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Marilis V. Marques - One of the best experts on this subject based on the ideXlab platform.

  • Regulation of Catalase-Peroxidase KatG Is OxyR Dependent and Fur Independent in Caulobacter crescentus
    Journal of bacteriology, 2011
    Co-Authors: Valéria C. S. Italiani, José F. Da Silva Neto, Vânia Santos Braz, Marilis V. Marques
    Abstract:

    Most organisms that grow in the presence of oxygen possess catalases and/or peroxidases, which are necessary for scavenging the H2O2 produced by aerobic metabolism. In this work we investigate the pathways that regulate the Caulobacter crescentus KatG gene, encoding the only enzyme with catalase-peroxidase function in this bacterium. The transcriptional start site of the KatG gene was determined, showing a short 5 untranslated region. The KatG regulatory region was mapped by serial deletions, and the results indicate that there is a single promoter, which is responsible for induction at stationary phase. An oxyR mutant strain was constructed; it showed decreased KatG expression, and no KatG Protein or catalase-peroxidase activity was detected in stationary-phase cell extracts, implying that OxyR is the main positive regulator of the C. crescentus KatG gene. Purified OxyR Protein bound to the KatG regulatory region between nucleotides 42 and 91 from the transcription start site, as determined by a DNase I footprinting assay, and a canonical OxyR binding site was found in this region. Moreover, OxyR binding was shown to be redox dependent, given that only oxidized Proteins bound adjacent to the 35 sequence of the promoter and the KatG P1 promoter was activated by OxyR in an H 2O2-dependent manner. On the other hand, this work showed that the iron-responsive regulator Fur does not regulate C. crescentus KatG, since a fur mutant strain presented wild-type levels of KatG transcription and catalase-peroxidase production and activity, and the purified Fur Protein was not able to bind to the KatG regulatory region.

  • Catalase-peroxidase activity is decreased in a Caulobacter crescentus rho mutant.
    FEMS microbiology letters, 2009
    Co-Authors: Valéria C. S. Italiani, Vânia S. Braz, Huifang Xiao, Howard M. Steinman, Marilis V. Marques
    Abstract:

    A Caulobacter crescentus rho::Tn5 mutant strain presenting a partially functional transcription termination factor Rho is highly sensitive to hydrogen peroxide in both exponential and stationary phases. The mutant was shown to be permanently under oxidative stress, based on fluorophore oxidation, and also to be sensitive to tert-butyl hydroperoxide and paraquat. However, the results showed that the activities of superoxide dismutases CuZnSOD and FeSOD and the alkylhydroperoxide reductase ahpC mRNA levels in the rho mutant were comparable to the wild-type control in the exponential and stationary phases. In contrast, the KatG catalase activity of the rho mutant strain was drastically decreased and did not show the expected increase in the stationary phase compared with the exponential phase. Transcription of the KatG gene was increased in the rho mutant and the levels of the immunoreactive KatG Protein do not differ considerably compared with the wild type in the stationary phase, suggesting that KatG activity is affected in a translational or a post-translational step.

Valéria C. S. Italiani - One of the best experts on this subject based on the ideXlab platform.

  • Regulation of Catalase-Peroxidase KatG Is OxyR Dependent and Fur Independent in Caulobacter crescentus
    Journal of bacteriology, 2011
    Co-Authors: Valéria C. S. Italiani, José F. Da Silva Neto, Vânia Santos Braz, Marilis V. Marques
    Abstract:

