CONTENTS

 Introduction

 Determinants of antigenicity

 Antigen specificity

 Biological classes of antigens

 Methods of identification of antigens and epitopes

 Actinibacillus actinomycetemcomitans antigens

 Treponema denticola antigens

 Tannerella forsythia antigens

 Porphyromomas gingivalis antigens

 Conclusion

 References
INTRODUCTION
An antigen has been defined as any substance which, when introduced
parenterally into the body, stimulates the production of an antibody with which it reacts
specifically, and in an observable manner.
The word ‘parenteral’ (meaning, outside the intestinal tract) is used in the
definition because orally administered antigens are usually hydrolyzed by digestive
enzymes, resulting in their antigenicity being destroyed; therefore there is no antibody
formation.
The word ‘specifically’ is important as specificity is the hallmark of all
immunological reactions. An antigen introduced into the body reacts only with those
particular immunocytes (B or T lymphocytes) which carry the specific marker for that
antigen and which produce antibody complementary to that antigen only. The antibody
so produced will react only with that particular antigen and with no other- through
immunological cross-reaction may occur between closely related antigens.
This traditional description of an antigen is no longer comprehensive enough in
the light of current concepts about the immune response. Some antigens may not induce
antibodies but may lead to cell mediated immunity or immunological tolerance.
The two attributes of antigenicity are:
1) induction of an immune response, and
2) specific reaction with antibodies. Based upon the ability of antigens to carry
out these two functions, they may be classified into different types.
A complete antigen is able to induce antibody formation and produce a specific and
observable reaction with the antibody so produced.
Haptens are substances which are incapable of inducing antibody formation by
themselves, but can react specifically with antibodies. Haptens become immunogenic
(capable of inducing antibodies) on combining with a larger molecule carrier. Haptens
may be –
 complex haptens can precipitate with specific antibodies,
 simple haptens are non- precipitating.
 The smallest unit of antigenicity is known as the antigenic determinant or
epitope. The epitope is that small area on the antigen, possessing a specific chemical
structure and steric (spatial) configuration, capable of sensitizing an immunocyte and of
reacting with its complementary site on the specific antibody.
The combining area on the antibody area, corresponding to the epitope is called the
paratope
Epitopes and paratopes determine the specificity of immunological reactions

DETERMINANTS OF ANTIGENICITY

A number of properties have been identified which make a substance antigenic,

but the exact basis of antigenicity is still not clear.

Size: Antigenicity bears a relation to molecular size. Very large molecules are highly
antigenic and particles with low molecular weight are nonantigenic or feebly so.

Chemical nature: Most naturally occurring antigens are proteins and polysaccharides.
Lipids and nucleic acids are less antigenic.
A certain degree of structural complexity is required for antigenicity. This explains why
proteins which are composed of 20 different aminoacids are better antigens than
polysaccharides which are only 4 or 5 monosaccharide units.
Not all proteins are however antigenic. A well known exception is gelatin.

Susceptibility to tissue enzymes: Only substances which are metabolised and are
susceptible to the action of tissue enzymes behave as antigens.

Foreignness: Only antigens which are 'foreign' to the individual (nonself) induce an
immune response. An individual does not normally mount an immune response against
his own normal constituent antigens. Tolerance of self antigens is conditioned by contact
with them during the development of the immune apparatus. Breakdown of this
homeostatic mechanism results in autoimmunisation and autoimmune disease.
In general, the antigenicity of a substance is related to the degree of its foreignness.

ANTIGENIC SPECIFICITY
The basis of antigenic specificity is stereochemical. Antigenic specificity is
determined by chemical groupings and even by a single acid radical. The specificity of
natural tissue antigens of animals may be considered under different categories as
species, iso-, auto-, and organ specificities.

Species specificity: Tissues of all individuals in a species contain species specific

antigens.

Isospecificity: Isoantigens are antigens found in some but all members of a species. Eg :
blood groups

Autospecificity: Autologous or self antigens

Organ specificity: Some organs, such as the brain, kidney and lens protein of different
species, share the same antigen. Such antigens characteristic for an organ or tissue,
found in different species, are called organ specific antigens.

