The research work presented in this thesis involved the examination of several fundamental questions concerning the role of oral anaerobic spirochetes (OAS) in the etiology of periodontal disease (PD). OAS are unusual, helical, Gram-negative bacteria that are considered putative periodontopathogens due to numerical association with diseased sites and the enzymatic arsenal available to OAS that appears consistent with disease symptoms. T. denticola is the most commonly isolated OAS from periodontal pockets and as such is the focus of most investigations into the role of OAS in PD. / As free iron is severely limited in humans the means by which OAS may obtain sufficient iron to prosper in the sub-gingiva was examined. The resultant model suggests T. denticola obtains iron through the $ beta$-hemolysis of erythrocytes and the binding of liberated hemin by a 47-kDa outer membrane sheath (OMS) protein. The kinetics of the ligand-receptor interaction are presented and the receptor has been purified to apparent homogeneity from T. denticola. $ sp3$H-labeled hemin is not transported into the cell. Evidence is presented to show that T. denticola produces an iron reductase, which may facilitate the transport of ferrous iron across biological membranes. It is also shown that T. denticola (Td), T. vincentii (Tv) and T. socranskii (Ts) do not produce siderophores. In growth assays, under conditions of iron-limitation, T. denticola may use inorganic iron, a source unlikely to be available in vivo. / Hyaluronidase (Hase) activity is elevated in the gingival crevice during episodes of disease. Hase, when injected into the periodontal cavity under experimental conditions has been shown to result in connective tissue degradation. It is also known that T. pallidum, the agent of syphilis, produces a Hase that is critical to pathogenesis. Evidence is presented herein to show that Td, Tv and Ts all produce a hyauronoglucosaminidase (HGase). The identity and specificity of the Td HGase is confirmed through the use of enzyme inhibitors and activators, by electron microscope observations of the enzyme using the Hase inhibitor gold sodium thiomalate and anti-Apis mellifera venom antibodies and examination of the purity of the HA substrate using other polysaccharide degrading enzymes. As the HGase of these OAS would not migrate through a substrate-SDS PAGE system, we have employed hyaluronate (HA)- and chondroitin sulfate (CS)-absorbed nitrocellulose membranes to visualize HGase activity. The 59 kDa HGase of Td has been purified to apparent homogeneity through the conjugation of HA and CS to Affigel 701 beads. / The last subject to be addressed by this thesis pertains to the ultrastructure of oral spirochetes. Using the copper-containing dye, Alcian blue, we have shown that T. denticola produces an exopolysaccharide layer, in an electron microscopy investigation. The development of a stain for dark-field microscopy has simplified the observation of this layer. The exopolysaccharide layer may have relevance to the evasion of phagocytosis, to protection against colicins, immunoglobulins and bacteriophages, to adherence and perhaps to the immunogenicity of OAS inhabiting the sub-gingiva.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.34446 |
| Date | January 1996 |
| Creators | Scott, David, 1964 Jan. 11- |
| Contributors | Chan, E. C. S. (advisor), Siboo, R. (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Microbiology and Immunology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001549763, proquestno: NQ30378, Theses scanned by UMI/ProQuest. |
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