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The Heterodimeric ABC Transporter EfrCD Mediates Multidrug Efflux in Enterococcus faecalis

View Article: PubMed Central - PubMed

ABSTRACT

Nosocomial infections with Enterococcus faecalis are an emerging health problem. However, drug efflux pumps contributing to intrinsic drug resistance are poorly studied in this Gram-positive pathogen. In this study, we functionally investigated seven heterodimeric ABC transporters of E. faecalis that are annotated as drug efflux pumps. Deletion of ef0789-ef0790 on the chromosome of E. faecalis resulted in increased susceptibility to daunorubicin, doxorubicin, ethidium, and Hoechst 33342, and the corresponding transporter was named EfrCD. Unexpectedly, the previously described heterodimeric multidrug ABC transporter EfrAB contributes marginally to drug efflux in the endogenous context of E. faecalis. In contrast, heterologous expression in Lactococcus lactis revealed that EfrAB, EfrCD, and the product of ef2226-ef2227 (EfrEF) mediate the efflux of fluorescent substrates and confer resistance to multiple dyes and drugs, including fluoroquinolones. Four of seven transporters failed to exhibit drug efflux activity for the set of drugs and dyes tested, even upon overexpression in L. lactis. Since all seven transporters were purified as heterodimers after overexpression in L. lactis, a lack of drug efflux activity is not attributed to poor expression or protein aggregation. Reconstitution of the purified multidrug transporters EfrAB, EfrCD, and EfrEF in proteoliposomes revealed functional coupling between ATP hydrolysis and drug binding. Our analysis creates an experimental basis for the accurate prediction of drug efflux transporters and indicates that many annotated multidrug efflux pumps might be incapable of drug transport and thus might fulfill other physiological functions in the cell.

No MeSH data available.


Fluorescent dye transport mediated by EfrAB, EfrCD, and EfrEF. Fluorescence spectroscopy was used to measure the accumulation of ethidium (a), Hoechst 33342 (b), and BCECF-AM (c) by L. lactis NZ9000 ΔlmrA ΔlmrCD cells expressing the respective wild-type or inactive E-to-Q mutant transporter. Active efflux manifests in a slower increase of fluorescence.
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Figure 3: Fluorescent dye transport mediated by EfrAB, EfrCD, and EfrEF. Fluorescence spectroscopy was used to measure the accumulation of ethidium (a), Hoechst 33342 (b), and BCECF-AM (c) by L. lactis NZ9000 ΔlmrA ΔlmrCD cells expressing the respective wild-type or inactive E-to-Q mutant transporter. Active efflux manifests in a slower increase of fluorescence.

Mentions: L. lactis NZ9000 ΔlmrA ΔlmrCD cells harboring plasmids encoding the seven transporters (in their wild-type or inactive E-to-Q mutant forms) were grown in GM17 containing 5 μg/ml chloramphenicol at 30°C. Expression was induced at an OD600 of 0.4 to 0.6 with a nisin-containing culture supernatant of L. lactis NZ9700 for 1 h (1:1,000 [vol/vol]). Cells were washed and were resuspended with fluorescence buffer (50 mM KPi at pH 7.0, 5 mM MgSO4). Cells were adjusted to an OD600 of 0.5 in 2 ml fluorescence buffer and were energized by adding 0.5% glucose. Nigericin and valinomycin (1 μM each) were added prior to the addition of 2′,7′-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) to avoid changes in BCECF fluorescence as a result of pH changes in the cytoplasm. The accumulation of 5 μM ethidium, 0.5 μM Hoechst 33342, or 0.2 μM BCECF-AM was monitored at 25°C for 600 s using an LS-55 fluorescence spectrometer (PerkinElmer). Excitation and emission wavelengths (with slit widths given in parentheses) were set at 520 nm (10 nm) and 595 nm (15 nm) for ethidium, 355 nm (5 nm) and 457 nm (5 nm) for Hoechst 33342, and 502 nm (2.5 nm) and 525 nm (4.0 nm) for BCECF, respectively (see Fig. 3 and Fig. S1 in the supplemental material).


