Efflux of cytoplasmically acting antibiotics from gram-negative bacteria: periplasmic substrate capture by multicomponent efflux pumps inferred from their cooperative action with single-component transporters

In gram-negative bacteria, coexpression of single- and multicomponent efflux pumps may result in multiplicative enhancement of the level of resistance against cytoplasmically acting antibiotics. Here, a simple model is presented to show that this cooperative effect can be accounted for only if substrate capture by the multicomponent efflux transporter occurs in the periplasm but not the cytosol.     

Funnel-like hexameric assembly of the periplasmic adapter protein in the tripartite multidrug efflux pump in gram-negative bacteria

Gram-negative bacteria expel diverse toxic chemicals through the tripartite efflux pumps spanning both the inner and outer membranes. The Escherichia coli AcrAB-TolC pump is the principal multidrug exporter that confers intrinsic drug tolerance to the bacteria. The inner membrane transporter AcrB requires the outer membrane factor TolC and the periplasmic adapter protein AcrA. However, it remains ambiguous how the three proteins are assembled. In this study, a hexameric model of the adapter protein was generated based on the propensity for trimerization of a dimeric unit, and this model was further validated by presenting its channel-forming property that determines the substrate specificity. Genetic, in vitro complementation, and electron microscopic studies provided evidence for the binding of the hexameric adapter protein to the outer membrane factor in an intermeshing cogwheel manner. Structural analyses suggested that the adapter covers the periplasmic region of the inner membrane transporter. Taken together, we propose an adapter bridging model for the assembly of the tripartite pump, where the adapter protein provides a bridging channel and induces the channel opening of the outer membrane factor in the intermeshing tip-to-tip manner.     

Alpha-periodicity analysis of small multidrug resistance (SMR) efflux transporters.

Proteins in the small multidrug resistance (SMR) family of transport proteins are about 110 amino acids in length and are predicted to have four transmembrane helices. This family is divided into a two groups, one of which we have referred to as small multidrug pumps (Smp) and confer resistance to a wide variety of quaternary ammonium compounds through a proton-drug efflux antiport mechanism. Members of the second group within this family have, as yet, not had their substrate profile characterized and are referred to as Sug proteins. Alpha-periodicity analysis was conducted on a set of six homologous proteins of the SMR family consisting of three established Smp and three Sug proteins. Several amino acid properties were used in the analysis including hydropathy, variability, and a substitution matrix for lipid exposed amino acids. The scanning window was varied between 8 and 14 residues and the alpha-periodicity was calculated from the peaks in the Fourier transform power spectra in the region between 3.0 and 4.3 residues/turn. This analysis adds to the hydropathy analysis to give a more confident prediction of which residues are within the lipid bilayer for each of the four transmembrane helices. Information was also obtained that allowed for the identification of zones within each transmembrane helix that face the interior of the helical bundle on one side and are lipid exposed on the other face.     

Essential oils against multidrug resistant gram-negative bacteria

Multi-drug resistant uropathogens are responsible for urinary tract infections. The antibacterial activity of seven essential oils, oregano, thyme, clove, arborvitae, cassia, lemongrass, tea tree) was investigated by agar diffusion method, followed by determination of minimum inhibitory (MIC) and bactericidal (MBC) concentrations against five multidrug resistant isolates namely Pseudomonas aeruginosa, Escherichia coli, Enterobacter cloaceae, Morganella morganii, Proteus mirabilis. Oregano, thyme, cassia had antibacterial activity with inhibition zones ranging 25–39 mm; clove, arborvitae, tea tree and lemongrass 12–15 mm. The essential oils showed antibacterial activities with MICs ranged from 0.005% (w/v) to 0.5% (w/v). Thyme had the same MIC and MBC on all strains. The effects of the vapors of the essential oils were also tested by placing the oils on the underside of the Petri dish lid. Thyme, oregano and cassia essential oils strongly inhibited the growth of the clinical strains of bacteria tested in vapor phase. This study demonstrates the potential of investigated essential oils as natural alternatives for further application in hospital therapies in order to retard or inhibit the bacterial growth. For the first time antibacterial effects of essential oils (clove, arborvitae, tea tree, lemongrass, and cassia) were evaluated against Enterobacter cloaceae and Morganella morganii clinical isolates.     

