Proton-dependent multidrug efflux systems.

Multidrug efflux systems display the ability to transport a variety of structurally unrelated drugs from a cell and consequently are capable of conferring resistance to a diverse range of chemotherapeutic agents. This review examines multidrug efflux systems which use the proton motive force to drive drug transport. These proteins are likely to operate as multidrug/proton antiporters and have been identified in both prokaryotes and eukaryotes. Such proton-dependent multidrug efflux proteins belong to three distinct families or superfamilies of transport proteins: the major facilitator superfamily (MFS), the small multidrug resistance (SMR) family, and the resistance/ nodulation/cell division (RND) family. The MFS consists of symporters, antiporters, and uniporters with either 12 or 14 transmembrane-spanning segments (TMS), and we show that within the MFS, three separate families include various multidrug/proton antiport proteins. The SMR family consists of proteins with four TMS, and the multidrug efflux proteins within this family are the smallest known secondary transporters. The RND family consists of 12-TMS transport proteins and includes a number of multidrug efflux proteins with particularly broad substrate specificity. In gram-negative bacteria, some multidrug efflux systems require two auxiliary constituents, which might enable drug transport to occur across both membranes of the cell envelope. These auxiliary constituents belong to the membrane fusion protein and the outer membrane factor families, respectively. This review examines in detail each of the characterized proton-linked multidrug efflux systems. The molecular basis of the broad substrate specificity of these transporters is discussed. The surprisingly wide distribution of multidrug efflux systems and their multiplicity in single organisms, with Escherichia coli, for instance, possessing at least nine proton-dependent multidrug efflux systems with overlapping specificities, is examined. We also discuss whether the normal physiological role of the multidrug efflux systems is to protect the cell from toxic compounds or whether they fulfil primary functions unrelated to drug resistance and only efflux multiple drugs fortuitously or opportunistically.     

The SMR family: a novel family of multidrug efflux proteins involved with the efflux of lipophilic drugs

The sequenced members of a novel family of small, hydrophobic, bacterial multidrug-resistance efflux proteins, which we have designated the small multidrug resistance (SMR) protein family, are identified and analysed. Two distinct clusters of proteins were identified within this family: (i) small multidrug efflux systems; and (ii) Sug proteins, potentially involved in the suppression of groEL mutations. Hydropathy and residue distribution analyses of this family suggest a structural model in which the polypeptide chain spans the membrane four times as mildly amphipathic α-helices. The roles of specific residues, a possible mechanistic model of drug efflux, and the primary physiological role(s) of the SMR proteins are discussed.     

A two-component multidrug efflux pump, EbrAB, in Bacillus subtilis

Genes (ebrAB) responsible for ethidium resistance were cloned from chromosomal DNA of Bacillus subtilis ATCC 9372. The recombinant plasmid produced elevated resistance against ethidium bromide, acriflavine, pyronine Y, and safranin O not only in Escherichia coli but also in B. subtilis. It also caused an elevated energy-dependent efflux of ethidium in E. coli. EbrA and EbrB showed high sequence similarity with members of the small multidrug resistance (SMR) family of multidrug efflux pumps. Neither ebrA nor ebrB was sufficient for resistance, but introduction of the two genes carried on different plasmids conferred drug resistance. Thus, both EbrA and EbrB appear to be necessary for activity of the multidrug efflux pump. In known members of the SMR family, only one gene produces drug efflux. Thus, EbrAB is a novel SMR family multidrug efflux pump with two components.     

Conservation of amino acid transporters in fungi, plants and animals

When comparing the transporters of three completely sequenced eukaryotic genomes--Saccharomyces cerevisiae, Arabidopsis thaliana and Homo sapiens--transporter types can be distinguished according to phylogeny, substrate spectrum, transport mechanism and cell specificity. The known amino acid transporters belong to five different superfamilies. Two preferentially Na(+)-coupled transporter superfamilies are not represented in the yeast and Arabidopsis genomes, whereas the other three groups, which often function as H(+)-coupled systems, have members in all investigated genomes. Additional superfamilies exist for organellar transport, including mitochondrial and plastidic carriers. When used in combination with phylogenetic analyses, functional comparison might aid our prediction of physiological functions for related but uncharacterized open reading frames.     

