RESUMO
Although vascular pathologies such as vasculitis, endocarditis and mycotic aneurysms have been described in brucellosis patients, the interaction of Brucella with the endothelium has not been characterized. In this study we show that Brucella abortus and Brucella suis can infect and replicate in primary human umbilical vein endothelial cells (HUVEC) and in the microvascular endothelial cell line HMEC-1. Infection led to an increased production of IL-8, MCP-1 and IL-6 in HUVEC and HMEC-1 cells, and an increased expression of adhesion molecules (CD54 in both cells, CD106 and CD62E in HUVEC). Experiments with purified antigens from the bacterial outer membrane revealed that lipoproteins (Omp19) but not lipopolysaccharide mediate these proinflammatory responses. Infection of polarized HMEC-1 cells resulted in an increased capacity of these cells to promote the transmigration of neutrophils from the apical to the basolateral side of the monolayer, and the same phenomenon was observed when the cells were stimulated with live bacteria from the basolateral side. Overall, these results suggest that Brucella spp. can infect and survive within endothelial cells, and can induce a proinflammatory response that might be involved in the vascular manifestations of brucellosis.
Assuntos
Brucella abortus/imunologia , Brucella abortus/patogenicidade , Brucella suis/imunologia , Brucella suis/patogenicidade , Citocinas/metabolismo , Células Endoteliais/imunologia , Células Endoteliais/microbiologia , Antígenos de Bactérias/imunologia , Antígenos CD/biossíntese , Proteínas da Membrana Bacteriana Externa/imunologia , Células Cultivadas , Humanos , Lipoproteínas/imunologia , Neutrófilos/imunologia , Migração Transendotelial e TransepitelialRESUMO
Clinical diagnosis of canine brucellosis is not sensitive enough and a negative blood culture cannot rule out the disease. Indirect methods of serological testing such as agar gel immunodiffusion (AGID), rapid slide agglutination test (RSAT) and indirect enzyme linked immunoassay (IELISA) are preferred for routine diagnosis. Since Brucella canis shares antigenic components with the Brucella ovis and Brucella abortus RB51 strain, it would seem that either strain could be used as antigen. We present data on AGID and IELISA tests using the B. ovis antigen, RSAT and IELISA using the B. canis antigen and IELISA using the B. abortus RB51 antigen. The cut-off values were adjusted by the ROC analysis; the IELISA-B. ovis cut-off value was 23 (%P) and the IELISA-B. abortus RB51, 24 (%P), with 100% sensitivity and 98.8% specificity. RSAT detected 100% of positive cases, while AGID was less sensitive. The sera from dogs treated with antibiotic showed that %P correlated well with the clinical course. Sera from dogs presumptively infected with B. suis were negative in all tests performed with the rough Brucella strains. RSAT is a very sensitive screening test and IELISA-B. canis, B. ovis and B. abortus RB51 could be used as confirmatory tests, since they show good specificity and sensitivity.
Assuntos
Testes de Aglutinação/veterinária , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Brucella canis/imunologia , Brucelose/veterinária , Doenças do Cão/diagnóstico , Ensaio de Imunoadsorção Enzimática/veterinária , Imunodifusão/veterinária , Aborto Animal/microbiologia , Animais , Antibacterianos/uso terapêutico , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/isolamento & purificação , Bacteriemia/microbiologia , Bacteriemia/veterinária , Cápsulas Bacterianas/análise , Brucella abortus/imunologia , Brucella canis/química , Brucella ovis/imunologia , Brucella suis/imunologia , Brucelose/sangue , Brucelose/diagnóstico , Brucelose/tratamento farmacológico , Brucelose/imunologia , Brucelose/microbiologia , Reações Cruzadas , Doenças do Cão/sangue , Doenças do Cão/tratamento farmacológico , Doenças do Cão/imunologia , Doenças do Cão/microbiologia , Cães , Estudos de Viabilidade , Feminino , Masculino , Gravidez , Especificidade da EspécieRESUMO
BACKGROUND: The sequenced genomes of the Brucella spp. have two urease operons, ure-1 and ure-2, but there is evidence that only one is responsible for encoding an active urease. The present work describes the purification and the enzymatic and phylogenomic characterization of urease from Brucella suis strain 1330. Additionally, the urease reactivity of sera from patients diagnosed with brucellosis was examined. RESULTS: Urease encoded by the ure-1 operon of Brucella suis strain 1330 was purified to homogeneity using ion exchange and hydrophobic interaction chromatographies. The urease was purified 51-fold with a recovery of 12% of the enzyme activity and 0.24% of the total protein. The enzyme had an isoelectric point of 5, and showed optimal activity at pH 7.0 and 28-35 degrees C. The purified enzyme exhibited a Michaelis-Menten saturation kinetics with a Km of 5.60 +/- 0.69 mM. Hydroxyurea and thiourea are competitive inhibitors of the enzyme with Ki of 1.04 +/- 0.31 mM and 26.12 +/- 2.30 mM, respectively. Acetohydroxamic acid also inhibits the enzyme in a competitive way. The molecular weight estimated for the native enzyme was between 130-135 kDa by gel filtration chromatography and 157 +/- 7 kDa using 5-10% polyacrylamide gradient non-denaturing gel. Only three subunits in SDS-PAGE were identified: two small subunits of 14,000 Da and 15,500 Da, and a major subunit of 66,000 Da. The amino terminal sequence of the purified large subunit corresponded to the predicted amino acid sequence encoded by ureC1. The UreC1 subunit was recognized by sera from patients with acute and chronic brucellosis. By phylogenetic and cluster structure analyses, ureC1 was related to the ureC typically present in the Rhizobiales; in contrast, the ureC2 encoded in the ure-2 operon is more related to distant species. CONCLUSION: We have for the first time purified and characterized an active urease from B. suis. The enzyme was characterized at the kinetic, immunological and phylogenetic levels. Our results confirm that the active urease of B. suis is a product of ure-1 operon.