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 review of the protein MmpL5, a transmembrane transporter of Mycobacterium, shows the importance of the RND superfamily of efflux pumps in drug resistance, and focuses on the mutation in the transcriptional regulator that can lead to resistance of antibiotics.
33 citations
TL;DR: A first functional HTS platform for identification of first-in-class inhibitors of APSR that can serve as molecular probes in unraveling the links between Mtb persistence, antibiotic tolerance, and sulfate assimilation, in addition to their potential therapeutic value is established.
Abstract: Development of effective therapies to eradicate persistent, slowly replicating M. tuberculosis (Mtb) represents a significant challenge to controlling the global TB epidemic. To develop such therapies, it is imperative to translate information from metabolome and proteome adaptations of persistent Mtb into the drug discovery screening platforms. To this end, reductive sulfur metabolism is genetically and pharmacologically implicated in survival, pathogenesis, and redox homeostasis of persistent Mtb. Therefore, inhibitors of this pathway are expected to serve as powerful tools in its preclinical and clinical validation as a therapeutic target for eradicating persisters. Here, we establish a first functional HTS platform for identification of APS reductase (APSR) inhibitors, a critical enzyme in the assimilation of sulfate for the biosynthesis of cysteine and other essential sulfur-containing molecules. Our HTS campaign involving 38 350 compounds led to the discovery of three distinct structural classes of ...
33 citations
TL;DR: It is demonstrated that redox active vitamin C triggers a multifaceted and robust adaptation response in Mycobacterium tuberculosis (Mtb) involving ~ 67% of the genome, suggesting a novel strategy of vit C adjunctive therapy by modulating bacterial physiology for enhanced efficacy of combination chemotherapy with existing drugs, and also possible synergies to guide new therapeutic combinations towards accelerating TB treatment.
Abstract: Bacterial dormancy is a major impediment to the eradication of tuberculosis (TB), because currently used drugs primarily target actively replicating bacteria. Therefore, decoding of the critical survival pathways in dormant tubercle bacilli is a research priority to formulate new approaches for killing these bacteria. Employing a network-based gene expression analysis approach, we demonstrate that redox active vitamin C (vit C) triggers a multifaceted and robust adaptation response in Mycobacterium tuberculosis (Mtb) involving ~ 67% of the genome. Vit C-adapted bacteria display well-described features of dormancy, including growth stasis and progression to a viable but non-culturable (VBNC) state, loss of acid-fastness and reduction in length, dissipation of reductive stress through triglyceride (TAG) accumulation, protective response to oxidative stress, and tolerance to first line TB drugs. VBNC bacteria are reactivatable upon removal of vit C and they recover drug susceptibility properties. Vit C synergizes with pyrazinamide, a unique TB drug with sterilizing activity, to kill dormant and replicating bacteria, negating any tolerance to rifampicin and isoniazid in combination treatment in both in-vitro and intracellular infection models. Finally, the vit C multi-stress redox models described here also offer a unique opportunity for concurrent screening of compounds/combinations active against heterogeneous subpopulations of Mtb. These findings suggest a novel strategy of vit C adjunctive therapy by modulating bacterial physiology for enhanced efficacy of combination chemotherapy with existing drugs, and also possible synergies to guide new therapeutic combinations towards accelerating TB treatment.
33 citations
Cites background from "Efflux Pumps of Mycobacterium tuber..."
...Efflux pumps are proposed to confer drug tolerance in Mtb [67,68]....
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TL;DR: An overview of the existing literature of nanocarrier-based DPIs of anti-TB drugs is provided and there is a need to investigate them for in vitro, ex vivo, in vivo and toxicity studies to achieve a market approval.
Abstract: Tuberculosis (TB) is a disease caused by the pathogen Mycobacterium tuberculosis. Prolonged administration of high dose antibiotics using oral and injectable routes and their associated side effects show limitations to successful treatment outcome of TB. Nanocarrier-based dry powder inhalers (DPIs) may provide a breakthrough as an alternative therapeutic approach because of their stable, non-invasive nature and ability to target the drug at the site of infection. The current review focuses on the roadmap of the respiratory system, drug deposition and targeting at the site of infection via the pulmonary route. This review will provide readers with an overview of the existing literature of nanocarrier-based DPIs of anti-TB drugs. Among different nanocarriers, results of most of the proliposomes and polymeric particles-based DPIs with respect to their characterisation parameters like encapsulation efficiency, drug loading, storage stability and aerodynamic properties are not encouraging, whereas surface engineered, nanostructured lipid carriers (NLCs), i.e. ligand attached-NLCs-based dry powder inhalers (NLCs-DPI) show promising results. But still, there is a need to investigate them for in vitro, ex vivo, in vivo and toxicity studies to achieve a market approval.
30 citations
TL;DR: In this article, a review aims to raise awareness of the rapidly increasing incidences of multidrug resistant (MDR) and extensively drug-resistant (XDR) tuberculosis, highlighting the significant modifications that were introduced in the TB treatment regimen over the past decade.
Abstract: Tuberculosis (TB) is a slow growing, potentially debilitating disease that has plagued humanity for centuries and has claimed numerous lives across the globe. Concerted efforts by researchers have culminated in the development of various strategies to combat this malady. This review aims to raise awareness of the rapidly increasing incidences of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis, highlighting the significant modifications that were introduced in the TB treatment regimen over the past decade. A description of the role of pathogen-host immune mechanisms together with strategies for prevention of the disease is discussed. The struggle to develop novel drug therapies has continued in an effort to reduce the treatment duration, improve patient compliance and outcomes, and circumvent TB resistance mechanisms. Herein, we give an overview of the extensive medicinal chemistry efforts made during the past decade toward the discovery of new chemotypes, which are potentially active against TB-resistant strains.
29 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