Fungal ATP-binding cassette (ABC) transporters in drug resistance & detoxification. - Related Documents




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76401.0000Fungal ATP-binding cassette (ABC) transporters in drug resistance & detoxification. Pleiotropic drug resistance (PDR) is a well-described phenomenon occurring in fungi. PDR shares several similarities with processes in bacteria and higher eukaryotes. In mammalian cells, multidrug resistance (MDR) develops from an initial single drug resistance, eventually leading to a broad cross-resistance to many structurally and functionally unrelated compounds. Notably, a number of membrane-embedded energy-consuming ATP-binding cassette (ABC) transporters have been implicated in the development of PDR/MDR phenotypes. The yeast Saccharomyces cerevisiae genome harbors some 30 genes encoding ABC proteins, several of which mediate PDR. Therefore, yeast served as an important model organism to study the functions of evolutionary conserved ABC genes, including those mediating clinical antifungal resistance in fungal pathogens. Moreover, yeast cells lacking endogenous ABC pumps are hypersensitive to many antifungal drugs, making them suitable for functional studies and cloning of ABC transporters from fungal pathogens such as Candida albicans. This review discusses drug resistance phenomena mediated by ABC transporters in the model system S. cerevisiae and certain fungal pathogens.200616611035
76510.9998Yeast ATP-binding cassette transporters: cellular cleaning pumps. Numerous ATP-binding cassette (ABC) proteins have been implicated in multidrug resistance, and some are also intimately connected to genetic diseases. For example, mammalian ABC proteins such as P-glycoproteins or multidrug resistance-associated proteins are associated with multidrug resistance phenomena (MDR), thus hampering anticancer therapy. Likewise, homologues in bacteria, fungi, or parasites are tightly associated with multidrug and antibiotic resistance. Several orthologues of mammalian MDR genes operate in the unicellular eukaryote Saccharomyces cerevisiae. Their functions have been linked to stress response, cellular detoxification, and drug resistance. This chapter discusses those yeast ABC transporters implicated in pleiotropic drug resistance and cellular detoxification. We describe strategies for their overexpression, biochemical purification, functional analysis, and a reconstitution in phospholipid vesicles, all of which are instrumental to better understanding their mechanisms of action and perhaps their physiological function.200516399365
828020.9998Regulation of the Expression of Bacterial Multidrug Exporters by Two-Component Signal Transduction Systems. Bacterial multidrug exporters confer resistance to a wide range of antibiotics, dyes, and biocides. Recent studies have shown that there are many multidrug exporters encoded in bacterial genome. For example, it was experimentally identified that E. coli has at least 20 multidrug exporters. Because many of these multidrug exporters have overlapping substrate spectra, it is intriguing that bacteria, with their economically organized genomes, harbor such large sets of multidrug exporter genes. The key to understanding how bacteria utilize these multiple exporters lies in the regulation of exporter expression. Bacteria have developed signaling systems for eliciting a variety of adaptive responses to their environments. These adaptive responses are often mediated by two-component regulatory systems. In this chapter, the method to identify response regulators that affect expression of multidrug exporters is described.201829177834
79630.9998The internal gene duplication and interrupted coding sequences in the MmpL genes of Mycobacterium tuberculosis: Towards understanding the multidrug transport in an evolutionary perspective. The multidrug resistance has emerged as a major problem in the treatment of many of the infectious diseases. Tuberculosis (TB) is one of such disease caused by Mycobacterium tuberculosis. There is short term chemotherapy to treat the infection, but the main hurdle is the development of the resistance to antibiotics. This resistance is primarily due to the impermeable mycolic acid rich cell wall of the bacteria and other factors such as efflux of antibiotics from the bacterial cell. The MmpL (Mycobacterial Membrane Protein Large) proteins of mycobacteria are involved in the lipid transport and antibiotic efflux as indicated by the preliminary reports. We present here, comprehensive comparative sequence and structural analysis, which revealed topological signatures shared by the MmpL proteins and RND (Resistance Nodulation Division) multidrug efflux transporters. This provides evidence in support of the notion that they belong to the extended RND permeases superfamily. In silico modelled tertiary structures are in homology with an integral membrane component present in all of the RND efflux pumps. We document internal gene duplication and gene splitting events happened in the MmpL genes, which further elucidate the molecular functions of these putative transporters in an evolutionary perspective.201525841626