Functional analysis of novel multidrug transporters from human pathogens

Proteins of the Smr family are the smallest multidrug transporters, about 110 amino acids long, that extrude various drugs in exchange with protons, thereby rendering bacteria resistant to these compounds. One of these proteins, EmrE, is an Escherichia coli protein, which has been cloned based on its ability to confer resistance to ethidium and methyl viologen and which has been extensively characterized. More than 60 genes coding for Smr proteins have been identified in several bacteria based on amino acid sequence similarity to the emrE gene. In this work we have analyzed the sequence similarity among these homologues and identified some distinct signature sequence elements and several fully conserved residues. Five of these homologues, from human pathogens Mycobacterium tuberculosis, Bordetella pertussis, and Pseudomonas aeruginosa and from Escherichia coli, were cloned into an E. coli expression system. The proteins were further characterized and show varying degrees of methyl viologen uptake into proteoliposomes and [(3)H]TPP binding in solubilized membranes. The homologues can also form mixed oligomers with EmrE that exhibit intermediate binding characteristics. A comparative study of various homologous proteins provides a tool for deciphering structure-function relationship and monomer-monomer interaction in multidrug transporters and in membrane proteins in general.     

ABC transporters involved in the biogenesis of the outer membrane in gram-negative bacteria

The outer membrane of gram-negative bacteria is an asymmetric lipid bilayer with phospholipids and lipopolysaccharides (LPSs). β-Barreled outer membrane proteins and lipoproteins are embedded in the outer membrane. All of these constituents are essential to the function of the outer membrane. The transport systems for lipoproteins have been characterized in detail. An ATP-binding cassette (ABC) transporter, LolCDE, initiates sorting by mediating the detachment of lipoproteins from the inner membrane to form a water-soluble lipoprotein-LolA complex in the periplasm. Lipoproteins are then transferred to LolB at the outer membrane and are incorporated into the lipid bilayer. A model analogous to the Lol system has been suggested for the transport of LPS, where an ABC transporter, LptBFG, mediates the detachment of LPS from the inner membrane. Recent developments in the functional characterization of ABC transporters involved in the biogenesis of the outer membrane in gram-negative bacteria are discussed.     

Chimeric analysis of AcrA function reveals the importance of its C-terminal domain in its interaction with the AcrB multidrug efflux pump

AcrAB-TolC is the major, constitutively expressed efflux protein complex that provides resistance to a variety of antimicrobial agents in Escherichia coli. Previous studies showed that AcrA, a periplasmic protein of the membrane fusion protein family, could function with at least two other resistance-nodulation-division family pumps, AcrD and AcrF, in addition to its cognate partner, AcrB. We found that, among other E. coli resistance-nodulation-division pumps, YhiV, but not MdtB or MdtC, could also function with AcrA. When AcrB was assessed for the capacity to function with AcrA homologs, only AcrE, but not YhiU or MdtA, could complement an AcrA deficiency. Since AcrA could, but YhiU could not, function with AcrB, we engineered a series of chimeric mutants of these proteins in order to determine the domain(s) of AcrA that is required for its support of AcrB function. The 290-residue N-terminal segment of the 398-residue protein AcrA could be replaced with a sequence coding for the corresponding region of YhiU, but replacement of the region between residues 290 and 357 produced a protein incapable of functioning with AcrB. In contrast, the replacement of residues 357 through 397 of AcrA still produced a functional protein. We conclude that a small region of AcrA close to, but not at, its C terminus is involved in the interaction with its cognate pump protein, AcrB.     

