p.gingivalis

p.gingivalis

1. PORPHYROMONAS GINGIVALIS

2. CONTENTS Introduction Taxonomy Morphological & Biochemical characteristics Microbiological tests Oral ecology & transmission Virulence factors Role in periodontal diseases Effect of periodontal treatment Nonoral infections Conclusion References

3. INTRODUCTION Periodontal disease comprises a group of inflammatory conditions of the supporting tissues of the teeth that are caused by bacteria. In 1 mm 3 of dental plaque weighing approximately 1 mg more than 10 8 bacteria

4. World Workshop in Periodontology (Consensus Report 1996) designated A.actinomycetemcomitans Porphyromonas gingivalis Tannerella forsythia

5. TAXONOMY Family Bacteroidaceae ( Bergey’s manual 1923) 3 Genera Bacteroides Leptotrichia Fusobacterium PORPHYROMONAS Prevotella 1989

6. Emergence of the genus Porphyromonas : Castellani & Chalmers Gram-negative, non- sporeforming , non-motile anaerobic rods Genus BACTEROIDES 2 years later Oliver & Wherry , Black pigmented colonies on blood agar BACTEROIDES MELANINOGENICUS Growth requirements - factors X and V Genus HAEMOPHILUS (early editions of Bergey’s manual)

7. Genus BACTEROIDES was restored in the fifth edition of Bergey’s manual Prevot , adopted different classification system RISTELLA Schwabacher et al, No melanin B. nigrescens

8. Wilson & Miles, Fusiformis F.nigrescens Bacteroides in the seventh edition of Bergey’s manual

9. Bacteroides melaninogenicus asaccharolyticus intermedius melaninogenicus Holdeman & Moore Non oral Oral DNA homology B. gingivalis (Shah & Hardie and Coykendall et al) B. asaccharolyticus ICSB Taxonomic Subcommittee P. gingivalis Prevotella melaninogenicus Prevotella intermedia

10. Bacterium melaninogenicum - 1921 Bacterium melaninogenicus - 1939 asaccharolyticus B melaninogenicus intermedius melaninogenicus B. loescheii B. denticola 1970 1982 Prevotella Melaninogenicus 1988, 1990 Prevotella Loescheii 1988, 1990 Prevotella Denticola 1988, 1990 Holdeman & Moore

11. Bacterium melaninogenicum - 1921 Bacterium melaninogenicus - 1939 asaccharolyticus B. intermedius-1983 intermedius melaninogenicus 1970 Prevotella Intermedia 1988, 1990 Holdeman & Moore

12. Bacterium melaninogenicum - 1921 Bacterium melaninogenicus - 1939 asaccharolyticus B. asaccharolyticus-1977 intermedius melaninogenicus B. gingivalis (oral) B. asaccharolyticus (non-oral) 1970 1980 Porphyromonas gingivalis 1988 Porphyromonas asaccharolyticus 1988 Holdeman & Moore Shah & Hardie and Coykendall et al

13. Genus PORPHYROMONAS Gram-negative, Obligately anaerobic, Nonfermentative , non- sporeforming , nonmotile rods Purple pigment in “black-pigmented” colonies Porphyromonas asaccharolytica (Gut) Porphyromonas gingivalis “of the gums” Porphyromonas endodontalis “within a tooth”

14. MORPHOLOGY Pleomorphic Nonmotile short rods 0.5 X 1 to 2 μ m Colonies: Smooth raised Blood agar- young colonies

15. BIOCHEMICAL PROPERTIES Low oxygen tension Nitrogenous substrates Subgingival ecosystem IDEAL ENVIRONMENT Redox potential= low Shah HN, Arginine may be the primary substrate Peptides Phenylacetic acid

16. MICROBIOLOGICAL TESTS

17. ORAL ECOLOGY & TRANSMISSION Natural habitat : Highest frequency—periodontal pocket ( Asikainen et al 1997) Supragingival plaque and oral mucosal surfaces ( muller et al 1993) Saliva (van Winkelhoff et al 1986) Pharynx (van steenberg et al 1993)

18. Initial colonization: Clean Tooth surface Inflammation – poor oral hygiene & sites harboring G+ve dental plaque bacteria X

19. Subgingival distribution: Widely distributed (Beck et al) Efficacy of antibody response IgG response are not able to control Pg ( Lamster et al) Rodenburg et al -- Pg absent in younger age group (less than 20 yrs) -- age 30 -70 yrs harbored 60% of pathogens

