Treponema Denticola 38

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Ann Arbor, MI 48106-1346 USA 800-521-0600 • PHYSIOLOGIC AND MOLECULAR STUDIES ON ORAL ANAEROBIC SPIROCHETES AND PROCARYOTES FOUND IN BLOOD by Richard McLaughlin Faculty ofDentistry McGill University, Montreal March, 1999 A Thesis Submitted to the Faculty ofGraduate Studies and Research in Partial Fulfillment ofthe Requirements ofthe Degree ofDoctor ofPhilosophy © Richard McLaughIin, 1999 • National Library Bibliothèque nationale 1+1 of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395. rue Wellington Ottawa ON K1 A ON4 Ottawa ON K1A ON4 Canada canada Your file Votre ré'ërenee Ourfile Notre référenee The author bas granted a non L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library ofCanada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies ofthis thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format électronique. The author retains ownership ofthe L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts from it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permISSIon. autorisation. Q-612-55421-X Canada • TABLE OF CONTENTS Page Abstract ix Resumé xi Acknowledgments xiii Claim ofcontribution to knowledge xiv List offigures xvi List of tables xi~ List ofAbbreviations xxi CHAPTER 1. Introduction and Literature Review 1 I. Morphology ofSpirochetes 2 1. Mucoid Layers 5 ii. Outer Membrane Sheath 5 iii. Axial Fibrils 7 IV. Protoplasmic Cylinder Il v. Peptidoglycan 12 vi. Spirochete Genomes 13 vii. Spherical Bodies 15 II. Taxonomy ofSpirochetes 17 • ii • III. Ecology ofSpirochetes 19 IV. Periodontal Disease 23 l. AnOverview 23 11. Spirochetes as Etiological Agents 24 iii. Classification ofNovel Isolates 24 IV. Spirochete Culturability 25 v. Treatment ofPeriodontal Disease 27 vi. Adherence 30 vu. Cytotoxic Effects 32 viii. Iron Sequestration 35 v. Role ofthe Immune System in Gingivitis and Periodontitis 35 1. Introduction 35 11. Interleukin-l 36 lU. Tumor-Necrosis Factor-a. and Lymphotoxin 36 vi. Arachidonic acid 37 v. Metalloproteinases 37 Vi. Stimulation ofthe Immune System by Treponema denticola 38 vii. Summary 39 • VI. Spirochetes as the Etiological Agent ofAizheimer's Disease 40 Hi i. Introduction 40 • Il. Alzheimer's disease 41 Ill. Pathology 41 iv. Biochemical Abnormalities 42 VII. Examination ofBlood from Healthy Rumans 43 i. Introduction 43 ii. Phylogenetic Identification ofMicrobial Cells with Cultivation 43 111. Nonculture Methods for Identification ofMicroorganisms 45 iv. Norma flora ofthe Human Body 48 v. Blood Culture 48 VIII. L-Forms 49 IX. Mycoplasma 50 x. Procaryotes or Eucaryotes 51 XI. Outline ofthe Thesis 52 Guidelines Regarding Doctoral Theses Containing Quotations • From Published or Submitted Manuscripts 55 IV • Chapter 2. An inexpensive soUd medium for obtainiog colony-forming uoits oforal spirochetes 60 Abstract 61 Paper 62 Acknowledgements 73 Chapter 3. Rapid identification oforal aoaerobic spirochetes using restriction fragment length polymorphism ofthe 168 ribosomal gene 74 Introduction to Chapter 3 75 Abstract 76 Introduction 77 Materials and methods 79 Bacterial strains 79 Genomic DNA isolation and PCR amplification 79 Restriction fragment length polymorphisms 79 DNA cloning 80 Results and Discussion 82 • Acknowledgements 85 v Chapter 4. Factors affecting the formation ofspherical bodies in the • spirochete Treponema denticola 86 Introduction to Chapter 4 87 Abstract 88 Paper 89 Acknowledgements 98 Chapter 5. Alzheimer's disease May not be a spirochetosis 99 Introduction to Chapter 5 100 Abstract 101 Introduction 102 Materials and Methods 104 Subjects 104 Blood work-up 104 Results 107 Discussion 109 Acknowledgements 109 • vi Chapter 6. Naturally-occurring pleomorphic microorganisms in • human blood 110 Introduction to Chapter 6 111 Abstract 112 Introduction 113 Materials and Methods 114 Blood 114 Electron rnicroscopy 114 Average microscopic field 115 DNA extraction 116 