Heterogenetic (heterophile) specificity: The same or closely related antigens may

sometimes occur in different biological species, classes and kingdoms. These are known
as heterogenetic or heterophile antigens. The best known example of such heterophile
antigen is the Forssman antigen which is a lipid carbohydrate complex widely
distributed in various animals.

BIOLOGICAL CLASSES OF ANTIGENS

Depending on their ability to induce antibody formation, antigens are classified
as
 T cell dependent (TD), and
 T cell independent (TI) antigens
Antibody production is the property of B lymphocytes. For the full expression of this
function, however, the cooperation of T-lymphocytes is necessary. Some antigens can
directly stimulate antibody production by B cells, without the apparent participation of
T cells. Such antigens are called T cell independent (TI) antigens. Others that require T
cell participation to generate an immune response are called T cell dependent (TD)
antigens.
Several important differences exist between TI and TD antigens.
TI antigens –
 Are structurally simple, being composed of a limited number of repeating
epitopes. Eg: bacterial LPS, flagellar protein (Flagellin)
 Their immune response is critically dose dependent, too little being non-
immunogenic, while too much results in immunological tolerance rather than
immunity
 Their antibody response is usually limited to IgM and IgG3
 They do not produce immunological memory
 TI antigens do not appear to require preliminary processing of macrophages
 They are metabolized very slowly and remain in the body for long periods

TD antigens –
 Structurally more complex Eg: erythrocytes, serum proteins
 They are immunogenic over a wide dose range and do not cause tolerance
readily
 They induce the full gamut of immunoglobulin isotypes- IgM, IgG, IgA and IgE
 They produce immunological memory,
 Require preliminary processing, and
 Are rapidly metabolized in the body
 METHODS OF IDENTIFICATION OF BACTERIAL PROTEIN ANTIGENS
AND EPITOPES

Identification of antigens by proteomic analysis

As antigens are very often surface-exposed proteins, one strategy for their
identification is to characterize the proteins present in surface extracts of the pathogen
of interest. Often, such a strategy involves analysis of the surface extract by Western
blots of one-dimensional (ID) Sodium Dodecyl Sulphate Polyacrylamide Gel
Electrophoresis (SDS-PAGE) gels to identify antigenic proteins, which subsequently
may be identified by N-terminal sequencing by Edman degradation. Identification of
SDS-PAGE separated proteins by N-terminal sequencing alone, however can be
problematic. Surface-associated proteins are often heavily glycosylated, and several
other kinds of modification, such as acetylation, formylation and pyroglutamine
formation, are known to affect the N-terminus of proteins in such a way that usually
precludes straightforward analysis by N-terminal sequencing. Proteins derived from
complex samples such as preparations of bacterial cell walls and membranes cannot be
adequately resolved by ID SDS-PAGE alone. These techniques have now been
surpassed by proteomic analysis those organisms whose genomes have been sequenced.
The genomes of P. gingivalis, T. denticola and A actinomycetemcomitans. The
genome of T. forsythia (listed as B. forsythus) is currently being sequenced.
.
The proteomic approach of using two dimensional (2D) -PAGE separation and
mass spectrometric techniques to identify the proteins overcomes, the problems
associated with ID SDS-PAGE separation of complex samples. 2D-PAGE is capable of
resolving several thousand proteins on a single gel by enabling them to be separated
over two dimensions. The first dimension used is most frequently isoelectric focussing
(IEF) which separates proteins by charge, while the second dimension is SDS-PAGE
that separates proteins according to their molecular size. After the second dimension,
the 2D-gel can be subjected to Western blotting, while a second 2D-gel can be stained,
and the protein spots subjected to mass spectrometric (MS) analysis. For MS analysis, a
protease (often trypsin) is added to the gel-spot to cleave the protein into peptide
fragments, these peptides are subsequently recovered and analysed by an MS technique
known as peptide mass fingerprinting (PMF). PMF generates a list of peptide masses
that can be used to identify the protein by reference to a peptide mass database derived
from all possible protein sequences encoded by the pathogen's genome. The advantages
of using PMF to identify proteins include its speed and sensitivity. In addition to the
detection of more proteins, IMF has the further advantage of detecting proteins that co-
migrate in a single spot in the gel.

