scholarly journals bis-Molybdopterin Guanine Dinucleotide Is Required for Persistence of Mycobacterium tuberculosis in Guinea Pigs

2014 ◽  
Vol 83 (2) ◽  
pp. 544-550 ◽  
Author(s):  
Monique J. Williams ◽  
Crystal A. Shanley ◽  
Andrew Zilavy ◽  
Blas Peixoto ◽  
Claudia Manca ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nitrate Reductase ◽  
Guinea Pigs ◽  
Redox Reactions ◽  
Reductase Activity ◽  
Sulfur Metabolism ◽  
Content Type ◽  
Mouse Lungs ◽  
Molybdopterin Guanine Dinucleotide

Mycobacterium tuberculosisis able to synthesize molybdopterin cofactor (MoCo), which is utilized by numerous enzymes that catalyze redox reactions in carbon, nitrogen, and sulfur metabolism. In bacteria, MoCo is further modified through the activity of a guanylyltransferase, MobA, which converts MoCo tobis-molybdopterin guanine dinucleotide (bis-MGD), a form of the cofactor that is required by the dimethylsulfoxide (DMSO) reductase family of enzymes, which includes the nitrate reductase NarGHI. In this study, the functionality of themobAhomolog inM. tuberculosiswas confirmed by demonstrating the loss of assimilatory and respiratory nitrate reductase activity in amobAdeletion mutant. This mutant displayed no survival defects in human monocytes or mouse lungs but failed to persist in the lungs of guinea pigs. These results implicate one or morebis-MGD-dependent enzymes in the persistence ofM. tuberculosisin guinea pig lungs and underscore the applicability of this animal model for assessing the role of molybdoenzymes in this pathogen.

scholarly journals Mutation ofRv2887, amarR-Like Gene, Confers Mycobacterium tuberculosis Resistance to an Imidazopyridine-Based Agent

2015 ◽  
Vol 59 (11) ◽  
pp. 6873-6881 ◽  
Author(s):  
Kathryn Winglee ◽  
Shichun Lun ◽  
Marco Pieroni ◽  
Alan Kozikowski ◽  
William Bishai
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Efflux Pump ◽  
Transcriptional Regulator ◽  
Cross Resistance ◽  
Loss Of Function ◽  
Strong Activity ◽  
Content Type ◽  
Novel Drug

ABSTRACTDrug resistance is a major problem inMycobacterium tuberculosiscontrol, and it is critical to identify novel drug targets and new antimycobacterial compounds. We have previously identified an imidazo[1,2-a]pyridine-4-carbonitrile-based agent, MP-III-71, with strong activity againstM. tuberculosis. In this study, we evaluated mechanisms of resistance to MP-III-71. We derived three independentM. tuberculosismutants resistant to MP-III-71 and conducted whole-genome sequencing of these mutants. Loss-of-function mutations inRv2887were common to all three MP-III-71-resistant mutants, and we confirmed the role ofRv2887as a gene required for MP-III-71 susceptibility using complementation. The Rv2887 protein was previously unannotated, but domain and homology analyses suggested it to be a transcriptional regulator in the MarR (multiple antibiotic resistance repressor) family, a group of proteins first identified inEscherichia colito negatively regulate efflux pumps and other mechanisms of multidrug resistance. We found that two efflux pump inhibitors, verapamil and chlorpromazine, potentiate the action of MP-III-71 and that mutation ofRv2887abrogates their activity. We also used transcriptome sequencing (RNA-seq) to identify genes which are differentially expressed in the presence and absence of a functional Rv2887 protein. We found that genes involved in benzoquinone and menaquinone biosynthesis were repressed by functional Rv2887. Thus, inactivating mutations ofRv2887, encoding a putative MarR-like transcriptional regulator, confer resistance to MP-III-71, an effective antimycobacterial compound that shows no cross-resistance to existing antituberculosis drugs. The mechanism of resistance ofM. tuberculosisRv2887mutants may involve efflux pump upregulation and also drug methylation.

scholarly journals Structural and Kinetic Characterization of Hyperthermophilic NADH-Dependent Persulfide Reductase from Archaeoglobus fulgidus