    Most organisms that grow in the presence of oxygen possess catalases and/or peroxidases, which are necessary for scavenging the H2O2 produced by aerobic metabolism. In this work we investigate the pathways that regulate the Caulobacter crescentus KatG gene, encoding the only enzyme with catalase-peroxidase function in this bacterium. The transcriptional start site of the KatG gene was determined, showing a short 5 untranslated region. The KatG regulatory region was mapped by serial deletions, and the results indicate that there is a single promoter, which is responsible for induction at stationary phase. An oxyR mutant strain was constructed; it showed decreased KatG expression, and no KatG Protein or catalase-peroxidase activity was detected in stationary-phase cell extracts, implying that OxyR is the main positive regulator of the C. crescentus KatG gene. Purified OxyR Protein bound to the KatG regulatory region between nucleotides 42 and 91 from the transcription start site, as determined by a DNase I footprinting assay, and a canonical OxyR binding site was found in this region. Moreover, OxyR binding was shown to be redox dependent, given that only oxidized Proteins bound adjacent to the 35 sequence of the promoter and the KatG P1 promoter was activated by OxyR in an H 2O2-dependent manner. On the other hand, this work showed that the iron-responsive regulator Fur does not regulate C. crescentus KatG, since a fur mutant strain presented wild-type levels of KatG transcription and catalase-peroxidase production and activity, and the purified Fur Protein was not able to bind to the KatG regulatory region.

  • Catalase-peroxidase activity is decreased in a Caulobacter crescentus rho mutant.
    FEMS microbiology letters, 2009
    Co-Authors: Valéria C. S. Italiani, Vânia S. Braz, Huifang Xiao, Howard M. Steinman, Marilis V. Marques
    Abstract:

    A Caulobacter crescentus rho::Tn5 mutant strain presenting a partially functional transcription termination factor Rho is highly sensitive to hydrogen peroxide in both exponential and stationary phases. The mutant was shown to be permanently under oxidative stress, based on fluorophore oxidation, and also to be sensitive to tert-butyl hydroperoxide and paraquat. However, the results showed that the activities of superoxide dismutases CuZnSOD and FeSOD and the alkylhydroperoxide reductase ahpC mRNA levels in the rho mutant were comparable to the wild-type control in the exponential and stationary phases. In contrast, the KatG catalase activity of the rho mutant strain was drastically decreased and did not show the expected increase in the stationary phase compared with the exponential phase. Transcription of the KatG gene was increased in the rho mutant and the levels of the immunoreactive KatG Protein do not differ considerably compared with the wild type in the stationary phase, suggesting that KatG activity is affected in a translational or a post-translational step.

Wladimir Sougakoff - One of the best experts on this subject based on the ideXlab platform.

  • Comparative study of enzymatic activities of new KatG mutants from low- and high-level isoniazid-resistant clinical isolates of Mycobacterium tuberculosis
    Tuberculosis, 2016
    Co-Authors: Florence Brossier, Marlène Boudinet, Vincent Jarlier, Stéphanie Petrella, Wladimir Sougakoff
    Abstract:

    Resistance to isoniazid (INH-R) in Mycobacterium tuberculosis is mainly due to mutations at position 315 (S315T) of the catalase-peroxidase KatG. We identified 16 mutations (including 13 biochemically uncharacterized mutations) in KatG from INH-R clinical isolates of M. tuberculosis showing mutations other than S315T. The KatG enzymatic activities (catalase, peroxidase, free radical production and isonicotinoyl-NAD formation) of wild-type KatG and the 16 mutants were determined and correlated to their spatial location in a KatG model structure. Of all mutations studied, H270R, which conferred a high level of INH-R and results in the disruption of a coordination bond with the heme, caused complete loss of all enzymatic KatG activities. The mutants generally associated with a very high level of INH-R were all characterized by a drastic reduction in catalase activity and a marked decrease in INH activation activities. One mutant, A162E, displayed a behavior similar to S315T, i.e. a moderate decrease in catalase activity and a drastic decrease in the formation of the radical form of INH. Finally, the mutants associated with a low level of INH-R showed a moderate reduction in the four catalytic activities, likely stemming from an overall alteration of the folding and/or stability of the KatG Protein.

