2. CONTENTS 1) Introduction 2) Structure of Gingipains 3)
Pathogenic activities of Gingipains a) Activation of
Kallikerin/Kinin system b) Activation of blood clotting system c)
Degradation of fibrinogen to fibrin d) Disturbances of host defense
systems 4) Gingipains as targets for periodontitis therapy 22 June
2015 2
3. 1) INTRODUCTION Porphyromonas gingivalis (P. gingivalis) is
a major causative agent of adult periodontitis. Gram-negative,
anaerobic, Non-motile, asaccharolytic rods that usually exhibit
coccal to short rod morphologies. P. gingivalis is tentatively
identified on the basis of greenish-black colony and trypsin like
activity on a non-selective agar supplemented with sheep blood,
yeast extract, hemin and menadione. 22 June 2015 3
4. Virulence components of this bacterial cell include cysteine
proteases (gingipains), fimbriae, capsular polysaccharide (CPS),
lipopolysaccharide, and outer membrane vesicles Gingipains are
cysteine proteinases produced by Porphyromonas gingivalis, a major
causative bacterium of adult periodontitis. 22 June 2015 4
5. STRUCTURE OF GINGIPAINS GINGIPAINS (CYSTEIN ENDOPEPTID ASE)
85% of general proteolyti c activity 100% of trypsin like activity
22 June 2015 5
6. Gingipains are the products of 3 genes which encode cystein
proteinases. Gingipains are of two types: a) Gingipain R : which
hydrolyse Arg-Xaa bond b) Gingipain- K: which hydrolyse Lys-Xaa
bond 22 June 2015 6 GENE GINGIPAIN Enzymes rgpA gene gingipain R
with hemagglutinin/adhesi on domains 1) RgpA(cat) 2) mt-RgpA 3)
HRgpA rgpB gene Gingipain R without adhesion domains 1) RgpB 2)
mt-RgpB kgp gingipain K 1) kgp
7. 1) RgpA(cat) is a form of the catalytic domain alone and is
made by either aberrant proteolytic processing of the initial
protein or by an interrupted transcription process 2) mt-RgpA(cat):
the catalytic domain is modified with lipopolysaccharides. 3) HRgpA
stable complex of the catalytic domain and a hemagglutinin/adhesin
domain(s) 22 June 2015 7
8. 1) RgpB form of only catalytic domain 2) mt-RgpB :
Attachment of (lipo)polysaccharides to RgpB 22 June 2015 8
9. 22 June 2015 9
10. Structural Relationship Between Gingipains and Other
Cysteine Proteinases mammalian legumain (family C13), caspases
(family C14), and clostripain (family C11). 22 June 2015 10
11. PATHOGENIC ACTIVITIES OF GINGIPAINS a) Activation of
Kallikerin/Kinin system b) Activation of blood clotting system c)
Degradation of fibrinogen to fibrin d) Disturbances of host defense
systems 22 June 2015 11
12. a) Activation of Kallikerin/Kinin system Kallikrein/kinin
pathway was first described by Hinode et al (1992) It was found
that gingipains R are very potent vascular permeability enhancement
(VPE) factors, inducing this activity through plasma prekallikrein
activation and subsequent BK release. 22 June 2015 12
13. Contribute to gingival crevicular fluid (GCF) production
and edema formation at periodontitis sites infected with P.
gingivalis. By this activity, gingipains provide a continuous
supply of nutrients necessary for bacterial growth and virulence.
BK may be involved in alveolar bone resorption by inducing
prostaglandin production in periodontal ligament cells and
osteoblasts 22 June 2015 13
14. b) Activation of blood clotting system In periodontitis
lesions it is likely that gingipain R generate a large quantity of
thrombin, which is not linked to hemostasis. The multiple
proinflammatory actions of thrombin will promote periodontal
disease. 22 June 2015 14
15. EFFECTS OF THROMBIN Enhances vascular permeability. Induces
leukocyte chemotaxis. Contribute to GCF production. Stimulates
prostaglandin secretion by osteoblastic cells. Stimulates
interleukin-1 production by macrophages. Stimulates bone resorption
by osteoclasts through a prostaglandin-dependent pathway. Thus
Uncontrolled generation of thrombin is likely to be associated with
the progression of periodontitis and alveolar bone resorption. 22
June 2015 15
16. c) Degradation of fibrinogen to fibrin Bleeding on probing
is a well-known sign of periodontitis. This sign significantly
correlates positively with the presence of P. gingivalis in the
periodontal pocket, suggesting an involvement of the bacterium-
associated substances in the bleeding tendency. 22 June 2015
16
17. Gingipains degraded fibrinogen within minutes, thus
rendering this physiological thrombin substrate non- clottable. 22
June 2015 17
18. Kgp prolonged plasma clotting time more efficiently,
indicating that in plasma Kgp inter-acts with fibrinogen more
specifically than gingipain R. Kgp exerts strong fibrinolytic
activity in vivo which cannot be effectively controlled by host
inhibitors. This action of Kgp would contribute to a bleeding
tendency at periodontitis sites. 22 June 2015 18
19. Disturbances of host defense systems P. gingivalis
proteinase(s), presumably gingipain R, cause complement system
activation in through C3 convertase production, resulting in
consumption of its components. Thus, bacteriolysis through
complement system activation on the surface of the bacterium would
be impaired. 22 June 2015 19
20. RgpB easily degrades C3, the central factor of the system,
thereby interfering with the production of the opsonin C3b,
resulting in the insufficient generation of the phagocyte
chemotactic factor C5a and the membrane attack complex. 22 June
2015 20
21. Kgp were found to cleave C5a receptor. Thus, gingipains
would disturb neutrophil migration to the site of P. gingivalis
infection and protect the bacterium from phagocytosis by the
leukocytes stimulated with C5a. 22 June 2015 21
22. EFFECTS ON MONOCYTES 22 June 2015 22 Gingipains degrade
monocyte CD14, a major receptor for bacterial lipopolysaccharides
(LPS) Activation signal by LPS of P. gingivalis is insufficient for
the monocytes/macrophages to act as phagocytes defective
elimination of P. gingivalis from the host, and consequently, the
chronic inflammation at periodontitis lesions.