20. Transmission: Vertical transmission: Tuite -McDonnell et al – intrafamilial transmission

21. Horizontal transmission: Siblings- Petit et al 1993 Saarela et al 1996 Spouses – Asikainen et al 1996:- 30-75% Identical genotype

22. Route of infection from person to person Saliva, mucosal contact and inanimate objects

23. VIRULENCE FACTORS Classical studies Recent observations Molecules that result in the establishment and maintenance of a species associated with or within the confines of the host Molecules that exerts a detrimental effect on a host cell

24. Capsule Anti- phagocytic virulence factor Ruthenium red & routine lead acetate staining Electron dense layer Polysaccharide capsule Some strains- devoid 6 distinct capsular serotypes (K1-K6) Laine et al 1996 seventh serotype (K7) - R. E. Schifferle

25. Chemical composition :

26. Biological function: More hydrophilic Increased resistance to - phagocytosis - serum resistance Decreased induction of PMN leukocyte

27. Schiffer et al - decreased ability to activate alternate complement pathway Sundqvist et al- capsule does not guarantee that specific strains will be virulent

28. Outer membrane protein SDS-PAGE analysis 20 Major Proteins Mw 20-90 kDa

29. Mihara & Holt 1993 : - 24-kDa thymidine - fibroblasts. “fibroblast-activating factor” Takahashi et al :- Bone assay- Ca ++ release from bone Watenabe et al :- 75kDa protein B-cell activation, IL-1 production

30. Role in Coaggregation Gibbons & Nygaard 1970 - bacteria attach to both hard & soft surfaces G+ve & G- ve bacteria= specific outer membrane proteins Pg & A. viscosus – initial event in formation of subgingival biofilm Kinder & Holt 1989 = specific adhesin - receptor molecule

31. P gingivalis & Hemin hemin (iron) = growth Karunakaran et al 1993 – 48 kDa & 18 kDa Cytochrome b subunit Fumarate Succinate ENERGY Protoporphyrin IX Exogenous suppliment Hemolysin Attack & Hemolyze RBC

32. GCF- hemoglobin hemin -binding proteins – haptoglobin , hemopexin & albumin Shizukuishi et al – hemoglobin as main source of iron

33. Lipopolysaccharide Larger molecules ranging from 10kDa & larger Amphipathic character Hydrophilic end- Polysaccharide (O antigen) Hydrophobic end- lipid A SDS-PAGE analysis- Ladder like band appearence

34. Endotoxicity – Lipid A (Ogawa et al 1993) Immunobiological activity – O antigen (Takada H 1992)

35. P.gingivalis lipid A, induced IL-1 receptor antagonist IL-6, IL-8, interferon- γ Granulocyte-macrophage colony-stimulating factor Poor inducer of IL- lβ and TNF-α. ( Yamaji et al 1995, Ogawa et al )

36. P. gingivalis LPS, capable of stimulating the host inflammatory response in the epithelial cells, endothelial cells, fibroblasts, macrophages etc via the induction of host derived cytokine production.

37. Bacterial fimbriae Fimbriae play important roles in the expression of virulence Peritrichous fashion 2 distinct fimbria molecules fim A gene mfa1 gene

38. Long/ Major fimbriae : ( Yoshimura  et al ., 1984 )   First recognized on outer surface from strain 381 FimA proteins – size 40.5 to 49 kDa Type 1-4 based on amino-terminal sequence

39. Classification of Pg strains into different genetic groups based on fimA variations: Amano A et al 1995 6 variants: Type I – V & Ib Type II fimA genotype- Periodontitis ( > 8 mm pockets) Followed by type IV, Ib or I depending on ethnic population type I & III – Healthy subjects Fujise et al 2005 – despite lower prevalence, type I are associated with diseased sites refractory to periodontal treatment.

40. Adherence ability to host proteins : Human salivary proteins- Statherin , Proline -rich proteins, proline -rich glycoprotein ECM- laminin , fibronectin , type I collagen, elastin , vitronectin Other bacterial components

41. Host cells: Macrophages, fibroblasts, epithelial & endothelial cells Interact with host components- α 5 β 1-integrin, β 2- integrin , Inflammatory response : release of cytokines like IL-1, IL-6, IL-8, and TNF- α , toll like receptor 2 (Amano et al 1998, Ogawa et al 2002) ICAM-1, VCAM-1, and P- and E- selectins , α5 β 1

42. Short / Minor fimbriae : ( Arai et al, 2000) Homopolymers of subunit protein mfa1 Molecular mass 75 kDa Visualized when long fimbriae are absent Induce IL-1 α , IL-1 β , IL-6, TNF- α