951340.9997Distribution and physiology of ABC-type transporters contributing to multidrug resistance in bacteria. Membrane proteins responsible for the active efflux of structurally and functionally unrelated drugs were first characterized in higher eukaryotes. To date, a vast number of transporters contributing to multidrug resistance (MDR transporters) have been reported for a large variety of organisms. Predictions about the functions of genes in the growing number of sequenced genomes indicate that MDR transporters are ubiquitous in nature. The majority of described MDR transporters in bacteria use ion motive force, while only a few systems have been shown to rely on ATP hydrolysis. However, recent reports on MDR proteins from gram-positive organisms, as well as genome analysis, indicate that the role of ABC-type MDR transporters in bacterial drug resistance might be underestimated. Detailed structural and mechanistic analyses of these proteins can help to understand their molecular mode of action and may eventually lead to the development of new strategies to counteract their actions, thereby increasing the effectiveness of drug-based therapies. This review focuses on recent advances in the analysis of ABC-type MDR transporters in bacteria.200717804667
79750.9997Increasing the PACE of characterising novel transporters by functional genomics. Since the late 1990's the genome sequences for thousands of species of bacteria have been released into public databases. The release of each new genome sequence typically revealed the presence of tens to hundreds of uncharacterised genes encoding putative membrane proteins and more recently, microbial metagenomics has revealed countless more of these uncharacterised genes. Given the importance of small molecule efflux in bacteria, it is likely that a significant proportion of these genes encode for novel efflux proteins, but the elucidation of these functions is challenging. We used transcriptomics to predict that the function of a gene encoding a hypothetical membrane protein is in efflux-mediated antimicrobial resistance. We subsequently confirmed this function and the likely native substrates of the pump by using detailed biochemical and biophysical analyses. Functional studies of homologs of the protein from other bacterial species determined that the protein is a prototype for a family of multidrug efflux pumps - the Proteobacterial Antimicrobial Compound Efflux (PACE) family. The general functional genomics approach used here, and its expansion to functional metagenomics, will very likely reveal the identities of more efflux pumps and other transport proteins of scientific, clinical and commercial interest in the future.202134492595
78260.9997Discovery of inhibitors of Pseudomonas aeruginosa virulence through the search for natural-like compounds with a dual role as inducers and substrates of efflux pumps. Multidrug efflux pumps are ancient elements encoded in every genome, from bacteria to humans. In bacteria, in addition to antibiotics, efflux pumps extrude a wide range of substrates, including quorum sensing signals, bacterial metabolites, or plant-produced compounds. This indicates that their original functions may differ from their recently acquired role in the extrusion of antibiotics during human infection. Concerning plant-produced compounds, some of them are substrates and inducers of the same efflux pump, suggesting a coordinated plant/bacteria coevolution. Herein we analyse the ability of 1243 compounds from a Natural Product-Like library to induce the expression of P. aeruginosa mexCD-oprJ or mexAB-oprM efflux pumps' encoding genes. We further characterized natural-like compounds that do not trigger antibiotic resistance in P. aeruginosa and that act as virulence inhibitors, choosing those that were not only inducers but substrates of the same efflux pump. Four compounds impair swarming motility, exotoxin secretion through the Type 3 Secretion System (T3SS) and the ability to kill Caenorhabditis elegans, which might be explained by the downregulation of genes encoding flagellum and T3SS. Our results emphasize the possibility of discovering new anti-virulence drugs by screening natural or natural-like libraries for compounds that behave as both, inducers and substrates of efflux pumps.202133818002
821570.9997Insight into Two ABC Transporter Families Involved in Lantibiotic Resistance. Antimicrobial peptides, which contain (methyl)-lanthionine-rings are called lantibiotics. They are produced by several Gram-positive bacteria and are mainly active against these bacteria. Although these are highly potent antimicrobials, some human pathogenic bacteria express specific ABC transporters that confer resistance and counteract their antimicrobial activity. Two distinct ABC transporter families are known to be involved in this process. These are the Cpr- and Bce-type ABC transporter families, named after their involvement in cationic peptide resistance in Clostridium difficile, and bacitracin efflux in Bacillus subtilis, respectively. Both resistance systems differentiate to each other in terms of the proteins involved. Here, we summarize the current knowledge and describe the divergence as well as the common features present in both the systems to confer lantibiotic resistance.201729404338
