Microarray Analysis of Efflux Pump Genes in Multidrug-Resistant<i>Mycobacterium tuberculosis</i>During Stress Induced by Common Anti-Tuberculous Drugs

Gupta, Anuj; Katoch, Vishwa Mohan; Chauhan, D. S.; Sharma, Rahul; Singh, Mukesh Vir; Venkatesan, K; Sharma, V. D.

doi:10.1089/mdr.2009.0054

Cited by 133 publications

(126 citation statements)

References 33 publications

(28 reference statements)

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“…Furthermore, in situ overexpression of several pps genes was confirmed using a CDC1551 strain, as well as a macrophage model for the Beijing isolate, indicating that the changes in expression of ppsA-ppsE are unlikely to be due to inherent characteristics of the two clinical strains used or the in vitro culture system. Our study is different from previous studies demonstrating that exposure to rifampin induces several changes in rifampin-resistant M. tuberculosis gene expression, including marked upregulation of drug efflux pump-related genes (30,51). Specifically, in this study, the impacts of rpoB mutations on the proteomes and metabolomes of rifampin-resistant and -susceptible strains were studied in the absence of rifampin in order to determine protein and metabolite abundance changes that are associated with rpoB mutations independent of drug exposure.…”

Section: Discussionmentioning

confidence: 65%

Upregulation of the Phthiocerol Dimycocerosate Biosynthetic Pathway by Rifampin-Resistant,rpoBMutant Mycobacterium tuberculosis

Bisson

Mehaffy²,

Broeckling

et al. 2012

J Bacteriol

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h Multidrug-resistant tuberculosis has emerged as a major threat to tuberculosis control. Phylogenetically related rifampin-resistant actinomycetes with mutations mapping to clinically dominant Mycobacterium tuberculosis mutations in the rpoB gene show upregulation of gene networks encoding secondary metabolites. We compared the expressed proteomes and metabolomes of two fully drug-susceptible clinical strains of M. tuberculosis (wild type) to those of their respective rifampin-resistant, rpoB mutant progeny strains with confirmed rifampin monoresistance following antitubercular therapy. Each of these strains was also used to infect gamma interferon-and lipopolysaccharide-activated murine J774A.1 macrophages to analyze transcriptional responses in a physiologically relevant model. Both rpoB mutants showed significant upregulation of the polyketide synthase genes ppsA-ppsE and drrA, which constitute an operon encoding multifunctional enzymes involved in the biosynthesis of phthiocerol dimycocerosate and other lipids in M. tuberculosis, but also of various secondary metabolites in related organisms, including antibiotics, such as erythromycin and rifamycins. ppsA (Rv2931), ppsB (Rv2932), and ppsC (Rv2933) were also found to be upregulated more than 10-fold in the Beijing rpoB mutant strain relative to its wild-type parent strain during infection of activated murine macrophages. In addition, metabolomics identified precursors of phthiocerol dimycocerosate, but not the intact molecule itself, in greater abundance in both rpoB mutant isolates. These data suggest that rpoB mutation in M. tuberculosis may trigger compensatory transcriptional changes in secondary metabolism genes analogous to those observed in related actinobacteria. These findings may assist in developing novel methods to diagnose and treat drug-resistant M. tuberculosis infections.A pproximately 9 million people develop active tuberculosis (TB) each year, resulting in nearly 2 million deaths annually (75). Recent progress controlling drug-susceptible TB has been made in many regions (50); however, drug resistance in Mycobacterium tuberculosis, including strains resistant to both first-line drugs, isoniazid and rifampin (multidrug-resistant [MDR] TB), has emerged as a threat to TB control worldwide (26, 65). Although international surveillance systems are inadequate, data from over 80 countries indicate that approximately 1 in 10 new cases of active TB have primary drug resistance (77), and MDR TB has been reported in essentially every country in which drug resistance has been studied (76). Treatment of MDR TB requires the use of costly and toxic second-line drugs for nearly 2 years and is associated with high rates of morbidity and mortality (65), which makes the spread of drug-resistant TB a major public health concern.Drug resistance in M. tuberculosis is due primarily to singlenucleotide polymorphisms in genes encoding key mycobacterial enzymes (6). The rpoB gene encodes the ␤-subunit of bacterial RNA polymerase, which is the target of rifampin (12...

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Section: Discussionmentioning

confidence: 65%

Upregulation of the Phthiocerol Dimycocerosate Biosynthetic Pathway by Rifampin-Resistant,rpoBMutant Mycobacterium tuberculosis

Bisson

Mehaffy²,

Broeckling

et al. 2012

J Bacteriol

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“…Based on homology analyses, both SNPs are located in conserved positions within the substrate-binding translocation pore. Rv2994 and Rv0783 have previously been reported to be overexpressed in MDR isolates (33) and RIF monoresistant isolates (13), respectively. WGS of isolate MDR-A also revealed two nonsynonymous mutations in the rpoC gene (Gly594Glu and Pro1040Ala).…”

