RESUMO
This study aims at genetic characterization and phylogenetic relationships of Nocardia brasiliensis focusing by using housekeeping rrs, hsp65, and sodA genes. N. brasiliensis is the species responsible for 80% of cases of actinomycetoma, one form of cutaneous nocardiosis which occurs mainly in tropical regions reaching immunocompetent patients in which the disease can lead to amputation. We analyze 36 indigenous cases of N. brasiliensis that happened in France. Phylogenetic analysis targeting rrs gene showed no robustness at phylogenetic nodes level. However, the use of a concatenation of hsp65 and sodA genes showed that the tested strains surprisingly ranked in 3 well-defined genotypes. Genotypes 2 and 3 were phylogenetically closer to each other and both diverged from genotype 1 sustained by a high bootstrap of 81%. This last genotype hosts all the cases of pulmonary forms (3), the sole cerebral form, and almost all the cases of immunocompromised patients (3 out of 4). Moreover, excepting one of them, all the strains belonging to this group present a susceptibility to imipenem which is not the case in the other genotypes that rarely count among them strains being susceptible to this drug. The haplotype diversity (Hd) of hsp65 (0.927) and sodA (0.885) genes was higher than that of rrs (0.824). For this gene, we obtained 16 polymorphic sites whereas, for hsp65 and sodA genes, up to 27 and 29 were identified, respectively. This study reveals that these two genes have an important genetic discriminatory power for the evaluation of the intraspecies genetic variability of N. brasiliensis and they may be useful for identification purposes at species level. This study also reveals the possible existence of a new species harbored by genotype 1.
Assuntos
Proteínas de Bactérias/genética , Variação Genética , Nocardiose/genética , Superóxido Dismutase-1/genética , França/epidemiologia , Humanos , Nocardia/genética , Nocardia/patogenicidade , Nocardiose/epidemiologia , Nocardiose/microbiologia , Nocardiose/patologia , FilogeniaRESUMO
Actinomycetoma, caused by the intracellular bacterium Nocardia brasiliensis, is characterized by an infiltration of several inflammatory cell populations. To explore aspects of the immune response in the pathogenesis of these bacteria we injected 10(6) CFU in footpads of BALB/c mice. After 1, 2, 3, 4, 7, 30 and 90 days immunohistochemistry was performed to compare presence and distribution of the inflammatory cytokines TNF-alpha, IL-1 beta, IL-6, IFN-gamma, IL-4, IL-10, and TGF-beta. Analysis of serial paraffin tissue sections showed strong participation and differences in distribution of cytokine-producing cells during the course of infection. Several TNF-alpha immunoreactive lymphocytes of the dermis were present during the course of the infection, but absent in the site of inflammation. During the first 4 days, IL-1 beta immunoreactivity was observed in dendritic epidermal cells and in cells surrounding the neutrophils around the grain. In later stages of infection, immunoreactive cells to this cytokine were mainly in the periphery of the microabscesses. Strong immunoreactivity was observed with IL-6 during the course of infection. Some cells in the epidermis and dermis, as well as muscle cells and several cells at the periphery of the microabscesses, showed strong IL-6 immunoreactivity. Cells immunoreactive to IL-4, IL-10, IFN-gamma and TGF-beta were present at the site of infection and, in later stages, in cells at the periphery of the microabscesses. In conclusion a mix of proinflammatory and antiinflammatory cytokines are produced at the same time by host cells. According to their distribution, inflammatory cytokines seems to have different functions during the course of infection with the intracellular bacterium N. brasiliensis.
Assuntos
Citocinas/metabolismo , Nocardiose/imunologia , Nocardia/imunologia , Animais , Biomarcadores/metabolismo , Citocinas/imunologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , Modelos Animais de Doenças , Feminino , Pé/microbiologia , Pé/patologia , Linfócitos/imunologia , Linfócitos/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Nocardia/patogenicidade , Nocardiose/etiologia , Nocardiose/patologia , Pele/imunologia , Pele/microbiologia , Pele/patologiaRESUMO
Anti-Nocardia brasiliensis antibodies quantification and its clinical utility was confirmed in this study. A protein cellular extract from a N. brasiliensis strain named HUJEG-1 and registered at the ATCC # 700358 was used in a western blot assay to identify the immunodominant antigens. The protein P24 was selected to set up an ELISA test because it exhibit no cross-reaction with sera from tuberculosis and leprosy patients. A purified protease was also used as antigen in the ELISA test to compare its utility. Sera from N. brasiliensis mycetoma persons gave absorbance values above 0.3 when the disease was active using the P24 as antigen, these values decreased after patients completed their medical treatment. Anti-protease antibodies showed great variation and absorbance values similar to the healthy controls. We confirmed the clinical usefulness of the ELISA test both in serodiagnosis and in assessing the response to medical treatment. This is the first sensitive and specific serologic test for routine clinical laboratory.
