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: The role of efflux transporters in TB drug disposition is reviewed and the promise ofefflux pump inhibition from a novel holistic perspective is evaluated.
Abstract: Insight into drug transport mechanisms is highly relevant to the efficacious treatment of tuberculosis (TB). Major problems in TB treatment are related to the transport of antituberculosis (anti-TB) drugs across human and mycobacterial membranes, affecting the concentrations of these drugs systemically and locally. Firstly, transporters located in the intestines, liver, and kidneys all determine the pharmacokinetics and pharmacodynamics of anti-TB drugs, with a high risk of drug-drug interactions in the setting of concurrent use of antimycobacterial, antiretroviral, and antidiabetic agents. Secondly, human efflux transporters limit the penetration of anti-TB drugs into the brain and cerebrospinal fluid, which is especially important in the treatment of TB meningitis. Finally, efflux transporters located in the macrophage and Mycobacterium tuberculosis cell membranes play a pivotal role in the emergence of phenotypic tolerance and drug resistance, respectively. We review the role of efflux transporters in TB drug disposition and evaluate the promise of efflux pump inhibition from a novel holistic perspective.
42 citations
TL;DR: Results from this study indicate that a 1599 clarithromycin combination is potentially viable, providing the drug exposures can be carefully monitored, and the importance of validating in vitro synergism and the challenge of matching drug exposures to obtain a synergistic outcome in vivo is highlighted.
Abstract: Spectinamides are new semi-synthetic spectinomycin derivatives with potent anti-tubercular activity. The reported synergism of the precursor spectinomycin with other antibiotics prompted us to examine whether spectinamides sensitize M. tuberculosis to other antibiotics not traditionally used in the treatment of tuberculosis to potentially expand therapeutic options for MDR/XDR Tuberculosis. Whole cell synergy checkerboard screens were performed using the laboratory strain M. tuberculosis H37Rv, lead spectinamide 1599, and a broad panel of 27 antibiotics. In vitro, 1599 synergized with 11 drugs from 6 antibiotic classes. The observed synergy was tested against clinical isolates confirming synergy with Clarithromycin, Doxycycline and Clindamycin, combinations of which were taken forward for in vivo efficacy determination. Co-administration of 1599 and clarithromycin provided additional bacterial killing in a mouse model of acute tuberculosis infection, but not in a chronic infection model. Further studies indicated that mismatched drug exposure profiles likely permitted induction of phenotypic clarithromycin resistance and subsequent loss of synergism. These studies highlight the importance of validating in vitro synergism and the challenge of matching drug exposures to obtain a synergistic outcome in vivo. Results from this study indicate that a 1599 clarithromycin combination is potentially viable, providing the drug exposures can be carefully monitored.
42 citations
TL;DR: Genome-wide expression profiling makes it possible to pinpoint genes differentially expressed between drug sensitive Mycobacterium and multidrug-resistant clinical isolates, and genes involved in adaptive responses and drug tolerance could become new targets for improving the efficacy of existing antibiotics.
Abstract: Antibiotics can stimulate or depress gene expression in bacteria. The analysis of transcriptional responses of Mycobacterium to antimycobacterial compounds has improved our understanding of the mode of action of various drug classes and the efficacy and effect of such compounds on the global metabolism of Mycobacterium. This approach can provide new insights for known antibiotics, for example those currently used for tuberculosis treatment, as well as help to identify the mode of action and predict the targets of new compounds identified by whole-cell screening assays. In addition, changes in gene expression profiles after antimycobacterial treatment can provide information about the adaptive ability of bacteria to escape the effects of antibiotics and allow monitoring of the physiology of the bacteria during treatment. Genome-wide expression profiling also makes it possible to pinpoint genes differentially expressed between drug sensitive Mycobacterium and multidrug-resistant clinical isolates. Finally, genes involved in adaptive responses and drug tolerance could become new targets for improving the efficacy of existing antibiotics.
41 citations
Cites background from "Efflux Pumps of Mycobacterium tuber..."
...Moreover, Mycobacterium can tolerate antibiotics due to their ability to reduce their intracellular accumulation by increasing active efflux of these molecules (Poole, 2007; Balganesh et al., 2012)....
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TL;DR: Recent high-throughput phenotypic screening strategies for anti-M.
Abstract: The rise of multi- and extensively drug-resistant Mycobacterium tuberculosis (M. tb) strains and co-infection with human immunodeficiency virus has escalated the need for new anti-M. tb drugs. Numerous challenges associated with the M. tb, in particular slow growth and pathogenicity level 3, discouraged use of this organism in past primary screening efforts. From current knowledge of the physiology and drug susceptibility of mycobacteria in general and M. tb specifically, it can be assumed that many potentially useful drug leads were missed by failing to screen directly against this pathogen. This review discusses recent high-throughput phenotypic screening strategies for anti-M. tb drug discovery. Emphasis is placed on prioritization of hits, including their extensive biological and chemical profiling, as well as the development status of promising drug candidates discovered with phenotypic screening.
40 citations
TL;DR: The study indicates that the efflux pump knockout strains are more susceptible to PSI than the wild type, strongly suggesting an important role for efflux pumps in the intrinsic resistance of M. tuberculosis toPSI.
Abstract: We evaluated the contributions of Mycobacterium tuberculosis efflux pumps towards intrinsic resistance to different classes of peptidoglycan synthesis inhibitors (PSI). Our study indicates that the efflux pump knockout strains are more susceptible to PSI than the wild type. Vancomycin and ceftriaxone exhibited up to 3 log increased kill on efflux pump mutants compared to the wild-type strain, strongly suggesting an important role for efflux pumps in the intrinsic resistance of M. tuberculosis to PSI.
38 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)....
[...]
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