1. 1. PRESENTED BY ANKITA JAIN 22 June 2015 1
2. 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. 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. 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. 5. STRUCTURE OF GINGIPAINS GINGIPAINS (CYSTEIN ENDOPEPTID ASE) 85% of general proteolyti c activity 100% of trypsin like activity 22 June 2015 5
6. 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. 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. 8. 1) RgpB form of only catalytic domain 2) mt-RgpB : Attachment of (lipo)polysaccharides to RgpB 22 June 2015 8
9. 9. 22 June 2015 9
10. 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. 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. 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. 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. 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. 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. 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. 17. Gingipains degraded fibrinogen within minutes, thus rendering this physiological thrombin substrate non- clottable. 22 June 2015 17
18. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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