The Heterodimeric ABC Transporter EfrCD Mediates Multidrug Efflux in Enterococcus faecalis
Fluorescent dye transport mediated by EfrAB, EfrCD, and EfrEF. Fluorescence spectroscopy was used to measure the accumulation of ethidium (a), Hoechst 33342 (b), and BCECF-AM (c) by L. lactis NZ9000 ΔlmrA ΔlmrCD cells expressing the respective wild-type or inactive E-to-Q mutant transporter. Active efflux manifests in a slower increase of fluorescence.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4997860&req=5

Figure 3: Fluorescent dye transport mediated by EfrAB, EfrCD, and EfrEF. Fluorescence spectroscopy was used to measure the accumulation of ethidium (a), Hoechst 33342 (b), and BCECF-AM (c) by L. lactis NZ9000 ΔlmrA ΔlmrCD cells expressing the respective wild-type or inactive E-to-Q mutant transporter. Active efflux manifests in a slower increase of fluorescence.
Mentions: L. lactis NZ9000 ΔlmrA ΔlmrCD cells harboring plasmids encoding the seven transporters (in their wild-type or inactive E-to-Q mutant forms) were grown in GM17 containing 5 μg/ml chloramphenicol at 30°C. Expression was induced at an OD600 of 0.4 to 0.6 with a nisin-containing culture supernatant of L. lactis NZ9700 for 1 h (1:1,000 [vol/vol]). Cells were washed and were resuspended with fluorescence buffer (50 mM KPi at pH 7.0, 5 mM MgSO4). Cells were adjusted to an OD600 of 0.5 in 2 ml fluorescence buffer and were energized by adding 0.5% glucose. Nigericin and valinomycin (1 μM each) were added prior to the addition of 2′,7′-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) to avoid changes in BCECF fluorescence as a result of pH changes in the cytoplasm. The accumulation of 5 μM ethidium, 0.5 μM Hoechst 33342, or 0.2 μM BCECF-AM was monitored at 25°C for 600 s using an LS-55 fluorescence spectrometer (PerkinElmer). Excitation and emission wavelengths (with slit widths given in parentheses) were set at 520 nm (10 nm) and 595 nm (15 nm) for ethidium, 355 nm (5 nm) and 457 nm (5 nm) for Hoechst 33342, and 502 nm (2.5 nm) and 525 nm (4.0 nm) for BCECF, respectively (see Fig. 3 and Fig. S1 in the supplemental material).

View Article: PubMed Central - PubMed

ABSTRACT

Nosocomial infections with Enterococcus faecalis are an emerging health problem. However, drug efflux pumps contributing to intrinsic drug resistance are poorly studied in this Gram-positive pathogen. In this study, we functionally investigated seven heterodimeric ABC transporters of E. faecalis that are annotated as drug efflux pumps. Deletion of ef0789-ef0790 on the chromosome of E. faecalis resulted in increased susceptibility to daunorubicin, doxorubicin, ethidium, and Hoechst 33342, and the corresponding transporter was named EfrCD. Unexpectedly, the previously described heterodimeric multidrug ABC transporter EfrAB contributes marginally to drug efflux in the endogenous context of E. faecalis. In contrast, heterologous expression in Lactococcus lactis revealed that EfrAB, EfrCD, and the product of ef2226-ef2227 (EfrEF) mediate the efflux of fluorescent substrates and confer resistance to multiple dyes and drugs, including fluoroquinolones. Four of seven transporters failed to exhibit drug efflux activity for the set of drugs and dyes tested, even upon overexpression in L. lactis. Since all seven transporters were purified as heterodimers after overexpression in L. lactis, a lack of drug efflux activity is not attributed to poor expression or protein aggregation. Reconstitution of the purified multidrug transporters EfrAB, EfrCD, and EfrEF in proteoliposomes revealed functional coupling between ATP hydrolysis and drug binding. Our analysis creates an experimental basis for the accurate prediction of drug efflux transporters and indicates that many annotated multidrug efflux pumps might be incapable of drug transport and thus might fulfill other physiological functions in the cell.

No MeSH data available.