VmrA, a Member of a Novel Class of Na+-Coupled Multidrug Efflux Pumps from Vibrio parahaemolyticus

Gene vmrA, cloned from Vibrio parahaemolyticus, made Escherichia coli resistant to 4',6-diamino-2-phenylindol, tetraphenylphosphonium chloride, acriflavine, and ethidium bromide. VmrA belongs to the DinF branch of MATE family efflux transporters. VmrA catalyzed acriflavine efflux and showed Na(+)/drug transporter activity because the addition of tetraphenylphosphonium to Na(+)-loaded cells caused Na(+) efflux.     

The RND permease superfamily: an ancient, ubiquitous and diverse family that includes human disease and development proteins

A previous report identified and classified a small family of gram-negative bacterial drug and heavy metal efflux permeases, now commonly referred to as the RND family (TC no. 2.6). We here show that this family is actually a ubiquitous superfamily with representation in all major kingdoms. We report phylogenetic analyses that define seven families within the RND superfamily as follows: (1) the heavy metal efflux (HME) family (gram negative bacteria), (2) the hydrophobe/amphiphile efflux-1 (HAE1) family (gram negative bacteria), (3) the nodulation factor exporter (NFE) family (gram negative bacteria), (4) the SecDF protein-secretion accessory protein (SecDF) family (gram negative and gram positive bacteria as well as archaea), (5) the hydrophobe/amphiphile efflux-2 (HAE2) family (gram positive bacteria), (6) the eukaryotic sterol homeostasis (ESH) family, and (7) the hydrophobe/amphiphile efflux-3 (HAE3) family (archaea and spirochetes). Functionally uncharacterized proteins were identified that are members of the RND superfamily but fall outside of these seven families. Some of the eukaryotic homologues function as enzymes and receptors instead of (or in addition to) transporters. The sizes and topological patterns exhibited by members of all seven families are shown to be strikingly similar, and statistical analyses establish common descent. Multiple alignments of proteins within each family allow derivation of family-specific signature sequences. Structural, functional, mechanistic and evolutionary implication of the reported results are discussed.     

Efflux in Fungi: La Pièce de Résistance

Pathogens must be able to overcome both host defenses and antimicrobial treatment in order to successfully infect and maintain colonization of the host. One way fungi accomplish this feat and overcome intercellular toxin accumulation is efflux pumps, in particular ATP-binding cassette transporters and transporters of the major facilitator superfamily. Members of these two superfamilies remove many toxic compounds by coupling transport with ATP hydrolysis or a proton gradient, respectively. Fungal genomes encode a plethora of members of these families of transporters compared to other organisms. In this review we discuss the role these two fungal superfamilies of transporters play in virulence and resistance to antifungal agents. These efflux transporters are responsible not only for export of compounds involved in pathogenesis such as secondary metabolites, but also export of host-derived antimicrobial compounds. In addition, we examine the current knowledge of these transporters in resistance of pathogens to clinically relevant antifungal agents.     

NorM of vibrio parahaemolyticus is an Na(+)-driven multidrug efflux pump

NorM of Vibrio parahaemolyticus apparently is a new type of multidrug efflux protein, with no significant sequence similarity to any known transport proteins. Based on the following experimental results, we conclude that NorM is an Na(+)-driven Na(+)/drug antiporter. (i) Energy-dependent ethidium efflux from cells possessing NorM was observed in the presence of Na(+) but not of K(+). (ii) An artificially imposed, inwardly directed Na(+) gradient elicited ethidium efflux from cells. (iii) The addition of ethidium to cells loaded with Na(+) elicited Na(+) efflux. Thus, NorM is an Na(+)/drug antiporting multidrug efflux pump, the first to be found in the biological world. Judging from the similarity of the NorM sequence to those of putative proteins in sequence databases, it seems that Na(+)/drug antiporters are present not only in V. parahaemolyticus but also in a wide range of other organisms.     

