Cytopathic Effects of the Major Surface Protein and the Chymotrypsinlike Protease ofTreponema denticola

Abstract

Prominent antigens of Treponema denticola have been suggested to be mediators of the cytopathic effects typically seen in periodontal disease. In the present study of the T. denticola major surface protein (Msp) and the surface-expressed chymotrypsinlike protease complex (CTLP), we characterized the ability of these proteins to adhere to and lyse epithelial cells. Msp and CTLP were closely associated in spirochete outer membranes. Purified Msp, both native and recombinant, and CTLP bound to glutaraldehyde-fixed periodontal ligament epithelial cells. Adherence of Msp was partially blocked by specific antibodies. Adherence of CTLP was partially blocked by serine protease inhibitors and was further inhibited by specific antibodies. Both native Msp and CTLP were cytotoxic toward periodontal ligament epithelial cells, and their cytotoxicity was inhibited by the same treatments that inhibited adherence. Msp, but not CTLP, lysed erythrocytes. Msp complex (partially purified outer membranes free of protease activity) was cytotoxic toward a variety of different cell types. Pore-forming activities of recombinant Msp in black lipid model membrane assays and in HeLa cell membranes were similar to those reported for the native protein, supporting the hypothesis that Msp cytotoxicity was due to its pore-forming activity.

Mutagenesis of outer membrane virulence determinants of the oral spirochete Treponema denticola

“…2) showed intriguing patterns of Msp and CTLP expression in the mutant strains that suggested structural interactions between these proteins. These data are consistent with previous studies demonstrating co-localization of Msp and CTLP in the outer membrane [5]. Antigenic epitopes of Msp were predicted to be in the amino terminal half of the molecule, encoded upstream of the HindIII and PstI sites [15].…”

“…3B, and data not shown). Earlier studies implied that the high molecular mass native Msp oligomeric complex might be structurally associated with the CTLP complex [5,18]. The lack of oligomeric Msp and hexagonal array in strain CKE suggested that CTLP expression might in£uence the production or assembly of the Msp hexagonal array.…”

“…Analysis of potential virulence factors of oral spirochetes has focused on surface-expressed and secreted proteins (reviewed in [4]). Two prominent surface antigens of T. denticola ATCC 35405, the poreforming major surface protein (Msp) and the chymotrypsin-like protease (CTLP), adhered to and were cytotoxic to epithelial cells [5]. Msp induced pore formation in HeLa cell membranes [6].…”

“…ORF2 and prtP appeared to be transcribed from a promoter directly upstream of ORF2 [9]. While recombinant Msp had essentially native activity [5], the factors required for CTLP activity are not known [9]. Similarly, the mechanisms of assembly and surface presentation of Msp oligomers and the CTLP enzyme complex are unknown.…”

New insights into the emerging role of oral spirochaetes in periodontal disease

“…Periodontal tissue cells T. denticola cells and individual virulence factors are cytotoxic to epithelial cells [75,92]; they perturb cytoskeletal dynamics [93][94][95][96][97][98] and cell-cell junctions [99,100]. T. denticola can penetrate epithelium [96,101], whereas T. medium also invades epithelial cells [102].…”