Identification of B-cell and T-cell antigenic determinants using epitope mapping

analysis
Once an antigenic protein has been identified and the amino acid sequence
deduced using proteomic and genomic techniques, those parts (antigenic epitopes) that
are important in the host immune response to the protein can be identified using a number
of different techniques. Computer algorithms are one technique that can be used to
predict potential B-cell and T-cell antigenic epitopes by analyzing the amino acid
sequence of a given protein. B-cell epitopes can be predicted by analyzing hydrophilic,
hydrophobic, segment mobility, -helical and β-turn segments within a protein and
assigning antigenic index values for amino acid sequences. Algorithms predicting T-cell
epitopes use structural data from MHC-peptide complexes and peptide-MHC binding
data to analyze the amino acid sequence of a given protein and predict those peptide
sequences that may bind to a particular MHC molecule. A number of computer
algorithms are available to predict cytotoxic T-cell (CD8+) MHC-class I epitopes and
MHC-class II epitopes. Although computer algorithms have been successfully used to
identify B-cell and T-cell epitopes, the predicted sequences still need to be tested in bio-
assays to evaluate whether they bind antibody or stimulate T-cells. Limitations of
computer algorithms in predicting antigenic epitopes centre around a limited data base to
make the predictions from, although the rapidly expanding information on protein
structure and function will enhance epitope prediction algorithms.
 A systematic method for identifying both B-cell epitopes and T-cell epitopes is
PEPSCAN. The principle of the method is to synthesize overlapping peptides of a given
length (e.g. 8 to 15 mers) covering the whole or part of a given protein sequence onto a
solid support. For B-cell epitope identification the amino acid side chain protecting
groups used in peptide synthesis are removed and the peptides are left on the solid
support. The solid support- bound peptides are then probed in an ELISA with antisera to
the whole organism or protein under investigation. For identification of T-cell epitopes,
the peptides need to be cleaved from the solid support and used in cytotoxic T-cell or T-
cell proliferation assays with bacteria- or protein-primed T-cells. Although the
PEPSCAN approach has been applied to identify epitopes on an A.
actinomycetemcomitans antigen, it has been mostly applied in identifying P. gingivalis
antigenic epitopes.
The identification of antigenic epitopes of a particular protein from a pathogenic
bacterium forms an essential part of the development and design of sub- unit/synthetic
vaccines and immunodiagnostics. This knowledge also enhances our understanding of the
role a particular protein antigen plays in the host immune response. Thus by combining
proteomic, genomic and epitope mapping techniques together with animal and human
studies it is possible to identify pathogen antigens and elucidate their immunological and
biological role in disease.
 Actinobacillus actinomycetemcomitans antigens
A. actinomycetemcomitans is a gram-negative, non-spore forming, non-motile,
capnophilic, facultative anaerobic coccobacillus.

1. Polysaccharide and lipopolysaccharide

A. actinomycetemcomitans can be classified into six distinct serotypes (a-f based
on surface polysaccharides located on the 0 side chains of lipopolysaccharide).
 The outer membrane of G-ve bacteria is assymetric, the outer leaflet of which
contains the LPS
 LPS -
Very large molecule
3 parts –
-The O antigen
-Core region
-Lipid A

The presence of A. actinomycetemcomitans in subgingival plaque has been

associated with aggressive periodontitis with serotype b twice as prevalent as serotype
a. These data are supported by a number of other studies that have shown serotype b to
be the predominant A. actinomycetemcomitans serotype associated with periodontitis.
Animal studies have shown that serotype b induces the pro-inflammatory cytokine
interleukin-6 (IL-6) from murine splenocytes. Furthermore, serotype b induces more IL-
1 (another pro-inflammatory cytokine) from thymocytes than serotype a or c. Ebersole
et al. (1995) have reported that the order of virulence for A. actinomycetemcomitans
serotypes in the murine lesion model was serotype b > a > c. The serotype-specific
polysaccharides are reported to be major antigenic targets of the immune response of
aggressive periodontitis patients infected with A. actinomycetemcomitans with the
predominant antibody subclass being IgG2 followed by IgG1 and IgG3.
 Pro-inflammatory cytokines such as IL-1ra, IL-Iβ and TNF-α induce bone
resorption by promoting differentiation of osteoclast precursor cells and by activating
osteoclast cells. Kawai et al. (2000) have shown that A. actinomycetemcomitans
lipopolysaccharide injected into the gingiva of rats could induce periodontal bone
resorption but only after the transfer of antigen-specific Th1 clone cells, but not after
the transfer of Th2 clone cells. These data support earlier studies which showed that an
A. actinomycetumcomitans-specific Th2 clone when adoptively transferred into
euthymic rats protected against periodontal bone loss induced by oral infection with A.
actinomycetemcomitans.

