Multidrug ATP‐binding cassette transporters are essential for hepatic development of Plasmodium sporozoites

Multidrug resistance‐associated proteins (MRPs) belong to the C‐family of ATP‐binding cassette (ABC) transport proteins and are known to transport a variety of physiologically important compounds and to be involved in the extrusion of pharmaceuticals. Rodent malaria parasites encode a single ABC transporter subfamily C protein, whereas human parasites encode two: MRP1 and MRP2. Although associated with drug resistance, their biological function and substrates remain unknown. To elucidate the role of MRP throughout the parasite life cycle, Plasmodium berghei and Plasmodium falciparum mutants lacking MRP expression were generated. P. berghei mutants lacking expression of the single MRP as well as P. falciparum mutants lacking MRP1, MRP2 or both proteins have similar blood stage growth kinetics and drug‐sensitivity profiles as wild type parasites. We show that MRP1‐deficient parasites readily invade primary human hepatocytes and develop into mature liver stages. In contrast, both P. falciparum MRP2‐deficient parasites and P. berghei mutants lacking MRP protein expression abort in mid to late liver stage development, failing to produce mature liver stages. The combined P. berghei and P. falciparum data are the first demonstration of a critical role of an ABC transporter during Plasmodium liver stage development.     

ABC proteins in yeast and fungal pathogens

All fungal genomes harbour numerous ABC (ATP-binding cassette) proteins located in various cellular compartments such as the plasma membrane, vacuoles, peroxisomes and mitochondria. Most of them have initially been discovered through their ability to confer resistance to a multitude of drugs, a phenomenon called PDR (pleiotropic drug resistance) or MDR (multidrug resistance). Studying the mechanisms underlying PDR/MDR in yeast is of importance in two ways: first, ABC proteins can confer drug resistance on pathogenic fungi such as Candida spp., Aspergillus spp. or Cryptococcus neoformans; secondly, the well-established genetic, biochemical and cell biological tractability of Saccharomyces cerevisiae makes it an ideal tool to study basic mechanisms of drug transport by ABC proteins. In the past, knowledge from yeast has complemented work on human ABC transporters involved in anticancer drug resistance or genetic diseases. Interestingly, increasing evidence available from yeast and other organisms suggests that ABC proteins play a physiological role in membrane homoeostasis and lipid distribution, although this is being intensely debated in the literature.     

Availability and applications of <Emphasis Type="Italic">A</Emphasis>TP-binding <Emphasis Type="Italic">c</Emphasis>assette (ABC) transporter blockers

ATP-binding cassette (ABC) transporters encompass membrane transport proteins that couple the energy derived from ATP hydrolysis to the translocation of solutes across biological membranes. The functions of these proteins include ancient and conserved mechanisms related to nutrition and pathogenesis in bacteria, spore formation in fungi, and signal transduction, protein secretion and antigen presentation in eukaryotes. Furthermore, one of the major causes of drug resistance and chemotherapeutic failure in both cancer and anti-infective therapies is the active movement of compounds across membranes carried out by ABC transporters. Thus, the clinical relevance of ABC transporters is enormous, and the membrane transporters related to chemoresistance are among the best-studied members of the ABC transporter superfamily. As ABC transporter blockers can be used in combination with current drugs to increase their efficacy, the (possible) impact of efflux pump inhibitors is of great clinical interest. The present review summarizes the progress made in recent years in the identification, design, availability, and applicability of ABC transporter blockers in experimental scenarios oriented towards improving the treatment of infectious diseases caused by microorganisms including parasites.     

Flavone-resistant Leishmania donovani Overexpresses LdMRP2 Transporter in the Parasite and Activates Host MRP2 on Macrophages to Circumvent the Flavone-mediated Cell Death

In parasites, ATP-binding cassette (ABC) transporters represent an important family of proteins related to 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 baicalein (BLN)-resistant parasites (pB²⁵R) show overexpression of a novel ABC transporter, which was classified as ABCC2 or Leishmania donovani multidrug resistance protein 2 (LdMRP2). The protein is primarily localized in the flagellar pocket region and in internal vesicles. Overexpressed LdABCC2 confers substantial BLN resistance to the parasites by rapid drug efflux. The BLN-resistant promastigotes when transformed into amastigotes in macrophage cells cannot be cured by treatment of macrophages with BLN. Amastigote resistance is concomitant with the overexpression of macrophage MRP2 transporter. Reporter analysis and site-directed mutagenesis assays demonstrated that antioxidant response element 1 is activated upon infection. The expression of this phase II detoxifying gene is regulated by NFE2-related factor 2 (Nrf2)-mediated antioxidant response element activation. In view of the fact that the signaling pathway of phosphoinositol 3-kinase controls microfilament rearrangement and translocation of actin-associated proteins, the current study correlates with the intricate pathway of phosphoinositol 3-kinase-mediated nuclear translocation of Nrf2, which activates MRP2 expression in macrophages upon infection by the parasites. In contrast, phalloidin, an agent that prevents depolymerization of actin filaments, inhibits Nrf2 translocation and Mrp2 gene activation by pB²⁵R infection. Taken together, these results provide insight into the mechanisms by which resistant clinical isolates of L. donovani induce intracellular events relevant to drug resistance.     

