Porphyromonas gingivalis - Dr Harshavardhan Patwal

Porphyromonas gingivalis belongs to the phylum Bacteroidetes and is a nonmotile, Gram-negative, rod-shaped, anaerobic, pathogenic bacterium. It forms black colonies on blood agar.
It is found in the oral cavity, where it is implicated in certain forms of periodontal disease, as well as in the upper gastrointestinal tract, the respiratory tract, and the colon. It has also been isolated from women with bacterial vaginosis. Collagen degradation observed in chronic periodontal disease results in part from the collagenase enzymes of this species. It has been shown in an in vitro study that P. gingivalis can invade human gingival fibroblasts and can survive in them in the presence of considerable concentrations of antibiotics.P. gingivalis also invades gingival epithelial cells in high numbers, in which cases both bacteria and epithelial cells survive for extended periods of time. High levels of specific antibodies can be detected in patients harboring P. gingivalis. Dr Harshavardhan Patwal , explains the various enzymes enzyme peptidyl-arginine deiminase, which is involved in citrullination.[4] Patients with rheumatoid arthritis have an increased incidence of periodontal disease, and antibodies against the bacterium are significantly more common in these patients.
P. gingivalis is divided into K-serotypes based upon capsular antigenicity of the various types.

1. PORPHYROMONAS
GINGIVALIS
Dr Harshavardhan PatwalDr Harshavardhan Patwal

2. INTRODUCTION:
Periodontitis is a multifactorial disease due to complex interaction
between the host and plaque bacteria. Although specificity has
been found for Actinobacillus actinomycetemcomitans in LJP,
it is difficult to obtain a specific etiological role of bacteria
associated with periodontal disease of adults.
The main anaerobic and gram negative bacteria implicated as
etiological agent for periodontal disease include P. gingivalis,
B. forsythus, P. intermedia, A. actinomycetmcomitans, C.
species, T .denticola, spirocetes, F .nucleatum, C. rectus and E.
corrodens – putative periodontal pathogens (Loesche.W 1992)

3. Bacteria must either colonize the gingival crevice by
evading host defenses, damaging the crevicular epithelial
barrier or producing substances, which can directly or
indirectly cause tissue damage.
P. Gingivalis produces by far the greatest proteolytic
activity of any periodontal bacterium. The hallmarks of
periodontitis including bleeding on probing, neutrophil
accumulation, attachment loss and increased crevicular
flow all involve proteolytic events (Travis J 1997)

4. P.Gingivalis
This species are gram negative, anaerobic, non-motile,
asaccharolytic rods that usually exhibit coccal to short rod
morphologies.
Is a member of “Black-Pigmented Bacteroides” group.
Organisms of this group from brown to black colonies on blood
agar plates and were initially grouped into a single species,
B.melaninogenicus (Oliver & Wherry 1921)
In 1970s, it was recognized that bacteroides contained species
that were asaccharolytic and either had an intermediate level of
carbohydrate fermentation or highly saccharolytic.

6. The role for P.g as periodontal pathogens
Haffajee & Socransky 1994
Association Elevated in lesions of periodontitis
Lower in sites of health, gingivitis and
edentulous subjects.
Elevated in actively progressing lesions
Elevated in subjects exhibiting periodontal
disease progression.
Detected in cells or tissues of periodontal
lesions.
Presence indicates increased risk for alveolar
bone loss and attachment level loss.

7. Elimination  Elimination resulted in
successful therapy.
 Recurrent lesions harbored the
species.
 Successful treatment lowered
level and / avidity of antibody.
Host response  Elevated antibody in serum or
saliva in subjects with various
forms of periodontitis.
 Altered local antibody in
periodontitis.

