PORPHYROMONAS GINGIVALIS

Dr Harshavardhan PatwalDr Harshavardhan Patwal

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)

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)

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.

The role for P.g as periodontal pathogens Haffajee & Socransky 1994

Association Elevated in lesions of periodontitisLower in sites of health, gingivitis and edentulous subjects.Elevated in actively progressing lesionsElevated 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.

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.

Collagenase, EndotoxinProteolytic trypsin-like activityFibrinolysinHemolysinGingipain, Phospholipase ADegrades 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

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.

P.GingivalisDESCRIPTION OF THE GENUS OF PORPHYROMONAS Species of the genus porphyromonas (Collier L 1998)Porphyromonas cangingivalisPorphyromonas canorisPorphyromonas catoniaePorphyromonas circumdentariaPorphyromonas crevioricanisPorphyromonas endodontalisPorphyromonas gingivalisPorphyromonas gingivicanisPorphyromonas leviiPorphyromonas macacae

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.

Biochemical properties of p.gingivalis:

Host defense mechanism

Bacterial species Bacterial property Biologic effect

Specific antibody p.gingivalisp.intermedia

IgA IgG degrading protease

Degradation of specific antibody

PMN p.gingivalisA.AT.Denticola

CapsuleInhibition of superoxide production

Inhibition of phagocytosisDecreased bacterial killing

Release of IL-8 p.gingivalis Inhibition of IL-8 production by epithelial cells

Impairment of PMN response to bacteria

Socransky & Haffajee 1994

P.GingivalisBacteroides ForsythusTreponema Denticola

Invasion of P.Gingivalis Into Gingival Epithelial Cells:Invasion and internalization mechanism of p.g provide

some fascinating insights into the molecular dialogue thatoccurs 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)

Model of p.g trafficking in endothelial cells

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 person2. Common vehicle – food / water3. Airborne – droplets4. Through a vector

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.

Virulence factorsDefinition:

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

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.

Virulence factors and host effectors produced by p.gingivalisTissue destruction Host evasion

CollagenaseTrypsin like proteasesGelatinaseAminopeptieasePhospholipase – AAlkaline phosphataseAcid phospataseChondroitin sulfataseHyaluronidaseKeratinaseHeparinaseEpitheliotoxinFibroblast growth inhibitorsEndotoxicityLps induced bone resorption

Degrdation of plasma protease inhibitors

Degradation of iron transport proteins

Inhibition of PMN leucocytesChemotaxis inhibitorsDecrease phagocytosis

Lysis and intracellular killing

Resistance to c’killingC-3 / c-5 degradationIg protease

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 .

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 fimbriae2. Sex pili

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.

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.

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.

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.

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.

CAPSULE:Is an important anti-phagocytic virulence factorElectron 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.

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

OUTER MEMBRANE PROTEINSThe cell envelop has three part:1. Inner cytoplasmic membrane2. A thin peptidoglycan3. Attached to which is the asymmetrical outer membraneThe 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

- 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.

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.

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.

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.

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.

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.

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.

Classification of proteases:1. Trypsin like protease2. Collagenolytic protease3. Other protease – dipeptidylpeptidaseTrypsin 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.

FOUR Pg PROTEINASES:- Serine- Aspartate- Thiol- MetalloproteinaseGINGIPAIN – 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.

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.

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 effects2. External effects - Inflammatory and immune system - Vascular system3. Host tissue destruction

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.

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..

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.

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.

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 R2. Gingipain KCurtis M.A 1999 suggested that gene encoding gingipain R

with hemagglutinin / adhesion domain – RgpAGene that encodes gingipain r without this carboxyl-terminal

domain – RgpB.The name Kgp was suggested as a reference to gene

encoding for gingipain k.

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.

Enzyme Gene Moleculat Wt Enzyme class

Cleavage specificity

Comments on function

Arg-gingipain

rgpA 50 to 110 Kda

cysteine Cleaves peptide bonds following an arginine residue

Gene also encodes an adhesin domain that appears to improve proteolytic efficiency

Arg-gingipain

rgpB 48 to 90 Kda cysteine Cleaves peptide bonds following a lysine residue

Gene also encodes an adhesin domain

Lys-gingipain

kgP 60 to 180 Kda

cysteine Only 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

Gingipain – K structure:The Kgp gene encodes a polyprotein consiting of a typical

leader sequence: A profragment A catalytic domain Hemagglutinin / adhesin domainThe 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.

THE ROLE OF PROTEOLYTIC SYSTEM OF P.GINGIVALIS IN THE ACQUISITION OF NUTRIENTS

Host plasma or tissue proteins

Proteins fragments andlarge 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

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 cpromotes 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

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

fimbriae75 Kda

OMP

75 Kda OMP precursor

HArep

HbR

Pro-Kgp

Kgp

Pro-PrtPPro-PrtT

PrtPPrtT

PATHOGENIC ACTIVITIES OF GINGIPAINS:1. Activation of the kallikrein/kinin system2. Activation of the blood clotting system3. Degradation of fibrinogen and fibrin4. Disturbance of host defense systems

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

Cytokine activation and inactivation

Phagocyte receptor cleavage

ComplementComponent degradation

Kallikrein/kininSystem 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 heartdisease

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.

Gingipains as virulence factors in the development of periodontitis;

Hallmarks of periodontitis include: Bleeding on probing Neutrophil accumulation Attachment loss Increased crevicular flowGingipains – pathophysiological roles in periodontitis:1. Role of gingipain is vascular permeability

enhancement2. Gingipains and neutrophil chemotaxis3. Gingipains and bleeding tendency in periodontitis4. Gingipains as adhesions.

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 thisactivity through production of plasma kallikrein andsubsequently bradykinin release.Responsible for GCF production at periodontitis sitesinfected with p.gII. Gingipains and neutrophil chemotaxis:Gingipain R found to be a very efficient enzyme of production

of potent chemotactic factor c5a1. 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.

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. Hemagglutination2. Hemolysis of erythrocytes3. Subsequent degradation of Hb by bacterial and host

proteinase

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.

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.

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.

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.

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.

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