Efflux-mediated antibiotics resistance in bacteria

Bacteria can resist to antibiotics by active exportation mediated by membrane transporters called efflux pumps. These proteins can be specific of a class of antibiotics or responsible for multidrug resistance (MDR). Energy required by efflux pumps can be provided by transmembrane electrochemical gradient of protons (MFS, RND, SMR families) or sodium ions (MATE family) or by ATP hydrolysis (ABC family). Several physiological functions have been described in prokaryotes, such as protection from environmental toxics and regulation of cell homeostasis, which can indirectly contributes to bacterial virulence. In Gram-negative bacteria, efflux transporters usually are organized as multicomponent systems in which the efflux pump located in the inner membrane works in conjunction with a periplasmic fusion protein and an outer membrane factor. The most frequently encountered pumps are of the RND-type such as AcrB in Escherichia coli or MexB in Pseudomonas aeruginosa. In Gram-positive bacteria, efflux is solely mediated by the pump protein, so described with MFS pumps such as NorA or QacA in Staphylococcus aureus and PmrA in Streptococcus pneumoniae. Efflux transporters have also been described in mycobacteria. Although numerous bacterial pumps have been characterized, the clinical consequences of efflux-mediated resistance are mostly unknown because of variable levels of expression and of the lack of specific markers in laboratory practice. Finally, associating pump-specific inhibitors to efflux-sensitive antibiotics might prove an interesting therapeutic perspective. However, inhibitors that are not toxic to eukaryotic cells remain to be identified.     

Multidrug ABC transporters in bacteria

Multidrug efflux transporters are a plague in the antibiotic resistance mechanisms as they confer the capacity of bacteria to evade most of current therapies. Although these transporters were initially discovered as proton-motive driven pumps, another class of multidrug efflux transporters has emerged in the mid-90s that are powered by ATP hydrolysis. This new class of transporters belongs to one of the largest families of proteins, the ATP-Binding Cassette (ABC) transporters, which are involved in the influx or efflux of a huge variety of molecules. Tremendous progresses have been made in the recent years regarding the functioning mechanism of multidrug efflux ABC transporters, in particular with the accumulation of 3D structures, but many questions remain unsolved. In this review, we will give an overview of our current knowledge on the structure and function of multidrug ABC transporters with an emphasis on bacterial pumps.     

Role of drug efflux transporters in the brain for drug disposition and treatment of brain diseases

The blood-brain barrier (BBB) serves as a protective mechanism for the brain by preventing entry of potentially harmful substances from free access to the central nervous system (CNS). Tight junctions present between the brain microvessel endothelial cells form a diffusion barrier, which selectively excludes most blood-borne substances from entering the brain. Astrocytic end-feet tightly ensheath the vessel wall and appear to be critical for the induction and maintenance of the barrier properties of the brain capillary endothelial cells. Because of these properties, the BBB only allows entry of lipophilic compounds with low molecular weights by passive diffusion. However, many lipophilic drugs show negligible brain uptake. They are substrates for drug efflux transporters such as P-glycoprotein (Pgp), multidrug resistance proteins (MRPs) or organic anion transporting polypeptides (OATPs) that are expressed at brain capillary endothelial cells and/or astrocytic end-feet and are key elements of the molecular machinery that confers the special permeability properties to the BBB. The combined action of these carrier systems results in rapid efflux of xenobiotics from the CNS. The objective of this review is to summarize transporter characteristics (cellular localization, specificity, regulation, and potential inhibition) for drug efflux transport systems identified in the BBB and blood-cerebrospinal fluid (CSF) barrier. A variety of experimental approaches available to ascertain or predict the impact of efflux transport on brain access of therapeutic drugs also are described and critically discussed. The potential impact of efflux transport on the pharmacodynamics of agents acting in the CNS is illustrated. Furthermore, the current knowledge about drug efflux transporters as a major determinant of multidrug resistance of brain diseases such as epilepsy is reviewed. Finally, we summarize strategies for modulating or by-passing drug efflux transporters at the BBB as novel therapeutic approaches to drug-resistant brain diseases.     