23. EFFECT ON INTERLEUKINS-8 (chemotactic factor of
neutrophills) 22 June 2015 23 Gingipain R and Kgp in soluble forms
cleave Interleukin-8 (chemotactic factor for neutrophills) at Arg5-
Ser6 and Lys8-Glu9, IL-86-77 and IL-89-77 are 2- to3-fold more
potent than the native gingipains associated with vesicles
instantly degrade this chemokine (IL), abolishing its chemotactic
activity.
24. Proximal position of plaque Rich in gingipains associated
with vesicles Abrogate IL-8 activity 22 June 2015 24 At the distal
part of plaque Free gingipains are more than the vescicle bound
Augments IL-8 activity
25. Distal position of plaque (point far away from point of
attachment) Concentration of IL-8 And neutrophills unable to
approach the bacteria in the plaque Proximal (point of attachment
of plaque) ( more vesicle bound gingipains) 22 June 2015 25
26. EFFECT ON IL-6 (mediator of inflammation) 22 June 2015 26
Gingipains degrade IL-6 Negative gradient of IL-6 created from
plaque to adjacent tissues in periodontal lesions a very low level
of IL-6 was detected in the gingival tissue adjacent to the
bacterial plaque elevated concentrations of this IL-6 are found in
tissues over 6 mm away from the infected pocket
27. The multiple pathogenic activities of gingipains and their
association with periodontitis and other diseases. (Modified from
Potempa et al.) 22 June 2015 27
28. GINGIPAINS AS TARGETS FOR PERIODONTITIS THERAPY The
potential contribution of gingipains to the pathophysiology of
periodontitis suggests availability of the enzymes as targets for
the therapy of periodontal disease. Two possibilities are there: a)
vaccination therapy using gingipains for periodontitis. b)
development of inhibitors specific for gingipains. 22 June 2015
28
29. a) Vaccination therapy using gingipains for periodontitis
Immunization of primates with RgpB appeared to reduce alveolar bone
loss slightly in the experimental gingivitis and ligature induced
periodontitis, but it did not suppress the emergence of P.
gingivalis in spite of the significantly elevated level of IgG
specific for both the bacterium and the gingipain. 22 June 2015
29
30. In contrast, the immunization of mice with a peptide
derived from the catalytic domain of gingipains R resulted in
protection from P. gingivalis invasion and subsequent cachexia and
death. 22 June 2015 30
31. The IgG titer obtained following immunization with the last
peptide and following immunization with the amino terminal peptide.
This result indicates that antibodies directed to catalytic domain
of gingipains R are capable of inducing a protective immune
response against P. gingivalis infection, suggesting a possible
availability of the vaccination with the peptide for human
periodontal disease. 22 June 2015 31
32. Development of inhibitors specific for gingipains:
DX-9065a, a proteinase inhibitor, selectively reduced P. gingivalis
growth, suggesting a potential therapeutic effect of gingipain
inhibitors on periodontitis. At concentrations as low as 10 M, this
highly safe compound inhibits both gingipain R and kallikrein
generated by the proteinases in plasma, attenuating the
gingipain-induced BK production. 22 June 2015 32
33. Tetracycline and its analogues, when administered to
periodontitis patients, have been observed to improve their
clinical parameters. However, it does not affect the P. gingivalis
load at periodontitis sites. But , these antibiotics have been
found to inhibit all 3 gingipains (HRgpA, RgpB, Kgp). This
indicates the improvement of the clinical parameters is due to
their ability to inhibit multiple gingipain actions rather than to
eradicate P. gingivalis. 22 June 2015 33
34. PLANTIBODIES A very recent approach for vaccination
strategies molecular biological techniques to express bacterial or
viral antigens in plants, which could be used as orally
administered vaccines. This suggests the potential use of plants in
synthesizing adjuvant fimbrial protein for the development of
adjuvant mucosal vaccines against P. gingivalis. (Kudyar N. et al
2011) 22 June 2015 34
35. Conclusion P. gingivalis is one of the major periodontal
pathogen which is highly prevalent in inflammatory sites with deep
periodontal pockets. P. gingivalis demonstrates multiple
components, particularly its cysteine proteases (i.e. gingipains)
that alter host cell and tissue functions, and decrease, or even,
negate host protective responses. 22 June 2015 35
36. As yet, there are no periodontal vaccine trials that have
been successful in satisfying all requirements to prevent the
colonization of porphyromonas gingivalis The trial of gingipain
inhibitors and vaccination against gingipains need to be conducted
because this approach may contribute to the therapy of periodontal
diseases. 22 June 2015 36