大肠杆菌的AcrAB-TolC多药外排泵及其调控研究进展

多药外排泵造成了细菌的多种药物的耐药现象, 这对感染性疾病的防治提出了挑战。对于多药外排泵的研究不仅使人们认识细菌耐药性机制, 而且为细菌耐药性的防治提供思路。大肠杆菌AcrAB-TolC外排泵系统的结构和调控机制研究取得了一些新进展, 这为病原菌的相关研究提供了参考, 本文对其进行了综述。     

大肠杆菌的AcrAB-To1C多药外排泵及其调控研究进展

多药外排泵造成了细菌的多种药物的耐药现象,这对感染性疾病的防治提出了挑战.对于多药外排泵的研究不仅使人们认识细菌耐药性机制,而且为细菌耐药性的防治提供思路.大肠杆菌AcrAB-To1C外排泵系统的结构和调控机制研究取得了一些新进展,这为病原菌的相关研究提供了参考,本文对其进行了综述.     

AcrA, AcrB, and TolC of Escherichia coli Form a Stable Intermembrane Multidrug Efflux Complex

Many transporters of Gram-negative bacteria involved in the extracellular secretion of proteins and the efflux of toxic molecules operate by forming intermembrane complexes. These complexes are proposed to span both inner and outer membranes and create a bridge across the periplasm. In this study, we analyzed interactions between the inner and outer membrane components of the tri-partite multidrug efflux pump AcrAB-TolC from Escherichia coli. We found that, once assembled, the intermembrane AcrAB-TolC complex is stable during the separation of the inner and outer membranes and subsequent purification. All three components of the complex co-purify when the affinity tag is attached to either of the proteins suggesting bi-partite interactions between AcrA, AcrB, and TolC. We show that antibiotics, the substrates of AcrAB-TolC, stabilize interactions within the complex. However, the formation of the AcrAB-TolC complex does not require an input of energy.     

Multidrug Efflux Pump MdtBC of Escherichia coli Is Active Only as a B2C Heterotrimer

RND (resistance-nodulation-division) family transporters in Gram-negative bacteria frequently pump out a wide range of inhibitors and often contribute to multidrug resistance to antibiotics and biocides. An archetypal RND pump of Escherichia coli, AcrB, is known to exist as a homotrimer, and this construction is essential for drug pumping through the functionally rotating mechanism. MdtBC, however, appears different because two pump genes coexist within a single operon, and genetic deletion data suggest that both pumps must be expressed in order for the drug efflux to occur. We have expressed the corresponding genes, with one of them in a His-tagged form. Copurification of MdtB and MdtC under these conditions showed that they form a complex, with an average stoichiometry of 2:1. Unequivocal evidence that only the trimer containing two B protomers and one C protomer is active was obtained by expressing all possible combinations of B and C in covalently linked forms. Finally, conversion into alanine of the residues, known to form a proton translocation pathway in AcrB, inactivated transport only when made in MdtB, not when made in MdtC, a result suggesting that MdtC plays a different role not directly involved in drug binding and extrusion.Bacterial multidrug resistance is a major public health problem (10, 17). One widespread resistance mechanism involves the multidrug resistance (MDR) transporters. Among these, the resistance-nodulation-cell division (RND) family transporters, such as the AcrAB-TolC system in Escherichia coli, play a major role in drug resistance in Gram-negative bacteria because they allow the direct extrusion of drug molecules into extracellular space, and because they sometimes confer an increased level of tolerance to an astonishingly wide range of toxic compounds (18). In general, an RND-type exporter protein (such as AcrB), located in the inner membrane, forms a tripartite complex with a periplasmic adaptor protein, such as AcrA, and a homotrimeric outer membrane channel, such as TolC (18). The drug efflux process requires the presence of all three components. The crystallographic structures of AcrB (13, 14, 22, 24), AcrA (11, 27), and TolC (2, 8) are known, and models of the tripartite complex have been proposed (6, 27).AcrB is a homotrimeric transporter (14) located in the inner membrane and uses the proton gradient as the energy source (31). The homotrimeric structure is thought to be functionally important, or even essential, as each protomer appears to undergo a series of mandatory conformational alterations during the process of drug export, often called “functionally rotating mechanism,” as deduced from the structure of the asymmetric trimers of AcrB (13, 22, 24). This mechanism was also supported by the observation that, in a trimer in which protomers were covalently linked to each other, inactivation of one protomer alone abolishes the activity of the entire trimeric complex (29).Not all RND-type transporters, however, follow this homotrimeric organization. The mdtABC genes of E. coli encode an RND system that is unusual in that it contains two different RND pump genes, mdtB and mdtC, in addition to its own adaptor gene, mdtA. Previous genetic studies have demonstrated that the deletion of either of the two RND pump genes abolishes (1) the resistance to β-lactams, novobiocin, and bile salt derivatives, like deoxycholate, or narrows the range of pump substrates (15), a result suggesting that the functional unit is likely a heteromultimeric pump formed by MdtB/MdtC proteins. However, no direct data have so far been presented supporting the interaction between these proteins or the stoichiometry of the complex. Because the heterooligomeric composition of this pump was unexpected based on the accepted notion of how the homotrimeric pump functions by the functionally rotating mechanism, we examined here the nature of the MdtBC complex in detail.In this study, we first purified the oligomeric transporter by labeling either MdtB or MdtC with a His tag. We obtained a trimeric complex(es) containing both MdtB and MdtC in an approximately 2:1 ratio. However, we could not rule out the possibility that there were mixtures of trimers containing different ratios of the B and C proteins. We therefore utilized the recently introduced technology of creating covalently linked trimers (29), and we show here that the only active trimers are those containing two units of MdtB and one unit of MdtC.     

