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: By far the most widespread mechanism of resistance to AGs is the inactivation of these antibiotics by AG-modifying enzymes, and an overview of these mechanisms is provided.
Abstract: Aminoglycoside (AG) antibiotics are used to treat many Gram-negative and some Gram-positive infections and, importantly, multidrug-resistant tuberculosis. Among various bacterial species, resistance to AGs arises through a variety of intrinsic and acquired mechanisms. The bacterial cell wall serves as a natural barrier for small molecules such as AGs and may be further fortified via acquired mutations. Efflux pumps work to expel AGs from bacterial cells, and modifications here too may cause further resistance to AGs. Mutations in the ribosomal target of AGs, while rare, also contribute to resistance. Of growing clinical prominence is resistance caused by ribosome methyltransferases. By far the most widespread mechanism of resistance to AGs is the inactivation of these antibiotics by AG-modifying enzymes. We provide here an overview of these mechanisms by which bacteria become resistant to AGs and discuss their prevalence and potential for clinical relevance.
329 citations
TL;DR: Mechanisms of phenotypic drug tolerance and resistance in bacteria are reviewed with the goal of providing a framework for understanding the similarities and differences in these cells.
Abstract: One of the challenges in clinical infectious diseases is the problem of chronic infections, which can require long durations of antibiotic treatment and often recur. An emerging explanation for the refractoriness of some infections to treatment is the existence of subpopulations of drug tolerant cells. While typically discussed as “persister” cells, it is becoming increasingly clear that there is significant heterogeneity in drug responses within a bacterial population and that multiple mechanisms underlie the emergence of drug tolerant and drug-resistant subpopulations. Many of these parallel mechanisms have been shown to affect drug susceptibility at the level of a whole population. Here we review mechanisms of phenotypic drug tolerance and resistance in bacteria with the goal of providing a framework for understanding the similarities and differences in these cells.
157 citations
Cites background from "Efflux Pumps of Mycobacterium tuber..."
...First, upregulation of three of the known classes of efflux pumps leads to increased MICs of several antibiotics including all first line drugs in vitro (Balganesh et al., 2012; Dinesh et al., 2013)....
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TL;DR: The antitubercular efficacy of spectinamides demonstrates that synthetic modifications to classical antibiotics can overcome the challenge of intrinsic efflux pump-mediated resistance and expands opportunities for target-based tuberculosis drug discovery.
Abstract: Although the classical antibiotic spectinomycin is a potent bacterial protein synthesis inhibitor, poor antimycobacterial activity limits its clinical application for treating tuberculosis. Using structure-based design, we generated a new semisynthetic series of spectinomycin analogs with selective ribosomal inhibition and excellent narrow-spectrum antitubercular activity. In multiple murine infection models, these spectinamides were well tolerated, significantly reduced lung mycobacterial burden and increased survival. In vitro studies demonstrated a lack of cross resistance with existing tuberculosis therapeutics, activity against multidrug-resistant (MDR) and extensively drug-resistant tuberculosis and an excellent pharmacological profile. Key to their potent antitubercular properties was their structural modification to evade the Rv1258c efflux pump, which is upregulated in MDR strains and is implicated in macrophage-induced drug tolerance. The antitubercular efficacy of spectinamides demonstrates that synthetic modifications to classical antibiotics can overcome the challenge of intrinsic efflux pump-mediated resistance and expands opportunities for target-based tuberculosis drug discovery.
151 citations
TL;DR: This study showed that the active efflux pump AdeABC appeared to play important roles in the tigecycline resistance of A. baumannii, and phenyl-arginine-β-naphthylamide (PAβN) and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) could partially reverse the resistance pattern of tigECYcline.
Abstract: For its remarkable ability to acquire antibiotic resistance and to survive in nosocomial environments, Acinetobacter baumannii has become a significant nosocomial infectious agent worldwide. Tigecycline is one of the few therapeutic options to treat infections caused by A. baumannii isolates. However, tigecycline resistance has been increasingly reported. Our aim was to assess the prevalence and characteristics of efflux-based tigecycline resistance in clinical isolates of A. baumannii collected from a hospital in China. A total of 74 A. baumannii isolates including 64 tigecycline non-susceptible A. baumannii (TNAB) and 10 tigecycline susceptible A. baumannii (TSAB) isolates were analyzed. The majority of them were detected to be positive for adeABC , adeRS , adeIJK and abeM, while the adeE gene was found in only one TSAB isolate. Compared with TSAB isolates, the mean expression level of adeB , adeJ, adeG and abeM in TNAB isolates were observed to increase by 29-, 3-, 0.7- and 1-fold, respectively. The efflux pump inhibitors (EPIs) PAβN and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) could partially reverse the resistance pattern of tigecycline. Moreover, tetX1 gene was detected in 12 (18.8%) TNAB isolates. To our knowledge, this is the first report that tetX1 gene was detected in the A. baumannii isolates. ST208 and ST191 which both clustered into clonal complex 92 (CC92) were the predominant sequence types (STs). This study showed that active efflux pump AdeABC appeared to play important roles in the tigecycline resistance of A. baumannii. The dissemination of TNAB isolates in our hospital is mainly attributable to the spread of CC92.