630880.9997A shotgun antisense approach to the identification of novel essential genes in Pseudomonas aeruginosa. BACKGROUND: Antibiotics in current use target a surprisingly small number of cellular functions: cell wall, DNA, RNA, and protein biosynthesis. Targeting of novel essential pathways is expected to play an important role in the discovery of new antibacterial agents against bacterial pathogens, such as Pseudomonas aeruginosa, that are difficult to control because of their ability to develop resistance, often multiple, to all current classes of clinical antibiotics. RESULTS: We aimed to identify novel essential genes in P. aeruginosa by shotgun antisense screening. This technique was developed in Staphylococcus aureus and, following a period of limited success in Gram-negative bacteria, has recently been used effectively in Escherichia coli. To also target low expressed essential genes, we included some variant steps that were expected to overcome the non-stringent regulation of the promoter carried by the expression vector used for the shotgun antisense libraries. Our antisense screenings identified 33 growth-impairing single-locus genomic inserts that allowed us to generate a list of 28 "essential-for-growth" genes: five were "classical" essential genes involved in DNA replication, transcription, translation, and cell division; seven were already reported as essential in other bacteria; and 16 were "novel" essential genes with no homologs reported to have an essential role in other bacterial species. Interestingly, the essential genes in our panel were suggested to take part in a broader range of cellular functions than those currently targeted by extant antibiotics, namely protein secretion, biosynthesis of cofactors, prosthetic groups and carriers, energy metabolism, central intermediary metabolism, transport of small molecules, translation, post-translational modification, non-ribosomal peptide synthesis, lipopolysaccharide synthesis/modification, and transcription regulation. This study also identified 43 growth-impairing inserts carrying multiple loci targeting 105 genes, of which 25 have homologs reported as essential in other bacteria. Finally, four multigenic growth-impairing inserts belonged to operons that have never been reported to play an essential role. CONCLUSIONS: For the first time in P. aeruginosa, we applied regulated antisense RNA expression and showed the feasibility of this technology for the identification of novel essential genes.201424499134
935790.9997The bifunctional enzymes of antibiotic resistance. The evolutionary union of two genes--each encoding proteins of complementary enzymatic activity--into a single gene so as to allow the coordinated expression of these activities as a fusion polypeptide, is an increasingly recognized biological occurrence. The result of this genetic union is the bifunctional enzyme. This fusion of separate catalytic activities into a single protein, whose gene is regulated by a single promoter, is seen especially where the coordinated expression of the separate activities is highly desirable. Increasingly, a circumstance driving the evolution of the bifunctional enzyme in bacteria is the resistance response of bacteria to antibiotic chemotherapy. We summarize the knowledge on bifunctional antibiotic-resistance enzymes, as possible harbingers of clinically significant resistance mechanisms of the future.200919615931
9105100.9997tRNA Methylation Is a Global Determinant of Bacterial Multi-drug Resistance. Gram-negative bacteria are intrinsically resistant to drugs because of their double-membrane envelope structure that acts as a permeability barrier and as an anchor for efflux pumps. Antibiotics are blocked and expelled from cells and cannot reach high-enough intracellular concentrations to exert a therapeutic effect. Efforts to target one membrane protein at a time have been ineffective. Here, we show that m(1)G37-tRNA methylation determines the synthesis of a multitude of membrane proteins via its control of translation at proline codons near the start of open reading frames. Decreases in m(1)G37 levels in Escherichia coli and Salmonella impair membrane structure and sensitize these bacteria to multiple classes of antibiotics, rendering them incapable of developing resistance or persistence. Codon engineering of membrane-associated genes reduces their translational dependence on m(1)G37 and confers resistance. These findings highlight the potential of tRNA methylation in codon-specific translation to control the development of multi-drug resistance in Gram-negative bacteria.201930981730
