Two novel families of bacterial membrane proteins concerned with nodulation, cell division and transport

Homology has been established for members of two families of functionally related bacterial membrane proteins. The first family (the resistance/nodulation/cell division (RND) family) Includes (i) two metal-resistance efflux pumps in Alcaligenes eutrophus (CzcA and CnrA), (ii) three proteins which function together in nodulation of alfalfa roots by Rhizobium meliloti (NoIGHI), and (iii) a cell division protein in Escherichia coli (EnvD). The second family (the putative membrane fusion protein (MFP) family) includes a nodulation protein (NoIF), a cell division protein (EnvC), and a multidrug resistance transport protein (EmrA). We propose that an MFP functions co-operatively with an RND protein to transport large or hydrophobic molecules across the two membranes of the Gram-negative bacterial cell envelope.     

The Cus efflux system removes toxic ions via a methionine shuttle

Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes in the resistance-nodulation-cell division (RND) family to expel diverse toxic compounds from the cell. These efflux systems span the entire cell envelope to mediate the phenomenon of bacterial multidrug resistance. The three parts of the efflux complexes are: (1) a membrane fusion protein (MFP) connecting (2) a substrate-binding inner membrane transporter to (3) an outer membrane-anchored channel in the periplasmic space. One such efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions. We recently determined the crystal structures of both the inner membrane transporter CusA and MFP CusB of the CusCBA tripartite efflux system from E. coli. These are the first structures of the heavy-metal efflux (HME) subfamily of the RND efflux pumps. Here, we summarize the structural information of these two efflux proteins and present the accumulated evidence that this efflux system utilizes methionine residues to bind and export Cu(I)/Ag(I). Genetic and structural analyses suggest that the CusA pump is capable of picking up the metal ions from both the periplasm and cytoplasm. We propose a stepwise shuttle mechanism for this pump to extrude metal ions from the cell.     

Crystal Structure of the Periplasmic Component of a Tripartite Macrolide-Specific Efflux Pump

In Gram-negative bacteria, type I protein secretion systems and tripartite drug efflux pumps have a periplasmic membrane fusion protein (MFP) as an essential component. MFPs bridge the outer membrane factor and an inner membrane transporter, although the oligomeric state of MFPs remains unclear. The most characterized MFP AcrA connects the outer membrane factor TolC and the resistance-nodulation-division-type efflux transporter AcrB, which is a major multidrug efflux pump in Escherichia coli. MacA is the periplasmic MFP in the MacAB-TolC pump, where MacB was characterized as a macrolide-specific ATP-binding-cassette-type efflux transporter. Here, we report the crystal structure of E. coli MacA and the experimentally phased map of Actinobacillus actinomycetemcomitans MacA, which reveal a domain orientation of MacA different from that of AcrA. Notably, a hexameric assembly of MacA was found in both crystals, exhibiting a funnel-like structure with a central channel and a conical mouth. The hexameric MacA assembly was further confirmed by electron microscopy and functional studies in vitro and in vivo. The hexameric structure of MacA provides insight into the oligomeric state in the functional complex of the drug efflux pump and type I secretion system.     

Structure–function analysis of the ATP-driven glycolipid efflux pump DevBCA reveals complex organization with TolC/HgdD

In Gram-negative bacteria, trans-envelope efflux pumps have periplasmic membrane fusion proteins (MFPs) as essential components. MFPs act as mediators between outer membrane factors (OMFs) and inner membrane factors (IMFs). In this study, structure–function relations of the ATP-driven glycolipid efflux pump DevBCA-TolC/HgdD from the cyanobacterium Anabaena sp. PCC 7120 were analyzed. The binding of the MFP DevB to the OMF TolC absolutely required the respective tip-regions. The interaction of DevB with the IMF DevAC mainly involved the β-barrel and the lipoyl domain. Efficient binding to DevAC and TolC, substrate recognition and export activity by DevAC were dependent on stable DevB hexamers.     