8. Collagenase,
Endotoxin
Proteolytic trypsin-like activity
Fibrinolysin
Hemolysin
Gingipain,
Phospholipase A
Degrades Ig,
Fibroblast inhibitory factor,
Capsule polysaccharide, bone Resorption inducing
factor,
Induction of cytokine production from various host
cells,
Generates chemotactic activities,
Inhibits migration of PMNs across epithelial
barriers,
Invades epithelial cells in vitro.
Virulence
factors

9. ANIMAL
STUDIES

Imporant in
experimental pure or
mixed subcutaneous
infections.
Induced disease in
gnotobiotic rats.
Studies in
sheep,monkeys and
dogs.
Immunisation
diminished disease in
experimental animals.

10. P.Gingivalis
DESCRIPTION OF THE GENUS OF PORPHYROMONAS
Species of the genus porphyromonas (Collier L 1998)
Porphyromonas cangingivalis
Porphyromonas canoris
Porphyromonas catoniae
Porphyromonas circumdentaria
Porphyromonas crevioricanis
Porphyromonas endodontalis
Porphyromonas gingivalis
Porphyromonas gingivicanis
Porphyromonas levii
Porphyromonas macacae

11. Members of the genus are 0.5 – 0.8 by 1.0 – 3.5µm diameter and
are obligately anaerobic, non-spore forming, non-motile
rods.
CHARACTERISTICS OF GENUS:
Production of large amounts of cell-associated protoheme.
When grown on complex carbohydrates and proteins the
major fermentation end products are n-butyrate, propionate
and acetate which accounts for much of the malodour
associated with oral infections.
Several of these strains possess significant proteolytic
activity the other strains are relatively nonproteolytic.

12. Biochemical properties of p.gingivalis:
Host defense
mechanism
Bacterial speciesBacterial propertyBiologic effect
Specific antibodyp.gingivalis
p.intermedia
IgA IgG
degrading protease
Degradation of
specific antibody
PMN p.gingivalis
A.A
T.Denticola
Capsule
Inhibition of
superoxide
production
Inhibition of
phagocytosis
Decreased
bacterial killing
Release of IL-8p.gingivalis Inhibition of IL-8
production by
epithelial cells
Impairment of
PMN response to
bacteria

13. Socransky & Haffajee 1994
P.Gingivalis
Bacteroides Forsythus
Treponema Denticola

14. Invasion of P.Gingivalis Into Gingival Epithelial Cells:
Invasion and internalization mechanism of p.g provide
some fascinating insights into the molecular dialogue that
occurs between bacterial and host cells.
P.G interaction with primary gingival epithelial cells:
Bind through adhesions such as fimbriae to the surface receptors
on gingival cells.
Microtubules and microfilaments are rearranged to facilitate
invagination of the membrane – engulfment of bacterial cells.
p.g cells rapidly locate in the perinuclear area . Ca ions are
released from intercellular stores and othr signaling molecules
such as the MAP-kinase family can be phosphorylated /
dephosphorylated or degraded by gene expression. (Lamout.RJ
1998)

15. Model of p.g trafficking in endothelial cells

16. Transmission of periodontal pathogens
The behaviour of a pathogenic microorganism depends
upon the interaction depends upon the interaction between
the host.
Four generally accepted routes of bacterial transmission are,
1.Contact - person to person
2.Common vehicle – food / water
3.Airborne – droplets
4.Through a vector

17. Vertical transmission: when transmission is directly from
parents to offspring.
Horizontal transmission: when an individual infects unrelated
individuals by contact, respiratory or faecal-oral spread.
Zambon et al 1983 – two major periodontal pathogens, A.A,
P.gingivalis.
They suggest that the bacteria are transmitted between family
members or that family members share susceptibility to
colonization by these bacteria or clonal types of these
species.

18. Virulence factors
Definition:
Factors which enable pathogen to adhere colonize invades host
tissue, evade the host defenses and induce tissue destruction.
Virulence attributes of microbial pathogens include the ability to
Enter a host
Find a unique ecological niche
Circumvent or subvert the host’s normal defenses
Replicate in new environment
Express specialized pathogenic traits

19. VIRULENCE FACTORS OF P. GINGIVALIS
P.g possesses an armamentarium on the cell surface
associated with extracellular activities.
Several are putative adhesions which interact with
other bacteria, epithelial cells, extra cellular matrix
proteins.
Secreted or cell bound enzymes, toxins and
hemolyisn may play a significant role in the spread
of organism through tissue, tissue destruction and in
evasion of host defenses.