Comparative analysis of sequences encoding ABC systems in the genome of the microsporidian Encephalitozoon cuniculi

Microsporidia are amitochondriate eukaryotic microbes with fungal affinities and a common status of obligate intracellular parasites. A set of 13 potential genes encoding ATP-binding cassette (ABC) systems was identified in the fully sequenced genome of Encephalitozoon cuniculi. Our analyses of multiple alignments, phylogenetic trees and conserved motifs support a distribution of E. cuniculi ABC systems within only four subfamilies. Six half transporters are homologous to the yeast ATM1 mitochondrial protein, a finding which is in agreement with the hypothesis of a cryptic mitochondrion-derived compartment playing a role in the synthesis and transport of Fe-S clusters. Five half transporters are similar to the human ABCG1 and ABCG2 proteins, involved in regulation of lipid trafficking and anthracyclin resistance respectively. Two proteins with duplicated ABC domains are clearly candidate to non-transport ABC systems: the first is homologous to mammalian RNase L inhibitor and the second to the yeast translation initiation regulator GCN20. An unusual feature of ABC systems in E. cuniculi is the lack of homologs of P-glycoprotein and other ABC transporters which are involved in multiple drug resistance in a large number of eukaryotic microorganisms.     

Multidrug resistance in parasites: ABC transporters, P-glycoproteins and molecular modelling

Parasitic diseases, caused by protozoa, helminths and arthropods, rank among the most important problems in human and veterinary medicine, and in agriculture, leading to debilitating sicknesses and loss of life. In the absence of vaccines and with the general failure of vector eradication programs, drugs are the main line of defence, but the newest drugs are being tracked by the emergence of resistance in parasites, sharing ominous parallels with multidrug resistance in bacterial pathogens. Any of a number of mechanisms will elicit a drug resistance phenotype in parasites, including: active efflux, reduced uptake, target modification, drug modification, drug sequestration, by-pass shunting, or substrate competition. The role of ABC transporters in parasitic multidrug resistance mechanisms is being subjected to more scrutiny, due in part to the established roles of certain ABC transporters in human diseases, and also to an increasing portfolio of ABC transporters from parasite genome sequencing projects. For example, over 100 ABC transporters have been identified in the Escherichia coli genome, but to date only about 65 in all parasitic genomes. Long established laboratory investigations are now being assisted by molecular biology, bioinformatics, and computational modelling, and it is in these areas that the role of ABC transporters in parasitic multidrug resistance mechanisms may be defined and put in perspective with that of other proteins. We discuss ABC transporters in parasites, and conclude with an example of molecular modelling that identifies a new interaction between the structural domains of a parasite P-glycoprotein.     

A novel ATP-binding cassette transporter from Leishmania is involved in transport of phosphatidylcholine analogues and resistance to alkyl-phospholipids

ATP-binding cassette (ABC) transporters represent an important family of membrane proteins involved in drug resistance and other biological activities. The present work reports the characterization of the first ABC subfamily G (ABCG)-like transporter, LiABCG4, in the protozoan parasite Leishmania. LiABCG4 localized mainly to the parasite plasma membrane. Overexpression of this half-transporter reduced the accumulation of phosphatidylcholine analogues and conferred resistance to alkyl-phospholipids. Likewise, when expressed in Saccharomyces cerevisiae, the protein localized to the yeast plasma membrane and conferred resistance to alkyl-phospholipids. Post-Golgi secretory vesicles isolated from a LiABCG4-overexpressing yeast mutant contained the leishmanial ABC transporter and exhibited ATP-dependent, vanadate-sensitive transport of phosphatidylcholine analogues from the cytosolic to the lumenal leaflet of the vesicle membrane. Cross-linking showed dimerization of LiABCG4. These results suggest that LiABCG4 is involved in the active transport of phosphatidylcholine and resistance to alkyl-phospholipids in Leishmania.     