2. Leukotoxin
A. actinomycetemcomitans produces a 116 kDa immunomodulating protein
antigen, termed leukotoxin. The leukotoxin is a pore-forming protein and is a member of
the repeats-in-toxin (RTX) family of bacterial toxins. The leukotoxin is not released by
A. actinornycetemcomitans but is anchored by a G-terminal hydrophobic domain to the
cellular outer membrane or membranous vesicles. A number of studies have shown that
the killing of T-cells, neutrophils, natural killer (NK) cells and polymorphonuclear
leukocytes by leukotoxin is by the formation of pores in the cell membrane resulting in
osmotic lysis and necrosis. Unlike other members of the RTX family, which kill a wide
variety of cells, A. actinomycetemcomitans leukotoxin is specific for cells that express
β2-integrin and lymphocyte functional antigen-1 (LFA-1). The binding of the leukotoxin
to LFA- 1, which is expressed on all immune cells, is suggested to facilitate insertion into
the cell membrane and pore formation. Aa leukotoxin at low doses induces apoptosjs,
whereas at high doses, leukotoxin has been reported to cause cell death by necrosis. At
high concentrations it is suggested that the toxin binds non-specifically to cell membranes
forming large pores allowing the rapid influx of Ca 2+ and loss of ATP resulting in
necrosis, while at low concentrations the toxin is thought to bind to specific cell surface
proteins on susceptible cells and form small diameter pores allowing the uncontrolled
influx of Na+ and the activation of apoptosis. Thus the A actinomycetemeomitans
leukotoxin is a potent cytotoxic antigen able to kill immune cells, resulting in the
dysregulation of the host immune response and the release of a variety of enzymes and
reactive molecules from phagocytic cells that may result in tissue damage and further
inflammation. Ability of serotypes b and a, but not c, to induce apoptosis is consistent
with the serotype virulence (b > a > c) in animal models.

3. Extracellular proteolydc enzymes

Proteolytic activity in subgingival plaque, in particular trypsin-like proteolytic

activity, has been highly correlated with clinical measurements of periodontitis. Culture
supernatants of A. actinomycetemcomitans serotype b, have been shown to have high
trypsin-like proteolytic activity. Protease inhibition assays indicated that this enzyme is a
serine- or metallo-protease. Furthermore, the trypsin-like protease reduced the
proliferation of human gingival epithelial cells and cell surface levels of fibronectin. As
increased proteolytic activity of other periodontal pathogens, especially P. gingivalis,
has been linked to enhanced virulence, the greater proteolytic activity, especially trypsin-
like activity, of A. actinomycetemcomitans serotype b compared with serotype c is
consistent therefore with the greater virulence of this serotype. As well as degrading
major components of connective tissue, proteolytic enzymes in A.
actinomycetemcomitans culture supernatants have been reported to degrade IgG (all four
subclasses), serum (but not secretory) lgA and IgM but not lgD or lgE in vitro. Thus the
proteolytic activity of A. actinomycetemcomitans may be an important virulence factor
involved in dysregulation of the host's immune response.

4. GroEL heat shock protein

A. actinomycetemcomitans produces an antigenic 64 kDa GroEL protein that is

equally expressed on the cell surface of all serotypes a-e. Heat shock proteins have been
suggested to be associated with the etiology and pathogenesis of both experimental and
naturally occurring autoimmune diseases such as juvenile chronic arthritis, rheumatoid
arthritis and atherosclerosis. Microbial heat shock proteins, despite the high homology
between prokaryotic and eukaryotic cells, are highly immunogenic and can lead to
antimicrobial immunity. It has been speculated, therefore that the long term infection
associated with chronic inflammatory diseases, such as periodontitis, results in the
constant exposure of microbial heat shock antigens to the immune system, which may
promote autoimmune disease.
Actinobacillus actinomycetemcomitans GroEL may also recruit and activate
osteoclasts. As well as activating osteoclasts, A. actinomycetemcomitans GroEL inducer
epithelial cell proliferation at low concentrations (0.4-1.0 g/mL) and is cytotoxic at
higher concentrations.

