Gene cloning and properties of the RND-type multidrug efflux pumps MexPQ-OpmE and MexMN-OprM from Pseudomonas aeruginosa

We cloned two operons for putative RND-type multidrug efflux pumps from Pseudomonas aeruginosa by a PCR method. We designated the genes in one operon mexPQ(-opmE) and in another operon mexMN. Introduction of the mexPQ-opmE into drug hypersensitive cells resulted in elevated MICs of macrolides, fluoroquinolones and some other drugs. Introduction of the mexMN into the hypersensitive cells possessing oprM, but not into cells not possessing oprM, resulted in elevated MICs of chloramphenicol and thiamphenicol. Thus, we conclude that MexPQ-OpmE and MexMN-OprM are functional multidrug efflux pumps when expressed in P. aeruginosa.     

The mexR repressor of the mexAB-oprM multidrug efflux operon in Pseudomonas aeruginosa: characterization of mutations compromising activity

Mutations in mexR yield a multidrug resistance phenotype in nalB mutants of Pseudomonas aeruginosa as a result of derepression of the mexAB-oprM multidrug efflux operon. MexR produced by several nalB strains carried single amino acid changes that compromised MexR stability or its ability to dimerize. Changes at residues L95 and R21, however, produced a stable MexR protein capable of dimerization and, thus, likely compromised DNA binding.     

P-glycoproteins: mediators of multidrug resistance

Multidrug resistance represents a major obstacle to successful chemotherapy of metastatic disease. Elevated levels in cancer cells if the product of the multidrug resistance gene, P-glycoprotein or the multidrug transporter, have been associated with the development of simultaneous resistance to a great variety of amphiphilic cytotoxic drugs. P-glycoproteins is an integral plasma membrane protein which contains 12 putative transmembrane regions and two ATP binding sites. It confers multidrug resistance by functioning as an energy-dependent drug efflux pump. Here we describe recent studies on the biosynthesis, structure, function, and mechanism of action of P-glycoprotein which have provided insights into the complexity of this multifunctional transport system and revealed an additional chloride channel activity. The physiological role of P-glycoprotein, however, still remains to be elucidated.     

A broad-specificity multidrug efflux pump requiring a pair of homologous SMR-type proteins

The Bacillus subtilis genome encodes seven homologues of the small multidrug resistance (SMR) family of drug efflux pumps. Six of these homologues are paired in three distinct operons, and coexpression in Escherichia coli of one such operon, ykkCD, but not expression of either ykkC or ykkD alone, gives rise to a broad specificity, multidrug-resistant phenotype including resistance to cationic, anionic, and neutral drugs.     

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.     

Use of fluorescent dyes as molecular probes for the study of multidrug resistance

Fluorescence microscopy has shown that 18 different fluorescent dyes, staining various intracellular structures in transformed hamster fibroblasts (DM-15), did not stain or stained weakly multidrug-resistant cells selected from DM-15 by colchicine. Reduced staining by fluorescent dyes was characteristic also of five other tested multidrug-resistant cell lines of hamster and mouse origin, selected by actinomycin D, colcemid, rubomycin, and ruboxyl. The intensity of staining of two revertant cell lines was similar to that of parental sensitive cells. All tested inhibitors of multidrug resistance, including weak detergent, metabolic inhibitors, calcium channel blockers, calmodulin inhibitors, and reserpine, restored normal staining of multidrug-resistant cells. The dyes accumulated in resistant cells in presence of these inhibitors left the cells several minutes after the removal of the inhibitor from the incubation medium. Sensitive cells retained the dyes for several hours. The efflux of the dyes from resistant cells is an active process since it occurred even in the presence of the dyes in the incubation medium. The efflux could be blocked by all tested inhibitors of multidrug resistance and it is possibly a basic mechanism of the reduced staining of resistant cells. These data support the idea that multidrug resistance is based on active nonspecific efflux of the drugs and indicate that the simple procedure of cell staining can be used for the detection of resistant cells and further study of the phenomenon of multidrug resistance.     

