Efflux Pumps of Mycobacterium tuberculosis Play a Significant Role in Antituberculosis Activity of Potential Drug Candidates
01 May 2012-Antimicrobial Agents and Chemotherapy (American Society for Microbiology)-Vol. 56, Iss: 5, pp 2643-2651
TL;DR: It is shown that these four efflux pump KO mutants of M. tuberculosis play a vital role in mediating efflux of different chemical scaffolds and inhibitors of one or several of these efflux pumps could have a significant impact in the treatment of tuberculosis.
Abstract: Active efflux of drugs mediated by efflux pumps that confer drug resistance is one of the mechanisms developed by bacteria to counter the adverse effects of antibiotics and chemicals. To understand these efflux mechanisms in Mycobacterium tuberculosis, we generated knockout (KO) mutants of four efflux pumps of the pathogen belonging to different classes. We measured the MICs and kill values of two different compound classes on the wild type (WT) and the efflux pump (EP) KO mutants in the presence and absence of the efflux inhibitors verapamil and l-phenylalanyl-l-arginyl-β-naphthylamide (PAβN). Among the pumps studied, the efflux pumps belonging to the ABC (ATP-binding cassette) class, encoded by Rv1218c, and the SMR (small multidrug resistance) class, encoded by Rv3065, appear to play important roles in mediating the efflux of different chemical classes and antibiotics. Efflux pumps encoded by Rv0849 and Rv1258c also mediate the efflux of these compounds, but to a lesser extent. Increased killing is observed in WT M. tuberculosis cells by these compounds in the presence of either verapamil or PAβN. The efflux pump KO mutants were more susceptible to these compounds in the presence of efflux inhibitors. We have shown that these four efflux pumps of M. tuberculosis play a vital role in mediating efflux of different chemical scaffolds. Inhibitors of one or several of these efflux pumps could have a significant impact in the treatment of tuberculosis. The identification and characterization of Rv0849, a new efflux pump belonging to the MFS (major facilitator superfamily) class, are reported.
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TL;DR: A fragment comprising the N-heptenoyldifluorophenylalanine side chain of the pharmacologically optimizedADEPs potentiates the antibacterial activity of the ADEPs against actinobacteria to a greater extent than reserpine, a well-known efflux inhibitor.
Abstract: Membrane protein-mediated drug efflux is a phenomenon that compromises our ability to treat both infectious diseases and cancer. Accordingly, there is much interest in the development of strategies for suppression of the mechanisms by which therapeutic agents are effluxed. Here, using resistance to the cyclic acyldepsipeptide (ADEP) antibacterial agents as a model, we demonstrate a new counter-efflux strategy wherein a fragment of an actively exported bioactive compound competitively interferes with its efflux and potentiates its activity. A fragment comprising the N-heptenoyldifluorophenylalanine side chain of the pharmacologically optimized ADEPs potentiates the antibacterial activity of the ADEPs against actinobacteria to a greater extent than reserpine, a well-known efflux inhibitor. Beyond their validation of a new approach to studying molecular recognition by drug efflux pumps, our findings have important implications for killing Mycobacterium tuberculosis with ADEPs and reclaiming the efficacies of therapeutic agents whose activity has been compromised by efflux pumps.
13 citations
TL;DR: First complete genomic investigation of extensive drug resistance (XDR) in a nosocomial Stenotrophomonas maltophilia complex strain that is resistant to mainstream drugs and reveals its exclusive fourteen dynamic regions and highly enriched resistome comprising of two sulfonamide resistance genes on two diverse super-integrons of chromosomal origin.
12 citations
TL;DR: By freeze fracture EM, this work found three different size distributions in three different lipid environments for TBsmr indicating different oligomeric states, of particular importance is the link between protein function, oligomersic state and lipid composition.
11 citations
Cites background from "Efflux Pumps of Mycobacterium tuber..."
...tuberculosis [21], which belongs to the clinically most challenging MDR organisms [22]....
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TL;DR: It is demonstrated that iron deprivation impairs drug efflux pump activity and confers synergism for anti-TB drugs in presence of efflux Pump inhibitors against MTB, and MmpL3 being a promiscuousAnti-TB target, metal chelation strategy could be adopted to boost the effectiveness of current anti- TB drug regimes to combat drug resistance TB.