20. Virulence factors and host effectors produced by p.gingivalis
Tissue destruction Host evasion
Collagenase
Trypsin like proteases
Gelatinase
Aminopeptiease
Phospholipase – A
Alkaline phosphatase
Acid phospatase
Chondroitin sulfatase
Hyaluronidase
Keratinase
Heparinase
Epitheliotoxin
Fibroblast growth inhibitors
Endotoxicity
Lps induced bone resorption
Degrdation of plasma protease inhibitors
Degradation of iron transport proteins
Inhibition of PMN leucocytes
Chemotaxis inhibitors
Decrease phagocytosis
Lysis and intracellular killing
Resistance to c’killing
C-3 / c-5 degradation
Ig protease

21. Bacterial protease – virulence factors:
Considered as unique molecular attributes of a pathogen used to
accomplish colonization and infection of host.
Certain “housekeeping” functions are required by the pathogen
for efficient multiplication on non-living substrates which in
considered as virulence factors.
To provide peptide nutrients for a bacterial pathogen
Ability to contribute to the pathogenesis of infectious diseases
They mediate direct damage to hot tissues by lysing host cell
surface and tissue proteins, mediate destruction indirectly by
activating MMP .

22. BACTERIAL FIMBRIAE:
Fimbriae were first reported on meners of
enterobacteriaceae, originally referred to as
pili and were shown to be important in RBC
agglutination.
2 major classes of fimbriae:
1.Type specific fimbriae
2.Sex pili

23. The type specific fimbriae have been found to play
important role in interaction between specific host
cell and delivery of selected toxins and as
colonization antigens.
Fimbriae may be involved in motility and in
chemotaxis.
It contain fimbriae arranged in a peritrichous
fashion over the cell surface. The mature fimbriae
are composed of at least 1000 protein subunits.
The fimA gene is resident on chromosome as a
single copy and all P.gingivalis strains contain this
gene.

24. BIOLOGICAL PROPERTIES OF FIMBRIAE OF P.GINGIVALIS:
binding of the bacterium to host cells and saliva-coated
hydroxyapatitie.
It bound well to epithelial host cells revealed abundant
peritrichously arranged fimbriae which is 1-1.5mm long and 0.5nm
wide on their surface.
Antibody to the fimbriae of P.g strain 381 found to inhibit adhesion
of bacteria to the host cells.
Interaction of the fimbriae with the 48 Kda protein – first step in
signaling process that mediates uptake of the bacteria into the host
cell.(Weinberg et al)
Highly immunogenic, eliciting both an antibody and cell mediated
immune response in serum and saliva.

25. Genetic examination of P.Gingivalis fimbriae formation:
The fim A found to be resident on chromosome as a single copy
gene in all P.g strains.
It exhibit heterogeneity in fim A and since only one fimA gene
copy is found on the chromosome, the variation in the fimbrillin
gene is most likely due to mutational events
GENETIC EXCHANGE BETN STRAINS.
Environmental effects on P.g fimbriae formation:
It include temperature, pH, hemin limitation, serum saliva,
galactidase activity,osmotic effects and the effects of Ca
limitation.

26. Characteristic of minor fimbriae species from P.G
strains:
Fimbrillin peptides were required to mediate
attachment of p.g to saliva coated
hydroxyapatite.
Involved in both adherence to epithelial cells
and fibroblasts as well as initiating
haemagglutination reactions.

27. Non –fimbrial proteins:
Involved in interaction with host cells and regulate
the expression of the fimbriae.
Non-fimbrial components were identical to pure
proteins and biochemical characterization revealed
them to be identical to arginine specific cysteine –
proteinases.
Study on non fimbrial P.g surface proteins – the
surface proteinase and cysteine proteinase are
putative adhesive and function in regulation and
expression of p.g fimbriae.

