RESUMO
An acid phosphatase activity was detected in the supernatant of Haemophilus parasuis, a Gram-negative pleomorphic bacillus and the causative agent of Glässers disease in pigs. To identify the gene responsible for the secreted activity, a genomic library of H. parasuis strain ER-6P was produced in Escherichia coli. Screening of the library allowed identification of two homologs to known phosphatases: PgpB and AphA. PgpB was predicted to be located in the bacterial membrane through six transmembrane domains while AphA was predicted to have a signal peptide. The aphA gene was cloned and expressed in E. coli. Characterization of H. parasuis AphA indicated that this protein belongs to the class B nonspecific acid phosphatases. AphA contained sequence signatures characteristic of this family of phosphatases and its activity was inhibited by EDTA. The optimal pH of recombinant AphA differed from that of the phosphatase activity found in H. parasuis supernatants. In addition, the phosphatase activity from H. parasuis supernatants was not inhibited by EDTA, indicating that H. parasuis AphA does not account for the phosphatase activity observed in the supernatants. Our results demonstrate the presence of a class B acid phosphatase (AphA) in H. parasuis and suggest that the bacterium would also secrete another, as yet unidentified phosphatase (AU)
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Assuntos
Animais , Fosfatase Ácida/isolamento & purificação , Haemophilus parasuis/imunologia , Fenômenos Fisiológicos Bacterianos/imunologia , Doenças dos Suínos/imunologia , Biomarcadores/análiseRESUMO
The bacterial thyA gene encodes the enzyme thymidylate synthase, which is essential for dTMP synthesis and, consequently, for DNA replication. In this work, a Haemophilus parasuis thyA mutant was constructed in order to analyze its colonization characteristics and its capacity to generate serum bactericidal activity in infected guinea pigs. The data showed that colonization by the H. parasuis thyA mutant was much less than that of the wild-type strain. Nevertheless, the mutant generated a strong immunogenic response in the host, as detected by measuring serum bactericidal activity (AU)
El gen bacteriano thyA codifica la enzima timidilato sintasa, que es esencial para la síntesis de dTMP y, en consecuencia, para la replicación del DNA. En este trabajo se ha construido un mutante thyA de Haemophilus parasuis para analizar sus características de colonización, así como la capacidad de generar actividad bactericida en suero en cobayas infectadas. Los datos obtenidos demuestran que la capacidad de colonización del mutante thyA de H. parasuis es muy reducida con respecto a la cepa tipo salvaje. Sin embargo, el mutante thyA de H. parasuis origina un elevado nivel de respuesta inmunogénica en el huésped, como se detecta midiendo la actividad bactericida del suero (AU)