Assuntos
Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/isolamento & purificação , Micetoma/imunologia , Nocardia/imunologia , Antígenos de Bactérias/imunologia , Reações Cruzadas/imunologia , Endopeptidases , Ensaio de Imunoadsorção Enzimática , Humanos , Micetoma/diagnóstico , Micetoma/microbiologia , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeAssuntos
Alergia e Imunologia/tendências , Biologia Molecular/tendências , Infecções Bacterianas/diagnóstico , Fenômenos Fisiológicos Celulares , Previsões , Doenças Genéticas Inatas/diagnóstico , Terapia Genética , Humanos , Infecções/terapia , Masculino , Neoplasias da Próstata/terapia , Vacinas/uso terapêuticoRESUMO
Nocardia brasiliensis is a Gram-positive bacterium that lives as a saprophyte in soil. In this article the physical properties, chemical composition and taxonomic position of this species is reviewed. Human infections and an experimental model of actinomycetoma in BALB/c mice as well as the host-immune response is described.
Assuntos
Micetoma , Nocardiose , Nocardia , Animais , Humanos , Camundongos , Micetoma/imunologia , Micetoma/microbiologia , Micetoma/patologia , Nocardia/química , Nocardia/classificação , Nocardia/citologia , Nocardia/patogenicidade , Nocardiose/imunologia , Nocardiose/microbiologiaRESUMO
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.
Assuntos
Actinomycetales/genética , Catalase/genética , Nocardia/enzimologia , Nocardia/genética , Rhodococcus/genética , Actinomycetales/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Catalase/química , Sequência Consenso , Primers do DNA , DNA Fúngico/genética , Genes Fúngicos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Rhodococcus/enzimologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Streptomyces/enzimologia , Streptomyces/genéticaRESUMO
Nine- to twelve-week-old BALB/c mice were injected in footpads with 10(7) CFU of a Nocardia brasiliensis cell suspension. Typical actinomycetoma lesions, characterized by severe local inflammation with abscess and fistula formation, were fully established by day 28 after infection. These changes presented for 90 days, and then tissue repair with scar formation slowly appeared, with complete healing after 150 days of infection. Some animals developed bone destruction in the affected area. Histopathology showed an intense inflammatory response, with polymorphonuclear cells and hyaloid material around the colonies of the bacteria, some of which were discharged from draining abscesses. Sera from experimental animals were analyzed by Western blotting, and immunodominant antigens P61 and P24 were found as major targets for antibody response. Anti-P24 immunoglobulin M (IgM) isotype antibodies were present as early as 7 days, IgG peaking 45 days after infection. Lymphocyte proliferation with spleen and popliteal lymph node cells demonstrated thymidine incorporation at 7 days after infection, the stimulation index decreasing by day 60. Levels of interleukin-1 (IL-1), IL-2, IL-4, IL-6, tumor necrosis factor alpha, and gamma interferon (IFN-gamma) were determined by enzyme-linked immunosorbent assay in the sera of infected animals. The circulating levels of IFN-gamma increased more than 10 times the basal levels; levels of IL-4, IL-6 and IL-10 also increased during the first 4 days of infection.
Assuntos
Antígenos de Bactérias/administração & dosagem , Micetoma/imunologia , Nocardiose/imunologia , Nocardia/imunologia , Animais , Anticorpos Antibacterianos/sangue , Citocinas/sangue , Modelos Animais de Doenças , Feminino , Humanos , Epitopos Imunodominantes , Técnicas In Vitro , Ativação Linfocitária , Masculino , Camundongos , Micetoma/etiologia , Micetoma/patologia , Nocardia/patogenicidade , Nocardiose/etiologia , Nocardiose/patologia , Células Th1/imunologia , Células Th2/imunologia , Fatores de TempoRESUMO
We prepared a Nocardia brasiliensis cell extract and purified two immunodominant antigens with molecular weights of 61,000 and 24,000. The isolated proteins were shown to be reasonably pure when analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8 to 18% polyacrylamide gradient) and stained with Coomassie blue and silver nitrate. By using an immunoelectrotransfer blot method (Western blotting), we demonstrated that these two purified proteins reacted strongly with serum from N. brasiliensis-infected mycetoma patients. To obtain anti-P61 and anti-P24 monoclonal antibodies (MAbs), we used an N. brasiliensis cell extract as the antigen for the first immunization; 2 weeks later female mice were reimmunized with a semipurified antigen containing the P24 or P61 fraction. A booster injection was given 3 days before the fusion was carried out. Two hybrids that reacted strongly with P24 were cloned by limiting dilution, the generated MAbs were analyzed for isotyping, and their specificity was tested in a Western blot assay with cell extracts from Nocardia asteroides and Mycobacterium tuberculosis cultures. Anti-P24 MAbs were shown to be specific for N. brasiliensis HUJEG-1 and did not cross-react with either the N. asteroides or M. tuberculosis strains used. However, additional studies with several N. asteroides and N. brasiliensis strains are needed to investigate whether there are cross-reactions between strains or species when these MAbs are used. The anti-P61 and anti-24 MAbs were used to locate the antigen in N. brasiliensis cells by immunofluorescence. The lack of reaction with intact cells suggests that the P24 and P61 antigens are not exposed in the complete bacterial cell surface or that the recognized epitopes are different. Only one anti-P61 MAb that reacted specifically with the N. brasiliensis cell extract was obtained.