28. CAPSULE:
Is an important anti-phagocytic virulence factor
Electron microscopic: P.g strains by ruthenium red staining
and routine lead staining – presence of an electron dense
layer external to the outer membrane- polysaccharide
capsule.
CHEMICAL COMPOSITION:
P.g 381 contains glucose, galactose and glucosamine
(Manshiem .B.J 1977)
Okuda et al determined the sugar composition of a similar
strain.

29. BIOLOGICAL FUNCTION:
It exhibited decreased auto-agglutination
Lower buoyant densities
More hydrophilic than the less encapsulated strains
Increased resistance to phagocytosis
Serum resistance
Decreased chemiluminescence
It conjugated to bovine serum albumin and also to p.g
fimbriae protein
Protection from host defenses

30. OUTER MEMBRANE PROTEINS
The cell envelop has three part:
1.Inner cytoplasmic membrane
2.A thin peptidoglycan
3.Attached to which is the asymmetrical outer membrane
The outer membrane contains:
-Complex LPS
-Lipoproteins
-Peripheral and transport proteins which connect OM to
peptidoglycan and provide structural integrity to the cell
envelop
-Porin proteins provide a transport mechanism for the
movement of selected proteins
-OM is covered by numerous thin, short fimbriae, if motile
long thick flagella

31. -LPS and heamagglutinins are intimately associated with the OMP
-OM of P.g studied by using shearing to separate the om from the
peptidoglycan showed that major OMP’s had varied effects on the
epithelial cells, fibroblasts and bone cells.
-It contains at least 20 major proteins ranging in size from app 20 to 90
Kda.
Mihara and holt purified a 24-Kda protein from the outer membrane
vesicles of P. strain W50. Because of its significant fibroblast
stimulating ability, - 24-Kda protein as “fibroblast activating factor”
75-Kda major outer membrane protein that exists as high molecular weight
oligomer.
Watanabe et al – protein canstimulate polyclonal – B-cell activation and
can elicit IL-1 production.

32. P.G outer membrane proteins and coaggregation:
Coaggregation between P.g and A.viscosus – found
to be important for the initial events in the
formation of the subgingival biofilm.
Kinder and holt – these coaggregating pairs of
resident gr negative members of the
periodontopatic microbiota also interacted with
each other via specific adhesin-receptor
molecules.

33. P.GINGIVALIS AND HEMIN:
P.g has an absolute growth requirement for hemin, the presence of an
active hemolysis associated with the bacterium was investigated
5 p.gingivalis strains from their ability to lyse red blood cells, and all
five strains produced a functional ‘hemolyisn” associated with the
outer membrane vesicles.
Two of these “hemin – regulated outer membrane proteins” app 83
and 26 Kda, recognized by SDB PAGE analysis as major hemin
regulated p.g W50 outer membrane proteins.

34. LIPOPOLYSACCHARIDE:
The outer membrane of gm negative bacteria lies external to the
peptidoglycan and is attached to it by lipoproteins.
Murein lipoproteins are attached by both covalent and non-covalent
bonds to protein units within P.g and to the outer membranes by their
lipid moieties.
Its very large molecule, which estimates 10 Kda.
Its amphipathic character is a result of one end of the molecule, the
hydrophilic and consisting of the polysaccharide
O-specific antigen which is exposed to the environment on the
exterior surface of the outer membrane and the core region
Within the outer leaflet which connects the O-antigen to the
hydrophobic end of the molecule or lipid A.

35. CHEMICAL COMPOSITION:
Hold and Bramanti – LPS composition rich in a C15 : 10 iso-branched
chain fatty acid ranging from 6 to 48% of the total acids depending
on the strain.
Sugar analysis of LPS from at least six different P.g strains indicates that
the LPS contains neutral sugars.
Biological properties:
Antibodies to fimbriae pg 381 found to inhibit adhesion of the
bacterium to host cells
Endotoxic property is confined to the lipid a, while significant
immunobiological activity is contained within the O-antigen.
Lipid A has an IL-1β antagonist activity in tissue cultures stimulated
with compound 506 or E.coli LPS.

