Distribution of aNocardia brasiliensisCatalase Gene Fragment in Members of the GeneraNocardia,Gordona, andRhodococcus

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 generaNocardia, Rhodococcus, Gordona, andStreptomyces. All of the N. brasiliensisstrains 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.

IgM but not IgG monoclonal anti-Nocardia brasiliensisantibodies confer protection against experimental actinomycetoma in BALB/c mice

“…Nocardia brasiliensis immunodominant antigens include P61, P26 and P24; sequence analysis of P61 showed similarity to eukaryotic and prokaryotic catalases (Vera-Cabrera et al, 1992, 1999. Catalases play an important role in survival and in the protection against oxygen-dependent microbicidal activities of phagocytes, and they have been claimed as a virulence factor for other Nocardia species (Beaman et al, 1985).…”

“…Gonzalez'. Bacteria were grown in brain heart infusion (BHI) medium (Difco Laboratories, Detroit, MI), as published elsewhere (Vera-Cabrera et al, 1992;Salinas-Carmona et al, 1999).…”

“…P61 is an immunodominant antigen with catalase activity (Vera-Cabrera et al, 1999;Salinas-Carmona et al, 2003). An experimental model of chronic N. brasiliensis infection in BALB/c mice demonstrated that infected mice sera react against the same immunodominant antigens as infected patients (Salinas-Carmona et al, 1999). Previous studies (Salinas-Carmona & Pérez-Rivera, 2004;Salinas-Carmona et al, 2006) revealed that hyperimmune serum with high titers of immunoglobulin (IgM) to N. brasiliensis immunodominant antigens is able to protect against actinomycetoma when passively transferred to naïve mice, and a lack of protective effect is observed with passive transfer of hyperimmune sera containing high titers of IgG.…”

In Vivo Activity of the Benzothiazinones PBTZ169 and BTZ043 against Nocardia brasiliensis

“…brasiliensis HUJEG-1 (ATCC700358) previously used in other in vitro and in vivo assays [ 7 , 8 ]. These isolates are from human mycetoma lesions and were identified by conventional biochemical methods as well as by sequence analysis of a portion of the 16S rRNA gene using the NOC-3 and NOC-4 primers [ 9 ].…”

Screening and Identification of B-Cell Epitopes in the P61 Protein of Nocardia brasiliensis

“…The P61 protein is encoded by the gene O3I_001640 and is the target of the humoral immune response in patients suffering from nocardial mycetoma (Gordon et al, 2013 ). This immunodominant protein is highly conserved in the Nocardia genus (Vera-Cabrera et al, 1999 ), furthering its potential as a tool for clinical diagnosis of nocardiosis.…”