VIRULENCE FACTORS OF ORAL ANAEROBIC SPIROCHETES David Scott Department of Microbiology and Immuoology McGiH University, Montreal JuneJ996 A Thesis Submitted to the Facuity of Graduate Studies and Research in Partial Fulfillment of the Requirements of the Degree of Doctor of Philosophy O David Scott, 1996 National Library Bibliothbque nationale du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395. rue Wellington OttawaON K1AW OttawaON K1AON4 Canada Canada The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la fome de microfiche/film, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otheniise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. TABLE OF CONTENTS Page Abstract vii Resumé ir Acknowledgements xi Claim of contribution to knowledge xii List of Figures xiv List of Tables xvii CHAPTER 1. Literature review and introduction 1. Taxonomy of Spirochetes II. General Characteristics of Spirochetes 5 (i) Mucoid Layer 5 (ii) Outer Membrane Sheath 6 (iii) Axial Fibrils 8 (iv) Peptidoglycan layer 13 (v) Cell Membrane 13 (vi) Cytoplasrn, Nucleoid and Extrachromosornal elements 14 III. Spirochete-Associated Diseases f 7 IV. The Etiology of Periodontal Disease 20 (i) C haracterization of Periodontal Disease 21 (ii) Treatment of Penodontal Disease 23 (iii) Events in the periodontium during Periodontitis 24 (iv) The Role of bacteria in Periodontal Disease 27 (v) The Role of the Immune Response in Periodontitis 32 V. The Role of Spirochetes in Periodontitis 39 (i) Association Studies of Spirochetes and Periodontal Disease 39 (ii) Aquisition of Spirochetes 42 (iii) Adherence and Colonization of Spirochetes 42 (iv) Invasion of Penodontal Tissue by Oral Spirochetes 47 (v) Virulence Factors and the Effects on the Immune System of Oral Spirochetes and Rationale for Research 48 Guidelines Regarding Doctoral Thesis 60 CHAPTER 2. Iron Aquisition by Oral Anaerobic Spirochetes PART 1. Binding of hemin and Congo red by oral hemolytic spirocheies Abstract Introduction Materials and methods Growth of oral spirochetes Congo red and hemin binding to colony-forming units Hemolytic activity Binding of Congo red and hemin to bacterial cells Binding of Congo red or hemin to treated cells E lectrophoresis Assay for siderophores Results Binding of hemin and Congo red to colony-forrning units Hemolysis of erythrocytes Siderophore production Binding of Congo red and hemin to cells Congo red and hemin binding to treated cells Electrophoresis Discussion References PART II. Iron aquisition by oral hemolytic spirochetes : isolation of a hemin-binding protein and identification of iron reductase activity. Introduction to Part II Abstract Introduction Materials and Methods Growth of Oral Spirochetes L ipoprotein extraction Purification of the 47-kDa Hemin-binding protein Preparation of 'H-labeled Hemin Binding assays for 'H-hemin by ir denticolu ATCC 35405 Grotvth of T. denticoln ATCC 35405 under iron t imitation and supplemented with hemin. protoporphynn IX. Congo red. ferric chloride and ferric ammonium citrate as iron sources Replacement of senun with hemin in NOS medium Photometric and solid-phase assays for iron reductase activity Results Purification of the 47 kDa hemin-binding protein Binding and cellular distribution of 'H-labeled hemin Growth of T. denticola ATCC 35405 under iron limitation and supplemented with hemin. protoporphyrin IX. Congo red. ferric chloride and femc ammonium citrate as iron sources Replacement of serum with hemin in NOS medium Iron reductase activity Discussion References CHAPTER 3. An extracellular enzyme of some oral anaerobic spirochaetes with hyaluronidase and chondroitinase activities Abstract Introduction Materials and Methods Reagents Bactena and culture conditions Assays for hyaluronidase and chondroitinase activities Inhibition assays Conjugation of hyaluronate and chondroitin sulfate to Affi Gel 701 beads Electrophoresis and electroblotting Elec tron Microscopy Results Hyaluronidase and chondroitinase activities in culture supematants II2 Affinity chromatography 112 Effect of inhibitors 115 SDS-PAGE, transblots and hydrolysis of HA and C4S 115 Electron rnicroscopy Discussion Refesences CWTER4. Visualization of an extracellular mucoid iayer of Treponema denticola ATCC 35405 and surface sugar lectin-analysis of some oral spirochete species introduction to Chapter 4 Abstract Introduction Materials and methods, results and discussion Electron microscopy of the extracellular mucoid layer of Treponema denticola ATCC 3 5405 Dark-field microscopy of the extracellular rnucoid layer of T. denticola ATCC 35405 Surface sugar lectin-analysis of T. denficola ATCC 35405. T. vincentii ATCC35580 and T. socranskii ATCC 35536 References CHAPTER 5. Summary and conclusions to the thesis Literature cited Appendix 1 : Further observations of the two morphotypes of T. denficola ATCC 35405 Appendix 2 : Abbreviations used in the thesis ABSTRACT The research work presented in this thesis involved the exarnination of several fundamental questions concerning the role of oral anaerobic spirochetes (OAS) in the etiology of penodontal disease (PD). OAS are unusual, helical, Gram-negative bactena that are considered putative periodontopathogens due to numerical association with diseased sites and the enzyrnatic arsenal available to OAS that appears consistent with disease symptoms. T. denticola is the most cornmonly isolated OAS fiom 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 suficient iron to prosper in the sub-gingiva was examined. The resultant mode1 suggests 7: denîicola obtains iron through the P-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 fiom T. denticola. 'H-labeled hemin is not transported into the cell. Evidence is presented to show that T. denticoh produces an iron reductase, which may facilitate the transport of ferrous iron across biological membranes. It is also show that T. denticola (Td), T. vincenîii (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 expenmental conditions has been shown to result in comective tissue degradation. It is also known that T. pallidurn, the agent of syphilis. produces a Hase that is critical to pathogenesis. Evidence is presented herein to show that Td, TV and Ts al1 produce a hyauronoglucosaminidase (HGase). The identity and specificity of the Td HGase is confirmed through the use of enzyme inhibiton and activators, by electron microscope observations of the enzyme using the Hase inhibitor gold sodium thiomalate and anti- pis mellifra venom antibodies vii 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 Mgel 701 beads. The last subject to be addressed by this thesis pertains to the ultrastmcnire 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, irnmunoglobulins and bacteriophages. to adherence and perhaps to the irnmunogenicity of OAS inhabiting the sub-gingiva. viii Le travail de recherche présenté dans cette thèse implique l'étude de plusieurs questions fondamentales concernant le rôle des spirochètes oraux anaérobies (OAS) dans l'étiologie de la maladie périodontale (PD). Les OAS sont des bactéries Gram-négatives en hélice soupçonnées d'être périodontopathogènes à cause de leurs nombreuses associations avec les sites atteints et l'arsenal enzymatique dont ils bénéficient (qui semble cohérent avec les symptômes de la maladie). Treponema denticola est I'OAS le plus souvent extrait des poches périodontales, et de ce fait, est le sujet de la plupart des recherches dans le rôle des OAS dans la PD. Étant donné que les sources de fer libre sont très limitées, nous avons examiné les moyens par lesquels les OAS peuvent se procurer le fer nécessaire