36. Ability to function as a chemokine inducer and antagonist.
Protection of host cells from cytopathic effects.
Yamaji et al – the induction of IL-6 and IL-8 in human PDL
fibroblasts when exposed to p.gingivalis LPS.
INTERACTION OF LPS WITH HOST CELLS:
MOLECULAR STUDIES:
LPS very effective in activating both myeloid and non-myeloid
cells via an interaction of LPS with a LPS binding protein.
It stimulates cytokine secretion in monocytes after binding to CD14
after it had interacted with soluble serum factors.
Low biological activity – low endotoxicity, may reflect the
organisms ability to colonize and grow in sterile tissue
undetected by host.

37. PROTEOLYTIC ENZYMES OF P.G:
It produces a variety of proteases which differ in size, ph,
sensitivity to inhibitors ability to hydrolyse specific
substrates and located within or in the surface of the
bacterial cell.
Extracellular proteases of P.g are critical survival
determinants and loss or reduction of these protease
activities by the use of protease inhibitors of gene
inactivation renders the bacteria susceptible to
phagocytosis and killing by neutrophils.

38. Classification of proteases:
1.Trypsin like protease
2.Collagenolytic protease
3.Other protease – dipeptidylpeptidase
Trypsin like proteinases:
Ability to cleave peptide substrates with arginine terminal groups
suchs as BANA or BAPNA – trypsin like activity.
Location of the proteinases:
Associated with bacterial membranes and the enzymes are
located at the inner cell membrane and the cell surface.
High activity has also been found in extracellular outer
membrane vesicle.
Other common cysteine proteinases like papain, and by analogy
it has beenproposed that it is either known as gingivpain or
gingivain.

39. FOUR Pg PROTEINASES:
-Serine
-Aspartate
-Thiol
-Metalloproteinase
GINGIPAIN – GINGIVAIN:
One group has separated the trypsin like activity from pg from
both outer membrane vesicles and culture supernatant.
It was a thiol dependent cysteine proteinase which cleaved
synthetic arginine substrates – gingivain.
Identical proteinase was isolated from the culture supernatant
by another group – gingipain.

40. Gingipain –R, gingipain-K:
One gingipain had a narrow spectrum of activity against peptide
bonds containg arginine, resistant to serine proteinase inhibitors
and was activated by glycine containing dipeptidase.
Molecular wt 50kda and a ph optimum of 6.0 – Arg-gingipain
or gingipain-R
De cario 1997 - Identified this lysine specific activity and called
the enzyme lys-gingivain.
It capable of cleaving high molecular wt kininogens to
bradykinin, and also degrade fibrinogen.
Capable of activating fibroblast and neutrophil interstitial
colagenases.

41. P.g appears to produce two cysteine proteinases with trypsin like
activity.
1.One is arginine specific and called as Arg-gingipain or Arg-
gingivain
2.The other is lysine specific and called as Lys-gingipain or Lys-
gingivain.
Effects of P.g proteases:
1. Internal effects
2. External effects
- Inflammatory and immune system
- Vascular system
3. Host tissue destruction

42. EXTERNAL EFFECTS:
a.Effects on inflammatory & immune system:
P.g proteinase are able to degrade Ig, IgA1, IgA2 and IgG,
complement components
RgpA is able to degrade IgG, C3 and this may reduce
opsonization function.
Rgpa and kgp – to degrade C5 and release C5a.
Rgpa can attenuate the respiratory burst characteristic of
PMN leucocyte bacterial killing.
P.g cycteine protease can degrade lysozyme found in GCF
and saliva.

43. b. EFFECTS ON VASCULAR SYSTEM :
Both RgpA and RgpB activate pre-kallikrein both in
addition to kgp may degrade high molecular
kininogen directly to bradykinin.
To produce vasodilation and increases vascular
permeability.
several protease related properties that prevent blood
clotting and hence promote bleeding.
Its proteases can alter the clotting by precipitation of
factor X
Fibronogen degradation increases the local clotting
time leading to gingival bleeding..