The bile/arsenite/riboflavin transporter (BART) superfamily

Secondary transmembrane transport carriers fall into families and superfamilies allowing prediction of structure and function. Here we describe hundreds of sequenced homologues that belong to six families within a novel superfamily, the bile/arsenite/riboflavin transporter (BART) superfamily, of transport systems and putative signalling proteins. Functional data for members of three of these families are available, and they transport bile salts and other organic anions, the bile acid:Na(+) symporter (BASS) family, inorganic anions such as arsenite and antimonite, the arsenical resistance-3 (Acr3) family, and the riboflavin transporter (RFT) family. The first two of these families, as well as one more family with no functionally characterized members, exhibit a probable 10 transmembrane spanner (TMS) topology that arose from a tandemly duplicated 5 TMS unit. Members of the RFT family have a 5 TMS topology, and are homologous to each of the repeat units in the 10 TMS proteins. The other two families [sensor histidine kinase (SHK) and kinase/phosphatase/synthetase/hydrolase (KPSH)] have a single 5 TMS unit preceded by an N-terminal TMS and followed by a hydrophilic sensor histidine kinase domain (the SHK family) or catalytic domains resembling sensor kinase, phosphatase, cyclic di-GMP synthetase and cyclic di-GMP hydrolase catalytic domains, as well as various noncatalytic domains (the KPSH family). Because functional data are not available for members of the SHK and KPSH families, it is not known if the transporter domains retain transport activity or have evolved exclusive functions in molecular reception and signal transmission. This report presents characteristics of a unique protein superfamily and provides guides for future studies concerning structural, functional and mechanistic properties of its constituent members.     

Folding studies of immunoglobulin-like beta-sandwich proteins suggest that they share a common folding pathway.

BACKGROUND: Are folding pathways conserved in protein families? To test this explicitly and ask to what extent structure specifies folding pathways requires comparison of proteins with a common fold. Our strategy is to choose members of a highly diverse protein family with no conservation of function and little or no sequence identity, but with structures that are essentially the same. The immunoglobulin-like fold is one of the most common structural families, and is subdivided into superfamilies with no detectable evolutionary or functional relationship. RESULTS: We compared the folding of a number of immunoglobulin-like proteins that have a common structural core and found a strong correlation between folding rate and stability. The results suggest that the folding pathways of these immunoglobulin-like proteins share common features. CONCLUSIONS: This study is the first to compare the folding of structurally related proteins that are members of different superfamilies. The most likely explanation for the results is that interactions that are important in defining the structure of immunoglobulin-like proteins are also used to guide folding.     

Evolution of the 12-spanner drug:H+ antiporter DHA1 family in hemiascomycetous yeasts

Frequently, although not exclusively, multidrug resistance (MDR) results from the action of drug-efflux pumps, which are thought to be able to catalyze the active expulsion of several unrelated cytotoxic compounds out of the cell or their intracellular partitioning. The transporters of the major facilitator superfamily (MFS) presumably involved in MDR belong to the 12-spanner drug:H(+) antiporter DHA1 or to the 14- spanner drug:H(+) antiporter DHA2 families. The expression of most Saccharomyces cerevisiae DHA1 family members was found to confer broad chemoprotection. The evolution of the hemiascomycetous DHA1 proteins, belonging to the Génolevures GL3C007 family, was studied using a combined phylogenetic and gene neighborhood approach. The phylogenetic analysis of 189 DHA1 proteins belonging to the genome of 13 hemiascomycetous species identified 20 clusters. Eleven clusters contained no S. cerevisiae members. The phylogenetic clusters were analyzed by the IONS method developed for Identification of Orthologues by Neighborhood and Similarity. This allowed reconstructing the evolutionary history of most DHA1 members within 10 main gene lineages, spanning the whole hemiascomycetes clade, encompassing an evolutionary history of about 350 million years. In addition, five other more species specific lineages, spanning only two hemiascomycetous species, were identified. It is concluded that 57 out of the 143 members of the DHA1 hemiascomycetous members originated from gene duplication events. In half of these duplicates, the two members belong to different phylogenetic clusters, indicating that at least one of them has sufficiently differentiated to provide potential novel functions to this complex family from which most physiological substrates remain unknown.     

