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Distribution of a Nocardia brasiliensis catalase gene fragment in members of the genera Nocardia, Gordona, and Rhodococcus.

ABSTRACT: An immunodominant protein from Nocardia brasiliensis, P61, was subjected to amino-terminal and internal sequence analysis. Three sequences of 22, 17, and 38 residues, respectively, were obtained and compared with the protein database from GenBank by using the BLAST system. The sequences showed homology to some eukaryotic catalases and to a bromoperoxidase-catalase from Streptomyces violaceus. Its identity as a catalase was confirmed by analysis of its enzymatic activity on H2O2 and by a double-staining method on a nondenaturing polyacrylamide gel with 3,3'-diaminobenzidine and ferricyanide; the result showed only catalase activity, but no peroxidase. By using one of the internal amino acid sequences and a consensus catalase motif (VGNNTP), we were able to design a PCR assay that generated a 500-bp PCR product. The amplicon was analyzed, and the nucleotide sequence was compared to the GenBank database with the observation of high homology to other bacterial and eukaryotic catalases. A PCR assay based on this target sequence was performed with primers NB10 and NB11 to confirm the presence of the NB10-NB11 gene fragment in several N. brasiliensis strains isolated from mycetoma. The same assay was used to determine whether there were homologous sequences in several type strains from the genera Nocardia, Rhodococcus, Gordona, and Streptomyces. All of the N. brasiliensis strains presented a positive result but only some of the actinomycetes species tested were positive in the PCR assay. In order to confirm these findings, genomic DNA was subjected to Southern blot analysis. A 1.7-kbp band was observed in the N. brasiliensis strains, and bands of different molecular weight were observed in cross-reacting actinomycetes. Sequence analysis of the amplicons of selected actinomycetes showed high homology in this catalase fragment, thus demonstrating that this protein is highly conserved in this group of bacteria.

SUBMITTER: Vera-Cabrera L

PROVIDER: S-EPMC84999 | biostudies-literature | 1999 Jun

REPOSITORIES: biostudies-literature

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Distribution of a Nocardia brasiliensis catalase gene fragment in members of the genera Nocardia, Gordona, and Rhodococcus.

Vera-Cabrera L L Johnson W M WM Welsh O O Resendiz-Uresti F L FL Salinas-Carmona M C MC

Journal of clinical microbiology 19990601 6

An immunodominant protein from Nocardia brasiliensis, P61, was subjected to amino-terminal and internal sequence analysis. Three sequences of 22, 17, and 38 residues, respectively, were obtained and compared with the protein database from GenBank by using the BLAST system. The sequences showed homology to some eukaryotic catalases and to a bromoperoxidase-catalase from Streptomyces violaceus. Its identity as a catalase was confirmed by analysis of its enzymatic activity on H2O2 and by a double-s ...[more]

PMID: 10325357

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Project description:BACKGROUND: Mycetoma is a chronic infectious disease of tropical and subtropical countries. It is produced by true fungi and actinobacteria. In México, Nocardia brasiliensis is the main causative agent of mycetoma, producing about 86% of the cases; the gold standard for the therapy of mycetoma by N. brasiliensis is the use of sulfonamides which give a 70% cure rate. The addition of amikacin to this regime increases to 95% the cure rate; however, the patients have to be monitored for creatinine clearance and audiometry studies because of the potential development of side effects. Because of that it is important to search for new active compounds. In the present work, we evaluated the in vivo effect of DA-7867, an experimental oxazolidinone, on the development of experimental mycetomas by N. brasiliensis in BALB/c mice. METHODOLOGY/PRINCIPAL FINDINGS: In order to determine the optimal dose utilized to apply to the animals, we first determined by HPLC the plasma levels using several concentrations of the compounds. Based on these results, we used 10 and 25 mg/kg subcutaneously every 24 hr; DA-7867 was also supplied in the drinking water at a calculated dose of 25 mg/kg. As a control we utilized linezolid at 25 mg/kg, a compound active in murine and human infections, three times a day. The mice were infected in the right footpad with a young culture of N. brasiliensis HUJEG-1, and one week later we started the application of the antimicrobials for six more weeks. After that we compared the development of lesions in the groups injected with saline solution or with the antimicrobials; the results were analyzed by the variance ANOVA test. DA-7867 was able to reduce the production of lesions at 25 mg/kg, when given either subcutaneously or in the drinking water. CONCLUSIONS/SIGNIFICANCE: The experimental oxazolidinone DA-7867 is active in vivo against N. brasiliensis, which opens the possibility of using this drug once it is accepted for human application. Since oxazolidinones seem to be active against a wide spectrum of actinobacteria, it is possible they could be used in human cases of mycetoma by other actinomycetales, such as Streptomyces somaliensis, highly prevalent in Sudan, or Actinomadura madurae and A. pelletieri, which are commonly observed in Africa and India.

Dataset Information

Distribution of a Nocardia brasiliensis catalase gene fragment in members of the genera Nocardia, Gordona, and Rhodococcus.

Publications

Distribution of a Nocardia brasiliensis catalase gene fragment in members of the genera Nocardia, Gordona, and Rhodococcus.

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