Characterization, cloning, and binding properties of the major 53- kilodalton Treponema denticola surface antigen

Abstract

Treponema denticola surface proteins were studied for their biochemical and biological characteristics. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of detergent extracts of whole cells revealed a major protein of 53 kDa and a number of minor proteins. Antiserum raised against whole cells of T. denticola ATCC 35405 reacted with the 53-kDa protein and a 72-kDa protein but not with the other proteins. Immunoelectron microscopy with anti-53-kDa-protein antibodies showed that the 53-kDa protein is located on the surface of the cell. SDS-PAGE analysis of unheated samples indicated that the 53-kDa protein is the major component of oligomers with molecular masses ranging from 130 to 300 kDa. Western blot (immunoblot) analysis showed that the high-molecular-mass oligomers reacted with whole- cell antiserum and anti-53-kDa-protein antibody. The aggregates dissociated into their subunits after heating to 70°C. Isoelectric focusing followed by SDS-PAGE indicated that the 53-kDa protein was separated into several forms with apparent pI values ranging from 8.0 to 5.5. The oligomeric forms were highly resistant to proteolysis by trypsin and proteinase K, whereas the monomeric proteins were readily digested. A clone expressing a 53-kDa antigen in Escherichia coli was isolated from a lambda ZAP II DNA library of T. denticola ATCC 35405. The recombinant protein had exactly the same molecular mass as the major 53-kDa T. denticola surface protein and reacted with antisera raised against this protein. The role of T. denticola ATCC 35405 surface proteins in attachment to laminin, fibronectin, gelatin, fibrinogen, and bovine serum albumin (BSA) was studied by a modified Western blot binding assay. Fibronectin, laminin, and fibrinogen attached to the 53-kDa surface protein of T. denticola as well as to a 72-kDa protein, whereas no attachment to gelatin or BSA was observed. Attachment could be inhibited by pretreating the blots with fibrinogen but not with gelatin or BSA. Our results suggest that the 53-kDa major surface protein of T. denticola may play a role in the attachment to host proteins and may thus be an important virulence determinant of this species.