Rapid Detection of Escherichia coli O157:H7 with Multiplex Real-Time PCR Assays

A SYBR Green LightCycler PCR assay using a single primer pair allowed simultaneous detection of stx1 and/or stx2 of Escherichia coli O157:H7. A distinct sequence of the Shiga-like toxin genes was amplified to yield products of 227 and/or 224 bp, respectively. The two products were distinguished by melting point curve analysis.     

Outer Membrane Proteins of Escherichia coli IV. Differences in Outer Membrane Proteins Due To Strain and Cultural Differences

When the 42,000-dalton major outer membrane protein of Escherichia coli O111 is examined on alkaline polyacrylamide gels containing sodium dodecyl sulfate, it is resolved into three distinct bands designated as proteins 1, 2, and 3. Band 3 consists of two distinct polypeptides, proteins 3a and 3b. E. coli K-12 does not make any protein 2, but makes proteins similar to 1, 3a, and 3b as indicated by comparison of cyanogen bromide peptide patterns. Several Shigella species and most other strains of E. coli resemble E. coli K-12 in that they lack protein 2, whereas Salmonella typhimurium is more similar to E. coli O111. In addition to these species and strain differences, cultural differences resulted in differences in the outer membrane protein profiles. Under conditions of catabolite repression, the level of protein 2 in E. coli O111 decreased while the level of protein 1 increased. An enterotoxin-producing strain similar to E. coli O111 produced no protein 1 and an elevated level of protein 2 under conditions of low catabolite repression. The levels of proteins 1 and 3 are also different in different phases of the growth curve, with protein 1 being the major species in the exponential-phase cells and protein 3 being the major species in stationary-phase cells. A multiply phage-resistant mutant of E. coli K-12 with no obvious cell wall defects produced no protein 1 or 2, but made increased amounts of protein 3. Thus, the major outer membrane proteins of E. coli and related species may vary considerably without affecting outer membrane integrity.     

Crystal Structure of Escherichia coli CusC,the Outer Membrane Component of a Heavy Metal Efflux Pump

Background

While copper has essential functions as an enzymatic co-factor, excess copper ions are toxic for cells, necessitating mechanisms for regulating its levels. The cusCBFA operon of E. coli encodes a four-component efflux pump dedicated to the extrusion of Cu(I) and Ag(I) ions.

Methodology/Principal Findings

We have solved the X-ray crystal structure of CusC, the outer membrane component of the Cus heavy metal efflux pump, to 2.3 Å resolution. The structure has the largest extracellular opening of any outer membrane factor (OMF) protein and suggests, for the first time, the presence of a tri-acylated N-terminal lipid anchor.

Conclusions/Significance

The CusC protein does not have any obvious features that would make it specific for metal ions, suggesting that the narrow substrate specificity of the pump is provided by other components of the pump, most likely by the inner membrane component CusA.     