44. INTERNAL EFFECTS:
This bacteria derives its energy and nutrition from the
breakdown of proteins. Therefore defective production of
one or more of its major proteinase is likely to affect its
growth and reproduction.
That mutations of the RgpA, prtT and tpr genes result in
growth reduction and reduction in production of fimbriae.
These mutants also have reduced levels of attachment to
epithelial cells and other bacteria.
The RpgA mutants also showed reduced production of
hemagglutinins.

45. HOST TISSUE DESTRUCTION:
involved in periodontal tissue degradation as they degrade
protein substrates such as albumin and iron binding
proteins.
Rgpa, shown to activate fibroblast and PMN leukocyte to
produce MMP-1 and 8, lead to the degradation of collagen
by host MMP.
To degrade plasma proteinase inhibitors, α1-proteinase
inhibitor and α2-macroglobulin – collagen degradation.
Rgpa shown to degrade basement membrane type IV
collagen and other components of ECM including
fibronectin, laminin.
Gingipains have a strong binding affinity for fibronectin
and laminin which inhibits hemagglutination.

46. GINGIPAINS:
STRUCTURE:
It constitute a group of cysteine endopeptidase that are
responsible for at least 85% of the general proteolytic
and 100% of so called-trypsin like activity produced by
p.g.
Gingipains are products of 2 genes encoding cysteine
proteinases:
1.Gingipain R
2.Gingipain K
Curtis M.A 1999 suggested that gene encoding gingipain R
with hemagglutinin / adhesion domain – RgpA
Gene that encodes gingipain r without this carboxyl-terminal
domain – RgpB.
The name Kgp was suggested as a reference to gene
encoding for gingipain k.

47. GINGIPAINS R STURCTURE:
The initial translation polyprotein is processed into at least
3 different molecular forms of the enzyme. Pavloff n 1995
Rgpa – is a form of the catalytic domain alone and is made
by either aberrant proteolytic processing of the initial
protein.
Mt-rgpa – a membrane assoicated form in which the
catalytic domain is modified with LPS.
Hrgpa – is a non-covalent but very stable complex of the
catalytic domain and hemagglutinin
The translated polypeptides of the rgpA and rgpB genes
share 72%, 93% and 40% identify within the profragments,
catalytic domains and the carboxy terminal extension.
The attachment of LPS to rgpB apparently leads to the
generation of membrane-associated form referred to as mt-
RgpB.

48. EnzymeGeneMoleculat WtEnzyme
class
Cleavage
specificity
Comments on function
Arg-
gingipain
rgpA50 to 110
Kda
cysteineCleaves
peptide bonds
following an
arginine
residue
Gene also encodes an
adhesin domain that
appears to improve
proteolytic efficiency
Arg-
gingipain
rgpB48 to 90 KdacysteineCleaves
peptide bonds
following a
lysine residue
Gene also encodes an
adhesin domain
Lys-
gingipain
kgP60 to 180
Kda
cysteineOnly cleaves
denatured
proteins; 55
Kda fragment
possesses
catalytic
domain
Rapidly inactivates host
plasma proteinase
inhibitors, such as ά-1
protease inhibitor, the
primary inhibitor of
human neutrohil
elastase.
SELECTED PROTEASES PRODUCED BY P.GINGIVALIS

49. Gingipain – K structure:
The Kgp gene encodes a polyprotein consiting of a typical
leader sequence:
A profragment
A catalytic domain
Hemagglutinin / adhesin domain
The Kgp and rgpA gene products from HG66 share 23% and
28% identity of amino acid sequence within the
progragments and catalytic domains.
The amino terminal portions of ha1, of both HRgpa and Kgp
share only 46% homology and in the case of Kgp – repeat
of amino-terminal sequence of HA4.
rgpA initial translation product, the nascent Kgp polyprotein is
a non-covalent heteromultimeric complex of the catalytic
and hemagglutin / adhesin domain.