43. Non fimbrial proteins Regulate the expression of the fimbriae Proteinase , Aminopeptidase , Caseinase , Collagenase etc

44. Proteinases Earlier thought - non-specific degradation enzyme Recent studies - cause specific activation/ inactivation of bioactive proteins. Exposed at the surface (in the outer membrane)- vesicles Within the periplasmic space

45. Functions of proteases: kuramitsu et al Internally directed Externally directed Internally Externally Growth rate Outer membrane protein processing Fimbrial expression Regulation of protease expression Processing of proteases Vascular permeability Blood clotting Complement inactivation Hemagglutination Binding to eukaryotic cells Binding to G+ve bacteria MMP activation Platelet aggregation Cytokine regulation Antibody degradation Cytokine receptor alterations Attenuate neutrophil activity

46. Classified: Collagenases , Aminopeptidases , trypsin -like proteinases

47. Collagenase Periodontal tissue destruction - specific proteolytic enzymes, especially the collagenases Host and periodontopathic microbiota P. gingivalis collagenase may participate with host-derived collagenase ( Mayrand and Grenier et al 1985)

48. Aminopeptidase Only member of periodontopathic microbiota that exhibits strong dipeptidyl arylaminopeptidase activity Acts on type I collagen 2 additional aminopeptidase Abiko et al N- CBz - glycyl - arginyl peptidase Glycyl-prolyl peptidase

49. Trypsine like proteinases

50. Structure of gingipains Endopeptidases = 85% of general proteolytic activity Potempa et al 1997 100% trypsin – like activity Potempa et al 1995

51. Lysine Arginine GINGIPAIN R GINGIPAIN K rgpA rgpB kgp

52. Gingipain R

53. Gingipain K

54. Pathogenic activity of Gingipains

55. Activation of kallikrein / Kinin system BRADYKININ

56. Activation of kallikrein / Kinin system Hinode et al.(1992)and Kaminishi et al.(1993) ; Imamura et al.(1994 ) Potent vascular permeability enhancement (VPE) factors GCF production and edema formation  continuous supply of nutrients. Bradykinin - Alveolar bone resorption by inducing prostaglandin production Gingipain R Gingipain K

57. Activation of blood clotting mechanism

58. Activation of blood clotting mechanism Potent platelet activator and converts fibrinogen to a fibrin clot, thus plugging damaged vessels. Enhances vascular permeability ( DeMichele et al 1990) Induces leukocyte chemotaxis (Bar- Shavit et al 1983)

59. Stimulates prostaglandin secretion by osteoblastic cells & potentiates LPS-stimulated IL- 1 production by macrophages HRgpA was more potent than RgpB Gingipain R

60. Degradation of fibrinogen & fibrin

61. Degradation of fibrinogen & fibrin Bleeding on probing Gingipains degraded fibrinogen within minutes (Pike et al 1996) Fibrinogenolytic activity of the bacterium is attributed mainly to the Kgp Cannot be effectively controlled by host proteinase inhibitors Gingipain R Gingipain K

62. Disturbance of host defence system Gingipain R complement system activation C3 convertase production bacteriolysis through complement system activation impaired consumption of its components

63. RgpB

64. Gingipains Degrade monocyte CD14, a major receptor for bacterial lipopolysaccharides Leukocytes hyporesponsive to LPS (Sugawara et al 2000) Signal by LPS insufficient for phagocytes to secrete cytokines and stimulate immune cells

65. GINGIPAINS Cytokine activation & inactivation Phagocytic receptor cleavage Complement component degradation Kallikrein / kinin System activation Fibrinolysis MMP synthesis stimulation & activation Clotting system activation Sustained P.g infection Inflammation Gingival swelling GCF production Bleeding tendency Alveolar bone resorption Periodontal bone destruction Gingival recession DIC IHD MULTIPLE PATHOGENIC ACTIVITIES Potempa et al 2000 PERIODONTITIS

66. Gingipains as a target for therapy Vaccination therapy immunization of primates with RgpB - reduce alveolar bone loss Amino terminal of catalytic domain Capable of inducing a protective immune response against P. gingivalis infection

67. Caseinase Hydrolyze the protein casein active against salivary and egg-white lysozyme & insulin chain B. Exists as three isoenzymes .