  • Performance of the Genotype MTBDR Line Probe Assay for Detection of Resistance to Rifampin and Isoniazid in Strains of Mycobacterium tuberculosis with Low- and High-Level Resistance
    Journal of clinical microbiology, 2006
    Co-Authors: Florence Brossier, Vincent Jarlier, Nicolas Veziris, Chantal Truffot-pernot, Wladimir Sougakoff
    Abstract:

    We assessed the performance of the Genotype MTBDR line probe assay that offers the simultaneous identification of Mycobacterium tuberculosis and its resistance to rifampin (RIF) and isoniazid (INH) by detecting the most commonly found mutations in the rpoB and KatG genes. One hundred thirteen M. tuberculosis isolates were tested. The nucleotide sequences of the KatG and inhA genes and the mabA-inhA promoter region were also determined. The MTBDR assay detected 100% and 67% (n = 64) of the strains resistant to RIF and INH, respectively. Among the latter, 62 strains carried a Ser315Thr mutation in KatG, 59 of them displaying a high level of resistance to INH. Two strains with a low level of INH resistance had a Ser315Asn mutation. No mutation was found by the MTBDR assay for 31 INH-resistant strains (33%), of which 24 showed a low level of resistance. By DNA sequencing, we found among them various mutations in the KatG Protein for 7 strains, a C→T mutation in position −15 of the mabA-inhA promoter in 17 strains, and a Ser94Ala mutation in InhA for 7 strains. In conclusion, the MTBDR assay, which fits easily in the workflow of a routine laboratory, enabled the detection of 100% of the RIF-resistant strains and 89% of the INH-resistant strains with a high level of resistance but only 17% of the strains characterized by a low level of INH resistance, indicating that the test can be used as a rapid method to detect in the same experiment the rifampin-resistant and the high-level isoniazid-resistant strains of M. tuberculosis.

Indu Verma - One of the best experts on this subject based on the ideXlab platform.

  • Specific amplification of gene encoding N-terminal region of catalase-peroxidase Protein (KatG-N) for diagnosis of disseminated MAC disease in HIV patients.
    Diagnostic microbiology and infectious disease, 2014
    Co-Authors: Romica Latawa, Krishna K. Singh, Ajay Wanchu, Sunil Sethi, Kusum Sharma, Aman Sharma, Suman Laal, Indu Verma
    Abstract:

    Disseminated Mycobacterium avium-intracellulare complex (MAC) infection is considered as severe complication of advanced HIV/AIDS disease. Currently available various laboratory investigations have not only limited ability to discriminate between MAC infection and tuberculosis but are also laborious and time consuming. The aim of this study was, therefore, to design a molecular-based strategy for specific detection of MAC and its differentiation from Mycobacterium tuberculosis (M. tb) isolated from the blood specimens of HIV patients. A simple PCR was developed based on the amplification of 120-bp KatG-N gene corresponding to the first 40 amino acids of N-terminal catalase-peroxidase (KatG) Protein of Mycobacterium avium that shows only ~13% sequence homology by clustal W alignment to N-terminal region of M. tb KatG Protein. This assay allowed the accurate and rapid detection of MAC bacteremia, distinguishing it from M. tb in a single PCR reaction without any need for sequencing or hybridization protocol to be performed thereafter. This study produced enough evidence that a significant proportion of Indian HIV patients have disseminated MAC bacteremia, suggesting the utility of M. avium KatG-N gene PCR for early detection of MAC disease in HIV patients.

  • KatG Protein: A novel marker for differential diagnosis of Myobacterium avium complex infection
    Indian journal of medical microbiology, 2010
    Co-Authors: Krishan Lal Gupta, Indu Verma, Gopal K. Khuller, R Mahajan
    Abstract:

    Purpose: Biochemical or nucleic acid based diagnostic techniques for MAC infection are unsatisfactory. This study aims to identify and evaluate M. avium secretory Protein(s) of diagnostic potential, so as to develop a rapid and simple method for diagnosis of MAC infection. Material and Methods: Initially, a specific Protein band of ~80-85 kDa was recognised by differential immunoblotting; which was subjected to anion exchange column chromatography for purification of Proteins. After fractionisation using SDS-PAGE and electroelution, blast search was carried out. Further immunoreactivity studies were done with M. avium and Mtb infected mice sera. Clinical utilisation of separated Protein was evaluated by conducting indirect ELISA with serum samples from mycobacterial infected patients. Results: A specific 81.6 kDa Protein, shown to be catalase-peroxidase Protein (KatG) by blast search was separated. Immunoreactivity studies of purified KatG Proteins with mice sera confirmed it to be specific for M. avium infection. Indirect ELISA with patient samples further confirmed it to be M. avium infection specific. Conclusion: KatG Protein is specifically recognised by MAC patients and can be used as a marker for simple and rapid ELISA based tests for differential diagnosis of M. avium infection.

