Role of Porphyromonas gingivalis in periodontal diseases

Introduction:

Periodontal diseases are the inflammatory diseases triggered specifically by some selected micro-organisms, i.e. periodontopathic bacteria, accumulated in and around the gingival crevice. Porphyromonas gingivalis is one of them. Porphyromonas gingivalis is a gram-negative, anaerobic, non-motile, asaccharolytic and black pigmented rod that form greenish-black colonies in blood agar plates1. It is one of the major pathogens of chronic periodontitis2-3 in the study done by Socransky et al. (1998)4, this organism has been included in red complex, which is strongly associated with periodontal destruction. In both periodontitis and healthy subjects, P gingivalis can be recovered in low frequency from the subgingival flora, tongue, buccal mucosa and tonsils and saliva5-7

Structural components of P.Gingivalis:

Capsule: It is anti phagocytosis virulence factor. Six distinct capsular serotypes have currently been described (K1-K6)8-9.  Capsules from 3 PG strains have been purified. They are mainly composed of sugars like galactose , glucose, glucasamine, rhannose, mannose, etc. A strong relationship exists between the extent P.G encapsulation and its ability to act as pathogen. Increased encapsulation is related with decreased auto agglutination, increased resistance to phagocytosis, serum resistance and decreased induction of PMN leukocyte chemiluminescence. Studies in a mouse infection model have revealed that encapsulated P. Gingivalis strains are more virulent than non-encapsulated strains10-13. Vaccination with the help of capsules have been tried.

Outer membrane of PG: Outer membrane of PG contains atleast 20 major proteins, ranging from 20-90 kDa . These proteins have significant effect on fibroblasts. They stimulate their proliferation. Because of this a 24 kDa protein has been given name “fibroblast activating factor”. PG also produces a 75 kDa major outer membrane protein that exists as a high molecular weight polymer. Watanabe et al found that protein can stimulate polyclonal B cell invasion and can elicit IL-1 production. These outer membrane proteins are also important in plaque formation and coaggregation. P.G plays an important role in formation and maintenance of plaque biofilm. Gibbson and Nygaard (1970)14 demonstrated that there was a specific interaction of PG with the various gram positive and gram negative bacteria. Coaggregation between PG and AAC was found to be important for initial sub gingival biofilm formation. PG has an absolute growth requirement of haemin (iron). It produces haemolysin associated with its outer membrane which lysis the blood cells.

Lipopolysaccharide(LPS): The LPS from PG is composed of sugar. Analysis of LPS from 6 different strains indicate that it contain sugars like rhannose ,mannose, galactose. LPS has been studies for various immunological properties. Chemically it is composed of 3 parts O antigen, core and lipid A. Endotoxin activity is confined to lipid A ,while significant immunological activities are due to O antigen. LPS also interacts with CD 14 receptor on cells and initiate the immune reaction.

Bacterial fimbrae: Fimbriae or pili are proteinaceous, filamentous appendages that protrude outwards from the bacterial cell surface and play a crucial role in virulence by stimulating bacterial attachment to host cells or tissues 15. Fimbriae of P. gingivalis were first recognized on the outer surface by electron microscopic observation 16-17. They are arranged in peritrichous pattern on PG. fimbrae is composed of 1000 units of fimbrillin. P. gingivalis fimbriae possess a strong ability to interact with host proteins such as salivary proteins, extracellular matrix proteins, epithelial cells, and fibroblast, which promote the colonization of P. gingivalis to the oral cavity 18-19. Animal experiments conducted strongly implicate fimbrae as an important virulence factor. Environmental factors like temperature, Ph, haemin limitation, serum , saliva , osmotic effects and effects of Ca++ limitation have significant effect on fimbrae formation. P. gingivalis fimbriae are classified into six genotypes based on the diversity of the fimA genes encoding each fimbrillin (types I to V, and type Ib), and that P. gingivalis with type II fimA is most closely associated with the progression of chronic periodontitis 20-22.

Proteinases: PG produces a large number of hydrolytic, proteolytic and lipolytic enzymes that are produced essentially by all of the known strains. Many of these enzymes are exposed on the surface of the bacterium where they come in contact with host cells and tissues. These play a significant role in PD disease progression including dissemination of PG into deeper tissues. Classification of proteinases is relied upon their catalytic functions. To date 4 proteinases have been recognised : serine, aspartate, thiol, metalloproteinases. The arg and lys – proteinases have been given a common name Gingipains. There are atleast 3 different genes which encode for the proteolytic activity of PG. These genes encode for 2 cystein proteinases , arginine- gingipain ( Arg-gingipain A & B; Rgp-A and Rgp-B) and lysine-gingipain (lys-gingipain ,Kgp). Arg-gingipain is encoded by 2 genes and lys- gingipain by one gene 23. Movement of cystein proteinases from the bacterial cytoplasm to membrane involves the secretory pathway. Arg- and lys proteinases or the gingipains belong to trypsin like proteinases.