Novel Macrolide-Specific ABC-Type Efflux Transporter in Escherichia coli

In the Escherichia coli genome, five putative open reading frame (ORF) clusters, mdlAB, ybjYZ, yddA, yojHI, and yhiH, have been assumed to be possible genes for ABC drug efflux transporters (I. T. Paulsen, M. K. Sliwinski, and M. H. Saier, Jr., J. Mol. Biol. 277:573-592, 1998). We cloned all of these ORFs in multicopy plasmids and investigated the drug resistance of drug-supersensitive host cells lacking constitutive multidrug efflux transporter genes acrAB. Among them, only ybjYZ gave significant erythromycin resistance and significantly decreased the accumulation of [(14)C]erythromycin. Therefore, ybjYZ was renamed macAB (macrolide-specific ABC-type efflux carrier). Plasmids carrying both the macA and -B genes conferred resistance against macrolides composed of 14- and 15-membered lactones but no or weak resistance against 16-membered ones. Neither of the two genes produced resistance alone. The DNA sequence suggests that MacB is an integral membrane protein with four transmembrane segments and one nucleotide-binding domain, while MacA belongs to a membrane fusion protein (MFP) family with a signal-like sequence at its N terminus. The expression of the histidine-tagged proteins confirmed that MacB is an integral membrane protein and MacA is a peripheral membrane protein. In addition, MacAB required TolC for its function in a way similar to that of most of the MFP-dependent transporters in E. coli. MacB is thus a novel ABC-type macrolide efflux transporter which functions by cooperating with the MFP MacA and the multifunctional outer membrane channel TolC. This is the first case of an experimentally identified ABC antibiotic efflux transporter in gram-negative organisms.     

Efflux of beta-galactosidase products from Escherichia coli.

Several different strains of Escherichia coli were grown on a variety of carbon sources under various growth conditions. Lactose was added (usually at mid-log phase), and the concentrations of the products of beta-galactosidase action on this sugar (galactose, glucose, and allolactose) were determined at various times thereafter in the total culture and in the medium. It was found that with each strain, with all carbon sources, and under all of the conditions studied, a very large proportion of the products were found in the medium. Control studies were carried out which showed that these results were not artifacts of the method of separating the cells from the medium. The results also did not arise from the secretion of beta-galactosidase into the medium, from the diffusion of substrates and products into and out of the cells due to leaks in the membrane, or from faults in the method of sugar analysis. In addition, the results showed that there were very high levels of products inside the cells under the conditions used and that the efflux of the products was rapid. The efflux might be energetically advantageous to the cell as well as being a means of storing excess products until needed.     

Outer Membrane Protein OmpW Participates with Small Multidrug Resistance Protein Member EmrE in Quaternary Cationic Compound Efflux

In Escherichia coli, the small multidrug resistance (SMR) transporter protein EmrE confers host resistance to a broad range of toxic quaternary cation compounds (QCC) via proton motive force in the plasma membrane. Biologically produced QCC also act as EmrE osmoprotectant substrates within the cell and participate in host pH regulation and osmotic tolerance. Although E. coli EmrE is one of the most well-characterized SMR members, it is unclear how the substrates it transports into the periplasm escape across the outer membrane (OM) in Gram-negative bacteria. We tested the hypothesis that E. coli EmrE relies on an unidentified OM protein (OMP) to complete the extracellular release of its QCC. Eleven OMP candidates were screened using an alkaline phenotypic growth assay to identify OMP involvement in EmrE-mediated QCC efflux. E. coli single-gene deletion strains were transformed with plasmid-carried copies of emrE to detect reduced-growth and rescued-growth phenotypes under alkaline conditions. Among the 11 candidates, only the ΔompW strain showed rescued alkaline growth tolerance when transformed with pEmrE, supporting the corresponding protein''s involvement in EmrE osmoprotectant efflux. Coexpression of plasmids carrying the ompW and emrE genes transformed into the E. coli ΔompW and ΔemrE strains demonstrated a functional complementation restoring the original alkaline loss-of-growth phenotype. Methyl viologen drug resistance assays of pEmrE and pOmpW plasmid-complemented E. coli ΔompW and wild-type strains found higher host drug resistance than with other plasmid combinations. This study confirms our hypothesis that the porin OmpW participates in the efflux of EmrE-specific substrates across the OM.     