Osmotic upshift transiently inhibits uptake via ABC transporters in gram-negative bacteria

ATP-binding cassette transporters from several rhizobia and Salmonella enterica serovar Typhimurium, but not secondarily coupled systems, were inhibited by high concentrations (100 to 500 mM) of various osmolytes, an effect reversed by the removal of the osmolyte. ABC systems were also inactivated in isolated pea bacteroids, probably due to the obligatory use of high-osmolarity isolation media. Measurement of nutrient cycling in isolated pea bacteroids is impeded by this effect.     

Functional analysis of alkane hydroxylases from gram-negative and gram-positive bacteria

We have cloned homologs of the Pseudomonas putida GPo1 alkane hydroxylase from Pseudomonas aeruginosa PAO1, Pseudomonas fluorescens CHA0, Alcanivorax borkumensis AP1, Mycobacterium tuberculosis H37Rv, and Prauserella rugosa NRRL B-2295. Sequence comparisons show that the level of protein sequence identity between the homologs is as low as 35%, and that the Pseudomonas alkane hydroxylases are as distantly related to each other as to the remaining alkane hydroxylases. Based on the observation that rubredoxin, an electron transfer component of the GPo1 alkane hydroxylase system, can be replaced by rubredoxins from other alkane hydroxylase systems, we have developed three recombinant host strains for the functional analysis of the novel alkane hydroxylase genes. Two hosts, Escherichia coli GEc137 and P. putida GPo12, were equipped with pGEc47 Delta B, which encodes all proteins necessary for growth on medium-chain-length alkanes (C(6) to C(12)), except a functional alkane hydroxylase. The third host was an alkB knockout derivative of P. fluorescens CHA0, which is no longer able to grow on C(12) to C(16) alkanes. All alkane hydroxylase homologs, except the Acinetobacter sp. ADP1 AlkM, allowed at least one of the three hosts to grow on n-alkanes.     

Structure-function analysis of multidrug transporters in Lactococcus lactis

The active extrusion of cytotoxic compounds from the cell by multidrug transporters is one of the major causes of failure of chemotherapeutic treatment of tumor cells and of infections by pathogenic microorganisms. A multidrug transporter in Lactococcus lactis, LmrA, is a member of the ATP-binding cassette (ABC) superfamily and a bacterial homolog of the human multidrug resistance P-glycoprotein. Another multidrug transporter in L. lactis, LmrP, belongs to the major facilitator superfamily, and is one example of a rapidly expanding group of secondary multidrug transporters in microorganisms. Thus, LmrA and LmrP are transport proteins with very different protein structures, which use different mechanisms of energy coupling to transport drugs out of the cell. Surprisingly, both proteins have overlapping specificities for drugs, are inhibited by the same set of modulators, and transport drugs via a similar transport mechanism. The structure-function relationships that dictate drug recognition and transport by LmrP and LmrA represent an intriguing area of research.     

Broad-specificity efflux pumps and their role in multidrug resistance of Gram-negative bacteria

Antibiotic resistance mechanisms reported in Gram-negative bacteria are causing a worldwide health problem. The continuous dissemination of 'multidrug-resistant' (MDR) bacteria drastically reduces the efficacy of our antibiotic 'arsenal' and consequently increases the frequency of therapeutic failure. In MDR bacteria, the overexpression of efflux pumps that expel structurally unrelated drugs contributes to the reduced susceptibility by decreasing the intracellular concentration of antibiotics. During the last decade, several clinical data have indicated an increasing involvement of efflux pumps in the emergence and dissemination of resistant Gram-negative bacteria. It is necessary to clearly define the molecular, functional and genetic bases of the efflux pump in order to understand the translocation of antibiotic molecules through the efflux transporter. The recent investigation on the efflux pump AcrB at its structural and physiological levels, including the identification of drug affinity sites and kinetic parameters for various antibiotics, may pave the way towards the rational development of an improved new generation of antibacterial agents as well as efflux inhibitors in order to efficiently combat efflux-based resistance mechanisms.     

Distribution and physiology of ABC-type transporters contributing to multidrug resistance in bacteria.