Abstract: Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) is a global threat to human health hence better understanding of the MTB pathogenesis for improved therapeutics requires immediate attention. Emergence of drug-resistant strains has stimulated an urgent need for adopting new strategies that could be implemented to control TB. One of the contributing mechanisms by which MTB evades drug doses is overexpression of drug efflux pumps. Thus blocking or modulating the functionality of efflux pumps represents an attractive approach to combat drug resistance. Iron is a critical micronutrient required for MTB survival and not freely available inside the host. In this study, we demonstrated that iron deprivation impairs drug efflux pump activity and confers synergism for anti-TB drugs in presence of efflux pump inhibitors against MTB. Mechanistic insights revealed that iron deprivation inhibit resistance nodulation division superfamily transporter activity. This was evident from enhanced Nile red accumulation and reduced expression of MmpL3, a transmembrane promising target involved in mycolic acid transport across membrane. Furthermore, iron deprivation led to abrogated MA transport particularly of class methoxy-MA which was confirmed by TLC and mass spectrometry based lipidome analysis. Additionally, iron deprivation leads to enhanced membrane fluidity in MTB. Together, MmpL3 being a promiscuous anti-TB target, metal chelation strategy could be adopted to boost the effectiveness of current anti-TB drug regimes to combat drug resistance TB.
11 citations
TL;DR: In these studies, a structural approach to the discovery, structure-guided design, and synthesis of a series of adenosine analogues that displayed inhibition constants ranging from 5 to 120 nM against MtbAdoK showed the molecular basis of inhibition, potency, and selectivity.
Abstract: Mycobacterium tuberculosis adenosine kinase (MtbAdoK) is an essential enzyme of Mtb and forms part of the purine salvage pathway within mycobacteria. Evidence suggests that the purine salvage pathway might play a crucial role in Mtb survival and persistence during its latent phase of infection. In these studies, we adopted a structural approach to the discovery, structure-guided design, and synthesis of a series of adenosine analogues that displayed inhibition constants ranging from 5 to 120 nM against the enzyme. Two of these compounds exhibited low micromolar activity against Mtb with half maximal effective inhibitory concentrations of 1.7 and 4.0 μM. Our selectivity and preliminary pharmacokinetic studies showed that the compounds possess a higher degree of specificity against MtbAdoK when compared with the human counterpart and are well tolerated in rodents, respectively. Finally, crystallographic studies showed the molecular basis of inhibition, potency, and selectivity and revealed the presence of a...
11 citations
References
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TL;DR: Evidence is presented that multidrug-resistance efflux pumps have roles in bacterial pathogenicity and it is proposed that these pumps therefore have greater clinical relevance than is usually attributed to them.
Abstract: 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.
1,367 citations
"Efflux Pumps of Mycobacterium tuber..." refers background in this paper
...tuberculosis within its human host, as well (25)....
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TL;DR: The microbial transcriptome served as a bioprobe of the MTB phagosomal environment, showing it to be nitrosative, oxidative, functionally hypoxic, carbohydrate poor, and capable of perturbing the pathogen's cell envelope.
Abstract: Little is known about the biochemical environment in phagosomes harboring an infectious agent. To assess the state of this organelle we captured the transcriptional responses of Mycobacterium tuberculosis (MTB) in macrophages from wild-type and nitric oxide (NO) synthase 2–deficient mice before and after immunologic activation. The intraphagosomal transcriptome was compared with the transcriptome of MTB in standard broth culture and during growth in diverse conditions designed to simulate features of the phagosomal environment. Genes expressed differentially as a consequence of intraphagosomal residence included an interferon γ– and NO-induced response that intensifies an iron-scavenging program, converts the microbe from aerobic to anaerobic respiration, and induces a dormancy regulon. Induction of genes involved in the activation and β-oxidation of fatty acids indicated that fatty acids furnish carbon and energy. Induction of σE-dependent, sodium dodecyl sulfate–regulated genes and genes involved in mycolic acid modification pointed to damage and repair of the cell envelope. Sentinel genes within the intraphagosomal transcriptome were induced similarly by MTB in the lungs of mice. The microbial transcriptome thus served as a bioprobe of the MTB phagosomal environment, showing it to be nitrosative, oxidative, functionally hypoxic, carbohydrate poor, and capable of perturbing the pathogen's cell envelope.
