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Variations ofPorphyromonas

gingivalis fimbriae in relation to

microbial pathogenesis

Amano A, Nakagawa I, Okahashi N, Hamada N

J Periodont Res 2004; 39; 136-142

Guided By: Dr. Rajesh Kr. Thakur

Presented By: Dr. Purushottam Singh

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INTRODUCTION

Periodontitis is a heterogenous disease that is intricately

influenced by host susceptible differences and/or diversities in

virulence among the organisms harboured by individuals.

Porphyromonas gingivalis, a gram-negative bacteria, though

detected in lower frequency in periodontally healthy

individuals, is frequently associated with various forms of

periodontal diseases.

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This microorganism produces a number of virulencefactors such as fimbriae, LPS, capsules and proteases.

Further, specific virulent clones exist in patients with

strongly developed chronic and aggressive periodontitis.

The clonal variations along with the major and minor

fimbrae as virulence factors are associated with

microbial pathogenesis of periodontal diseases.

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1.Cytoplasm with organelles: Genome (N), plasmid (P), ribosomes.

2. Cytoplasmic membrane: This phospholipid bilayer functions as an osmoticbarrier.

3. Peptidoglycan: This large molecule provides protection.

4. Periplasmatic space: This is gram-negative specific.

5. Outer membrane: Found onl in ram-ne ative or anisms with inner and9/27/2013 4


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Description: This electron microscope view of vesicles and fimbriae of the

strain

ATCC 3327


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Distinct Virulence

The heterogenic virulence properties of P. gingivaliswere examined using animal models among which

mouse and guinea pig abscess model were extensively

employed.

Following subcutaneous infection of rodents with P.

gingivalis, virulence was evaluated in relation to the size

of the abscesses and/or eroded skin lesions along with

cachexia and death.

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In those studies, many strains ofP. gingivalis wereclassified as either avirulent/non-invasive or

virulent/invasive.

Avirulent strains were ATCC 33277,381,2561 and HG1694.

Virulent strains were ATCC 53977, A7a2-10, HG1690

and W83.

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Encapsulated strains appeared to be more virulent.

Further 6 serotypes (K-antigen types; K1 to K6) of

P. gingivalis were recognized based on the capsular

antigens linked to pathogenicity in animal models.

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Genotypic characterization is also performed to isolatespecific periodontitis-related clones from periodontitis

patients.

This characterization is based on restriction fragmantlength polymerization, multilocus enzyme

electrophoresis, arbitrarily primed polymerase chain

reaction and amplified fragment length polymorphism

methods.

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Results from these studies reveal an extensiveheterogenicity and as many as 100 different clonal types

ofP. gingivalis isolates have been found.

The consensus of these studies is that there isconsiderable heterogenecity among P. gingivalis

isolates, whereas intra-individual heterogenecity is very

low.

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P.gingivalis has a non-clonal population structure, withits genotypic diversity derived from an accumulation of

genetic changes or mutations that are subject to

ecological selective pressures in periodontal lesions.

These genetic changes may be caused by a variety of

genetic mechanisms, including the movement of

insertion sequence elements and/or recombination

between non-mobile repeated DNA sequences.

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It was suggested that the virulence ofP.gingivalis wasnot confined to a distinct evolutionary linease.

The particular genotypes, possibly with increased

pathogenic potential, are able to spread successfully inhumans.

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Major Fimbriae

Major fimbriae were first determined in 1984 byYoshimura et al. and are recognized as a critical

virulence factor influencing disease initiation and

progression.

These are filamentous components on the cell surface

and their subunit protein, fimbrillin (Fim A), reportedly

acts on bacterial interactions with host tissues by

mediating bacterial adhesion and colonization intargeted sites. (Ref 1)

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Ref 1- Amano A. Molecular interactions of

Porphyromonas gingivalis with host cells:

Implications for the Microbial Pathogenesis of

periodontal Disease. J periodontol 2003; 74,90-96

Fimbriae are capable of binding to human salivary

components, commensal bacteria, and a variety of hostcells including macrophages, epithelial cells, and

fibroblasts.

Human extracellular matrix (ECM) protiens such asvitronectin and fibronectin play important roles in cellular

signal transduction via binding to receptor integrins.

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Major fimbriae are capable of binding specifically to and

activating various host cells such as human epithelial cells,

endothelial cells, spleen cells, and peripheral bloodmonocytes.

These result in the release of cytokines including

interleukin-1(IL-1), IL-6, IL-8, and tumor necrosis factor-(TNF-) 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.