MgMfs1, a major facilitator superfamily transporter from the fungal wheat pathogen Mycosphaerella graminicola, is a strong protectant against natural toxic compounds and fungicides

MgMfs1, a major facilitator superfamily (MFS) gene from the wheat pathogenic fungus Mycosphaerella graminicola, was identified in expressed sequence tag (EST) libraries. The encoded protein has high homology to members of the drug:H(+) antiporter efflux family of MFS transporters with 14 predicted transmembrane spanners (DHA14), implicated in mycotoxin secretion and multidrug resistance. Heterologous expression of MgMfs1 in a hypersensitive Saccharomyces cerevisiae strain resulted in a strong decrease in sensitivity of this organism to a broad range of unrelated synthetic and natural toxic compounds. The sensitivity of MgMfs1 disruption mutants of M. graminicola to most of these compounds was similar when compared to the wild-type but the sensitivity to strobilurin fungicides and the mycotoxin cercosporin was increased. Virulence of the disruption mutants on wheat seedlings was not affected. The results indicate that MgMfs1 is a true multidrug transporter that can function as a determinant of pathogen sensitivity and resistance to fungal toxins and fungicides.     

Small multidrug resistance proteins: a multidrug transporter family that continues to grow

The small multidrug resistance (SMR) protein family is a bacterial multidrug transporter family. As suggested by their title, SMR proteins are composed of four transmembrane alpha-helices of approximately 100-140 amino acids in length. Since their designation as a family, many homologues have been identified and characterized both structurally and functionally. In this review the topology, structure, drug resistance, drug binding, and transport mechanisms of the entire SMR protein family are examined. Additionally, updated bioinformatic analysis of predicted and characterized SMR protein family members was also conducted. Based on SMR sequence alignments and phylogenetic analysis of current members, we propose that this small multidrug resistance transporter family should be expanded into three subclasses: (i) the small multidrug pumps (SMP), (ii) suppressor of groEL mutation proteins (SUG), and a third group (iii) paired small multidrug resistance proteins (PSMR). The roles of these three SMR subclasses are examined, and the well-characterized members, such as Escherichia coli EmrE and SugE, are described in terms of their function and structural organization.     

The identification of a new family of sugar efflux pumps in Escherichia coli

Using a functional cloning strategy with an Escherichia coli genomic plasmid library, we have identified a new family of sugar efflux proteins with three highly homologous members in the E. coli genome. In addition, two open reading frames, one present in Yersinia pestis and the other in Deinococcus radiodurans, appear to encode closely related proteins. An in vitro transport assay using inside-out membrane vesicles prepared from overproducing strains was used to demonstrate that members of this new family can efflux [14C]-lactose. As sugar efflux phenomena have been reported previously in several bacterial species including E. coli, the identification of a new family of sugar efflux proteins may help to reveal the physiological role of sugar efflux in metabolism. It is proposed that the E. coli members of this family, whose functions were previously unknown, be given the gene family designation SET for sugar efflux transporter.     

The cadherin superfamily database

The cadherin superfamily is a large protein family with diverse structures and functions. Because of this diversity and the growing biological interest in cell adhesion and signaling processes, in which many members of the cadherin superfamily play a crucial role, it is becoming increasingly important to develop tools to manage, distribute and analyze sequences in this protein family. Current profile and motif databases classify protein sequences into a broad spectrum of protein superfamilies, however to provide a more specific functional annotation, the next step should include classification of subfamilies of these protein superfamilies. Here, we present a tool that classified greater than 90% of the proteins belonging to the cadherin superfamily found in the SWISS PROT database. Therefore, for most members of the cadherin superfamily, this tool can assist in adding more specific functional annotations than can be achieved with current profile and motif databases. Finally, the classification tool and the results of our analysis were integrated into a web-accessible database (http://calcium.uhnres. utoronto.ca/cadherin).     