1,352 citations
"Efflux Pumps of Mycobacterium tuber..." refers background in this paper
...Several efflux pumps and their regulators are also induced during macrophage infection (1, 20, 32, 34)....
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TL;DR: This review focuses on chromosomally encoded pumps in bacteria that cause infections in humans, and suggests that resistance nodulation division systems are important in pathogenicity and/or survival in a particular ecological niche.
Abstract: 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.
1,078 citations
"Efflux Pumps of Mycobacterium tuber..." refers background in this paper
...No efflux pump inhibitor has yet reached clinical practice, but it is clear that this area of drug development offers a lot of promise, as it will further enhance the effective use of several drugs that have previously been considered to be of great clinical value and also new molecules that are currently under development (24)....
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...This is possible because of the redundancy of their functions, which may overlap extensively (24, 27)....
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TL;DR: Given the clinical significance of multidrug (and drug-specific) exporters, efflux must be considered in formulating strategies/approaches to treating drug-resistant infections, both in the development of new agents less impacted by efflux and in targeting efflux directly with efflux inhibitors.
Abstract: Antibiotic resistance continues to plague antimicrobial chemotherapy of infectious disease. And while true biocide resistance is as yet unrealized, in vitro and in vivo episodes of reduced biocide susceptibility are common and the history of antibiotic resistance should not be ignored in the development and use of biocidal agents. Efflux mechanisms of resistance, both drug specific and multidrug, are important determinants of intrinsic and/or acquired resistance to these antimicrobials, with some accommodating both antibiotics and biocides. This latter raises the spectre (as yet generally unrealized) of biocide selection of multiple antibiotic-resistant organisms. Multidrug efflux mechanisms are broadly conserved in bacteria, are almost invariably chromosome-encoded and their expression in many instances results from mutations in regulatory genes. In contrast, drug-specific efflux mechanisms are generally encoded by plasmids and/or other mobile genetic elements (transposons, integrons) that carry additional resistance genes, and so their ready acquisition is compounded by their association with multidrug resistance. While there is some support for the latter efflux systems arising from efflux determinants of self-protection in antibiotic-producing Streptomyces spp. and, thus, intended as drug exporters, increasingly, chromosomal multidrug efflux determinants, at least in Gram-negative bacteria, appear not to be intended as drug exporters but as exporters with, perhaps, a variety of other roles in bacterial cells. Still, given the clinical significance of multidrug (and drug-specific) exporters, efflux must be considered in formulating strategies/approaches to treating drug-resistant infections, both in the development of new agents, for example, less impacted by efflux and in targeting efflux directly with efflux inhibitors.
979 citations
"Efflux Pumps of Mycobacterium tuber..." refers background in this paper
...This is possible because of the redundancy of their functions, which may overlap extensively (24, 27)....
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TL;DR: The multifaceted implications of drug efflux transporters warrant novel strategies to combat multidrug resistance in bacteria.
Abstract: Drug efflux pumps play a key role in drug resistance and also serve other functions in bacteria. There has been a growing list of multidrug and drug-specific efflux pumps characterized from bacteria of human, animal, plant and environmental origins. These pumps are mostly encoded on the chromosome, although they can also be plasmid-encoded. A previous article in this journal provided a comprehensive review regarding efflux-mediated drug resistance in bacteria. In the past 5 years, significant progress has been achieved in further understanding of drug resistance-related efflux transporters and this review focuses on the latest studies in this field since 2003. This has been demonstrated in multiple aspects that include but are not limited to: further molecular and biochemical characterization of the known drug efflux pumps and identification of novel drug efflux pumps; structural elucidation of the transport mechanisms of drug transporters; regulatory mechanisms of drug efflux pumps; determining the role of the drug efflux pumps in other functions such as stress responses, virulence and cell communication; and development of efflux pump inhibitors. Overall, the multifaceted implications of drug efflux transporters warrant novel strategies to combat multidrug resistance in bacteria.
755 citations