A novel transporter,Pfcrt, confers antimalarial drug resistance

The elucidation of the molecular details of drug resistance phenomena is a very active area of research that crosses many disciplinary boundaries. Drug resistance is due to altered drug-target interaction, and/or dysregulated signaling related to cell growth and death. Since many drugs need to rapidly diffuse into and within cells in order to find their targets, and since transmembrane ion transport is an important facet of cellular signaling, it is not surprising that membrane transport phenomena have been implicated in the evolution of drug resistance in tumor cells, bacteria, and intracellular parasites such as Plasmodium falciparum, the causative agent of the most lethal form of human malaria. The most infamous membrane transport protein involved in drug resistance is "MDR protein" or "P-glycoprotein" (Pgp),1 which was found to be overexpressed in drug-resistant tumor cells over 15 years ago, and which is representative of the ATP-binding cassette (ABC) superfamily that also includes the important cystic fibrosis transmembrane conductance regulator (CFTR) and sulfonyl urea receptor (SUR) ion channels. Availability of mouse and human Pgp cDNA rather quickly led to the identification of homologues in many species, including P. falciparum, and these were de facto assumed to be the ultimate determinants of drug resistance in these systems as well. However, research over the past 10 years has taught us that this assumption likely is wrong and that the situation is more complex. We now know that human Pgp plays a relatively minor role in clinically relevant tumor drug resistance, and that an integral membrane protein with no homology to the ABC superfamily, Pfcrt, ultimately confers chloroquine resistance in P. falciparum. Thus, the general hypothesis that membrane transport and membrane transport proteins are important in drug resistance phenomena remains correct, but at a genetic, biochemical, and physiological level we have recently witnessed a few very interesting surprises.     

ABC细胞膜转运蛋白及其介导的细胞多药耐药研究进展

ABC细胞膜转运蛋白是一个能转运多种底物的蛋白质家族,其在宿主对异物的防御机制和肿瘤细胞对抗癌药物的耐药性中发挥重要作用。ABC转运蛋白能将已进人细胞的外源性物质从胞内泵出胞外,是造成肿瘤细胞多药耐药的主要原因,其基因表达水平与细胞内药物浓度和耐药程度密切相关。近年来,肿瘤细胞多药耐药性研究炙手可热。我们简要综述ABC细胞膜转运蛋白的特点、分布、表达及其介导的细胞多药耐药方面的研究进展。     

Yeast ABC transporters-- a tale of sex, stress, drugs and aging

Yeast ATP-binding cassette (ABC) proteins are implicated in many biological phenomena, often acting at crossroads of vital cellular processes. Their functions encompass peptide pheromone secretion, regulation of mitochondrial function, vacuolar detoxification, as well as pleiotropic drug resistance and stress adaptation. Because yeast harbors several homologues of mammalian ABC proteins with medical importance, understanding their molecular mechanisms, substrate interaction and three-dimensional structure of yeast ABC proteins might help identifying new approaches aimed at combating drug resistance or other ABC-mediated diseases. This review provides a comprehensive discussion on the functions of the ABC protein family in the yeast Saccharomyces cerevisiae.     

ABC转运蛋白结构及在植物病原真菌中的功能研究进展

ABC (ATP-binding cassette)转运蛋白是最大的膜转运蛋白超家族之一,其主要功能是利用ATP水解产生的能量将底物进行逆浓度梯度运输.所有生物体都含有大量ABC蛋白. ABC蛋白位于细胞的不同空间,如细胞膜、液泡、线粒体和过氧化物酶体.通常,ABC转运蛋白由跨膜结构域(TMD)和核苷酸结合结构域(NBD)组成,分别与底物和ATP结合.NBD执行与ATP结合和水解,是ABC转运蛋白的动力引擎,TMD识别特异性配体.大多数ABC转运蛋白最初是通过研究生物体耐药性而被发现的,包括多效耐药(PDR)和多药耐药(MDR).本文对ABC转运蛋白的结构及作用机制,以及植物病原真菌中ABC转运蛋白功能的研究进展进行综述.     

PstS和PstB调控无机磷酸盐转运和介导细菌耐药的机制

无机磷酸盐(Pi)在菌体遗传、能量代谢及细胞内的信号传导等生物过程中发挥重要的作用。在细菌中,主要由磷酸盐特殊转运系统(Pst)和磷酸盐转运系统(Pit)来完成对Pi的吸收和利用。其中,Pst是在低磷胁迫下转运Pi的关键系统。近年来的研究表明,Pst系统除在调控Pi的代谢和平衡中发挥重要作用外,还介导细菌耐药、产毒和侵袭等。Pst系统是ABC转运蛋白家族的一种,一般由PstS、PstC、PstA、PstB和PhoU5个蛋白组成。其中,PstS和PstB蛋白是该系统中的关键蛋白。本文重点对PstS和PstB调控Pi转运和介导细菌耐药的分子机制进行综述,旨在为深入研究该系统与细菌耐药的关系,以及研发以PstS和PstB为靶点的新药提供参考。     