Section: Discussionmentioning

confidence: 98%

Profiling of rpoB Mutations and MICs for Rifampin and Rifabutin in Mycobacterium tuberculosis

et al. 2014

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bResistance to rifampin (RIF) and rifabutin (RFB) in Mycobacterium tuberculosis is associated with mutations within an 81-bp region of the rpoB gene (RIF resistance-determining region [RRDR]). Previous studies have shown that certain mutations in this region are more likely to confer high levels of RIF resistance, while others may be found in phenotypically susceptible isolates. In this study, we sought to determine the relationship between the MICs of RIF and RFB and rpoB RRDR mutations in 32 multidrug-resistant (MDR), 4 RIF-monoresistant, and 5 susceptible M. tuberculosis clinical isolates. The MICs were determined using the MGIT 960 system. Mutations in the rpoB RRDR were determined by Sanger sequencing. RpoB proteins with mutations S531L (a change of S to L at position 531), S531W, H526Y, and H526D and the double mutation D516A-R529Q were associated with high MICs for RIF and RFB. Five isolates carrying the mutations L511P, H526L, H526N, and D516G-S522L were found to be susceptible to RIF. Several mutations were associated with resistance to RIF and susceptibility to RFB (F514FF, D516V, and S522L). Whole-genome sequencing of two MDR isolates without rpoB RRDR mutations revealed a mutation outside the RRDR (V146F; RIF MIC of 50 g/ml). The implications of the polymorphisms identified in the second of these isolates in RIF resistance need to be further explored. Our study further establishes a correlation between the mutations and the MICs of RIF and, also, RFB in M. tuberculosis. Several rpoB mutations were identified in RIF-and RFB-susceptible isolates. The clinical significance of these findings requires further exploration. Until then, a combination of phenotypic and molecular testing is advisable for drug susceptibility testing.

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“…Interestingly, it has been shown previously that blocking calcium channels in vivo in M. tuberculosis-infected mice using specific antibodies significantly reduced bacterial loads (6). Induction of efflux pump expression or mutations enhancing pump activity may explain the newly attained multidrug resistance and tolerance induced by antimicrobial drug exposure (7)(8)(9)(10)(11)(12). Furthermore, several mycobacterial efflux pumps, including Rv1258c, and their regulators are induced during macrophage infection (10,(13)(14)(15)(16)(17)(18).…”

mentioning

confidence: 95%

Acceleration of Tuberculosis Treatment by Adjunctive Therapy with Verapamil as an Efflux Inhibitor

Gupta

Tyagi

Almeida

et al. 2013

Am J Respir Crit Care Med

150

124

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Rationale: A major priority in tuberculosis (TB) is to reduce effective treatment times and emergence of resistance. Recent studies in macrophages and zebrafish show that inhibition of mycobacterial efflux pumps with verapamil reduces the bacterial drug tolerance and may enhance drug efficacy. Objectives: Using mice, a mammalian model known to predict human treatment responses, and selecting conservative human bioequivalent doses, we tested verapamil as an adjunctive drug together with standard TB chemotherapy. As verapamil is a substrate for CYP3A4, which is induced by rifampin, we evaluated the pharmacokinetic/ pharmacodynamic relationships of verapamil and rifampin coadministration in mice. Methods: Using doses that achieve human bioequivalent levels matched to those of standard verapamil, but lower than those of extended release verapamil, we evaluated the activity of verapamil added to standard chemotherapy in both C3HeB/FeJ (which produce necrotic granulomas) and the wild-type background C3H/HeJ mouse strains. Relapse rates were assessed after 16, 20, and 24 weeks of treatment in mice. Measurements and Main Results: We determined that a dose adjustment of verapamil by 1.5-fold is required to compensate for concurrent use of rifampin during TB treatment. We found that standard TB chemotherapy plus verapamil accelerates bacterial clearance in C3HeB/FeJ mice with near sterilization, and significantly lowers relapse rates in just 4 months of treatment when compared with mice receiving standard therapy alone.Conclusions: These data demonstrate treatment shortening by verapamil adjunctive therapy in mice, and strongly support further study of verapamil and other efflux pump inhibitors in human TB.Keywords: human equivalent doses; verapamil; efflux; Mycobacterium tuberculosis Tuberculosis (TB), a disease affecting millions of people worldwide, requires treatment of patients with multiple drugs for several months. The complexity and length of therapy has led to the emergence of extensively drug-resistant Mycobacterium tuberculosis, which poses a global threat (1). The limited number of new antimicrobials in the TB drug development pipeline further emphasizes the need for fresh approaches in TB therapy. Repurposing existing approved drugs that may serve as treatmentshortening adjuvants is a promising and relatively efficient approach (2). Efflux pump inhibitors (EPIs) are potential agents in this category, and there have been initial promising reports for licensed agents, such as verapamil, reserpine, and piperine (3).Recently, Adams and colleagues (4) showed that the bacterial efflux pump-encoding gene, Rv1258c promotes intracellular bacterial survival, and may mediate drug tolerance. Addition of verapamil reduced tolerance to rifampin in both M. tuberculosis and Mycobacterium marinum, and incubation of M. marinuminfected macrophages with verapamil reduced intracellular bacterial growth. When verapamil was added to isoniazidand rifampin-treated M. marinum-infected macrophages, it restored antibiotic killing by red...

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Microarray Analysis of Efflux Pump Genes in Multidrug-ResistantMycobacterium tuberculosisDuring Stress Induced by Common Anti-Tuberculous Drugs

Cited by 133 publications

References 33 publications

Upregulation of the Phthiocerol Dimycocerosate Biosynthetic Pathway by Rifampin-Resistant,rpoBMutant Mycobacterium tuberculosis

Upregulation of the Phthiocerol Dimycocerosate Biosynthetic Pathway by Rifampin-Resistant,rpoBMutant Mycobacterium tuberculosis

Profiling of rpoB Mutations and MICs for Rifampin and Rifabutin in Mycobacterium tuberculosis

Acceleration of Tuberculosis Treatment by Adjunctive Therapy with Verapamil as an Efflux Inhibitor

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