772110.9997A Transcriptomic Approach to Identify Novel Drug Efflux Pumps in Bacteria. The core genomes of most bacterial species include a large number of genes encoding putative efflux pumps. The functional roles of most of these pumps are unknown, however, they are often under tight regulatory control and expressed in response to their substrates. Therefore, one way to identify pumps that function in antimicrobial resistance is to examine the transcriptional responses of efflux pump genes to antimicrobial shock. By conducting complete transcriptomic experiments following antimicrobial shock treatments, it may be possible to identify novel drug efflux pumps encoded in bacterial genomes. In this chapter we describe a complete workflow for conducting transcriptomic analyses by RNA sequencing, to determine transcriptional changes in bacteria responding to antimicrobials.201829177833
787120.9997Multidrug-resistance efflux pumps - not just for resistance. It is well established that multidrug-resistance efflux pumps encoded by bacteria can confer clinically relevant resistance to antibiotics. It is now understood that these efflux pumps also have a physiological role(s). They can confer resistance to natural substances produced by the host, including bile, hormones and host-defence molecules. In addition, some efflux pumps of the resistance nodulation division (RND) family have been shown to have a role in the colonization and the persistence of bacteria in the host. Here, I present the accumulating evidence that multidrug-resistance efflux pumps have roles in bacterial pathogenicity and propose that these pumps therefore have greater clinical relevance than is usually attributed to them.200616845433
6307130.9997High-density transposon libraries utilising outward-oriented promoters identify mechanisms of action and resistance to antimicrobials. The use of bacterial transposon mutant libraries in phenotypic screens is a well-established technique for determining which genes are essential or advantageous for growth in conditions of interest. Standard, inactivating, transposon libraries cannot give direct information about genes whose over-expression gives a selective advantage. We report the development of a system wherein outward-oriented promoters are included in mini-transposons, generation of transposon mutant libraries in Escherichia coli and Pseudomonas aeruginosa and their use to probe genes important for growth under selection with the antimicrobial fosfomycin, and a recently-developed leucyl-tRNA synthase inhibitor. In addition to the identification of known mechanisms of action and resistance, we identify the carbon-phosphorous lyase complex as a potential resistance liability for fosfomycin in E. coli and P. aeruginosa. The use of this technology can facilitate the development of novel mechanism-of-action antimicrobials that are urgently required to combat the increasing threat worldwide from antimicrobial-resistant pathogenic bacteria.202033186989
788140.9996Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria. Efflux pump genes and proteins are present in both antibiotic-susceptible and antibiotic-resistant bacteria. Pumps may be specific for one substrate or may transport a range of structurally dissimilar compounds (including antibiotics of multiple classes); such pumps can be associated with multiple drug (antibiotic) resistance (MDR). However, the clinical relevance of efflux-mediated resistance is species, drug, and infection dependent. This review focuses on chromosomally encoded pumps in bacteria that cause infections in humans. Recent structural data provide valuable insights into the mechanisms of drug transport. MDR efflux pumps contribute to antibiotic resistance in bacteria in several ways: (i) inherent resistance to an entire class of agents, (ii) inherent resistance to specific agents, and (iii) resistance conferred by overexpression of an efflux pump. Enhanced efflux can be mediated by mutations in (i) the local repressor gene, (ii) a global regulatory gene, (iii) the promoter region of the transporter gene, or (iv) insertion elements upstream of the transporter gene. Some data suggest that resistance nodulation division systems are important in pathogenicity and/or survival in a particular ecological niche. Inhibitors of various efflux pump systems have been described; typically these are plant alkaloids, but as yet no product has been marketed.200616614254
9338150.9996Polyamines in bacteria: pleiotropic effects yet specific mechanisms. Extensive data in a wide range of organisms point to the importance of polyamine homeostasis for growth. The two most common polyamines found in bacteria are putrescine and spermidine. The investigation of polyamine function in bacteria has revealed that they are involved in a number of functions other than growth, which include incorporation into the cell wall and biosynthesis of siderophores. They are also important in acid resistance and can act as a free radical ion scavenger. More recently it has been suggested that polyamines play a potential role in signaling cellular differentiation in Proteus mirabilis. Polyamines have also been shown to be essential in biofilm formation in Yersinia pestis. The pleiotropic nature of polyamines has made their investigation difficult, particularly in discerning any specific effect from more global growth effects. Here we describe key developments in the investigation of the function of polyamines in bacteria that have revealed new roles for polyamines distinct from growth. We describe the bacterial genes necessary for biosynthesis and transport, with a focus on Y. pestis. Finally we review a novel role for polyamines in the regulation of biofilm development in Y. pestis and provide evidence that the investigation of polyamines in Y. pestis may provide a model for understanding the mechanism through which polyamines regulate biofilm formation.200717966408