Mutagenesis and Modeling To Predict Structural and Functional Characteristics of the Staphylococcus aureus MepA Multidrug Efflux Pump

MepA is a multidrug and toxin extrusion (MATE) family protein and the only MATE protein encoded within the Staphylococcus aureus genome. Structural data for MATE proteins are limited to a single high-resolution example, NorM of Vibrio cholerae. Substitution mutations were created in MepA using gradient plates containing both a substrate and reserpine as an efflux pump inhibitor. Site-directed mutagenesis of plasmid-based mepA was used to reproduce these mutations, as well as unique or low-frequency mutations identified in mepA-overexpressing clinical strains, and to mutagenize conserved acidic residues. The effect of these changes on protein function was quantitated in a norA-disrupted host strain by susceptibility testing with and without inhibitors and by determining the proficiency of ethidium efflux. Up-function substitutions clustered in the carboxy half of MepA, near the cytoplasmic face of the protein. Repeated application of the same gradient plate conditions frequently reproduced identical substitution mutations, suggesting that individual residues are required for interaction with specific substrates. Acidic residues critical to protein function were identified in helices 4 and 5. In silico modeling revealed an outward-facing molecule, with helices 1, 2, 4, 7, 8, and 10 having contact with a central cavity that may represent a substrate translocation pathway. Functionally important residues within this cavity included S81, A161, M291, and A302. These data provide a critical starting point for understanding how MATE multidrug efflux proteins function and will be useful in refining crystallographic data when they are available.     

Identification and Analysis of the Putative Pentose Sugar Efflux Transporters in Escherichia coli

Escherichia coli possesses a number of proteins that transport sugars out of the cell. We identified 31 candidate sugar efflux transporters based on their similarity to known sugar efflux transporters. We then tested whether these transporters affect arabinose and xylose metabolism. We identified 13 transporters - setC, cmr, ynfM, mdtD, yfcJ, yhhS, emrD, ydhC, ydeA, ybdA, ydeE, mhpT, and kgtP - that appeared to increase or decrease intracellular arabinose concentrations when respectively deleted or over-expressed. None of the candidate transporters affected xylose concentrations. These results indicate that E. coli possesses multiple arabinose efflux transporters. They also provide a novel target for future metabolic engineering.     

Efflux of choline and glycine betaine from osmoregulating cells of Escherichia coli

We present evidence that glycine betaine (betaine) which was synthesized from choline was excreted and reaccumulated in osmoregulating cells of Escherichia coli. Choline which was accumulated in bet mutants defective in betaine synthesis was shown to be excreted in response to betaine uptake. Our data suggest that E. coli has efflux systems for betaine and choline which are independent of the uptake systems for these metabolites. The ProU system of E. coli, but not that of Salmonella typhimurium, can mediate low-affinity choline uptake.     

Identification of Natural Compound Inhibitors for Multidrug Efflux Pumps of Escherichia coli and Pseudomonas aeruginosa Using In Silico High-Throughput Virtual Screening and In Vitro Validation

Pseudomonas aeruginosa and Escherichia coli are resistant to wide range of antibiotics rendering the treatment of infections very difficult. A main mechanism attributed to the resistance is the function of efflux pumps. MexAB-OprM and AcrAB-TolC are the tripartite efflux pump assemblies, responsible for multidrug resistance in P. aeruginosa and E. coli respectively. Substrates that are more susceptible for efflux are predicted to have a common pharmacophore feature map. In this study, a new criterion of excluding compounds with efflux substrate-like features was used, thereby refining the selection process and enriching the inhibitor identification process. An in-house database of phytochemicals was created and screened using high-throughput virtual screening against AcrB and MexB proteins and filtered by matching with the common pharmacophore models (AADHR, ADHNR, AAHNR, AADHN, AADNR, AAADN, AAADR, AAANR, AAAHN, AAADD and AAADH) generated using known efflux substrates. Phytochemical hits that matched with any one or more of the efflux substrate models were excluded from the study. Hits that do not have features similar to the efflux substrate models were docked using XP docking against the AcrB and MexB proteins. The best hits of the XP docking were validated by checkerboard synergy assay and ethidium bromide accumulation assay for their efflux inhibition potency. Lanatoside C and diadzein were filtered based on the synergistic potential and validated for their efflux inhibition potency using ethidium bromide accumulation study. These compounds exhibited the ability to increase the accumulation of ethidium bromide inside the bacterial cell as evidenced by these increase in fluorescence in the presence of the compounds. With this good correlation between in silico screening and positive efflux inhibitory activity in vitro, the two compounds, lanatoside C and diadzein could be promising efflux pump inhibitors and effective to use in combination therapy against drug resistant strains of P. aeruginosa and E. coli.     

A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli

Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM.