Helen I. Zgurskaya

Tackling antibiotic resistance

The development and spread of antibiotic resistance in bacteria is a universal threat to both humans and animals that is generally not preventable but can nevertheless be controlled, and it must be tackled in the most effective ways possible. To explore how the problem of antibiotic resistance might best be addressed, a group of 30 scientists from academia and industry gathered at the Banbury Conference Centre in Cold Spring Harbor, New York, USA, from 16 to 18 May 2011. From these discussions there emerged a priority list of steps that need to be taken to resolve this global crisis.

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.

Waltzing transporters and 'the dance macabre' between humans and bacteria

Multidrug-resistance efflux pumps — in particular those belonging to the resistance-nodulation-cell-division (RND) family of transporters, with their unusually high degree of substrate promiscuity — significantly restrict the effectiveness of antibacterial therapy. Recent years have heralded remarkable insights into the structure and mechanisms of these fascinating molecular machines. Here, we review recent advances in the field and describe various approaches used in combating efflux-mediated resistance.

Chimeric Analysis of the Multicomponent Multidrug Efflux Transporters from Gram-Negative Bacteria

Many multidrug transporters from gram-negative bacteria belong to the resistance-nodulation-cell division (RND) superfamily of transporters. RND-type multidrug transporters have an extremely broad substrate specificity and protect bacterial cells from the actions of antibiotics on both sides of the cytoplasmic membrane. They usually function as three-component assemblies spanning the outer and cytoplasmic membranes and the periplasmic space of gram-negative bacteria. The structural determinants of RND transporters responsible for multidrug recognition and complex assembly remain unknown. We constructed chimeric RND transporters composed of N-terminal residues of AcrB and C-terminal residues of MexB, the major RND-type transporters from Escherichia coli and Pseudomonas aeruginosa, respectively. The assembly of complexes and multidrug efflux activities of chimeric transporters were determined by coexpression of hybrid genes either with AcrA, the periplasmic component of the AcrAB transporter from E. coli, or with MexA and OprM, the accessory proteins of the MexAB-OprM pump from P. aeruginosa. We found that the specificity of interaction with the corresponding periplasmic component is encoded in the T60-V612 region of transporters. Our results also suggest that the large periplasmic loops of RND-type transporters are involved in multidrug recognition and efflux.

Mechanism and Function of the Outer Membrane Channel TolC in Multidrug Resistance and Physiology of Enterobacteria

TolC is an archetypal member of the outer membrane efflux protein (OEP) family. These proteins are involved in export of small molecules and toxins across the outer membrane of Gram-negative bacteria. Genomes of some bacteria such as Pseudomonas species contain multiple copies of OEPs. In contrast, enterobacteria contain a single tolC gene, the product of which functions with multiple transporters. Inactivation of tolC has a major impact on enterobacterial physiology and virulence. Recent studies suggest that the role of TolC in physiology of enterobacteria is very broad and affects almost all aspects of cell adaptation to adverse environments. We review the current state of understanding TolC structure and present an integrated view of TolC function in enterobacteria. We propose that seemingly unrelated phenotypes of tolC mutants are linked together by a single most common condition – an oxidative damage to membranes.

Reconstitution of the Escherichia coli macrolide transporter: the periplasmic membrane fusion protein MacA stimulates the ATPase activity of MacB.

Periplasmic membrane fusion proteins (MFPs) are essential components of the type I protein secretion systems and drug efflux pumps in Gram‐negative bacteria. Previous studies suggested that MFPs connect the inner and outer membrane components of the transport systems and by this means co‐ordinate the transfer of substrates across the two membranes. In this study, we purified and reconstituted the macrolide transporter MacAB from Escherichia coli. Here, MacA is a periplasmic MFP and MacB is an ABC‐type transporter. Similar to other MFP‐dependent transporters from E. coli, the in vivo function of MacAB requires the outer membrane channel TolC. The purified MacB displayed a basal ATPase activity in detergent micelles. This activity conformed to Michaelis‐Menten kinetics but was unresponsive to substrates or accessory proteins. Upon reconstitution into proteoliposomes, the ATPase activity of MacB was strictly dependent on MacA. The catalytic efficiency of MacAB ATPase was more than 45‐fold higher than the activity of MacB alone. Both the N‐ and C‐terminal regions of MacA were essential for this activity. MacA stimulated MacB ATPase only in phospholipid bilayers and did not need the presence of macrolides. Our results suggest that MacA is a functional subunit of the MacB transporter.

Sequential Mechanism of Assembly of Multidrug Efflux Pump AcrAB-TolC

Multidrug efflux pumps adversely affect both the clinical effectiveness of existing antibiotics and the discovery process to find new ones. In this study, we reconstituted and characterized by surface plasmon resonance the assembly of AcrAB-TolC, the archetypal multidrug efflux pump from Escherichia coli. We report that the periplasmic AcrA and the outer membrane channel TolC assemble high-affinity complexes with AcrB transporter independently from each other. Antibiotic novobiocin and MC-207,110 inhibitor bind to the immobilized AcrB but do not affect interactions between components of the complex. In contrast, DARPin inhibits interactions between AcrA and AcrB. Mutational opening of TolC channel decreases stability of interactions and promotes disassembly of the complex. The conformation of the membrane proximal domain of AcrA is critical for the formation of AcrAB-TolC and could be targeted for the development of new inhibitors.

Structural and functional diversity of bacterial membrane fusion proteins.

Membrane Fusion Proteins (MFPs) are functional subunits of multi-component transporters that perform diverse physiological functions in both Gram-positive and Gram-negative bacteria. MFPs associate with transporters belonging to Resistance-Nodulation-cell Division (RND), ATP-Binding Cassette (ABC) and Major Facilitator (MF) superfamilies of proteins. Recent studies suggested that MFPs interact with substrates and play an active role in transport reactions. In addition, the MFP-dependent transporters from Gram-negative bacteria recruit the outer membrane channels to expel various substrates across the outer membrane into external medium. This review is focused on the diversity, structure and molecular mechanism of MFPs that function in multidrug efflux. Using phylogenetic approaches we analyzed diversity and representation of multidrug MFPs in sequenced bacterial genomes. In addition to previously characterized MFPs from Gram-negative bacteria, we identified MFPs that associate with RND-, MF- and ABC-type transporters in Gram-positive bacteria. Sequence analyses showed that MFPs vary significantly in size (200-650 amino acid residues) with some of them lacking the signature alpha-helical domain of multidrug MFPs. Furthermore, many transport operons contain two- or three genes encoding distinct MFPs. We further discuss the diversity of MFPs in the context of current views on the mechanism and structure of MFP-dependent transporters.

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.