG (lane 3); AG100AΩ (ΔlacY) containing pKY9790 alone was included in lane 1; (lanes 4 to 6) to express similar levels of AdeJ and AcrB, cells containing pKY-adeIJ-6His were induced with 5 μM IPTG (lane 5) and cells containing pHSG-acrAB-4His were induced with 500 μM IPTG (lane 6); cells containing pKY9790 alone (lane 4) and pHSG576 alone (lane 7) were included as controls. The inner membrane proteins (10 μg) were separated by SDS-PAGE, and the expression levels of the AdeB, AdeJ, and AcrB proteins were analyzed by using immunoblotting with monoclonal antibody against tetrahistidine.
FIG 4
FIG 4
Expression levels of periplasmic adaptor proteins (PAPs) in AG100AΩ (ΔlacY). Plasmid-borne 6His-tagged PAP genes were expressed under the same conditions used in the assay whose results are presented in Fig. 3. Cells containing pKY-adeA-6His were induced with 10 μM IPTG (lane 2), and those containing pKY-acrA-6His were grown without IPTG (lane 3). Cells containing pKY-adeI-6His were induced with 5 μM IPTG (lane 4), and cells containing pHSG-acrA-6His were induced with 500 μM IPTG. Cells containing pKY9790 alone (lane 1) and pHSG576 alone (lane 6) were included as controls. The crude cell extracts (5 μg of protein) were separated by SDS-PAGE, and the expression levels of the AdeA, AdeI, and AcrA proteins were analyzed by using immunoblotting with a monoclonal antibody against tetrahistidine.

References

    1. Bonomo RA, Szabo D. 2006. Mechanisms of multidrug resistance in Acinetobacter species and Pseudomonas aeruginosa. Clin. Infect. Dis. 43(Suppl 2):S49–S56. 10.1086/504477. - DOI - PubMed
    1. Peleg AY, Seifer H, Paterson DL. 2008. Acinetobacter baumannii: emergence of successful pathogen. Clin. Microbiol. Rev. 21:538–582. 10.1128/CMR.00058-07. - DOI - PMC - PubMed
    1. Sugawara E, Nikaido H. 2012. OmpA is the principal nonspecific slow porin of Acinetobacter baumannii. J. Bacteriol. 194:4089–4096. 10.1128/JB.00435-12. - DOI - PMC - PubMed
    1. Sugawara E, Nikaido H. 2004. OmpA/OprF: slow porins or channels produced by alternative folding of outer membrane proteins, p 119–138 In Benz R. (ed), Bacterial and eukaryotic porins: structure, function, mechanism. Wiley-VCH, Weinheim, Germany.
    1. Nikaido H, Rosenberg EY, Foulds J. 1983. Porin channels in Escherichia coli: studies with β-lactams in intact cells. J. Bacteriol. 153:232–240. - PMC - PubMed

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