Complete Characterization of the Human ABC Gene Family

The human ATP-binding cassette (ABC) transporters comprise a large family of membrane transport proteins and play a vital role in many cellular processes. The genes provide functions as diverse as peptide transport, cholesterol and sterol transport, bile acid, retinoid, and iron transport. In addition some ABC genes play a role as regulatory elements. Many ABC genes play a role in human genetic diseases, and several are critical drug transport proteins overexpressed in drug resistant cells. Analysis of the gene products allows the genes to be grouped into seven different subfamilies.     

The ABC gene family in arthropods: Comparative genomics and role in insecticide transport and resistance

About a 100 years ago, the Drosophila white mutant marked the birth of Drosophila genetics. The white gene turned out to encode the first well studied ABC transporter in arthropods. The ABC gene family is now recognized as one of the largest transporter families in all kingdoms of life. The majority of ABC proteins function as primary-active transporters that bind and hydrolyze ATP while transporting a large diversity of substrates across lipid membranes. Although extremely well studied in vertebrates for their role in drug resistance, less is known about the role of this family in the transport of endogenous and exogenous substances in arthropods. The ABC families of five insect species, a crustacean and a chelicerate have been annotated in some detail. We conducted a thorough phylogenetic analysis of the seven arthropod and human ABC protein subfamilies, to infer orthologous relationships that might suggest conserved function. Most orthologous relationships were found in the ABCB half transporter, ABCD, ABCE and ABCF subfamilies, but specific expansions within species and lineages are frequently observed and discussed. We next surveyed the role of ABC transporters in the transport of xenobiotics/plant allelochemicals and their involvement in insecticide resistance. The involvement of ABC transporters in xenobiotic resistance in arthropods is historically not well documented, but an increasing number of studies using unbiased differential gene expression analysis now points to their importance. We give an overview of methods that can be used to link ABC transporters to resistance. ABC proteins have also recently been implicated in the mode of action and resistance to Bt toxins in Lepidoptera. Given the enormous interest in Bt toxicology in transgenic crops, such findings will provide an impetus to further reveal the role of ABC transporters in arthropods.     

Structural, mechanistic and clinical aspects of MRP1

The cDNA encoding ATP-binding cassette (ABC) multidrug resistance protein MRP1 was originally cloned from a drug-selected lung cancer cell line resistant to multiple natural product chemotherapeutic agents. MRP1 is the founder of a branch of the ABC superfamily whose members (from species as diverse as plants and yeast to mammals) share several distinguishing structural features that may contribute to functional and mechanistic similarities among this subgroup of transport proteins. In addition to its role in resistance to natural product drugs, MRP1 (and related proteins) functions as a primary active transporter of structurally diverse organic anions, many of which are formed by the biotransformation of various endo- and xenobiotics by Phase II conjugating enzymes, such as the glutathione S-transferases. MRP1 is involved in a number of glutathione-related cellular processes. Glutathione also appears to play a key role in MRP1-mediated drug resistance. This article reviews the discovery of MRP1 and its relationships with other ABC superfamily members, and summarizes current knowledge of the structure, transport functions and relevance of this protein to in vitro and clinical multidrug resistance.     

Multidrug resistance ABC transporters

Clinical multidrug resistance is caused by a group of integral membrane proteins that transport hydrophobic drugs and lipids across the cell membrane. One class of these permeases, known as multidrug resistance ATP binding cassette (ABC) transporters, translocate these molecules by coupling drug/lipid efflux with energy derived from the hydrolysis of ATP. In this review, we examine both the structures and conformational changes of multidrug resistance ABC transporters. Together with the available biochemical and structural evidence, we propose a general mechanism for hydrophobic substrate transport coupled to ATP hydrolysis.     