Multidrug resistance activity in human lymphocytes.

The multidrug resistance gene (mdr1) is a member of the recently described ATP binding cassette (ABC) superfamily of transporters. Family members include: (1) the cystic fibrosis transmembrane conductance regulator gene; (2) the hlyB gene of bacteria, and (3) the histocompatibility antigen modifier (HAM) gene. The level of expression of mdr1 correlates with multidrug resistance (MDR), the ability of cells to efflux otherwise toxic doses of several chemotherapeutic agents. MDR activity is also associated with the efflux of cationic lipophilic compounds such as the fluorescent dye rhodamine 123. Recently it was reported that normal lymphocytes efflux rhodamine 123, suggesting that these cells possess MDR-like activity due to the expression of mdr1. In this study, using two-color flow cytometric analysis, we observed that the ability to efflux rhodamine 123 was heterogeneous among human lymphocyte subsets in the order of CD8 greater than CD4 greater than CD2O. Rhodamine 123 efflux and accumulation in lymphocytes was sensitive to the known MDR reversing agents, verapamil and Solutol HS 15. Collectively, these data suggest that an MDR-like transport system is present in normal lymphocytes and may be important for trafficking of molecules involved in lymphocyte function.     

Amino acid residues affecting drug pump function in Candida albicans--C. albicans drug pump function.

Membrane-located drug transporters are important components in the multidrug resistance of microbial cells and human tissues. In fungi, clinically important resistance to antifungal drugs most often results from the over-expression of efflux pump proteins in the plasma membrane of the resistant cell. This review describes studies of the ATP binding cassette (ABC) family of membrane efflux pumps in the opportunistic human pathogen Candida albicans and, in particular, examines how changes in the polypeptide sequence can affect pump function. The identification of amino acid residues affecting pump function can provide new insights into efflux pump mechanisms and the relationship between structure and function. Such information will be important for the design of pump inhibitors which could supplement existing antifungal drugs.     

Determination of P-gp and MRP1 expression and function in peripheral blood mononuclear cells in vivo

P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) mediate the efflux of many therapeutic agents and have been implicated in the treatment failure of many infectious diseases and cancers. The ability to characterise the expression and function of these transporters in vivo is important when assessing the pharmacological activity of drugs. We investigated some of the problems involved in screening the multidrug resistance status of individuals using flow cytometry. Expression of P-gp and MRP1 on the surface of lymphocytes isolated from blood samples (30 ml) was determined by indirect immunofluorescence. Functional ability was assessed by measuring the efflux of specific fluorescent dyes. Results were expressed as a mean fold increase in fluorescence from the isotype control (expression) and a change in fluorescence compared to the load (function). Using these assays, we determined the expression of P-gp to be 2.01+/-0.40, n=30 and MRP1 to be 1.46+/-0.23, n=25. Functional ability was 6.98+/-4.97, n=25 for P-gp and 1.55+/-0.25, n=25 for MRP1. The dye efflux studies were associated with a lack of specificity and a number of methodological difficulties. There was no correlation between the expression and function of P-gp (r=0.338; p=0.10) or MRP1 (r=0.283; p=0.17). Therefore, we considered determination of P-gp and MRP1 expression to be a more reproducible and accurate approach to clinical investigation into the role of multidrug resistance.     