150 citations
TL;DR: Advances in the path of EPI discovery are summarized, potential avenues of E PI implementation and development are discussed, and the need for highly informative and comprehensive translational approaches is underlined.
Abstract: Conventional antimicrobials are increasingly ineffective due to the emergence of multidrug-resistance among pathogenic microorganisms. The need to overcome these deficiencies has triggered exploration for novel and unconventional approaches to controlling microbial infections. Multidrug efflux systems (MES) have been a profound obstacle in the successful deployment of antimicrobials. The discovery of small molecule efflux system blockers has been an active and rapidly expanding research discipline. A major theme in this platform involves efflux pump inhibitors (EPIs) from natural sources. The discovery methodologies and the available number of natural EPI-chemotypes are increasing. Advances in our understanding of microbial physiology have shed light on a series of pathways and phenotypes where the role of efflux systems is pivotal. Complementing existing antimicrobial discovery platforms such as photodynamic therapy (PDT) with efflux inhibition is a subject under investigation. This core information is a stepping stone in the challenge of highlighting an effective drug development path for EPIs since the puzzle of clinical implementation remains unsolved. This review summarizes advances in the path of EPI discovery, discusses potential avenues of EPI implementation and development, and underlines the need for highly informative and comprehensive translational approaches.
134 citations
Cites background from "Efflux Pumps of Mycobacterium tuber..."
...Although resistance to TB is multifactorial, the importance of efflux has been identified and documented in a variety of occasions [51, 143]....
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...The list is expanding to include ABC Superfamily members such as DrrAB [52] Bcg0231 (M. bovis BCG) Rv0194 [53] Rv1218c[51] Rv1456c-Rv1457c-Rv1458c [54] and the SMR systems Rv3065 [51] LfrA in M. smegmatis [55] as well as the homologues of M. tuberculosis Rv1145, Rv1146, Rv1877, Rv2846c (efpA), [56] and Rv3065 (mmr and emrE) in both M. tuberculosis and M. smegmatis [57]....
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...bovis BCG) Rv0194 [53] Rv1218c[51] Rv1456c-Rv1457c-Rv1458c [54] and the SMR systems Rv3065 [51] LfrA in M....
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References
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TL;DR: Recent advances in the field of multidrug-resistance efflux pumps are reviewed and various approaches used in combating efflux-mediated resistance are described.
Abstract: Multidrug-resistance efflux pumps — in particular those belonging to the resistance-nodulation-cell-division (RND) family of transporters, with their unusually high degree of substrate promiscuity — significantly restrict the effectiveness of antibacterial therapy. Recent years have heralded remarkable insights into the structure and mechanisms of these fascinating molecular machines. Here, we review recent advances in the field and describe various approaches used in combating efflux-mediated resistance.
176 citations
"Efflux Pumps of Mycobacterium tuber..." refers background in this paper
...Several investigators are actively involved in the search for such inhibitors (14, 19) to treat various bacterial infections....
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TL;DR: This work highlights the growing pool of information about internal, antibiotic-responsive regulatory proteins and corresponding resistance genes, and presents new concepts that rationalize how they might have evolved and could make many previously available antibiotics active against M. tuberculosis.
161 citations
"Efflux Pumps of Mycobacterium tuber..." refers background in this paper
...It could be due to the accumulation of mutations in the target genes over a period of time (21), exclusion of antibiotics by the highly impermeable cell wall (18, 22), the wide array of efflux mechanisms mediated by several ABC (ATP-binding cassette) transporters and major facilitator superfamily (MFS) proteins (9, 10), or antibiotic-modifying and -degrading enzymes (3), to name a few possibilities....
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TL;DR: Improved infection control, rapid diagnostic tools, enhanced screening strategies, and pharmacokinetic studies are needed to improve screening strategies and improve infection control.