Florence Brossier - One of the best experts on this subject based on the ideXlab platform.

  • Comparative study of enzymatic activities of new KatG mutants from low- and high-level isoniazid-resistant clinical isolates of Mycobacterium tuberculosis
    Tuberculosis, 2016
    Co-Authors: Florence Brossier, Marlène Boudinet, Vincent Jarlier, Stéphanie Petrella, Wladimir Sougakoff
    Abstract:

    Resistance to isoniazid (INH-R) in Mycobacterium tuberculosis is mainly due to mutations at position 315 (S315T) of the catalase-peroxidase KatG. We identified 16 mutations (including 13 biochemically uncharacterized mutations) in KatG from INH-R clinical isolates of M. tuberculosis showing mutations other than S315T. The KatG enzymatic activities (catalase, peroxidase, free radical production and isonicotinoyl-NAD formation) of wild-type KatG and the 16 mutants were determined and correlated to their spatial location in a KatG model structure. Of all mutations studied, H270R, which conferred a high level of INH-R and results in the disruption of a coordination bond with the heme, caused complete loss of all enzymatic KatG activities. The mutants generally associated with a very high level of INH-R were all characterized by a drastic reduction in catalase activity and a marked decrease in INH activation activities. One mutant, A162E, displayed a behavior similar to S315T, i.e. a moderate decrease in catalase activity and a drastic decrease in the formation of the radical form of INH. Finally, the mutants associated with a low level of INH-R showed a moderate reduction in the four catalytic activities, likely stemming from an overall alteration of the folding and/or stability of the KatG Protein.

  • Performance of the Genotype MTBDR Line Probe Assay for Detection of Resistance to Rifampin and Isoniazid in Strains of Mycobacterium tuberculosis with Low- and High-Level Resistance
    Journal of clinical microbiology, 2006
    Co-Authors: Florence Brossier, Vincent Jarlier, Nicolas Veziris, Chantal Truffot-pernot, Wladimir Sougakoff
    Abstract:

    We assessed the performance of the Genotype MTBDR line probe assay that offers the simultaneous identification of Mycobacterium tuberculosis and its resistance to rifampin (RIF) and isoniazid (INH) by detecting the most commonly found mutations in the rpoB and KatG genes. One hundred thirteen M. tuberculosis isolates were tested. The nucleotide sequences of the KatG and inhA genes and the mabA-inhA promoter region were also determined. The MTBDR assay detected 100% and 67% (n = 64) of the strains resistant to RIF and INH, respectively. Among the latter, 62 strains carried a Ser315Thr mutation in KatG, 59 of them displaying a high level of resistance to INH. Two strains with a low level of INH resistance had a Ser315Asn mutation. No mutation was found by the MTBDR assay for 31 INH-resistant strains (33%), of which 24 showed a low level of resistance. By DNA sequencing, we found among them various mutations in the KatG Protein for 7 strains, a C→T mutation in position −15 of the mabA-inhA promoter in 17 strains, and a Ser94Ala mutation in InhA for 7 strains. In conclusion, the MTBDR assay, which fits easily in the workflow of a routine laboratory, enabled the detection of 100% of the RIF-resistant strains and 89% of the INH-resistant strains with a high level of resistance but only 17% of the strains characterized by a low level of INH resistance, indicating that the test can be used as a rapid method to detect in the same experiment the rifampin-resistant and the high-level isoniazid-resistant strains of M. tuberculosis.