Elena B. Tikhonova

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.

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.

Kinetic control of TolC recruitment by multidrug efflux complexes

In Gram-negative pathogens, multidrug efflux pumps that provide clinically significant levels of antibiotic resistance function as three-component complexes. They are composed of the inner membrane transporters belonging to one of three superfamilies of proteins, RND, ABC, or MF; periplasmic proteins belonging to the membrane fusion protein (MFP) family; and outer membrane channels exemplified by the Escherichia coli TolC. The three-component complexes span the entire two-membrane envelope of Gram-negative bacteria and expel toxic molecules from the cytoplasmic membrane to the medium. The architecture of these complexes is expected to vary significantly because of the structural diversity of the inner membrane transporters. How the three-component pumps are assembled, their architecture, and their dynamics remain unclear. In this study, we reconstituted interactions and compared binding kinetics of the E. coli TolC with AcrA, MacA, and EmrA, the periplasmic MFPs that function in multidrug efflux with transporters from the RND, ABC, and MF superfamilies, respectively. By using surface plasmon resonance, we demonstrate that TolC interactions with MFPs are highly dynamic and sensitive to pH. The affinity of TolC to MFPs decreases in the order MacA > EmrA > AcrA. We further show that MFPs are prone to oligomerization, but differ dramatically from each other in oligomerization kinetics and stability of oligomers. The propensity of MFPs to oligomerize correlates with the stability of MFP–TolC complexes and structural features of inner membrane transporters. We propose that recruitment of TolC by various MFPs is determined not only by kinetics of MFP–TolC interactions but also by oligomerization kinetics of MFPs and pH.

Highly Branched Pentasaccharide-Bearing Amphiphiles for Membrane Protein Studies

Detergents are essential tools for membrane protein manipulation. Micelles formed by detergent molecules have the ability to encapsulate the hydrophobic domains of membrane proteins. The resulting protein-detergent complexes (PDCs) are compatible with the polar environments of aqueous media, making structural and functional analysis feasible. Although a number of novel agents have been developed to overcome the limitations of conventional detergents, most have traditional head groups such as glucoside or maltoside. In this study, we introduce a class of amphiphiles, the PSA/Es with a novel highly branched pentasaccharide hydrophilic group. The PSA/Es conferred markedly increased stability to a diverse range of membrane proteins compared to conventional detergents, indicating a positive role for the new hydrophilic group in maintaining the native protein integrity. In addition, PDCs formed by PSA/Es were smaller and more suitable for electron microscopic analysis than those formed by DDM, indicating that the new agents have significant potential for the structure-function studies of membrane proteins.

On the role of TolC in multidrug efflux: the function and assembly of AcrAB–TolC tolerate significant depletion of intracellular TolC protein

TolC channel provides a route for the expelled drugs and toxins to cross the outer membrane of Escherichia coli. The puzzling feature of TolC structure is that the periplasmic entrance of the channel is closed by dense packing of 12 α‐helices. Efflux pumps exemplified by AcrAB are proposed to drive the opening of TolC channel. How interactions with AcrAB promote the close‐to‐open transition in TolC remains unclear. In this study, we investigated in vivo the functional and physical interactions of AcrAB with the closed TolC and its conformer opened by mutations in the periplasmic entrance. We found that the two conformers of TolC are readily distinguishable in vivo by characteristic drug susceptibility, thiol modification and proteolytic profiles. However, these profiles of TolC variants respond neither to the in vivo stoichiometry of AcrAB:TolC nor to the presence of vancomycin, which is used often to assess the permeability of TolC channel. We further found that the activity and assembly of AcrAB–TolC tolerates significant changes in amounts of TolC and that only a small fraction of intracellular TolC is likely used to support efflux needs of E. coli. Our findings explain why TolC is not a good target for inhibition of multidrug efflux.

Thiodiglycol Metabolism in Alcaligenes xylosoxydans subsp. denitrificans

The investigation of the degradation of thiodiglycol (the major product of mustard gas hydrolysis) by Alcaligenes xylosoxydans subsp. denitrificans strain TD2 showed that thiodiglycol is metabolized through the oxidation of its primary alcohol groups and the subsequent cleavage of C–S bonds in the intermediate products, thiodiglycolic and thioglycolic acids. The end products of these reactions are SO42– ions and acetate, the latter being involved in the central metabolism of strain TD2. The oxidation of the sulfur atom gives rise to diglycolsulfoxide, which is recalcitrant to further microbial degradation. Based on the data obtained, a metabolic pathway of thiodiglycol transformation by A. xylosoxydans subsp. denitrificans strain TD2 is proposed.

Bioutilization of thiodiglycol, the product of mustard detoxification: isolation of degrading strains, study of biodegradation process and metabolic pathways

Alcaligenes xylosoxydans subsp. denitrificans TD1, possessing degrading activity against thiodiglycol (TDG), was isolated from soil samples contaminated by the products of mustard detoxification. Using long-term selection, the most active strain A. xylosoxydans TD2 was obtained. The effect of cultivation conditions—pH, specific substrate loading (SSL) and substrate concentration on the efficiency of TDG destruction process were determined. The initial microbial attack on the TDG molecule involved oxidation of both sulphur atom and primary alcohol groups with the formation of diglycolsulphoxide (DGSO) and thiodiglycolic acid (TDGA), respectively. The transformation to DGSO is a catabolic deadlock since this compound is not oxidized by bacterial cells or used by them as a sole carbon source for growth. The key metabolic reaction of TDG degradation is the uncoupling of the C–S bond of intermediates—TDGA and thioglycolic acid (TGA). This reaction leads to the formation of SO42− ions and acetate, which is involved in the reactions of central metabolic pathways. A scheme for TDG metabolism by A. xylosoxydans TD2 was suggested.

Mechanism of antibiotic efflux in Gram-negative bacteria.

Active efflux of antibiotics mediated by multidrug transporters is a mechanistic basis of multidrug resistance in bacteria. The most versatile multidrug transporters are those found in Gram-negative bacteria. They have a high level of constitutive expression and provide an immediate response to structurally diverse antimicrobial agents including clinically important antibiotics. The versatility and efficiency of multidrug transporters in Gram-negative bacteria heavily depend on coupling of drug efflux with the transport across the outer membrane. The coupling is achieved through the assembly of multi-component protein complexes that span both the inner and the outer membranes of Gram-negative bacteria. In this review we discuss the mechanistic and structural features of multidrug efflux complexes with the major focus on the tight coupling of drug efflux with transport across the outer membrane.