p.gingivalis

2. PORPHYROMONAS
GINGIVALIS

3. 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

4. INTRODUCTION
 Periodontal disease comprises a group of inflammatory
conditions of the supporting tissues of the teeth that are
caused by bacteria.
 In 1 mm3 of dental plaque
weighing approximately 1 mg
more than 108 bacteria

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

6. TAXONOMY
Family Bacteroidaceae (Bergey’s manual
1923)
3 Genera
Bacteroides LeptotrichiaFusobacterium
PORPHYROMONAS Prevotella
1989

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

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

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

10. 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”

11. MORPHOLOGY
 Pleomorphic
 Nonmotile short rods 0.5 X 1 to 2 μm
 Colonies: Smooth raised
Blood agar- young colonies

12. 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

13. MICROBIOLOGICAL TESTS
Immunodiag
nostic
methods
Nucleic acid
probe
Polymerase
chain reaction
Culture
methods

14. 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)

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

16.  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

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

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

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

20. 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

22. 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

23.  Chemical composition:
Mansheim &
Kasper
• Galactose
• Glucose
• glucosamine
Okuda et al
• Rhamnose
• Glucose
• Galactose
• Mannose
• methylpentose
Schifferle et
al
• Absence of
galactose
• Amino sugars

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

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

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

27.  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

28. 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

29. 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

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

31. 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

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

33.  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 )

34.  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.

35. Bacterial fimbriae
 Fimbriae play important roles in the expression of
virulence
 Peritrichous fashion
 2 distinct fimbria molecules
LONG / MAJOR
Subunit Fim
protein SHORT/ MINOR
Subunit Mfa
fimA gene
mfa1 gene

36.  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

37.  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.

38.  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

39.  Host cells:
 Macrophages, fibroblasts, epithelial & endothelial cells
 Interact with host components- α5β1-integrin, β2- 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,
α5β1

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

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

42. 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

43.  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

44.  Classified:
 Collagenases, Aminopeptidases, trypsin-like proteinases
Serine Apartate Thiol
Metallo-
proteinase

45. 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)

46. 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

47. Trypsine like proteinases
ARGININE
LYSINE
GINGIPAINS

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

49. Lysine
Arginine GINGIPAIN R
GINGIPAIN K
rgpA
rgpB
kgp

51. Pathogenic activity of Gingipains

52. Activation of kallikrein/Kinin system
BRADYKININ

53. 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

54. Activation of blood clotting mechanism

55. 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)

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

57. Degradation of fibrinogen & fibrin

58. 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

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

60. RgpB

61. 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

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

63. Enzymes
 Alkaline phosphatase
 Superoxide dismutase
 Sulfatase
 Heparinase
 Chondroitinase

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

67. PORPHYROMONAS
GINGIVALIS

68. 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

69. Adaptation strategies to environmental changes

70. 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

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

72. 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)

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

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

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

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

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

78. Adhesin molecules
 Fimbriae
 Major adherence-mediating determinant

79. 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

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

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

84. 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

85. RgpB

86.  Cytokines & chemokines:

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

88. 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

89.  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

90.  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

91. NONORAL INFECTIONS
 Occasionally been recovered from non - oral sites

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

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

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

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

96. 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?

97.  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

98.  Diabetes mellitus
 Hypertension
 Hyperlipidemia
 Smoking

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

101. 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

102. 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

103. 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

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

105. 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

106. CONCLUSION

107. REFERENCES
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