Characterization of the RND-type multidrug efflux pump MexAB-OprM of the plant pathogen Pseudomonas syringae

In gram-negative bacteria, transporters belonging to the RND family are the transporters most relevant for resistance to antimicrobial compounds. In Pseudomonas aeruginosa, a clinically important pathogen, the RND-type pump MexAB-OprM has been recognized as one of the major multidrug efflux systems. Here, homologues of MexAB-OprM in the plant pathogens Pseudomonas syringae pv. phaseolicola 1448A, P. syringae pv. syringae B728a, and P. syringae pv. tomato DC3000 were identified, and mexAB-oprM-deficient mutants were generated. Determination of MICs revealed that mutation of MexAB-OprM dramatically reduced the tolerance to a broad range of antimicrobials. Moreover, the ability of the mexAB-oprM-deficient mutants to multiply in planta was reduced. RNA dot blot hybridization revealed growth-dependent regulation of the mexAB-oprM operon in P. syringae; the expression of this operon was maximal in early exponential phase and decreased gradually during further growth.     

Membrane homoeostasis and multidrug resistance in yeast

The development of MDR (multidrug resistance) in yeast is due to a number of mechanisms. The most documented mechanism is enhanced extrusion of drugs mediated by efflux pump proteins belonging to either the ABC (ATP-binding cassette) superfamily or MFS (major facilitator superfamily). These drug-efflux pump proteins are localized on the plasma membrane, and the milieu therein affects their proper functioning. Several recent studies demonstrate that fluctuations in membrane lipid composition affect the localization and proper functioning of the MDR efflux pump proteins. Interestingly, the efflux pumps of the ABC superfamily are particularly susceptible to imbalances in membrane-raft lipid constituents. This review focuses on the importance of the membrane environment in functioning of the drug-efflux pumps and explores a correlation between MDR and membrane lipid homoeostasis.     

Pharmacologic circumvention of multidrug resistance

The ability of malignant cells to develop resistance to chemotherapeutic drugs is a major obstacle to the successful treatment of clinical tumors. The phenomenon multidrug resistance (MDR) in cancer cells results in cross-resistance to a broad range of structurally diverse antineoplastic agents, due to outward efflux of cytotoxic substrates by themdr1 gene product, P-glycoprotein (P-gp). Numerous pharmacologic agents have been identified which inhibit the efflux pump and modulate MDR. The biochemical, cellular and clinical pharmacology of agents used to circumvent MDR is analyzed in terms of their mechanism of action and potential clinical utility. MDR antagonists, termed chemosensitizers, may be grouped into several classes, and include calcium channel blockers, calmodulin antagonists, anthracycline andVinca alkaloid analogs, cyclosporines, dipyridamole, and other hydrophobic, cationic compounds. Structural features important for chemosensitizer activity have been identified, and a model for the interaction of these drugs with P-gp is proposed. Other possible cellular targets for the reversal of MDR are also discussed, such as protein kinase C. Strategies for the clinical modulation of MDR and trials combining chemosensitizers with chemotherapeutic drugs in humans are reviewed. Several novel approaches for the modulation of MDR are examined.Abbreviations ALL acute lymphocytic leukemia - AML acute myelogenous leukemia - CaM calmodulin - CsA cyclosporin A - MDR multidrug resistance - P-gp P-glycoprotein - PMA phorbol 12-myristate 13-acetate - PKC protein kinase C     

Isolation and characterization of a putative multidrug resistance pump from Vibrio cholerae

Multidrug-resistant strains of Vibrio cholerae (the causative agent of the diarrhoeal disease cholera) have recently been described. In an attempt to identify a homologue of the Escherichia coli TolC in V . cholerae , we isolated a DNA fragment (pVC) that enabled an E . coli tolC mutant to grow in the presence of 0.05% deoxycholate (DOC). However, other TolC defects were not complemented. Nucleotide sequence analysis of this fragment revealed the presence of two open reading frames (ORF1 and ORF2) separated by 9 bp and encoding 42.4 and 55.8 kDa proteins respectively. The translational products of these two ORFs correlated closely with the molecular weights of the predicted proteins. The deduced amino acid sequences of ORF1 and ORF2 showed a high degree of similarity with conserved regions of the E . coli efflux pump proteins, EmrA and EmrB. The presence of pVC2 within the E . coli efflux pump mutants defective in either the emrAB or the acrAB genes provided the mutants with resistance against several antibiotics. A V . cholerae isogenic mutant defective in ORF2 was constructed by gene replacement. Characterization of this mutant has shown it to be more sensitive to CCCP, PMA, PCP, nalidixic acid and DOC than the parent strain. These results suggest that ORF1 and ORF2 constitute an operon encoding two components of a putative multidrug resistance pump in V . cholerae . In addition, the presence of both structural and functional similarities between VceAB and EmrAB suggests that VceAB is a homologue of EmrAB.     