PfMDR1: mechanisms of transport modulation by functional polymorphisms

ATP-Binding Cassette (ABC) transporters are efflux pumps frequently associated with multidrug resistance in many biological systems, including malaria. Antimalarial drug-resistance involves an ABC transporter, PfMDR1, a homologue of P-glycoprotein in humans. Twenty years of research have shown that several single nucleotide polymorphisms in pfmdr1 modulate in vivo and/or in vitro drug susceptibility. The underlying physiological mechanism of the effect of these mutations remains unclear. Here we develop structural models for PfMDR1 in different predicted conformations, enabling the study of transporter motion. Such analysis of functional polymorphisms allows determination of their potential role in transport and resistance. The bacterial MsbA ABC pump is a PfMDR1 homologue. MsbA crystals in different conformations were used to create PfMDR1 models with Modeller software. Sequences were aligned with ClustalW and analysed by Ali2D revealing a high level of secondary structure conservation. To validate a potential drug binding pocket we performed antimalarial docking simulations. Using aminoquinoline as probe drugs in PfMDR1 mutated parasites we evaluated the physiology underlying the mechanisms of resistance mediated by PfMDR1 polymorphisms. We focused on the analysis of well known functional polymorphisms in PfMDR1 amino acid residues 86, 184, 1034, 1042 and 1246. Our structural analysis suggested the existence of two different biophysical mechanisms of PfMDR1 drug resistance modulation. Polymorphisms in residues 86/184/1246 act by internal allosteric modulation and residues 1034 and 1042 interact directly in a drug pocket. Parasites containing mutated PfMDR1 variants had a significant altered aminoquinoline susceptibility that appears to be dependent on the aminoquinoline lipophobicity characteristics as well as vacuolar efflux by PfCRT. We previously described the in vivo selection of PfMDR1 polymorphisms under antimalarial drug pressure. Now, together with recent PfMDR1 functional reports, we contribute to the understanding of the specific structural role of these polymorphisms in parasite antimalarial drug response.     

ABC multidrug transporters in schistosomes and other parasitic flatworms

Schistosomiasis, a neglected tropical disease affecting hundreds of millions, is caused by parasitic flatworms of the genus Schistosoma. Treatment and control of schistosomiasis relies almost exclusively on a single drug, praziquantel (PZQ), a dangerous situation for a disease of this magnitude. Though PZQ is highly effective overall, it has drawbacks, and reports of worms showing PZQ resistance, either induced in the laboratory or isolated from the field, are disconcerting. Multidrug transporters underlie multidrug resistance (MDR), a phenomenon in which resistance to a single drug is accompanied by unexpected cross-resistance to several structurally unrelated compounds. Some of the best studied multidrug transporters are members of the ancient and very large ATP-binding cassette (ABC) superfamily of efflux transporters. ABC multidrug transporters such as P-glycoprotein (Pgp; ABCB1) are also associated with drug resistance in parasites, including helminths such as schistosomes. In addition to their association with drug resistance, however, ABC transporters also function in a wide variety of physiological processes in metazoans. In this review, we examine recent studies that help define the role of schistosome ABC transporters in regulating drug susceptibility, and in normal schistosome physiology, including reproduction and excretory activity. We postulate that schistosome ABC transporters could be useful targets for compounds that enhance the effectiveness of current therapeutics as well as for agents that act as antischistosomals on their own.     

Changing the Transport of a Cell

Abstract

The review deals with some of the transport functions of different systems that have been implicated with several pathological disorders. Membrane transport role in parasitic diseases and metal resistance is discussed as a few selected examples. Among various limitations that are encountered in recombinant technology and in heterologous expression of proteins, transport functions of the host organisms cannot be ignored. Recently, membrane transport has acquired a new emerging role in multidrug resistance. Several membrane transporters, particularly ATP binding cassette (ABC) proteins that are involved in drug resistance, have been identified throughout the evolutionary scale. The review briefly emphasizes that membranes are not only important as structural elements but are also adopted to perform diverse functions.     

Expression of ATP-binding cassette multidrug transporters in the giant liver fluke Fasciola gigantica and their possible involvement in the transport of bile salts and anthelmintics

ATP-binding cassette (ABC) transporters belong to one of the largest protein families that either import or export a wide spectrum of different substrates. Certain members of this superfamily have been implicated in multidrug resistance in various types of cancer as well as in pathogenic microorganisms. The role of ABC proteins in parasitic multidrug resistance becomes increasingly evident. However, studies on ABC transporters in helminths have been limited to MDR1 and MRP orthologues. In the present study, we reported, for the first time, the expression and localization of ABC proteins including orthologues of MDR1, MRP1, BCRP, and BSEP in the giant liver fluke Fasciola gigantica. Furthermore, the functional activities of these ABC transporters were characterized in isolated fluke cells using a fluorescent substrate, rhodamine. The results revealed the inhibition of rhodamine efflux by cyclosporin A, a potent inhibitor of ABC transporters. Interestingly, our data suggested that these proteins might play a role in the export of bile salts, in particular, taurocholate. Although, we did not observe any substantial changes in rhodamine transport in the presence of anthelmintics under experimental conditions, however, our findings altogether shed light on the possible involvement of several members of ABC proteins in the mechanism of drug resistance as well as detoxification process in helminths to survive inside their hosts.