777160.9996Multiantibiotic resistance caused by active drug extrusion in Pseudomonas aeruginosa and other gram-negative bacteria. All living organisms have been exposed to noxious compounds throughout their long evolutionary history and those surviving have evolved to fabricate devices that detoxicate and extrude these life threatening substances. It is likely, therefore, that all viable organisms, from bacteria to mammals, are equipped with active extrusion machinery. When bacteria are attacked by antibiotics, they use these tactics to combat the drugs and to develop resistance. Drugs extrusion machinery in Gram-negative bacteria is complex, consisting of the inner membrane transporter which acts as an energy-dependent extrusion pump; a binding protein which presumably connect both membranes; and the outer membrane exit channel. The extrusion pump assemblies are often encoded by chromosomal genes and might be expressed by mutation(s) or induced in the presence of drug(s).19979353746
6326170.9996Identification of novel metronidazole-inducible genes in Mycobacterium smegmatis using a customized amplification library. The incidence of antibiotic resistance in pathogenic bacteria is rising. Bacterial resistance may be a natural defense of organisms, or it may result from spontaneous mutations or the acquisition of exogenous resistance genes. We grew spontaneous metronidazole-resistant Mycobacterium smegmatis mutants on solid medium cultures and employed differential expression using a customized amplification library to analyze the global gene profiles of metronidazole-resistant mutants under hypoxic conditions. In total, 66 genes involved in metronidazole resistance were identified and functionally characterized using the gene role category of M. smegmatis. Overall, genes associated with cell wall synthesis, such as methyltransferase and glycosyltransferase, and genes encoding drug transporters were highly expressed. The genes may be involved in the natural drug resistance of mycobacteria by increasing mycobacterial cell wall permeability and the efflux pumps of active drugs. In addition, the genes may play a role in dormancy. The genes identified in this study may lead to a better understanding of the mechanisms of metronidazole resistance during dormancy.200818373646
789180.9996Antibiotic efflux mechanisms. Bacterial genomes sequenced to date almost invariably contain genes apparently coding for multidrug efflux pumps, and the yeast genome contains more than 30 putative multidrug efflux genes. Thus it is not surprising that multidrug efflux is a major cause of intrinsic drug resistance in many microorganisms, and plays an even more prominent role in organisms with a low-permeability cell wall, such as Gram negative bacteria in general and Pseudomonas aeruginosa in particular, as well as Mycobacterium species. Furthermore, overproduction of intrinsic pumps, or acquisition of pump genes from external sources, often results in high levels of resistance. This review discusses the classification of efflux proteins, their mechanism of action, the regulation of their expression, and the clinical significance of efflux pumps.199917035817
9291190.9996Highlights of Streptomyces genetics. Sixty years ago, the actinomycetes, which include members of the genus Streptomyces, with their bacterial cellular dimensions but a mycelial growth habit like fungi, were generally regarded as a possible intermediate group, and virtually nothing was known about their genetics. We now know that they are bacteria, but with many original features. Their genome is linear with a unique mode of replication, not circular like those of nearly all other bacteria. They transfer their chromosome from donor to recipient by a conjugation mechanism, but this is radically different from the E. coli paradigm. They have twice as many genes as a typical rod-shaped bacterium like Escherichia coli or Bacillus subtilis, and the genome typically carries 20 or more gene clusters encoding the biosynthesis of antibiotics and other specialised metabolites, only a small proportion of which are expressed under typical laboratory screening conditions. This means that there is a vast number of potentially valuable compounds to be discovered when these 'sleeping' genes are activated. Streptomyces genetics has revolutionised natural product chemistry by facilitating the analysis of novel biosynthetic steps and has led to the ability to engineer novel biosynthetic pathways and hence 'unnatural natural products', with potential to generate lead compounds for use in the struggle to combat the rise of antimicrobial resistance.201931189905