N-Terminal Region of CusB Is Sufficient for Metal Binding and Metal Transfer with the Metallochaperone CusF

Gram-negative bacteria, such as Escherichia coli, utilize efflux resistance systems in order to expel toxins from their cells. Heavy-metal resistance is mediated by resistance nodulation cell division (RND)-based efflux pumps composed of a tripartite complex that includes an RND-transporter, an outer-membrane factor (OMF), and a membrane fusion protein (MFP) that spans the periplasmic space. MFPs are necessary for complex assembly and have been hypothesized to play an active role in substrate efflux. Crystal structures of MFPs are available, however incomplete, as large portions of the apparently disordered N- and C-termini are unresolved. Such is the case for CusB, the MFP of the E. coli Cu(I)/Ag(I) efflux pump CusCFBA. In this work, we have investigated the structure and function of the N-terminal region of CusB, which includes the metal-binding site and is missing from previously determined crystal structures. Results from mass spectrometry and X-ray absorption spectroscopy show that the isolated N-terminal 61 residues (CusB-NT) bind metal in a 1:1 stoichiometry with a coordination site composed of M21, M36, and M38, consistent with full-length CusB. NMR spectra show that CusB-NT is mostly disordered in the apo state; however, some slight structure is adopted upon metal binding. Much of the intact protein's function is maintained in this fragment as CusB-NT binds metal in vivo and in vitro, and metal is transferred between the metallochaperone CusF and CusB-NT in vitro. Functional analysis in vivo shows that full-length CusB is necessary in an intact polypeptide for full metal resistance, though CusB-NT alone can contribute partial metal resistance. These findings reinforce the theory that the role of CusB is not only to bind metal but also to play an active role in efflux.     

Crystal Structure of the Membrane Fusion Protein CusB from Escherichia coli

Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes belonging to the resistance-nodulation-division family to expel diverse toxic compounds from the cell. These systems contain a periplasmic membrane fusion protein (MFP) that is critical for substrate transport. We here present the x-ray structures of the CusB MFP from the copper/silver efflux system of E. coli. This is the first structure of any MFPs associated with heavy-metal efflux transporters. CusB bridges the inner-membrane efflux pump CusA and outer-membrane channel CusC to mediate resistance to Cu⁺ and Ag⁺ ions. Two distinct structures of the elongated molecules of CusB were found in the asymmetric unit of a single crystal, which suggests the flexible nature of this protein. Each protomer of CusB can be divided into four different domains, whereby the first three domains are mostly β-strands and the last domain adopts an entirely helical architecture. Unlike other known structures of MFPs, the α-helical domain of CusB is folded into a three-helix bundle. This three-helix bundle presumably interacts with the periplasmic domain of CusC. The N- and C-termini of CusB form the first β-strand domain, which is found to interact with the periplasmic domain of the CusA efflux pump. Atomic details of how this efflux protein binds Cu⁺ and Ag⁺ were revealed by the crystals of the CusB-Cu(I) and CusB-Ag(I) complexes. The structures indicate that CusB consists of multiple binding sites for these metal ions. These findings reveal novel structural features of an MFP in the resistance-nodulation-division efflux system and provide direct evidence that this protein specifically interacts with transported substrates.     

革兰氏阴性菌AcrAB-TolC多药外排泵结构数据对抑制剂研发的启示

革兰氏阴性菌的多重耐药性已成为全球广泛聚焦的问题。近年研究发现,耐药结节细胞分化(resistance-nodulation-cell division,RND)家族外排泵的过表达,与革兰氏阴性菌的多重耐药性密切相关。在RND家族中,广泛存在于革兰氏阴性菌中的AcrAB-TolC外排泵被认为是导致多重耐药性的主要原因之一。为了开发有效的抑制剂,需要对AcrAB-TolC外排泵的结构有一个清晰的认识。以往对该外排泵结构的研究主要局限于体外采用X射线晶体学技术或冷冻电镜单颗粒分析技术来解析其单个组分或全泵的结构。细胞冷冻电子断层扫描技术为揭示AcrAB-TolC外排泵在天然细胞膜环境中的组装和运行机制提供了新的见解,本文综述了AcrAB-TolC不同层级的结构数据在研发外排泵抑制剂方面的贡献。     

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.     