Identification of an organic anion transport system in the human colon carcinoma cell line HT29 clone 19A

The human colon carcinoma cell line HT29 c1.19A was studied for organic anion transporter activity by determining intracellular fluo-3 and fura-red accumulation and by measuring fluo-3 efflux. Modulators of organic anion transport systems were used to identify the transporters that are involved in dye extrusion. Addition of probenecid to the dye-loading medium, containing 10 microM fluo-3/AM and fura-red/AM, resulted in a dose-dependent increase in fluo-3 and fura-red accumulation in the cells. The increase in fluo-3 accumulation in the cells in the presence of probenecid was explained by the inhibitory effect of this compound on fluo-3 efflux. Fluo-3 efflux from the cells was also inhibited by sulfinpyrazone, another inhibitor of organic anion transport. Substrates of renal probenecid-sensitive organic anion exchange mechanisms as well as modulators of multidrug resistance associated protein (MRP) activity did not influence fluo-3 extrusion rates. However, reducing intracellular ATP contents completely blocked fluo-3 extrusion. Moreover, MK571, an inhibitor of MRP, significantly stimulated dye accumulation, whereas inhibitors of the multidrug resistance gene (MDR1) product Pglycoprotein, cyclosporin A and verapamil, did not. As probenecid inhibits fluo-3 efflux across the apical membrane of cells grown on permeable supports, we conclude that a probenecid-sensitive organic anion transporter is present in the apical membrane of HT29 c1.19A cells. This organic anion transport system differs from MDRI and MRP2.     

ATP竞争性GSK-3抑制剂对人食管鳞癌细胞多药耐药的影响

目的: 研究ATP竞争性糖原合成酶激酶3(GSK-3)抑制剂6-溴靛玉红-3'-肟(BIO)作用于食管鳞癌细胞后, 对ATP结合盒(ABC)转运蛋白——P-糖蛋白(P-gp)和多药耐药相关蛋白2(MRP2)、凋亡抑制蛋白Bcl-2以及β-catenin表达的影响,探讨它们表达的相互关系,以及对细胞内游离ATP水平和P-gp、MRP2转运功能的影响,进而初步探讨GSK-3抑制剂对食管鳞癌细胞多药耐药的影响。方法: BIO作用于食管鳞癌EC-109细胞后,运用免疫荧光细胞化学法、流式细胞术以及ATP检测试剂盒分别检测细胞内 MRP2、P-gp、β-catenin和Bcl-2表达的改变、ABC转运蛋白的转运功能以及细胞内游离ATP水平的改变。结果: BIO作用食管鳞癌EC-109细胞后,加药组与对照组相比,β-catenin和Bcl-2在胞浆中的表达增强,并且β-catenin出现胞核内累积,MRP2在胞浆和胞膜中表达增强,P-gp在胞浆和胞膜中表达减弱;细胞内游离ATP水平增加;ABC转运蛋白的转运功能增强。结论: BIO处理食管鳞癌细胞后增强了细胞的多药耐药。     

Human multidrug resistance ABCB and ABCG transporters: participation in a chemoimmunity defense system

In this review we give an overview of the physiological functions of a group of ATP binding cassette (ABC) transporter proteins, which were discovered, and still referred to, as multidrug resistance (MDR) transporters. Although they indeed play an important role in cancer drug resistance, their major physiological function is to provide general protection against hydrophobic xenobiotics. With a highly conserved structure, membrane topology, and mechanism of action, these essential transporters are preserved throughout all living systems, from bacteria to human. We describe the general structural and mechanistic features of the human MDR-ABC transporters and introduce some of the basic methods that can be applied for the analysis of their expression, function, regulation, and modulation. We treat in detail the biochemistry, cell biology, and physiology of the ABCB1 (MDR1/P-glycoprotein) and the ABCG2 (MXR/BCRP) proteins and describe emerging information related to additional ABCB- and ABCG-type transporters with a potential role in drug and xenobiotic resistance. Throughout this review we demonstrate and emphasize the general network characteristics of the MDR-ABC transporters, functioning at the cellular and physiological tissue barriers. In addition, we suggest that multidrug transporters are essential parts of an innate defense system, the "chemoimmunity" network, which has a number of features reminiscent of classical immunology.     