Archaea ◽  
2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Sherwin Shabdar ◽  
Bukuru Anaclet ◽  
Ana Garcia Castineiras ◽  
Neyissa Desir ◽  
Nicholas Choe ◽  
...  
Keyword(s):  
Nadh Oxidase ◽  
Reductase Activity ◽  
Sulfur Metabolism ◽  
Archaeoglobus Fulgidus ◽  
Bacterial Enzymes ◽  
Detectable Activity ◽  
Metabolic Role ◽  
Nadh Oxidase Activity

NADH-dependent persulfide reductase (Npsr) has been proposed to facilitate dissimilatory sulfur respiration by reducing persulfide or sulfane sulfur-containing substrates to H2S. The presence of this gene in the sulfate and thiosulfate-reducing Archaeoglobus fulgidus DSM 4304 and other hyperthermophilic Archaeoglobales appears anomalous, as A. fulgidus is unable to respire S0 and grow in the presence of elemental sulfur. To assess the role of Npsr in the sulfur metabolism of A. fulgidus DSM 4304, the Npsr from A. fulgidus was characterized. AfNpsr is specific for persulfide and polysulfide as substrates in the oxidative half-reaction, exhibiting k cat / K m on the order of 104 M-1 s-1, which is similar to the kinetic parameters observed for hyperthermophilic CoA persulfide reductases. In contrast to the bacterial Npsr, AfNpsr exhibits low disulfide reductase activity with DTNB; however, similar to the bacterial enzymes, it does not show detectable activity with CoA-disulfide, oxidized glutathione, or cystine. The 3.1 Å X-ray structure of AfNpsr reveals access to the tightly bound catalytic CoA, and the active site Cys 42 is restricted by a flexible loop (residues 60-66) that is not seen in the bacterial homologs from Shewanella loihica PV-4 and Bacillus anthracis. Unlike the bacterial enzymes, AfNpsr exhibits NADH oxidase activity and also shows no detectable activity with NADPH. Models suggest steric and electrostatic repulsions of the NADPH 2 ′ -phosphate account for the strong preference for NADH. The presence of Npsr in the nonsulfur-reducing A. fulgidus suggests that the enzyme may offer some protection against S0 or serve in another metabolic role that has yet to be identified.

scholarly journals Extending the Definition of the GyrB Quinolone Resistance-Determining Region in Mycobacterium tuberculosis DNA Gyrase for Assessing Fluoroquinolone Resistance in M. tuberculosis

2012 ◽  
Vol 56 (4) ◽  
pp. 1990-1996 ◽  
Author(s):  
Alix Pantel ◽  
Stéphanie Petrella ◽  
Nicolas Veziris ◽  
Florence Brossier ◽  
Sylvaine Bastian ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Hot Spot ◽  
Dna Gyrase ◽  
Quinolone Resistance ◽  
Fluoroquinolone Resistance ◽  
Wild Type ◽  
Content Type ◽  
Highly Active ◽  
Definition Of

ABSTRACTFluoroquinolone (FQ) resistance is emerging inMycobacterium tuberculosis. The main mechanism of FQ resistance is amino acid substitution within the quinolone resistance-determining region (QRDR) of the GyrA subunit of DNA gyrase, the sole FQ target inM. tuberculosis. However, substitutions in GyrB whose implication in FQ resistance is unknown are increasingly being reported. The present study clarified the role of four GyrB substitutions identified inM. tuberculosisclinical strains, two located in the QRDR (D500A and N538T) and two outside the QRDR (T539P and E540V), in FQ resistance. We measured FQ MICs and also DNA gyrase inhibition by FQs in order to unequivocally clarify the role of these mutations in FQ resistance. Wild-type GyrA, wild-type GyrB, and mutant GyrB subunits produced from engineeredgyrBalleles by mutagenesis were overexpressed inEscherichia coli, purified to homogeneity, and used to reconstitute highly active gyrase complexes. MICs and DNA gyrase inhibition were determined for moxifloxacin, gatifloxacin, ofloxacin, levofloxacin, and enoxacin. All these substitutions are clearly implicated in FQ resistance, underlining the presence of a hot spot region housing most of the GyrB substitutions implicated in FQ resistance (residues NTE, 538 to 540). These findings help us to refine the definition of GyrB QRDR, which is extended to positions 500 to 540.

