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The Heterodimeric ABC Transporter EfrCD Mediates Multidrug Efflux in Enterococcus faecalis

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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.

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Drug-modulated ATPase activities of reconstituted enterococcal ABC exporters. The ATPase activities of EfrAB, EfrCD, EfrEF, and EF0942/41 were measured in the presence of daunorubicin (a), ethidium (b), and Hoechst 33342 (c) at drug concentrations ranging from 0.1 μM to 200 μM. ATPase activities were normalized to the basal activity of the respective transporter in the absence of drugs (set at 100%). The error bars correspond to the standard deviations for three technical replicates. The x axis has a logarithmic scale.
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Figure 5: Drug-modulated ATPase activities of reconstituted enterococcal ABC exporters. The ATPase activities of EfrAB, EfrCD, EfrEF, and EF0942/41 were measured in the presence of daunorubicin (a), ethidium (b), and Hoechst 33342 (c) at drug concentrations ranging from 0.1 μM to 200 μM. ATPase activities were normalized to the basal activity of the respective transporter in the absence of drugs (set at 100%). The error bars correspond to the standard deviations for three technical replicates. The x axis has a logarithmic scale.

Mentions: For the determination of basal ATPase activity, detergent-purified or reconstituted protein was mixed with 1 mM ATP and was incubated at 30°C for 15 min. A malachite green–molybdate solution was added as described previously (21). This solution forms a complex with the released inorganic phosphate and is detected colorimetrically by measuring A640. ATPase reactions for detergent-purified proteins were carried out in 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10 mM MgSO4, and 0.03% β-DDM (see Table 4). For reconstituted proteins, 50 mM K-HEPES (pH 7.0) and 10 mM MgSO4 without the addition of β-DDM was used (see Table 4 and Fig. 5). Daunorubicin, ethidium, and Hoechst 33342 were added at final concentrations ranging from 0.1 μM to 200 μM.


The Heterodimeric ABC Transporter EfrCD Mediates Multidrug Efflux in Enterococcus faecalis
Drug-modulated ATPase activities of reconstituted enterococcal ABC exporters. The ATPase activities of EfrAB, EfrCD, EfrEF, and EF0942/41 were measured in the presence of daunorubicin (a), ethidium (b), and Hoechst 33342 (c) at drug concentrations ranging from 0.1 μM to 200 μM. ATPase activities were normalized to the basal activity of the respective transporter in the absence of drugs (set at 100%). The error bars correspond to the standard deviations for three technical replicates. The x axis has a logarithmic scale.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4997860&req=5

Figure 5: Drug-modulated ATPase activities of reconstituted enterococcal ABC exporters. The ATPase activities of EfrAB, EfrCD, EfrEF, and EF0942/41 were measured in the presence of daunorubicin (a), ethidium (b), and Hoechst 33342 (c) at drug concentrations ranging from 0.1 μM to 200 μM. ATPase activities were normalized to the basal activity of the respective transporter in the absence of drugs (set at 100%). The error bars correspond to the standard deviations for three technical replicates. The x axis has a logarithmic scale.
Mentions: For the determination of basal ATPase activity, detergent-purified or reconstituted protein was mixed with 1 mM ATP and was incubated at 30°C for 15 min. A malachite green–molybdate solution was added as described previously (21). This solution forms a complex with the released inorganic phosphate and is detected colorimetrically by measuring A640. ATPase reactions for detergent-purified proteins were carried out in 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10 mM MgSO4, and 0.03% β-DDM (see Table 4). For reconstituted proteins, 50 mM K-HEPES (pH 7.0) and 10 mM MgSO4 without the addition of β-DDM was used (see Table 4 and Fig. 5). Daunorubicin, ethidium, and Hoechst 33342 were added at final concentrations ranging from 0.1 μM to 200 μM.

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.


Related in: MedlinePlus