The limitations of renal epithelial cell line HK-2 as a model of drug transporter expression and function in the proximal tubule

Acquiring a mechanistic understanding of the processes underlying the renal clearance of drug molecules in man has been hampered by a lack of robust in vitro models of human proximal tubules. Several human renal epithelial cell lines derived from the renal cortex are available, but few have been characterised in detail in terms of transporter expression. This includes the HK-2 proximal tubule cell line, which has been used extensively as a model of nephrotoxicity. The aim of this study was to investigate the expression and function of drug transporters in HK-2 cells and their suitability as an in vitro model of the human proximal tubule. qPCR showed no mRNA expression of the SLC22 transporter family (OAT1, OAT3, OCT2) in HK-2 cells compared to renal cortex samples. In contrast, SLC16A1 (MCT1), which is important in the uptake of monocarboxylates, and SLCO4C1 (OATP4C1) were expressed in HK-2 cells. The functional expression of these transporters was confirmed by uptake studies using radiolabelled prototypic substrates dl-lactate and digoxin, respectively. The mRNA expression of apical membrane efflux transporters ABCB1 (MDR1) and several members of the ABCC family (multidrug resistance proteins, MRPs) was shown by qPCR. ABCG1 (BCRP) was not detected. The efflux of Hoechst 33342, a substrate for MDR1, was blocked by MDR1 inhibitor cyclosporin A, suggesting the functional expression of this transporter. Similarly, the efflux of the MRP-specific fluorescent dye glutathione methylfluorescein was inhibited by the MRP inhibitor MK571. Taken together, the results of this study suggest that HK-2 cells are of limited value as an in vitro model of drug transporter expression in the human proximal tubule.     

Sensitizing multi drug resistant Staphylococcus aureus isolated from surgical site infections to antimicrobials by efflux pump inhibitors

BackgroundStaphylococcus aureus is a common hospital acquired infections pathogen. Multidrug-resistant Methicillin-resistant Staphylococcus aureus represents a major problem in Egyptian hospitals. The over-expression of efflux pumps is a main cause of multidrug resistance. The discovery of efflux pump inhibitors may help fight multidrug resistance by sensitizing bacteria to antibiotics. This study aimed to investigate the role of efflux pumps in multidrug resistance.MethodsTwenty multidrug resistant S. aureus isolates were selected. Efflux pumps were screened by ethidium bromide agar cartwheel method and polymerase chain reaction. The efflux pump inhibition by seven agents was tested by ethidium bromide agar cartwheel method and the effect on sensitivity to selected antimicrobials was investigated by broth microdilution method.ResultsSeventy percent of isolates showed strong efflux activity, while 30% showed intermediate activity. The efflux genes mdeA, norB, norC, norA and sepA were found to play the major role in efflux, while genes mepA, smr and qacA/B had a minor role. Verapamil and metformin showed significant efflux inhibition and increased the sensitivity to tested antimicrobials, while vildagliptin, atorvastatin, domperidone, mebeverine and nifuroxazide showed no effect.ConclusionEfflux pumps are involved in multidrug resistance in Staphylococcus aureus. Efflux pump inhibitors could increase the sensitivity to antimicrobials.     

Tackling drug resistance with efflux pump inhibitors: from bacteria to cancerous cells

Abstract

Drug resistance is a serious concern in a clinical setting jeopardizing treatment for both infectious agents and cancers alike. The wide-spread emergence of multi-drug resistant (MDR) phenotypes from bacteria to cancerous cells necessitates the need to target resistance mechanisms and prevent the emergence of resistant mutants. Drug efflux seems to be one of the preferred approaches embraced by both microbial and mammalian cells alike, to thwart the action of chemotherapeutic agents thereby leading to a drug resistant phenotype. Relative to microbes, which predominantly employs proton motive force (PMF) powered, Major Facilitator Superfamily (MFS)/Resistance Nodulation and Division (RND) classes of efflux pumps to efflux drugs, cancerous cells preferentially use ATP fuelled ATP binding cassette (ABC) transporters to extrude chemotherapeutic agents. The prevalence, evolutionary characteristics and overlapping functions of ABC transporters have been highlighted in this review. Additionally, we outline the role of ABC pumps in conferring MDR phenotype to both bacteria and cancerous cells and underscore the importance of efflux pump inhibitors (EPI) to mitigate drug resistance. Based on the literature reports and analysis, we reason out feasibility of employing bacteria as a tool to screen for EPI’s targeting ABC pumps of cancerous cells.     