Dissociation of FAD from the NAD(P)H-Nitrate Reductase Complex from Ankistrodesmus braunii and Role of Flavin in Catalysis

1982 ◽  
Vol 37 (1-2) ◽  
pp. 24-30 ◽  
Author(s):  
Miguel A. De la Rosa ◽  
Antonio J. Márquez ◽  
José M. Vega
Keyword(s):  
Nitrate Reductase ◽  
Reductase Activity ◽  
Prosthetic Group ◽  
Original Activity ◽  
Enzyme Preparations ◽  
Fluorescence Studies ◽  
Transport Chain ◽  
Sulphydryl Groups ◽  
Nadh Cytochrome

Ankistrodesmus braunii NAD(P)H-nitrate reductase is a complex hemoflavomolybdoprotein composed by eight similar subunits. The flavin prosthetic group, identified as FAD, is essential for the NAD(P)H-dependent activities of the complex, and is located before the heme chromo- phore in the enzyme electron transport chain from reduced pyridine nucleotides to nitrate. Fluorescence studies indicate that nitrate reductase can dissociate about 80% of its FAD by incubation at room temperature, the flavin dissociation being followed by a parallel decrease of NADH-nitrate reductase activity. Dissociation of FAD from the protein is easily increased by dilution or prolonged dialysis of the enzyme preparations. However, exogenous FAD specifically prevents the dissociation of enzyme-bound flavin, and protects the NAD(P)H-dependent activities. The Km for FAD, as a protector of NADH-cytochrome c reductase activity, is 4 nᴍ. In addition, dithioerythritol also prevents the flavin dissociation, and therefore the presence of free sulphydryl groups in the FAD-domain is suggested. FAD-depleted nitrate reductase, obtained by several methods, is unable to recover its original activity when incubated in the presence of FAD alone or with thiols.

scholarly journals Metabolic Switching of Mycobacterium tuberculosis during Hypoxia Is Controlled by the Virulence Regulator PhoP

2020 ◽  
Vol 202 (7) ◽  
Author(s):  
Prabhat Ranjan Singh ◽  
Anil Kumar Vijjamarri ◽  
Dibyendu Sarkar
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nitrogen Metabolism ◽  
Electron Acceptor ◽  
Latent Infection ◽  
Critical Role ◽  
Hypoxic Stress ◽  
Content Type ◽  
Metabolic Switching ◽  
Virulence Regulator

ABSTRACT Mycobacterium tuberculosis retains the ability to establish an asymptomatic latent infection. A fundamental question in mycobacterial physiology is to understand the mechanisms involved in hypoxic stress, a critical player in persistence. Here, we show that the virulence regulator PhoP responds to hypoxia, the dormancy signal, and effectively integrates hypoxia with nitrogen metabolism. We also provide evidence to demonstrate that both under nitrogen limiting conditions and during hypoxia, phoP locus controls key genes involved in nitrogen metabolism. Consistently, under hypoxia a ΔphoP strain shows growth attenuation even with surplus nitrogen, the alternate electron acceptor, and complementation of the mutant restores bacterial growth. Together, our observations provide new biological insights into the role of PhoP in integrating nitrogen metabolism with hypoxia by the assistance of the hypoxia regulator DosR. The results have significant implications on the mechanism of intracellular survival and growth of the tubercle bacilli under a hypoxic environment within the phagosome. IMPORTANCE M. tuberculosis retains the unique ability to establish an asymptomatic latent infection. To understand the mechanisms involved in hypoxic stress which play a critical role in persistence, we show that the virulence regulator PhoP is linked to hypoxia, the dormancy signal. In keeping with this, phoP was shown to play a major role in M. tuberculosis growth under hypoxia even in the presence of surplus nitrogen, the alternate electron acceptor. Our results showing regulation of hypoxia-responsive genes provide new biological insights into role of the virulence regulator in metabolic switching by sensing hypoxia and integrating nitrogen metabolism with hypoxia by the assistance of the hypoxia regulator DosR.