The molecular basis of multidrug resistance in cancer: the early years of P-glycoprotein research

The discovery and characterization of P-glycoprotein, an energy-dependent multidrug efflux pump, as a mechanism of multidrug resistance in cancer is generally accepted as a significant contribution to the ongoing effort to end death and suffering from this disease. The historical reflections of Victor Ling and Michael Gottesman concerning the early years of this research highlight the important contributions of the multidisciplinary teams involved in these studies, and illustrate how technological developments in biochemistry and molecular and cell biology enabled this discovery.     

Control of the AcrAB multidrug efflux pump by quorum-sensing regulator SdiA

SdiA is an Escherichia coli protein that regulates cell division in a cell density-dependent, or quorum-sensing, manner. We report that SdiA also controls multidrug resistance by positively regulating the multidrug resistance pump AcrAB. Overproduction of SdiA confers multidrug resistance and increased levels of AcrAB. Conversely, sdiA null mutants are hypersensitive to drugs and have decreased levels of AcrB protein. Our findings provide a link between quorum sensing and multidrug efflux. Combined with previously published reports, our data support a model in which a role of drug efflux pumps is to mediate cell-cell communication in response to cell density. Xenobiotics expelled by pumps may resemble the communication molecules that they normally efflux.     

Development of dual-acting prodrugs for circumventing multidrug resistance

We have developed a novel dual-acting maleimide-bearing prodrug that incorporates the anticancer agent doxorubicin and an inhibitor of the P-glycoprotein efflux pump that is over-expressed in multidrug resistant tumor cells. Additionally, the prodrug contains a 1,6-self-immolative spacer coupled to the dipeptide Phe-Lys that acts as a substrate for cathepsin B. The prodrug, once bound through its maleimide moiety to the cysteine-34 group of human serum albumin, was cleaved by cathepsin B and in tumor homogenates demonstrating a release of the anticancer agent doxorubicin and the inhibitor.     

Molecular mechanism of multidrug resistance in tumor cells

The ability of tumor cells to develop simultaneous resistance to multiple lipophilic cytotoxic compounds represents a major problem in cancer chemotherapy. This review describes recent molecular biological studies which resulted in the identification and cloning of the gene responsible for multidrug resistance in human tumor cells. This gene, designated mdr1, is overexpressed in all and amplified in many of the multidrug-resistant cell lines analyzed. Gene transfer and expression assays have indicated that the mdr1 gene is both necessary and sufficient for multidrug resistance. The product of the mdr1 gene is P-glycoprotein, a transmembrane protein which shares homology with several bacterial proteins involved in active membrane transport. P-glycoprotein appears to function as an energy-dependent efflux pump responsible for the removal of drugs from multidrug-resistant cells. The functions of the mdr system in normal cells and its potential clinical implications are discussed.     

Role of multidrug resistance P-glycoprotein in the secretion of aldosterone by human adrenal NCI-H295 cells

We determinedthe role of the multidrug resistance (MDR1) gene product,P-glycoprotein (PGP), in the secretion of aldosterone by the adrenalcell line NCI-H295. Aldosterone secretion is significantly decreased bythe PGP inhibitors verapamil, cyclosporin A (CSA), PSC-833, andvinblastine. Aldosterone inhibits the efflux of the PGP substraterhodamine 123 from NCI-H295 cells and from human mesangial cells(expressing PGP). CSA, verapamil, and the monoclonal antibody UIC2significantly decreased the efflux of fluorescein-labeled (FL)-aldosterone microinjected into NCI-H295 cells. In MCF-7/VP cells,expressing multidrug resistance-associated protein (MRP) but not PGP,and in the parental cell line MCF7 (expressing no MRP andno PGP), the efflux of microinjected FL-aldosterone was slow. In BC19/3cells (MCF7 cells transfected with MDR1), the efflux of FL-aldosteronewas rapid and it was inhibited by verapamil, indicating thattransfection with MDR1 cDNA confers the ability to transportFL-aldosterone. These results strongly indicate that PGP plays a rolein the secretion of aldosterone by NCI-H295 cells and in other cellsexpressing MDR1, including normal adrenal cells.