Colonization of P. Gingivalis:

 Despite the host defense mechanisms in saliva and GCF, P. gingivalis can adhere and then colonize in gingival crevices to a variety of surface components lining the gingival crevicular cells and the tooth surface. The adhesionis mainly mediated by the fimbriae, although other bacterial components such as vesicles, hemagglutinin, and proteases may play an adjunctive role 24. P. gingivalis is capable of coaggregating with Actinomyces naeslundii 2 (Actinomyces viscosus), Streptococcus gordonii, and S. Mitis 25-26.

The concept of Virulence:

Virulence is defined as the relative capacity of a microbe to cause disease. Holt and Ebersole27 have proposed that virulence factors have multiple functions such as :

1) the ability to participate in microbe-host interactions (adhesion);

2) the ability to invade the host;

3) the ability to grow in the host cells;

4) the ability to evade/interfere with the host defense system.

The pathogenic factors of P. gingivalis include the following

  • Fimbriae,
  • Hemagglutinin,
  • Capsule lipopolysaccharide (LPS),
  • Outer membrane vesicles (OMV’s),
  • Organic metabolites such as butyric acid,
  • Various enzymes such as Arg- and Lys-gingipains, collagenase, gelatinase and hyaluronidase.

Pathogenic process of P. Gingivalis:

The initial event in the pathogenicity of P. Gingivalis is its interaction (adherence) in the oral cavity 28-29. This process is facilitated by fimbriae, proteases, hemagglutinins and lipopolysaccharide 30. P. Gingivalis fimbriae help the bacterial interactions with the host cells and also with other bacteria. Bacterial adherence to mucosal and tooth surfaces, as well as bacterial coaggregation are essential steps for colonization of various oral bacterial species. Fimbriae are involved in each of these processes 31 as well as cell adhesion molecules including intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and P- and E-selectins. P. gingivalis major fimbriae have been shown necessary for bacterial invasion to host cells 32.

P. gingivalis Proteinases:

P. gingivalis proteinases have been reported to exhibit enzymatic activity against a broad range of host proteins, including host proteinase inhibitors, immunoglobulins, iron transporting/sequestering proteins, extracellular matrix proteins, bactericidal proteins and peptides, and proteins involved in the coagulation, complement, and kallikrein/kinin cascades 33. The majority of this activity is due to cysteine proteinases referred to as gingipains

Gingipains:  

Gingipains were originally considered as “trypsin-like proteases”, but actually they comprise a group of cysteine endopeptidases 34  that have been reported to account for at least 85% of the general proteolytic activity displayed by P. gingivalis, and 100% of the expressed “trypsin-like activity.” They are either secreted or membrane boundand and are arginine or lysine specific. Gingipains are present in large quantities on the cell surface of P. gingivalis, and they can significantly contribute to the virulence exhibited by this species. They help in binding of the bacterium to host tissues. They are classified into two groups based on substrate specificity. Gingipains R (RgpA and RgpB) cleaves proteins after arginine residue and are encoded by two similar genes, RgpA and RgpB, while gingipain K (Kgp) cleaves proteins after lysine residue. RgpA together with RgpB, account for all trypsin-like activity of P. gingivalis. Kgp plays an important role in Fe acquisition by binding to hemoglobin 34. They have been shown to have role in controlling the expression of virulence factors and the stability and/or processing of extracellular and cell-surface proteins 35. As gingipains are expressed on the outer membrane of P. Gingivalis, they may be useful as vaccine against periodontal diseases.

Structural description of gingipains:

The mature form of RgpA possesses both a catalytic domain and a hemagglutinin domain, while RgpB possesses only a catalytic domain. Hemagglutinin domain is responsible for the adherence of microorganism to erythrocytes. There is a high degree of homology between the catalytic domains of RgpA and RgpB at both the DNA and protein levels, while the hemagglutinin domain of RgpA is similar to the P. gingivalis hemagglutinin domain of Kgp.

Mechanism of action of gingipains:

Vascular permeability enhancement: R-gingipains are very potent factors of vascular permeability enhancement. This activity is induced through plasma prekallikrein activation and subsequent bradykinin release 36-37. Gingipain K by itself is not able to induce vascular permeability in human plasma, but working in concert with R gingipains, it can induce vascular permeability by cleaving bradykinin directly from high-molecular-weight kininogen 37. So, gingipains are responsible for increase in gingival crevicular fluid production at periodontitis sites infected with P. gingivalis.