51. THE ROLE OF PROTEOLYTIC SYSTEM OF P.GINGIVALIS
IN THE ACQUISITION OF NUTRIENTS
Host plasma or tissue
proteins
Proteins fragments and
large peptides
Small peptides
Di-and tripeptides
nutrients indispensable
for bacterium growth and
proliferation
Direct action of gingipains
and/or neutrophil elastase
and activated host MMPs
Periodontain,
gelatinases, PrtT
and Tpr
Oligopeptidase Di- and
tripeptidyl peptidases

52. A POSSIBLE CONNECTION BETWEEN GINGIPAIN-INDUCED ACTIVATION
OF THE COAGULATION AND KALLIKREIN / KININ CASCADES AND
ALVEOLAR BONE RESORPTION
prothrombin
thrombin
bradykinin
HMW
kininogen
Factor XaFactor X
Rgp’s
Kgp
kalikrein
Protein C
Activated protein C
prekalikrein
Consumption of protein c
promotes thrombin
Activation of Pg
synthesis in
human osteoblasts,
endothelium and
PDL cells
Alveolar bone
erosion at the
periodontal
lesion
Consumption of
protein C promotes
thrombin generation

53. THE CENTRAL ROLE OF GINGIPAINS IN THE PROTEOLYTIC
PROCESSING OF SEVERAL SURFACE-ASSOCIATED AND
SECRETED PROTEINS OF P.GINGIVALIS
Rgp’s
Pro-Rgp
Pro-Tpr
Tpr
Profimbrilin
Mature
fimbrilin
fimbriae
75 Kda
OMP
75 Kda OMP
precursor
HArep
HbR
Pro-Kgp
Kgp
Pro-PrtP
Pro-PrtT
PrtP
PrtT

54. PATHOGENIC ACTIVITIES OF GINGIPAINS:
1.Activation of the kallikrein/kinin system
2.Activation of the blood clotting system
3.Degradation of fibrinogen and fibrin
4.Disturbance of host defense systems

55. Role of gingipain-r and gingipain-k in the dysregulation of
coagulation, complement and kallikrein/kinin cascade
pathway during the development of periodontitis
Hageman Factor Activated Hageman
Factor
Complement C5
KalliKrein Prekallikrein
C5a like peptides
leukocyte
accumulation
GINIGIPAIN - R
GINIGIPAIN - K
High molecular
weight kninogen
Bradykinin edema,
pain GCF
production
Fibrinogen
degradation bleeding
tendency

56. Cytokine activation
and inactivation
Phagocyte receptor
cleavage
Complement
Component degradation
Kallikrein/kinin
System activation
Fibrin lysins
MMP synthesis
stimulation and
activation
Clotting system
activation
GINGIPAINS
periodontitis
Sustained p.g.
infection
inflammation
Gingival swelling
Gcf production
Bleeding tendency
Alveolar bone
resorption
Periodontal tissue
destruction
Gingival recession
Disseminated intravascular
coagulation
Ischemic heart
disease

57. Effect of p.g gingipains on host biologic system:
The proinflammatory pathways are hypothesized to be
mediated by soluble gingipains whose effect includes
activation of IL-8.
The anti-inflammatory effects are hypothesized to be
mediated by membrane associated gingipain such as
vesicles released by the bacterium.
That p.g benefits from the presence of neutrophils in the
area bcos neutrophils will release a wealth of proteolytic
enzyme that undoubtedly contribute to tissue protein
degradation and thus assist in the acquisition of nutrients
for the bacterium.

58. Gingipains as virulence factors in the development of
periodontitis;
Hallmarks of periodontitis include:
Bleeding on probing
Neutrophil accumulation
Attachment loss
Increased crevicular flow
Gingipains – pathophysiological roles in periodontitis:
1.Role of gingipain is vascular permeability
enhancement
2.Gingipains and neutrophil chemotaxis
3.Gingipains and bleeding tendency in periodontitis
4.Gingipains as adhesions.