68. Enzymes Alkaline phosphatase Superoxide dismutase Sulfatase Heparinase Chondroitinase

69. Capsule Outer membrane proteins Hemin Lipopolysaccharide Fimbriae – major & minor Proteinase – serine, aspartate , thiol , metalloproteinase Collagenase Aminopeptidase trypsin - like proteins- gingipains Caseinase Enzymes

70. Development of inhibitors specific for gingipains DX-9065a, a proteinase inhibitor selectively reduces P. gingivalis growth (Matsushita et al 2006) Tetracycline and its analogues Ability to inhibit multiple gingipain actions rather than eradication of P.gingivalis

71. PORPHYROMONAS GINGIVALIS

72. CONTENTS Introduction Taxonomy Morphological & Biochemical characteristics Microbiological tests Oral ecology & transmission Virulence factors Adaptation strategies to environmental changes Role in periodontal diseases Effect of periodontal treatment Nonoral infections Immunization Conclusion References

73. Adaptation strategies to environmental changes

74. Temperature Mean temperature of the gingival sulcus during health - 35°C (30°C to 38°C ) Socransky SS, Haffajee AD, 1991 P. gingivalis when exposed to elevated temperature - heat shock response Lu et al 1994 Heat shock proteins function as molecular chaperones - involved in protein folding and oligomerization of structural proteins and DNA replication

75. GroEL (HSP6O family) and DnaK (HSP70 family) homologs have been described in P. gingivalis Vayssier et al 1994 temperature - fimbrillin expression superoxide dismutase activity A mano et al 1994

76. pH pH range within gingival sulcus during health -7.0 to 8.5 ( Cimasoni 1983) As disease progresses Periodontal pockets deepen and host inflammatory response is induced pH increases ( Cimasoni 1985) Gram + ve facultative →Gram – ve anaerobic (Marsh et al 1994)

77. Optimal pH for P. gingivalis - 7.5 (7.5 to 8.5) Marsh et al 1994 Trypsin -like activity with pH

78. Oxygen Oxygen concentration - induce the HSP6O-like stress protein in P. gingivalis Vayssier et al

79. ROLE IN PERIODONTAL DISEASE INITIAL COLONIZATION OF THE ORAL ENVIRONMENT INTERACTIONS WITH EPITHELIAL CELLS ENCOUNTER WITH HOST DEFENCE MECHANISMS

80. Entry into oral cavity Transmission from infected individuals Saliva = vector

81. Adherence to oral surfaces requires antecedent organism to create necessary environmental conditions

82. Adhesin molecules Fimbriae Major adherence-mediating determinant

83. Hemin : Growth Promote colonization-binding of bacteria to receptors on human cells

84. INTERACTIONS WITH EPITHELIAL CELLS Gingival epithelium- stratified squamous epithelium Junctional epithelium Sulcular epithelium Interaction- bacteria & epithelial cells CELLULAR MICROBIOLOGY ( Cossart et al 1996) Invasion- primary cultures of gingival epithelial cells, oral epithelial cell lines & cultures of multilayered pocket epithelium NON KERATINIZED

85. Impact on bone metabolism Alveolar bone loss- stimulating bone resorption , inducing bone destruction & inhibiting bone formation Pg LPS Osteoclasts PGE2 IL-1 β , TNF α Alveolar bone resorption Macrophages, monocytes fibroblasts

86. Heat-stable polysaccharide antigens 24 kDa outer membrane protein Major fimbriae

87. ENCOUNTER WITH HOST DEFENCE MECHANISM Bacterial modulation of host immune processes Innate & Acquired defence mechanism Pg impinges – PMN recruitment & activity Neutrophil chemotaxis X LMW fatty acid- succinic acid Immobilize PMN – depolarizing PMN membrane Inhibit E- selectin secretion - neutrophil adhesion & diapedesis

88. RgpB

89. Cytokines & chemokines :

90. Proinflammatory cytokines: IL-1 β , TNF- α , IL-6 & IL-8  promote inflammation Anti-inflammatory cytokines Degrading existing cytokines Antagonize IL-8 production

91. EFFECT ON PERIODONTAL TREATMENT Scaling and root planing - temporary decrease in levels but not capable of eradicating the organism from subgingival sites. Non - resective periodontal surgery - not effective in removing P. gingivalis

92. Elimination of periodontal pockets along with proper oral hygiene ( Mombelli A, 1995) Systemic antibiotic therapy + scaling and root planing may not ensure subgingival eradication of P. gingivalis Topical antimicrobial therapy - not very useful

93. Periodontal surgery + systemic antibiotic therapy + good oral hygiene: Zarkesh et al. (1999) – coating PTFE barrier membranes with tetracycline They permitted less P. gingivalis colonization and more clinical attachment gain