外排转运蛋白介导的抗真菌药物耐药研究进展

近年来,随着广谱抗生素,免疫抑制剂,抗肿瘤化疗药物的广泛应用,器官移植的普遍开展以及AIDS患者的逐年增加,各系统侵袭性真菌感染日益增多。抗真菌药物的大量应用使得真菌耐药现象日渐严重。大量研究表明,耐药真菌细胞膜上外排转运蛋白的过量表达对抗真菌药物耐药形成起到重要作用。ATP结合盒式蛋白(ABC转运体)和易化扩散载体超家族蛋白(MFS转运体)便是其中最重要的两种。本文从ABC及MFS转运体的结构和功能出发,分析其在抗真菌药物耐药形成中的作用,并对相关研究进展进行综述。     

Small multidrug resistance proteins: A multidrug transporter family that continues to grow

The small multidrug resistance (SMR) protein family is a bacterial multidrug transporter family. As suggested by their title, SMR proteins are composed of four transmembrane α-helices of approximately 100-140 amino acids in length. Since their designation as a family, many homologues have been identified and characterized both structurally and functionally. In this review the topology, structure, drug resistance, drug binding, and transport mechanisms of the entire SMR protein family are examined. Additionally, updated bioinformatic analysis of predicted and characterized SMR protein family members was also conducted. Based on SMR sequence alignments and phylogenetic analysis of current members, we propose that this small multidrug resistance transporter family should be expanded into three subclasses: (i) the small multidrug pumps (SMP), (ii) suppressor of groEL mutation proteins (SUG), and a third group (iii) paired small multidrug resistance proteins (PSMR). The roles of these three SMR subclasses are examined, and the well-characterized members, such as Escherichia coli EmrE and SugE, are described in terms of their function and structural organization.     

Comparison of three structures of the multidrug transporter EmrE

The small multidrug resistance proteins constitute a family of bacterial antiporters that confer multidrug resistance by H(+)-linked drug efflux across the bacterial cytoplasmic membrane. The structure of EmrE, the family archetype, has been determined by electron crystallography and shows that EmrE in the membrane is an asymmetric homodimer composed of a tightly packed bundle of eight alpha helices, six of which form the substrate-binding site, which has a single molecule of tetraphenylphosphonium at its centre. Two X-ray structures of EmrE have been determined; the first structure was of a non-native conformation of EmrE that formed a crystallographic tetramer, whereas EmrE in the second structure was an asymmetric dimer containing a single molecule of bound tetraphenylphosphonium. This recent EmrE structure bears a superficial resemblance to the electron crystallographic structure and the differences were ascribed to conformational changes. However, the biological relevance of these conformational differences is questionable.     

Topological and phylogenetic analyses of bacterial holin families and superfamilies

Holins are small “hole-forming” transmembrane proteins that mediate bacterial cell lysis during programmed cell death or following phage infection. We have identified fifty two families of established or putative holins and have included representative members of these proteins in the Transporter Classification Database (TCDB; www.tcdb.org). We have identified the organismal sources of members of these families, calculated their average protein sizes, estimated their topologies and determined their relative family sizes. Topological analyses suggest that these proteins can have 1, 2, 3 or 4 transmembrane α-helical segments (TMSs), and members of a single family are frequently, but not always, of a single topology. In one case, proteins of a family proved to have either 2 or 4 TMSs, and the latter arose by intragenic duplication of a primordial 2 TMS protein-encoding gene resembling the former. Using established statistical approaches, some of these families have been shown to be related by common descent. Seven superfamilies, including 21 of the 52 recognized families were identified. Conserved motif and Pfam analyses confirmed most superfamily assignments. These results serve to expand upon the scope of channel-forming bacterial holins.