Vibrio cholerae VexH encodes a multiple drug efflux pump that contributes to the production of cholera toxin and the toxin co-regulated pilus

The resistance-nodulation-division (RND) efflux systems are ubiquitous transporters that function in antimicrobial resistance. Recent studies showed that RND systems were required for virulence factor production in Vibrio cholerae. The V. cholerae genome encodes six RND efflux systems. Three of the RND systems (VexB, VexD, and VexK) were previously shown to be redundant for in vitro resistance to bile acids and detergents. A mutant lacking the VexB, VexD, and VexK RND pumps produced wild-type levels of cholera toxin (CT) and the toxin co-regulated pilus (TCP) and was moderately attenuated for intestinal colonization. In contrast, a RND negative mutant produced significantly reduced amounts of CT and TCP and displayed a severe colonization defect. This suggested that one or more of the three uncharacterized RND efflux systems (i.e. VexF, VexH, and VexM) were required for pathogenesis. In this study, a genetic approach was used to generate a panel of V. cholerae RND efflux pump mutants in order to determine the function of VexH in antimicrobial resistance, virulence factor production, and intestinal colonization. VexH contributed to in vitro antimicrobial resistance and exhibited a broad substrate specificity that was redundant with the VexB, VexD, and VexK RND efflux pumps. These four efflux pumps were responsible for in vitro antimicrobial resistance and were required for virulence factor production and intestinal colonization. Mutation of the VexF and/or VexM efflux pumps did not affect in vitro antimicrobial resistance, but did negatively affect CT and TCP production. Collectively, our results demonstrate that the V. cholerae RND efflux pumps have redundant functions in antimicrobial resistance and virulence factor production. This suggests that the RND efflux systems contribute to V. cholerae pathogenesis by providing the bacterium with protection against antimicrobial compounds that are present in the host and by contributing to the regulated expression of virulence factors.     

Multidrug efflux pumps and antimicrobial resistance in Pseudomonas aeruginosa and related organisms

Pseudomonas aeruginosa is an opportunistic human pathogen characterized by an innate resistance to multiple antimicrobial agents. A major contribution to this intrinsic multidrug resistance is provided by a number of broadly-specific multidrug efflux systems, including MexAB-OprM and MexXY-OprM. In addition, these and two additional tripartite efflux systems, MexCD-OprJ and MexEF-OprN, promote acquired multidrug resistance as a result of mutational hyperexpression of the efflux genes. In addition to antibiotics, these pumps promote export of numerous dyes, detergents, inhibitors, disinfectants, organic solvents and homoserine lactones involved in quorum sensing. The efflux pump proteins are highly homologous and consist of a cytoplasmic membrane-associated drug-proton antiporter of the Resistance-Nodulation-Division (RND) family, an outer membrane channel-forming protein [sometimes called outer membrane factor (OMF)] and a periplasmic membrane fusion protein (MFP). Homologues of these systems have been described in Stenotrophomonas maltophilia, Burkholderia cepacia, Burkholderia pseudomallei and the non-pathogen Pseudomonas putida, where they play a role in export of and resistance to multiple antimicrobial agents and/or organic solvents. Although the natural function of these multidrug efflux systems is largely unknown, their contribution to antibiotic resistance and their conservation in a number of important human pathogens makes them logical targets for therapeutic intervention.     

Assembly of the MexAB-OprM multidrug efflux system of Pseudomonas aeruginosa: identification and characterization of mutations in mexA compromising MexA multimerization and interaction with MexB

The membrane fusion protein (MFP) component, MexA, of the MexAB-OprM multidrug efflux system of P. aeruginosa is proposed to link the inner (MexB) and outer (OprM) membrane components of this pump as a probable oligomer. A cross-linking approach confirmed the in vivo interaction of MexA and MexB, while a LexA-based assay for assessing protein-protein interaction similarly confirmed MexA multimerization. Mutations compromising the MexA contribution to antibiotic resistance but yielding wild-type levels of MexA were recovered and shown to map to two distinct regions within the N- and C-terminal halves of the protein. Most of the N-terminal mutations occurred at residues that are highly conserved in the MFP family (P68, G72, L91, A108, L110, and V129), consistent with these playing roles in a common feature of these proteins (e.g., oligomerization). In contrast, the majority of the C-terminal mutations occurred at residues poorly conserved in the MFP family (V264, N270, H279, V286, and G297), with many mapping to a region of MexA that corresponds to a region in the related MFP of Escherichia coli, AcrA, that is implicated in binding to its RND component, AcrB (C. A. Elkins and H. Nikaido, J. Bacteriol. 185:5349-5356, 2003). Given the noted specificity of MFP-RND interaction in this family of pumps, residues unique to MexA may well be important for and define the MexA interaction with its RND component, MexB. Still, all but one of the MexA mutations studied compromised MexA-MexB association, suggesting that native structure and/or proper assembly of the protein may be necessary for this.     