scholarly journals Generation of Attenuated Mycobacterium bovis Strains by Signature-Tagged Mutagenesis for Discovery of Novel Vaccine Candidates

2005 ◽  
Vol 73 (4) ◽  
pp. 2379-2386 ◽  
Author(s):  
Desmond M. Collins ◽  
Bronwyn Skou ◽  
Stefan White ◽  
Shalome Bassett ◽  
Lauren Collins ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Bovis ◽  
Guinea Pigs ◽  
Essential Role ◽  
Immunoblot Analysis ◽  
Infection Model ◽  
Vaccine Candidates ◽  
Mouse Infection Model ◽  
Potential Vaccine

ABSTRACT Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex, has a particularly wide host range and causes tuberculosis in most mammals, including humans. A signature tag mutagenesis approach, which employed illegitimate recombination and infection of guinea pigs, was applied to M. bovis to discover genes important for virulence and to find potential vaccine candidates. Fifteen attenuated mutants were identified, four of which produced no lesions when inoculated separately into guinea pigs. One of these four mutants had nine deleted genes including mmpL4 and sigK and, in guinea pigs with aerosol challenge, provided protection against tuberculosis at least equal to that of M. bovis BCG. Seven mutants had mutations near the esxA (esat-6) locus, and immunoblot analysis of these confirmed the essential role of other genes at this locus in the secretion of EsxA (ESAT-6) and EsxB (CFP10). Mutations in the eight other attenuated mutants were widely spread through the chromosome and included pks1, which is naturally inactivated in clinical strains of M. tuberculosis. Many genes identified were different from those found by signature tag mutagenesis of M. tuberculosis by use of a mouse infection model and illustrate how the use of different approaches enables identification of a wider range of attenuating mutants.

scholarly journals Requirement of the mymA Operon for Appropriate Cell Wall Ultrastructure and Persistence of Mycobacterium tuberculosis in the Spleens of Guinea Pigs

2005 ◽  
Vol 187 (12) ◽  
pp. 4173-4186 ◽  
Author(s):  
Amit Singh ◽  
Radhika Gupta ◽  
R. A. Vishwakarma ◽  
P. R. Narayanan ◽  
C. N. Paramasivan ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Parental Strain ◽  
Guinea Pigs ◽  
Cell Envelope ◽  
Sodium Dodecyl ◽  
Phenotypic Traits ◽  
Antitubercular Drugs ◽  
Mycolic Acids ◽  
Increased Sensitivity

ABSTRACT We had recently reported that the mymA operon (Rv3083 to Rv3089) of Mycobacterium tuberculosis is regulated by AraC/XylS transcriptional regulator VirS (Rv3082c) and is important for the cell envelope of M. tuberculosis. In this study, we further show that a virS mutant (MtbΔvirS) and a mymA mutant (Mtbmym::hyg) of M. tuberculosis exhibit reduced contents and altered composition of mycolic acids along with the accumulation of saturated C24 and C26 fatty acids compared to the parental strain. These mutants were markedly more susceptible to major antitubercular drugs at acidic pH and also showed increased sensitivity to detergent (sodium dodecyl sulfate) and to acidic stress than the parental strain. We show that disruption of virS and mymA genes impairs the ability of M. tuberculosis to survive in activated macrophages, but not in resting macrophages, suggesting the importance of the mymA operon in protecting the bacterium against harsher conditions. Infection of guinea pigs with MtbΔvirS, Mtbmym::hyg, and the parental strain resulted in an ∼800-fold-reduced bacillary load of the mutant strains compared with the parental strain in spleens, but not in the lungs, of animals at 20 weeks postinfection. Phenotypic traits were fully complemented upon reintroduction of the virS gene into MtbΔvirS. These observations show the important role of the mymA operon in the pathogenesis of M. tuberculosis at later stages of the disease.

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