A novel ATP-binding cassette transporter, ABCG6 is involved in chemoresistance of Leishmania

ATP-binding cassette (ABC) transporters constitute the biggest family of membrane proteins involved in drug resistance and other biological activities. Resistance of leishmanial parasites to therapeutic drugs continues to escalate in developing countries and in many instances it is due to overexpressed ABC efflux pumps. Progressively adapted camptothecin (CPT)-resistant parasites show overexpression of a novel ABC transporter, which was classified as ABCG6. Transfection and overexpression of LdABCG6 in wild type parasites, shows its localization primarily in the plasma membrane and flagellar pocket region. Overexpressed LdABCG6 confers substantial CPT resistance to the parasites by rapid drug efflux. Various inhibitors have been tested for their ability to revert the CPT-resistant phenotype to specifically understand the inhibition of LdABCG6 transporter. Transport experiments using everted membrane vesicles were carried out to gain an insight into the kinetics of drug transport. This study provides further knowledge of specific membrane traffic ATPase and its involvement in the chemoresistance of Leishmania.     

Circumvention of multi-drug resistance of cancer cells by Chinese herbal medicines

Multi-drug resistance (MDR) of cancer cells severely limits therapeutic outcomes. A proposed mechanism for MDR involves the efflux of anti-cancer drugs from cancer cells, primarily mediated by ATP-binding cassette (ABC) membrane transporters including P-glycoprotein. This article reviews the recent progress of using active ingredients, extracts and formulae from Chinese medicine (CM) in circumventing ABC transporters-mediated MDR. Among the ABC transporters, Pgp is the most extensively studied for its role in MDR reversal effects. While other MDR reversal mechanisms remain unclear, Pgp inhibition is a criterion for further mechanistic study. More mechanistic studies are needed to fully establish the pharmacological effects of potential MDR reversing agents.     

Energy coupling in ABC exporters

Multidrug transporters are important and interesting molecular machines that extrude a wide variety of cytotoxic drugs from target cells. This review summarizes novel insights in the energetics and mechanisms of bacterial ATP-binding cassette multidrug transporters as well as recent advances connecting multidrug transport to ion and lipid translocation processes in other membrane proteins.     

Expression and localization of ATP binding cassette (ABC) family of drug transporters in gastric hepatoid adenocarcinomas

Aims:  Hepatoid adenocarcinomas are clinically chemoresistant; however, data concerning the mechanisms of chemoresistance are lacking. ATP-binding cassette (ABC) transporters play a role in the ATP-dependent outward efflux of drugs, and their function renders tumour cells multidrug resistant. We assumed that the expression of ABC transporters may be accompanied by hepatic differentiation because most ABC transporters are dominantly expressed in hepatocytes. The aim of this study was to investigate the expression of ABC transporters in hepatoid adenocarcinomas.
Methods and results:  The expression of P-glycoprotein, multidrug resistant-associated protein (MRP) 1, MRP2, MRP3 and MRP6 was investigated using immunohistochemistry in 13 cases of gastric hepatoid adenocarcinoma and 32 cases of control adenocarcinoma. P-glycoprotein was expressed in all cases examined. The positive rates of MRP1 (84.6% versus 21.9%), MRP2 (100% versus 43.8%) and MRP6 (61.5% versus 15.6%) were significantly higher in hepatoid than in control adenocarcinoma. MRP3 was negative in all hepatoid adenocarcinomas. Immunohistochemistry by polyclonal anti-carcinoembryonic antigen antibody, which detects bile canaliculi, suggested that MRP2 and MRP6 are located on bile canalicular-like structures in sheet or trabecular nests.
Conclusions:  These results support the hypothesis that ABC transporters participate in the mechanisms of multidrug resistance in hepatoid adenocarcinomas.