The AcrAB RND efflux system from the live vaccine strain of Francisella tularensis is a multiple drug efflux system that is required for virulence in mice

The ability of bacterial pathogens to infect and cause disease is dependent upon their ability to resist antimicrobial components produced by their host, such as bile acids, fatty acids and other detergent-like molecules, and products of the innate immune system (e.g. cationic antimicrobial peptides). Bacterial resistance to the antimicrobial effects of such compounds is often mediated by active efflux systems belonging to the resistance-nodulation-division (RND) family of transporters. RND efflux systems have been implicated in antibiotic resistance and virulence extending their clinical relevance. In this report the hypothesis that the Francisella tularensis AcrAB RND efflux system contributes to antimicrobial resistance and pathogenesis has been tested. A null mutation was generated in the gene encoding the AcrB RND efflux pump protein of the live vaccine strain of F. tularensis. The resulting mutant exhibited increased sensitivity to multiple antibiotics and antimicrobial compounds. Murine challenge experiments revealed that the acrB mutant was attenuated. Collectively these results suggest that the F. tularensis AcrAB RND efflux system encodes a multiple drug efflux system that is important for virulence.     

Non‐equivalent roles of two periplasmic subunits in the function and assembly of triclosan pump TriABC from Pseudomonas aeruginosa

In Gram‐negative bacteria, multidrug efflux transporters function in complexes with periplasmic membrane fusion proteins (MFPs) that enable antibiotic efflux across the outer membrane. In this study, we analyzed the function, composition and assembly of the triclosan efflux transporter TriABC–OpmH from Pseudomonas aeruginosa. We report that this transporter possesses a surprising substrate specificity that encompasses not only triclosan but the detergent SDS, which are often used together in antibacterial soaps. These two compounds interact antagonistically in a TriABC‐dependent manner and negate antibacterial properties of each other. Unlike other efflux pumps that rely on a single MFP for their activities, two different MFPs, TriA and TriB, are required for triclosan/SDS resistance mediated by TriABC–OpmH. We found that analogous mutations in the α‐helical hairpin and membrane proximal domains of TriA and TriB differentially affect triclosan efflux and assembly of the complex. Furthermore, our results show that TriA and TriB function as a dimer, in which TriA is primarily responsible for stabilizing interactions with the outer membrane channel, whereas TriB is important for the stimulation of the transporter. We conclude that MFPs are engaged into complexes as asymmetric dimers, in which each protomer plays a specific role.     

The Rap and Hor proteins of Erwinia, Serratia and Yersinia: a novel subgroup in a growing superfamily of proteins regulating diverse physiological processes in bacterial pathogens

The enteric bacterium Serratia marcescens is an opportunistic human pathogen. The strain ATCC39006 makes the red pigment, prodigiosin (Pig), and the β-lactam antibiotic carbapenem (Car). Mutants were isolated that were concomitantly defective for Pig and Car production. These mutants were found to have a mutation in the rap gene (regulation of antibiotic and pigment). Sequence analysis of the rap gene revealed a predicted protein product showing strong homology to SlyA, originally thought to be a haemolytic virulence determinant in Salmonella typhimurium. Homologues of rap were detected in several bacterial genera, including Salmonella, Yersinia, Enterobacter , and species of the plant pathogen, Erwinia. The Erwinia hor_Er (homologue of rap ) and the Yersinia hor_Ye genes were also found to be very similar to rap and slyA. Marker exchange mutagenesis of hor_Er revealed that it encoded a regulatory protein controlling the production of antibiotic and exoenzyme virulence determinants in the phytopathogen, Erwinia carotovora subspecies carotovora. We have shown that these new homologues of SlyA form a highly conserved subgroup of a growing superfamily of bacterial regulatory proteins controlling diverse physiological processes in human, animal and plant pathogens.     