In addition, P. gingivalis major fimbriae have been shown

necessary for bacterial invasion to host cells (Ref 2).

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Ref 2. Lamont RJ, Jenkinson HF. Subgingival

colonization by Porphyromonas gingivalis.

Oral Microbiol Immunol 2000: 15: 341349.

In order to cause periodontal disease,P. gingivalis must

colonize the subgingival region, a process that involves

several distinct steps and multiple gene products.

The organism must first navigate within the oral fluids

in order to reach the hard or soft tissues of the mouth.

Retention and growth of bacteria on these surfaces is

facilitated by a repertoire of adhesins including fimbriae,

hemagglutinins and proteinases.

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Once established subgingivally, P. gingivalis cells

participate in intercellular communication networks with

other oral prokaryotic cells and with eukaryotic cells.

The establishment of these multiple interactive interfaces

can lead to biofilm formation, invasion of root dentin and

internalization within gingival epithelial cells.

The resulting bacterial and host cellular locations,

products and fate contribute to the success ofP. gingivalis

in colonizing the periodontal region.

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Clonal variations of major fimbriae in relation

to bacterial virulence

Lee et al. first reported the variation of FimA proteins

and divided a number ofP. gingivalis strains into four

types based on their N-terminal amino acid sequences.

P. gingivalis fimA genes have been further classified into

six variants (types I to V, and Ib) on the basis of

nucleotide sequences

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The strains evaluated as virulent/invasive consisted of alarge number of type II fimA strains, such as ATCC

53977, A7A2-10, HG1690, HG184, and HW24D1, and

type IVfimA strains including W50, W83, and 9-14K-1.

Avirulent traits were expressed by type I fimA strains

such as ATCC 33277, 381, 2561, 1432, and 1112.

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Prevalance of specific fimbriae

genotypes and periodontal health status

A sensitive polymerase chain reaction assay using fimA

type-specific primer sets was developed to differentiate the

six types of fimA genes found in organisms insaliva and

dental plaque samples.

A majority of the patients were found to carry type IIfimA

organisms, followed by type IV, and the occurrence of type

II fimA organisms was significantly increased with themore severe forms of periodontitis.

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In contrast, the most prevalentfimA type ofP. gingivalisin the healthy adults was type I.

Similar findings were observed in both Down syndrome

patients, who are congenitally susceptible to periodontaldiseases, and young adults with mental disability, which

is a major factor in determining oral hygiene

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Other reports have also shown that type IIfimAorganisms are predominantly prevalant in periodontitis

patitents.

These findings indicate that there are disease-associatedand non-disease-associatedP.gingivalis organisms, and

that clonalfimA variations are related to the bacterial

infections traits that influence disease development.

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Influence of fimA variations on bacterial

interactions with host cells

Scant biological explanation was given for the differencesin pathhogenic potential of variousP.gingivalis strains with

differentfimA genotypes.

Recombinant FimA (rFimA) protiens were generatedcorresponding to their clonal variants and of their

capabilities of adhesion/invasion to human gingival

fibroblasts (HGF) and a human epithelial cell line (Hep-2

cells) were characterized.

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There was no significant differences in adhesion ability of

microspheres(MS) coated with these rFimAs to HGF.

However, adhesion ability of type II rFimA-MS to Hep-2cells was significantly greater than those of other rFimA

types.

The adhesion/invasion activities of type II rFimA wereabrogated by the additionof antibodies against type II rFimA

or51-integrin.

The results suggested that type II FimA is more able toefficiently promote bacterial invasion to the cells through

specific host receptors including 51-integrin.

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P. gingivalis can internalize in norml non-phagoyticgingival epithelial cells, then uniformly accumulate in

the perinuclear region.

Invasion ofP.gingivalis is associated with thephosphorylation of c-jun- N-terminal kinase (JNK) and

down-regulation of extra-cellular signal-regulated kinase

(ERK ) as well as transient elevation of intracellular

ca2+

ion levels.

However nuclear factor kappa B (NF-kappa B)is not

activated and secretion of IL-8 is inhibited.

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The major fimbriae ofP. gingivalis are involved inboth adhesion to epithelial cells and the subsequent

signalling events associated with invasion.

The organism is also known to cause proteolysis of

focal contact components such as paxillin and focal

adhesion kinase (FAK), which are signaling

molecules that regulate adhesion, survival,

proliferation, differentiation, and migration.

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These bacterial effects are suggested to be mainly due togingipains and, in part, fimbriae.