Membrane proteins responsible for the active efflux of structurally and functionally unrelated drugs were first characterized in higher eukaryotes. To date, a vast number of transporters contributing to multidrug resistance (MDR transporters) have been reported for a large variety of organisms. Predictions about the functions of genes in the growing number of sequenced genomes indicate that MDR transporters are ubiquitous in nature. The majority of described MDR transporters in bacteria use ion motive force, while only a few systems have been shown to rely on ATP hydrolysis. However, recent reports on MDR proteins from gram-positive organisms, as well as genome analysis, indicate that the role of ABC-type MDR transporters in bacterial drug resistance might be underestimated. Detailed structural and mechanistic analyses of these proteins can help to understand their molecular mode of action and may eventually lead to the development of new strategies to counteract their actions, thereby increasing the effectiveness of drug-based therapies. This review focuses on recent advances in the analysis of ABC-type MDR transporters in bacteria.     

Optimized production and analysis of the staphylococcal multidrug efflux protein QacA

The plasmid-encoded QacA multidrug transport protein confers high-level resistance to a range of commonly used antimicrobials and is carried by widespread clinical strains of the human pathogen Staphylococcus aureus making it a potential target for future drug therapies. In order to obtain a sufficient yield of QacA protein for structural and biophysical studies, an optimized strategy for QacA overexpression was developed. QacA expression, directed from several vector systems in Escherichia coli, was tested under various growth and induction conditions and a synthetic qacA gene, codon-optimized for expression in E. coli was developed. Despite the extreme hydrophobicity and potential toxicity of the QacA secondary transport protein, a strategy based on the pBAD expression system, yielding up to four milligrams of approximately 95% pure QacA protein per litre of liquid culture, was devised. Purified QacA protein was examined using circular dichroism spectroscopy and displayed a secondary structure akin to that predicted from in silico analyses. Additionally, detergent solubilized QacA protein was shown to bind its fluorescent substrate rhodamine 6G with micro-molar affinity using a fluorescence polarization-based binding assay, similar to other multidrug transport proteins. To check the applicability of the expression/purification system described for QacA to other staphylococcal secondary transporters, the gene encoding the TetA(K) tetracycline efflux protein, which was previously recalcitrant to overexpression, was incorporated into the pBAD-based system and shown to be readily produced at easily detectable levels. Therefore, this expression system could be of general use for the production of secondary transport proteins in E. coli.     

An H(+)-coupled multidrug efflux pump, PmpM, a member of the MATE family of transporters, from Pseudomonas aeruginosa

We cloned the gene PA1361 (we designated the gene pmpM), which seemed to encode a multidrug efflux pump belonging to the MATE family, of Pseudomonas aeruginosa by the PCR method using the drug-hypersensitive Escherichia coli KAM32 strain as a host. Cells of E. coli possessing the pmpM gene showed elevated resistance to several antimicrobial agents. We observed energy-dependent efflux of ethidium from cells possessing the pmpM gene. We found that PmpM is an H(+)-drug antiporter, and this finding is the first reported case of an H(+)-coupled efflux pump in the MATE family. Disruption and reintroduction of the pmpM gene in P. aeruginosa revealed that PmpM is functional and that benzalkonium chloride, fluoroquinolones, ethidium bromide, acriflavine, and tetraphenylphosphonium chloride are substrates for PmpM in this microorganism.     

Phylogeny of multidrug transporters.

We currently recognize five large ubiquitous superfamilies and one small eukaryotic-specific family in which cellular multidrug efflux pumps occur. One, the ABC superfamily, includes members that use ATP hydrolysis to drive drug efflux, but the MFS, RND, MATE and DMT superfamilies include members that are secondary carriers, functioning by drug:H(+)or drug:Na(+)antiport mechanisms. The small MET family seems to be restricted to endosomal membranes of eukaryotes, and only a single such system has been functionally characterized. In this review article, these families of drug transporters are discussed and evaluated from phylogenetic standpoints.