59. I. Role of Gingipains in vascular permeability enhancement:
Both membrane bound and secreted forms of gingipain R
have been found to be potent VPE factors inducing this
activity through production of plasma kallikrein and
subsequently bradykinin release.
Responsible for GCF production at periodontitis sites
infected with p.g
II. Gingipains and neutrophil chemotaxis:
Gingipain R found to be a very efficient enzyme of production
of potent chemotactic factor c5a
1.massive accumulation of neutrophils was found in p.g
infected periodontal tissue.
2.High concentration of neutrohil derived proteinases,
including elastase, cathepsin g, proteinase 3, gelatinase
and collagenase – connective tissue destruction.

60. III. Gingipains and bleeding tendency in periodontitis:
Gingipain R is the most potent fibrinogen degrading
proteinase.
The bleeding at periodontal sites is of primary importance
for p.g bcos it ensures delivery through the Hb
erythrocytes, the richest source of heme and iron.
The proteinase – hemagglutinin complexes may be vitaly
important in the uptake of these growth factors through
1.Hemagglutination
2.Hemolysis of erythrocytes
3.Subsequent degradation of Hb by bacterial and host
proteinase

61. GINGIPAINS AS ADHESIONS:
Gingipain has found have a strong binding affinity
for fibrinogen, fibronectin and laminin and this
interaction has been found to inhibit
hemagglutination.
All bound proteins are easily degraded by the
functional proteinase domain, with gingipain-r
being more active on laminin and fibronectin and
gingipain-k more effective in digestion of
fibrinogen.

62. Detection of Gingipains in Gingival Crevicular Fluid:
The bacterial proteases are released into the gingival crevice
or periodontal pocket and can be detected in the GCF
collected on filter paper strips.
The GCF arg-gingipain appears to be an excellent predictor
of future progressive attachment loss
a.Fluorescence detection system:
The intensity of fluorescence can be detected under ul light,
and is proportional to the amount of enzyme present in
the sample.
b. Color detection system:
Can be used by adding p-dimethylamino cinnamaldehyde to
the wells.
This molecule couples to AFC leaving group, forming a
purple color with schiff reagent.

63. INACTIVATION OF GINGIPAIN GENES:
A powerful tool in molecular studies is to inactivate
or “knock-out” specific target genes at molecular
level.
This is typically achieved by inserting an unrelated
“marker” gene such as an antibiotic resistance gene,
into the coding sequence of the target gene on the ch
of the native microorganism.
Transcription of the target gene is disrupted, and the
presence of the “marker” antibiotic resistance gene is
easily assessed by growth of the bacterium on media
containing relevant antibiotic – isogenic mutant.

64. A mutant strain in which both the genes have been
inactivated indicates that these two genes account
for all of the arg – gingipain activity found in p.g
Loss of hemagglutination in mutants with the Rgpa
or kgp genes inactivaed supports a role for the
protease associated adhesion domains in
hemagglutination.

65. Gingipains as targets for periodontal therapy:
The potential contribution of gingipains to the
pathophysiology of periodontitis suggest availability of
enzymes as targets for the therapy of periodontal disease.
A. VACCINES:
The immunization of primates with Rgpb appeared to reduce
bone loss in experimental gingivitis and ligature induced
periodontitis, but it did not suppress emergence of p.g
inspite of significantly elevated levels of IgG specific for
both bacterium and gingipain.
Antibodies directed against the aminoterminal region of the
catalytic domain of gingipain r are capable of inducing
protective immune response against p.g infection.

66. B. INHIBITORS FOR GINGIPAINS:
Matsushita et al found that dx-9065 a, a proteinase
inhibitor primarily specific for activated coagulation
factor X significantly reduced p.g growth
Tetracycline and its analogues when administered in
periodontitis pts, have been observed to improve their
clinical parameters.
These antibiotics have been found to inhibit all 3
gingipains (HRgpA, rgpB, KgP). The improvement of
clinical parameters is due to the ability to inhibit
multiple gingipain actions rather than eradicate p.g