Type II fimA degrade both paxillin and FAK more

quickly than otherfimA type strains, resulting in aninhibition of phosphorylation by these molecules

(Nagakawa et al., pers.comm).

These findings provide an explanation for therelationship offimA variations to bacterial virulence.

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Internalization ofP. gingivalis

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P. gingialis interactions with primary gingival

epithelial cells.

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Minor fimbriae ofP. gingivalis

Minor fimbriae was found in 1996 and shown to beshort fimbria like appendages in a fimA (major fimbria-

deficient) mutant of strain ATCC 33277.

A subunit protein of a minor fimbriae (Mfa1) encodingthe mfa1 gene was shown to be different in size (67 kDa

in contrast to 41 kDa of major fimbria subunit) and

antigenicity from that of major fimbriae. (Ref 3)

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Ref 3- Hamada N Watanabe K Arai M Hiramine H


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Ref 3 Hamada N, Watanabe K, Arai M, Hiramine H,

Umemoto T.Cytokine production induced by a

67-kDa fimbrial protein fromPorphyromonas

gingivalis. Oral Microbiol Immunol2002;17:197200.

In an orally infected rat model, a fimA mutant revealed a

significant reduction of adhesion potential to saliva-coated

hydroxyapatite, gingival epithelial cells, and fibroblasts aswell as bone adsorption capability.

Minor fimbriae purified from P. gingivalis ATCC 33277

markedly induced IL-1, IL-, IL-6, and TNF- cytokineexpression in mouse periotoneal macrophages.

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These results suggested that P.gingivalis 67-kDafimbriae may play a part during the development of

periodontal diseases.

To identify the influence of major and minor fimbriae on

bacterial virulence, isogenic mutants of P. gingivalis

were constructed and inoculated into the oral cavities of

rats. (Ref 4)

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Ref 4- Memoto T, Hamada N. Characterization of biologically


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Ref 4 Memoto T, Hamada N. Characterization of biologically

active cell surface components of a periodontal

pathogen. The roles of major and minor fimbriae of

Porphyromonas gingivalis. Jperiodontol 2003;74:119

122.

Inactivated mutants of 41- k fimbrillin gene fimA (major

fimbriae) and fimbrillin gene (mfa1) were constructed by a

homologous recombination technique and compared among

fimA mutant knockout (MPG1)[major fimbriae deficient

mutant], mfa1 KO (MPG67)[minor fimbriae deficient

mutant], and double KO (MPG4167) mutants [deficient in

both major and minor fimbriae].

The adherence and invasion levels of the mutants were lower

than the wild-type strain.

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The bone loss of rats infected with the MPG1 was higher than thatof those infected with MFG67.

The bone loss of rats infected with the double knockout mutant wassignificantly decreased compared to those of rats infected with

wild-type strain.

Minor fimbriae-deficient mutant MPG67 showed a more than 3-fold increase in adherence to wild-type strain 33277.

In contrast, no adherence was detected infimA mfa1double mutantMPG 4167.

These findings indicate that production of both major and minorfimbriae is required for the expression of pathogenic traits by P.

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Clonal variations of minor

fimbriae

Recent analyses showed that the Mfa1 molecule is the same

as that of the 75 kDa outer membrane protein , the 67 kDa

major outer membrane protein ,and Pg-II (a 72 kDa cell

surface protein) in strain ATCC 33277.

However, a 53 kDa protein isolated from strain 381 was

shown to be another minor fimbriae , which was

demonstrated to be the same molecule as a 53 kDa major

outer membrane protein in other reports, and a majorimmunodominant protein likely to contribute to hostbacterial

interaction.

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Those two types of minor fimbrial proteins showedno immunological cross-reactivity.

Thus, strain 33277 has 67 kDa minor fimbriae and

strain 381 has 53 kDa minor fimbriae.

These distinct molecules are being investigated for

their clonal heterogrnrcity in relation to bacterial

virulence.

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Conclusion

Genomic variations of the fimbria structures of P.gingivalis are likely involved in the initiation and

progression of human periodontitis.

It would be of value for periodontal therapy andassessment of prognosis if the disease contributing

strains could be differentiated based on clonal variations

of thefimA gene.

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A number of reports have demonstrated a wide varietyof chromosomal genotypes ofP. gingivalis, suggestingthat possible variations of other pathogenic genes areinvolved in its pathogenicity.

In addition, environmental conditions also seem to alterits virulence and the expression of virulence factors,including several proteolytic enzymes such asgingipains, is influenced by several factors.

Further studies regarding these aspects are necessary tobetter understand the virulence variations ofP. gingivalisclones.

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