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Journal ArticleDOI

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.
Citations
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Journal ArticleDOI
TL;DR: The percent growth values averaged over all 60 cell lines indicates the isoniazid-sucrose-copper-PEG-3350 complex has no anti-cancer activity, which suggests a lack of general toxicity and provides data for the complexes specificity against Mycobacterium tuberculosis.

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TL;DR: In this article, the authors proposed a novel approach to defeat the tuberculosis infection in humans, which is the most common cause of human tuberculosis infection due to the rapid proliferation of the human population worldwide along with increasing globalisation.

9 citations

Journal ArticleDOI
TL;DR: A two-pronged medicinal chemistry workflow for identifying new potent and safe NTM EPIs and/or new scaffolds to be optimized in terms of EPI activity is reported.
Abstract: Nontuberculous mycobacteria (NTM) are ubiquitous microbes belonging to the Mycobacterium genus. Among all NTM pathogens, M. avium is one of the most frequent agents causing pulmonary disease, especially in immunocompromised individuals and cystic fibrosis patients. Recently, we reported the first ad hoc designed M. avium efflux pump inhibitor (EPI; 1b) able to strongly boost clarithromycin (CLA) MIC against different M. avium strains. Since the 3-phenylquinolone derivative 1b suffered from toxicity issues toward human macrophages, herein we report a two-pronged medicinal chemistry workflow for identifying new potent and safe NTM EPIs. Initially, we followed a computational approach exploiting our pharmacophore models to screen FDA approved drugs and in-house compounds to identify "ready-to-use" NTM EPIs and/or new scaffolds to be optimized in terms of EPI activity. Although nicardipine 2 was identified as a new NTM EPI, all identified molecules still suffered from toxicity issues. Therefore, based on the promising NTM EPI activity of 1b, we undertook the design, synthesis, and biological evaluation of new 3-phenylquinolones differently functionalized at the C6/C7 as well as N1 positions. Among the 27 synthesized 3-phenylquinolone analogues, compounds 11b, 12b, and 16a exerted excellent NTM EPI activity at concentrations below their CC50 on human cells, with derivative 16a being the most promising compound. Interestingly, 16a also showed good activity in M. avium-infected macrophages both alone as well as in combination with CLA. The antimycobacterial activity observed for 16a only when tested in the ex vivo model suggests a high therapeutic potential of EPIs against M. avium, which seems to need functional efflux pumps to establish intracellular infections.

9 citations

Journal ArticleDOI
21 Aug 2015-PLOS ONE
TL;DR: An epigenetic drug-tolerance phenotype that appears when mycobacteria are cultured in space-confined bioreactors and disappears in larger volume growth contexts is uncovered and may be an effect of confined growth among other macrophage-specific mechanisms.
Abstract: Tuberculosis (TB) is the world’s deadliest curable disease, responsible for an estimated 1.5 million deaths annually. A considerable challenge in controlling this disease is the prolonged multidrug chemotherapy (6 to 9 months) required to overcome drug-tolerant mycobacteria that persist in human tissues, although the same drugs can sterilize genetically identical mycobacteria growing in axenic culture within days. An essential component of TB infection involves intracellular Mycobacterium tuberculosis bacteria that multiply within macrophages and are significantly more tolerant to antibiotics compared to extracellular mycobacteria. To investigate this aspect of human TB, we created a physical cell culture system that mimics confinement of replicating mycobacteria, such as in a macrophage during infection. Using this system, we uncovered an epigenetic drug-tolerance phenotype that appears when mycobacteria are cultured in space-confined bioreactors and disappears in larger volume growth contexts. Efflux mechanisms that are induced in space-confined growth environments contribute to this drug-tolerance phenotype. Therefore, macrophage-induced drug tolerance by mycobacteria may be an effect of confined growth among other macrophage-specific mechanisms.

9 citations

References
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Journal ArticleDOI
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)....

    [...]

Journal ArticleDOI
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)....

    [...]

Journal ArticleDOI
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)....

    [...]

  • ...This is possible because of the redundancy of their functions, which may overlap extensively (24, 27)....

    [...]

Journal ArticleDOI
Keith Poole1
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)....

    [...]

Journal ArticleDOI
20 Aug 2009-Drugs
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