Abstract: We investigated the emergence and evolution of drug-resistant tuberculosis (TB) in an HIV co-infected population at a South African gold mine with a well-functioning TB control program. Of 128 patients with drug-resistant TB diagnosed during January 2003-November 2005, a total of 77 had multidrug-resistant (MDR) TB, 26 had pre-extensively drug-resistant TB (XDR TB), and 5 had XDR TB. Genotyping suggested ongoing transmission of drug-resistant TB, and contact tracing among case-patients in the largest cluster demonstrated multiple possible points of contact. Phylogenetic analysis demonstrated stepwise evolution of drug resistance, despite stringent treatment adherence. These findings suggested that existing TB control measures were inadequate to control the spread of drug-resistant TB in this HIV co-infected population. Diagnosis delay and inappropriate therapy facilitated disease transmission and drug-resistance. These data call for improved infection control measures, implementation of rapid diagnostics, enhanced active screening strategies, and pharmacokinetic studies to determine optimal dosages and treatment regimens.
153 citations
"Efflux Pumps of Mycobacterium tuber..." refers background in this paper
...Multidrug-resistant (MDR) TB and the highly lethal form called extensively drug-resistant (XDR) TB are adding a new challenge to this old and persistent disease (7, 13)....
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TL;DR: It is demonstrated that Rv0194 is a novel multidrug efflux pump of M. tuberculosis that significantly reduced accumulation of ethidium bromide and conferred multidrog resistance to Mycobacterium smegmatis.
Abstract: The impermeability of the outer membrane in combination with drug efflux are major determinants of the natural drug resistance of mycobacteria. beta-Lactams are the most widely used antibiotics for treatment of bacterial infections. However, it is unknown how beta-lactams enter Mycobacterium tuberculosis and whether efflux pumps exist that can export these drugs out of the cell. To identify the molecular mechanisms of M. tuberculosis resistance to beta-lactams, a library of 7,500 transposon mutants was generated in the model organism Mycobacterium bovis BCG. Thirty-three unique insertion sites were determined that conferred medium or high-level (> or =2,000 microg/ml) resistance to ampicillin. Three mutants in sulfolipid synthesis or transport were highly resistant to ampicillin, indicating an indirect effect of the lipid composition on the outer membrane permeability of M. bovis BCG to ampicillin. Mutants with insertions in genes encoding surface molecules such as PPE proteins or lipoarabinomannan were also completely resistant to ampicillin, thus suggesting a lack of transport across the outer membrane. Insertion of the transposon in front of bcg0231 increased transcription of the gene and concomitantly the resistance of M. bovis BCG to ampicillin, streptomycin, and chloramphenicol by 32- to 64-fold. Resistance to vancomycin and tetracycline was increased four- to eightfold. Bcg0231 and Rv0194 are almost identical ATP-binding cassette transporters. Expression of rv0194 significantly reduced accumulation of ethidium bromide and conferred multidrug resistance to Mycobacterium smegmatis. Both effects were abrogated in the presence of the efflux pump inhibitor reserpine. These results demonstrate that Rv0194 is a novel multidrug efflux pump of M. tuberculosis.
150 citations
"Efflux Pumps of Mycobacterium tuber..." refers background in this paper
...tuberculosis belonging to different classes have been described in the last few years, and some pumps have been well characterized (2, 6, 8, 11, 29, 30)....
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TL;DR: It was observed that a simultaneous overexpression of efflux pump genes Rv2459, Rv3728, and Rv3065 was associated with resistance to the combination of isoniazid and ethambutol, and these drugs, along with streptomycin, were identified to group together, where efflux-mediated drug resistance appears to be important in M. tuberculosis.
Abstract: Treatment of multidrug-resistant tuberculosis has become one of the major problems in public health. Understanding the molecular mechanisms of drug resistance has been central to tuberculosis research in recent times. DNA microarray technology provides the platform to study the genomic variations related to these mechanisms on a comprehensive level. To investigate the role of efflux pumps in drug resistance, we have constructed a custom DNA microarray containing 25 drug efflux pump genes of Mycobacterium tuberculosis (Indian Patent file no. 2071/DEL/2007) and monitored changes in the expression of these genes on exposure of common anti-tuberculous drugs. Expression profiling of efflux pump genes in multidrug-resistant M. tuberculosis isolates showed overexpression of 10 genes following exposure to various anti-tuberculous drugs. Although two of these genes (Rv3065 and Rv2938) have already been reported to be active drug efflux pumps in M. tuberculosis in earlier studies, the increased activities of other ...
137 citations
"Efflux Pumps of Mycobacterium tuber..." refers background in this paper
...pAZI0290 Derived from pGOAL19 (15) by deleting...
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...Drug resistance arising out of efflux pump overexpression has been an issue in clinical settings, as well (15, 16, 35), which poses serious problems in the treatment of tuberculosis; this needs to be addressed as a priority....
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