94. NONORAL INFECTIONS Occasionally been recovered from non - oral sites

95. Cardiovascular disease: Atherosclerosis Arteriosclerotic aneurysms Peripheral arterial disease Coronary heart disease Heart valves of endocarditis Buerger’s disease

96. Transient bacteremia - 17-100% After preventive dental procedures & periodontal therapy Tooth brushing Chewing Subgingival irrigation Periodontal treatment Dental extraction

97. Important factors in Pg mediated atherosclerosis: Blood cholesterol levels Toll-like receptors Soluble inflammatory mediators Chiu et al 1999

98. P. gingivalis Soluble Inflammatory mediators Adhesion molecules oxLDL Scavenger receptors Indirect action Direct action monocytes Macrophage Foam cell Smooth muscle cell Transformation Transformation

99. Questions regarding Pg mediated mechanisms in vascular disease How can Pg, an obligate anaerobic bacterium, safely travel through the bloodstream from small vessels in the oral cavity to reach the central arteries in which atherosclerotic lesions develop? How can Pg adhere to normal endothelial cells given the extremely rapid blood flow in the abdominal or thoracic aorta? How can Pg invade normal endothelial cells of large-sized arteries?

100. Under normal physiologic conditions, not possible for anaerobic bacterium to invade normal endothelial cells An indirect mechanism However Direct invasion – endothelial function/ structure is destroyed Hokamura et al 2009

101. Diabetes mellitus Hypertension Hyperlipidemia Smoking

102. IMMUNIZATION Attenuated and inactivated bacterial vaccines Live bacterial vectors Passive immunization Purified antigen (subunit) vaccines Synthetic antigen vaccines

103. Attenuated and inactivated bacterial vaccines Production of serum antibody, which correlated with immune protection from the virulence properties of P.gingivalis ( Ebersole 1997, Genco CA, 1992, Kesavalu,1992) Active immunization of mice (Baker et al 1997) or rats ( Taubman et al 1983) with P. gingivalis - ability to alter disease manifestations of periodontitis in these animals

104. Live bacterial vectors The hemagglutinin gene of P. gingivalis has been cloned into an avirulent strains of S. typhimurium Dusek DM 1995 Used to orally immunize mice and resulted in a systemic and mucosal response to this antigen

105. Passive immunization Booth et al. (1996) produced a murine monoclonal antibody to P. gingivalis which prevented recolonization of deep pockets in periodontitis patients Laboratory tests revealed that this antibody inhibited the hemagglutination of red blood cells

106. Purified antigen (subunit) vaccines Bird et al. (1995) used the mouse abscess model and immunized it with an outer membrane – induction of protective immunity

107. Synthetic antigen vaccines Requires synthesis of linear & branched polymers of 3-10 amino acids based on known sequences of microbial antigens. Weakly immunogenic Coupled to large proteins  antibody response Safe, cheap, easy to store & handle & ideally suited to specific targets

108. CONCLUSION

109. REFERENCES Ingar Olsen, Haroun Shah & Saheer Gharbia . Taxonomy and biochemical characteristics of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis . Periodontology 2000, Vol. 20, 1999, 14-52 Jorgen Slots . Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in periodontal disease: introduction. Periodontology 2000, Vol. 20, 1999, 7-13 Sigmund Socransky and Haffajee . Periodontal microbial ecology. Periodontology 2000; Vol 38, 2005, 135-187

110. Stanley Holt, Lakshmyya Kaesavalu , Stephen Walker & C.A. Genco . Virulence factors of Porphyromonas gingivalis . Periodontology 2000, Vol. 20, 1999, 168-238 Takahisa Imamura . The role of gingipains in the pathogenesis of periodontal diseases. J Periodontol 2003;74:111-118 Newman, Takei, Klokkevold , Carranza. 10 th edition. Carranza’s Clinical Periodontology . W. B. Saunders Company H.k.Kuramitsu . Proteases of Porphyromonas gingivalis : what don’t they do?. Oral Microbiol Immunol 1998:13:263-270

111. Jorgen Slots & Miriam Ting . Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in human periodontal disease: occurrence and treatment . Periodontology 2000, Vol. 20, 1999, 82-121 Arie J. Van Winkelhoff & Jorgen Slots . Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in non oral infections. Periodontology 2000, Vol. 20, 1999, 122-135 Jorgen Slots & Taubman . Contemporary Oral Microbiology & Immunolgy