PCR amplification and cloning 116 Sequencing 117 Indirect immunofiuorescence 117 Fluorescence in situ hybridization 118 Cell culture 118 Results and discussion 119 Acknowledgements 131 • vii • Chapter 7. Summary and Conclusions 132 References 138 • viii • ABSTRACT Spirochetes are helical bacteria consisting of an outer sheath, a protoplasmic cylinder and periplasmic flagella. AlI oral anaerobic spirochetes (OAS) are species within the genus Treponema. They are important causative agents ofperiodontitis. This thesis examines sorne aspects of the physiology of OAS. As weil, a novel symbiotic bacteriurn found in the blood of healthy hurnans was studied as a consequence of my work with OAS. Our lab has been instrumental in rendering routine and reliable growth of OAS in vitro. An inexpensive medium which remains molten at 37°C and solidifies at 25°C was found for the enumeration ofcolony-forming units ofOAS. New Oral Spirochete (NOS) medium with the addition of0.5% gelatin-0.5% Noble agar met the above criteria. Clinical isolates of spirochetes from the periodontal pocket need to be readily identified. Restriction fragment length polymorphism (RFLP) analysis was used on reference strains ofTreponema denticola, T. vincentii, T. phagedenis, and T. socransldi as weil as a number of clinical isolates in our laboratory collection. The banding patterns observed allowed discrimination between the different spirochete species. Morphological variations such as spherical-shaped cells of T. denticola, termed "spherical bodies" are occasionally observed. The omission of the severa! individual components from NOS medium (brain heart infusion, yeast extract, rabbit serum, volatile fatty acids, or thiamine pyrophosphate), the age of the culture and the addition of lactic • acid, enhanced the formation ofthese bodies. ix J. MikIossy (NeuroRepo~ 1993) reported that spirochetes were found in blood, • cerebral cortex and cerebral spinal fluid in autopsied A1zheimer's Disease (AD) subjects. ft was suggested by her that spirochetes could be a causative factor in AD. Our laboratory attempted to duplicate these results and found spirochetes in the blood ofonly one late stage AD patient suggesting that spirochetes are not one ofthe causes ofAD. During the examination of blood by darkfield microscopy, we observed pleomorphic microorganisms. Blood of a healthy human is a sterile environment. Evidence for the existence of bacteria in blood includes light and electron micrographs of their morphology, and molecular analysis oftheir 16S ribosomal RNA and their gyrB gene. • x RESUMÉ • Les spirochètes sont des bactéries hélicoidales formèes d~une membrane exteme~ d'un cylindre protoplasmique et d'un flagelle périplasmique. Tous les spirochètes anaérobes oraux (OAS) sont des espèces du "'genus" Treponema. Ils sont une cause importante de la périodontite. Cette thèse explore certains aspects de la physiologie des OAS. De plus, cet analyse des OAS a conduit à l'identification et 1étude d'une nouvelle bactérie symbiotique retrouvée dans le sang de sujets sains. Les membres de notre laboratoire se sont révélés capables de promouvoir la croissance des OAS de façon routinière et hautement reproductible. Un milieu de culture non-dispendieux~ qui se maintient sous fonne liquide à 37°C mais se solidifie à 25°C, servant à l'énumération d'unités formatrices de colonies, a été élaboré par nous. Le milieu NOS (New Oral Spirochete) additionné de 0.5% gélatine-O.5% agar Noble recontre les caractéristiques énumérées ci-dessus. Des isolats cliniques de spirochètes provenant de la poche périodontale doivent être identifiés. L'analyse par RFLP (Restriction fragment length polymorphism) de souches de référence de Treponema denticola, T. vincentii, T. phagedenis et de T. socranskii ainsi que de nombreux isolats cliniques provenant de notre propre collection a été faite. Les différents patrons de bandes

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