Submitted to: Journal of Medical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/3/2003
Publication Date: 4/1/2004
Citation: Dassanayake, R.P., Caceres, N.E., Sarath, G., Duhamel, G.E. 2004. Biochemical properties of membrane-associated proteases of brachyspira pilosicoli isolated from humans with intestinal disorders. Journal of Medical Microbiology. 53:319-323. Interpretive Summary: Brachyspira is an important microbial pathogen in several mammals, including humans. This genus consists of both pathogenic and non-pathogenic species. To cause infection, the spirochete has to attach to the intestinal mucosa of the host. The mechanisms which control this attachment are not clear. It has been postulated that proteolytic enzymes might be involved in the attachment process, by degrading cell surface proteins. In earlier studies, it was documented that both pathogenic and non-pathogenic members of Brachyspira contain a membrane associated subtilisin-like protease. This study was undertaken to further characterize the membrane protease and specifically test if there was one or more enzymes present in outer-membrane extracts of B. pilosicoli. Using fluorescently-labeled peptide substrates, we established that there were at least three enzymes present in membrane extracts. Future studies will evaluate the role of these enzymes in the spirochete-host interactions.
Technical Abstract: A membrane-associated, subtilisin-like, serine protease activity has been found in both pathogenic and non-pathogenic strains of Brachyspira species in a previous study, but the biochemical properties of the enzyme were not investigated. The purpose of the present study was to further characterize the biochemical properties, including substrate specificity of membrane-associated protease of B. pilosicoli isolated from humans with intestinal disorders. Protease activity of detergent-enriched membrane protein extracts of B. pilosicoli was assessed using fluorescent dye-labeled synthetic peptides as substrates and determination of electrophoretic mobility of cleavage products in agarose gels. Each activity was further confirmed with class-specific protease inhibitors and thermal denaturation. The presence of a hydrophilic membrane-associated thermolabile serine endopeptidase with specificity for leucine was confirmed. Two additional hydrophilic membrane-associated thermostable proteolytic activities, one with a putative alanine specificity and a carboxypeptidase were identified. Taken together these data suggest that in addition to a previously described membrane-associated subtilisin-like serine protease, the membrane of B. pilosicoli contains proteins with at least two other proteolytic activities.