Cleavage of complement components: RgpA is a very efficient enzyme in generation of C5a through direct cleavage of C5. C5a is a potent chemotactic factor that likely contributes to the significant leukocyte infiltration at P. gingivalis–induced periodontitis lesions. It also degrades C3 and in this way eliminates the creation of C3-derived opsonins, thus rendering P. gingivalis resistant to phagocytosis 38-39 this ultimately results in massive accumulation of neutrophils in the inflamed periodontal tissue which contributes to the very high levels of active granular proteinases (elastase, cathepsin G, gelatinase, and collagen inhibiase) in gingival crevicular fluid 40-41 that may be responsible for connective tissue destruction.

Change in the subgingival environment: Massive accumulation of neutrophils leads to generation of high level of active granular proteinases which enables subgingival plaque bacteria to thrive due to the presence of high concentrations of peptides and amino acids, thus further aggravating tissue destruction 42.

Degradation of clotting factors: Recent studies indicate that RgpA is capable of activating factor X and suggest that this gingipain could be responsible for the production of thrombin 43. fibrinogen is major target for Kgp. In vitro, this enzyme degrades the fibrinogen A alpha-chain within minutes44, thus rendering it nonclottable. It has been postulated that the nonrestricted activity of gingipain K in periodontal pockets contributes to a bleeding tendency, especially since it also very efficiently destroys the pro-coagulant portion of high-molecular-weight kininogen 44.

Stimulation of different cell types to produce inflammatory mediators: More recently, it has been reported that P. gingivalis gingipains can activate different cell types leading to the secretion of inflammatory mediators 45-47. In one recent study, it has been found that  P. gingivalis gingipains induced an inflammatory response in macrophages through activation of intracellular kinases. Along with that, it was shown that both the Arg- and Lys-gingipain preparations induced the production of TNF-α and IL-8 by macrophages 48.

Haemolysis of erythrocytes: P. gingivalis RgpA and Kgp has hemagglutinin domains. Their ability to hemagglutinate erythrocytes has been well documented 49-50. Proteinase-hemagglutinin complexes may thus be important in the uptake of essential growth factors, via hemagglutination, thus hemolysis of erythrocytes 51.

Recent research:

As it is well known that Gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDLF) are functionally different cell types in the periodontium that can participate in the host immune response in periodontitis.  A study was done to find out the effect of viable P. gingivalis W83 on Gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDLF). Authors concluded that there was a considerable heterogeneity in responsiveness of these fibroblasts to P. gingivalis between individuals and that they may be crucial determinants for the susceptibility to develop periodontitis 52.

The invasive and evasive strategies of P. gingivalis and the role of its major virulence factors namely lipopolysaccharide, capsule, gingipains and fimbriae has been discussed in a recent review 53.

As resolvins have anti-inflammatory properties prevention of P. gingivalis-induced periodontitis by topically applied resolvins was evaluated in a 6-week experimental study. Results showed that resolvin E1 application prevented the onset and progression of periodontal destruction in experimental group as compared to the placebo group where significant progression of periodontal disease, including bone and attachment loss was observed 54.

 As Gingipains are responsible for evasion of host response, thus it indicates that inhibition of gingipains should be useful tools both to assess the contribution of their proteolytic activities to the virulence of the bacterium and to facilitate the development of new therapeutic approaches to periodontal diseases. Recently research has been done to find out therapeutic agents that inhibit gingipain activity. Among this series of compounds used for gingipain inhibition it has been found that KYT-1 and KYT-36 had the most potent and selective inhibitory activities of Rgp and Kgp, respectively 55..

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Vaccination against P. Gingivalis:

Many researchers have investigated a group of cell surface carbohydrates designated as K-antigens 56-57 lipopolysaccharides 58-59 and various proteins including fimbriae 60 the 53-KDa and 67K-Da cell surface proteins 61 hemagglutinin 62  and cysteine proteases referred to as gingipains 63. Gingipains are present in large quantities on the cell surface of P. Gingivalis and have highest potential to be used as vaccine antigen

Gingipains as candidate for periodontal vaccine:

As gingipains are expressed on the outer membrane of P. Gingivalis, they are potential candidates for periodontal vaccine. Gingipain vaccines are mainly DNA vaccines. Arg-gingipain-encoding genes have been cloned from various P. gingivalis strains 64-65.  The sequence analysis shows that two separate genes located on the chromosome of P. Gingivalis encode Rgp (rgpA and rgpB). As different gingipains contain catalytic domain and haemagglutinin domain or one of them, studies have been done to find out the protective host response against these after immunisation 66-69. The antibody response has been documented against these antigens. Further studies are required in this direction to analyse the effectiveness of vaccine and its practical implications.

 

 

References:

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