Downloaded from www.microbiologyresearch.org by IP: 54.166.55.152 On: Thu, 04 May 2017 20:32:27 Journal of Medical Microbiology (2004), 53, 319–323 DOI 10.1099/jmm.0.45549-0 45549 & 2004 SGM Printed in Great Britain 319 Correspondence Gerald E. Duhamel gduhamel1@unl.edu Gautam Sarath gsarath1@unl.edu Received 19 September 2003 Accepted 3 December 2003 Biochemical properties of membrane-associated proteases of Brachyspira pilosicoli isolated from humans with intestinal disorders Rohana P. Dassanayake, 1 Nancy E. Caceres, 2 Gautam Sarath 2,3 and Gerald E. Duhamel 1,2 Department of Veterinary and Biomedical Sciences 1 , Center for Biotechnology 2 and Department of Biochemistry 3 , University of Nebraska-Lincoln, Lincoln, NE 68583-0905, USA A membrane-associated, subtilisin-like, serine protease activity was 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 characterize further the biochemical properties, including substrate specificity, of the membrane-associated protease of Brachyspira pilosicoli isolated from humans with intestinal disorders. Protease activity of detergent- enriched membrane protein extracts of B. pilosicoli was assessed using fluorescent dye-labelled 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 Leu was confirmed. Two additional hydrophilic membrane- associated thermostable proteolytic activities were identified, one with a putative Ala specificity, and one a carboxypeptidase. 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. INTRODUCTION Brachyspira pilosicoli has been implicated as a cause of colonic spirochaetosis, an inflammatory bowel disease that affects a broad range of hosts, including humans (Duhamel, 2001; Trott et al., 1997). Infection with B. pilosicoli is characterized by colonization of the large intestine followed by polar attachment of the spirochaetes along the apical membrane of the colonic epithelium (Duhamel, 2001). Damage to epithelial cells and penetration of the epithelial layer have also been seen, but the mechanism(s) of invasion has not been investigated. Demonstration of a subtilisin-like serine protease activity in detergent-enriched membrane fractions of pathogenic and non-pathogenic Brachyspira species sug- gested that the enzyme might be important for survival in the intestinal environment (Muniappa & Duhamel, 1997). Alternatively, the membrane-associated serine protease might indirectly contribute to damage caused by pathogenic spirochaetes after intimate association with the colonic epithelial surface. Proteases have been found in spirochaetes and a role in damage to host cells and the mucosal barrier has been proposed (Ellen et al., 2000; Grenier et al., 1990). Among spirochaetes, trypsin- and chymotrypsin-like proteases of Treponema denticola, an oral spirochaete associated with periodontitis, have been characterized (Ellen et al., 2000; Grenier et al., 1990). A role for T. denticola proteases in periodontal tissue invasion by degradation of host cell proteins and inactivation of bioactive peptides has been suggested (Grenier et al., 1990; Reijntjens et al., 1986). The purpose of the present study was to characterize the biochemical properties of the membrane-associated protease activity present in human strains of B. pilosicoli. The substrate specificities of the proteases were identified using synthetic peptides representative of all predicted target amino acid residues designed on the basis of cleavage patterns obtained in a previous study (Muniappa & Duhamel, 1997). METHODS Bacterial strains and growth conditions. The human B. pilosicoli isolates SP16 (ATCC 49776) and SP13 (Jones et al., 1986; Ramanathan et al., 1993; Lee & Hampson, 1994; Duhamel et al., 1995; Fisher et al., 1997) and HRM-5B and HRM-7 (Coene et al., 1989; Lee & Hampson, 1994; Fisher et al., 1997), respectively isolated from human beings with intestinal disorders in the United States and Italy, were investigated in This paper was presented at the Second International Conference on Colonic Spirochaetal Infections in Animals and Humans, Edinburgh, UK, 2–4 April 2003. Abbreviation: ODG, n-octyl â-D-glucopyranoside.