Mechanistic parallels in bacterial and human multidrug efflux transporters.

Bacteria carry a battery of multidrug transporters, which belong to six families of transporters. Members of at least three families the ATP-Binding Cassette superfamily, the Major Facilitator Superfamily and the Multidrug Endosomal Transporter family have been shown to contribute to multidrug resistance phenotype in eukaryotic cells. This review is focused on comparison of bacterial and eukaryotic transporters that do not have a common evolutionary trait and use different sources of energy to perform the transport. Yet they demonstrate an impressive resemblance. All multidrug transporters are capable of recognizing a broad spectrum of structurally diverse compounds. The accumulated data suggest that structural and mechanistic determinants of such ability are similar among unrelated proteins. Despite the apparent similarity, many features are still unique for different classes of transporters. Intriguingly, some cells appear to simultaneously express transporters belonging to different classes. Depending on mechanistic particularities of transporters such concurrent expression can result in synergistic or non-synergistic effects.

AcrAB and related multidrug efflux pumps of Escherichia coli.

The AcrAB system of Escherichia coli is a multidrug efflux system composed of an RND-type transporter AcrB and a periplasmic accessory protein AcrA, and pumps out a wide variety of lipophilic and amphiphilic inhibitors directly into the medium, presumably through the TolC outer membrane channel. AcrA, a highly elongated protein, is thought to bring the outer and inner membranes closer. It forms a trimer that interacts with a monomeric AcrB, which was shown by in vitro reconstitution to be a proton antiporter. Details of interaction between the

Multidrug resistance mechanisms: drug efflux across two membranes.

A set of multidrug efflux systems enables Gram-negative bacteria to survive in a hostile environment. This review focuses on the structural features and the mechanism of major efflux pumps of Gram-negative bacteria, which expel from the cells a remarkably broad range of antimicrobial compounds and produce the characteristic intrinsic resistance of these bacteria to antibiotics, detergents, dyes and organic solvents. Each efflux pump consists of three components: the inner membrane transporter, the outer membrane channel and the periplasmic lipoprotein. Similar to the multidrug transporters from eukaryotic cells and Gram-positive bacteria, the inner membrane transporters from Gram-negative bacteria recognize and expel their substrates often from within the phospholipid bilayer. This efflux occurs without drug accumulation in the periplasm, implying that substrates are pumped out across the two membranes directly into the medium. Recent data suggest that the molecular mechanism of the drug extrusion across a two-membrane envelope of Gram-negative bacteria may involve the formation of the membrane adhesion sites between the inner and the outer membranes. The periplasmic components of these pumps are proposed to cause a close membrane apposition as the complexes are assembled for the transport.

Cross-linked complex between oligomeric periplasmic lipoprotein AcrA and the inner-membrane-associated multidrug efflux pump AcrB from Escherichia coli.

In Escherichia coli, the intrinsic levels of resistance to multiple antimicrobial agents are produced through expression of the three-component multidrug efflux system AcrAB-TolC. AcrB is a proton-motive-force-dependent transporter located in the inner membrane, and AcrA and TolC are accessory proteins located in the periplasm and the outer membrane, respectively. In this study, these three proteins were expressed separately, and the interactions between them were analyzed by chemical cross-linking in intact cells. We show that AcrA protein forms oligomers, most probably trimers. In this oligomeric form, AcrA interacts specifically with AcrB transporter independently of substrate and TolC.

Bypassing the periplasm: reconstitution of the AcrAB multidrug efflux pump of Escherichia coli.

AcrAB is a constitutively expressed, major multidrug efflux system of Escherichia coli. We have purified the cytoplasmic membrane component, AcrB, to near homogeneity, and reconstituted the protein into proteoliposomes. In the presence of DeltapH (outside acid), the protein catalyzed the extrusion of fluorescent phospholipids, which were then trapped by protein-free acceptor vesicles. Known substrates of AcrAB, such as bile acids, erythromycin, and cloxacillin, inhibited this activity. Addition of various drugs to AcrB-containing proteoliposomes, in the presence of DeltapH (inside acid) resulted in proton efflux, suggesting that AcrB is a proton antiporter. Interestingly, fluorescent lipid extrusion was accelerated strongly by the periplasmic protein AcrA in the presence of Mg2+, and at pH 5.0 AcrA alone produced a slow mixing of lipids of different vesicles, without causing the mixing of intravesicular material. These results suggest that AcrA brings two membranes together, and under certain conditions may even cause the fusion of at least the outer leaflets of the membranes, contributing to the ability of the AcrAB-TolC system to pump drugs out directly into the medium.

AcrA is a highly asymmetric protein capable of spanning the periplasm.

AcrA protein is a component of the multi-drug efflux complex AcrAB-TolC of Escherichia coli. Judged by the hypersusceptibility phenotype of acrA mutants, the AcrAB-TolC system pumps out an extraordinarily wide variety of antibiotics, chemotherapeutic agents, detergents and dyes. This complex traverses both the inner and outer membranes of E. coli and catalyzes efflux of the drugs directly into the medium. The coordinated operation of the inner membrane transporter AcrB and outer membrane channel TolC is thought to be mediated by AcrA. The latter is a lipoprotein located in the periplasmic space. We show here that a lipid-deficient derivative of AcrA is functionally active as demonstrated by the complementation of the hypersusceptibility phenotype of the acrA mutant. Purified non-lipidated and intact forms of AcrA were able to restore, with similar efficiency, the activity of AcrA-dependent efflux of erythromycin in Ca2+-sucrose-treated E. coli cells. Using analytical ultracentrifugation and dynamic light scattering techniques we determined hydrodynamic properties of the non-lipidated AcrA and found that AcrA exists in solution as a highly asymmetric monomeric molecule with an axial ratio of 8. This elongated shape of AcrA is compatible with the hypothesis that this protein spans the periplasmic space coordinating the concerted operation of inner and outer membrane components of the complex.

Antibiotic efflux mechanisms.

Bacterial genomes sequenced to date almost invariably contain genes apparently coding for multidrug efflux pumps, and the yeast genome contains more than 30 putative multidrug efflux genes. Thus it is not surprising that multidrug efflux is a major cause of intrinsic drug resistance in many microorganisms, and plays an even more prominent role in organisms with a low-permeability cell wall, such as Gram negative bacteria in general and Pseudomonas aeruginosa in particular, as well as Mycobacterium species. Furthermore, overproduction of intrinsic pumps, or acquisition of pump genes from external sources, often results in high levels of resistance. This review discusses the classification of efflux proteins, their mechanism of action, the regulation of their expression, and the clinical significance of efflux pumps.

The sigma S level in starving Escherichia coli cells increases solely as a result of its increased stability, despite decreased synthesis.

The sigma S level in starving (stationary phase) Escherichia coli cells increases four-to sixfold following growth in a defined or a complex medium. Chemostat-grown cells, subjected to increasing carbon starvation, also become progressively richer in sigma S content. These increases occur despite reduced transcription of the sigma S-encoding gene, rpoS, and translation of rpoS mRNA, and result solely from a large increase in the stability of the sigma protein. Previous results, based on rpoS::lacZ transcriptional and translational fusions, and on methionine incorporation in sigma S, had suggested increased synthesis of sigma S in starving cells. Alternative explanations for these results consistent with the conclusions of this paper are discussed.