Structure and mechanism of the tripartite CusCBA heavy-metal efflux complex

Gram-negative bacteria frequently expel toxic chemicals through tripartite efflux pumps that span both the inner and outer membranes. The three parts are the inner membrane, substrate-binding transporter (or pump); a periplasmic membrane fusion protein (MFP, or adaptor); and an outer membrane-anchored channel. The fusion protein connects the transporter to the channel within the periplasmic space. One such efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions. We previously described the crystal structures of both the inner membrane transporter CusA and the MFP CusB of Escherichia coli. We also determined the co-crystal structure of the CusBA adaptor-transporter efflux complex, showing that the transporter CusA, which is present as a trimer, interacts with six CusB protomers and that the periplasmic domain of CusA is involved in these interactions. Here, we summarize the structural information of these efflux proteins, and present the accumulated evidence that this efflux system uses methionine residues to bind and export Cu(I) and Ag(I). Genetic and structural analyses suggest that the CusA pump is capable of picking up the metal ions from both the periplasm and the cytoplasm. We propose a stepwise shuttle mechanism for this pump to export metal ions from the cell.     

Mutations in MexB that affect the efflux of antibiotics with cytoplasmic targets

Drug efflux pumps such as MexAB-OprM from Pseudomonas aeruginosa confer resistance to a wide range of chemically different compounds. Within the tripartite assembly, the inner membrane protein MexB is mainly responsible for substrate recognition. Recently, considerable advances have been made in elucidating the drug efflux pathway through the large periplasmic domains of resistance-nodulation-division (RND) transporters. However, little is known about the role of amino acids in other parts of the protein. We have investigated the role of two conserved phenylalanine residues that are aligned around the cytoplasmic side of the central cavity of MexB. The two conserved phenylalanine residues have been mutated to alanine residues (FAFA MexB). The interaction of the wild-type and mutant proteins with a variety of drugs from different classes was investigated by assays of cytotoxicity and drug transport. The FAFA mutation affected the efflux of compounds that have targets inside the cell, but antibiotics that act on cell wall synthesis and membrane probes were unaffected. Combined, our results indicate the presence of a hitherto unidentified cytoplasmic-binding site in RND drug transporters and enhance our understanding of the molecular mechanisms that govern drug resistance in Gram-negative pathogens.     

Mutations in the central cavity and periplasmic domain affect efflux activity of the resistance-nodulation-division pump EmhB from Pseudomonas fluorescens cLP6a

The EmhABC efflux system in Pseudomonas fluorescens cLP6a is homologous to the multidrug and solvent efflux systems belonging to the resistance-nodulation-division (RND) family and is responsible for polycyclic aromatic hydrocarbon transport, antibiotic resistance, and toluene efflux. To gain a better understanding of substrate transport in RND efflux pumps, the EmhB pump was subjected to mutational analysis. Mutagenesis of amino acids within the central cavity of the predicted three-dimensional structure of EmhB showed selective activity towards antibiotic substrates. An A384P/A385Y double mutant showed increased susceptibility toward rhodamine 6G compared to the wild type, and F386A and N99A single mutants showed increased susceptibility to dequalinium compared to the wild type. As well, the carboxylic acid side chain of D101, located in the central cavity region, was found to be essential for polycyclic aromatic hydrocarbon transport and resistance to all antibiotic substrates of EmhB. Phenylalanine residues located within the periplasmic pore domain were also targeted for mutagenesis, and the F325A and F281A mutations significantly impaired efflux activity for all EmhB substrates. One mutation (A206S) in the outer membrane protein docking domain increased antibiotic resistance and toluene tolerance, demonstrating the important role of this domain in transport activity. These data demonstrate the roles of the central cavity and periplasmic domains in the function of the RND efflux pump EmhB.