Treponema denticola is a member of the oral flora with its primary habitat being the gingival sulcus/periodontal pocket. This spirochete's potential for causing periodontal destruction along with being associated with anaerobic infections of the oral cavity is well documented. Movement of T. denticola through crevicular fluid and possibly periodontal tissue may not only be necessary for the survival of the organism, but also contribute to its virulence. A fundamental understanding of motility would be helpful in defining T. denticola's role as a pathogen in oral disease. Spirochete swimming is mediated by the periplasmic flagella (PFs) which reside between the protoplasmic cylinder and outer membrane sheath. It was recently shown that the PFs of T. denticola are antigenically similar to those of the well studied but genetically different spirochete T. phagedenis. The goals of the present investigation are to analyze the PFs of T. denticola in detail, and compare these results to those of other spirochetes. In this way, a better understanding of spirochete motility will be obtained. We propose the following: (1) Isolate the PFs of denticola; (2) characterize the major structural proteins comprising the PFs and their arrangement on the PFs; (3) clone and sequence the PF genes of T. denticola, and compare these results to those of T. phagedenis and other spirochetes. PFs will be isolated using established procedures. The separation and analysis of PF proteins will be done using sodium dodecyl sulfate- polyacrylamide gel electrophoresis, two dimensional gel electrophoresis, and Western blotting using both polyclonal and monoclonal antibody. The distribution of these proteins on the PFs will be determined by immunocytochemical methods using either monoclonal antibodies conjugated to protein A-colloidal gold, or undecagold-Fab probes. The PF genes of T. denticola will be cloned into Escherichia coli using PF specific monoclonal and polyclonal antisera and lambda gtll. Restriction enzyme maps along with Southern blotting and chromosome walking of cloned gene fragments will be used to determine the number of PF genes. The PF gene will be sequenced by the dideoxy chain termination method. Nucleotide and deduced amino acid sequences will be compared to data-bases, with a special emphasis on the PF genes from other spirochetes. Regions of homology will define conserved sequences among the spirochetes. These regions are likely to be necessary for PF structure and motility.
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| Li, H; Ruby, J; Charon, N et al. (1996) Gene inactivation in the oral spirochete Treponema denticola: construction of an flgE mutant. J Bacteriol 178:3664-7 |