5. Fimbriae
 Thin, straight appendage originally referred to as Pili
 2 MAJOR CLASSES-
-Type-specific fimbriae
-F- or sex pili
 Approx 3 to 25nm in diameter & 3 to 25 µm long
 Vary in distribution & number from 10 to 100/cell
A. actinomycetemcomitans produces highly antigenic bundle-forming fimbriae
that are 5 nm in diameter and several m in length. The flmbriae are suggested to play an
important role in colonization as fimbriated A. actinomycetemcomitans strains have
greater affinity for epithelial cells and saliva-coated hydroxyapatite than non-fimbriated
strains. Fimbriae may also have a role in A. actinomycetumcomitans invasion of
epithelial cells by, receptor-mediated endocytosis.

6. Other antigenic outer membrane proteins

An analysis of the frequency of patient sera that bound to antigenic outer membrane
proteins showed that the IgG subclass distribution was IgG2 > IgG3 > IGGI >> IgG4.
Beikler et al. (1999) monitored the antibody subclass responses to a 110 kDa A.
actinomycetemcomitans outer membrane protein in periodontitis patients 24 months
after receiving full mouth scaling and/or antimicrobial therapy followed by supportive
periodontal therapy. Patients, harboring .A. actinomycetemcomitansintraorally, with a
high IgG4 response to the 110 kDa proteins had significantly increased tooth survival
rates. No significant association with clinical treatment outcome was found for antigen-
specific lgA, IGGI, IgG2 or IgG3 serum levels, indicating that a serum IgG4 antibody
response to the 110 kDa protein may have a protective effect in A.
actinomycetemcomitans- associated periodontitis.
Outer membrane and secreted proteins from A. actinomycetemcomitans have also been
reported to be major inducers of inflammatory cytokines.
Treponema denticola antigens

1. Lipopolysaccharide

T. denticola is a gram-negative, motile, asaccharolytic, anaerobic spirochete with

typical helical morphology. Unlike lipopolysaccharide of other Gram- negative bacteria
the outer membrane sheath of T. denticola lacks 3-deoxy-D-manno-octulosonic acids,
heptoses and β-hydroxy fatty acids.

The cytoplasmic membrane and outer membrane sheath have similar fatty acyl
chain composition indicating that the membrane fluidity of T. denticola is similar to the
membranes containing lipoteichoic acid of gram-positive bacteria. Furthermore, the
membrane anchor consists of phospholipids and glycolipid-like structures containing 2
fatty acids (structurally similar to lipoteichoic acid) and not 6 as in lipid A of typical LPS.

These data indicate that the ultrastructure of the outer membrane sheath of T.
denticola is similar to the outer membrane of other gram-negative bacteria; however, the
lipid composition of the outer sheath is similar to lipoteichoic acid of the cell surface of
gram-positive bacteria.

Although there are differences in the outer membrane of T. denticola and other
gram-negative bacteria the outer membrane lipids are reported to be highly antigenic

Purified outer membrane lipid and enriched outer membrane lipoprotein preparations of
T. denticola have been reported to induce, in a dose dependent manner, the inflammatory
mediators, nitric oxide, TNF- and IL-1 from murine macrophages.

Gopalsami et al. (1993) have reported that the outer membrane lipid of T.
denticola was able to induce bone resorption in the radii and ulnae of 19-day-old fetal
rats. However, the addition of parathyroid hormone or prostaglandin E2, which are known
to enhance lipopolysaccharide-induced bone resorption from other gram-negative
bacteria, had no additive effect. These data suggest that the outer membrane lipids of T.
denticola induce bone resorption and activation of inflammatory cytokines by a separate
mechanism to lipopolysaccharide.

2. Major sheath protein

A major surface protein and antigen of T. denticola is the major sheath protein
(Msp), which has been reported to have different molecular masses in different T.
denticola strains. The protein exists as a 53-kDa and a 64-kDa protein in 2 different
strains. Calmano et al. (1999) has more recently suggested that Msp is predominately a
periplasmic protein that has limited surface exposure. This limited surface exposure
may explain the differences in the antigenicity between strains
The 53-kDa Msp protein binds fibrinogen, fibronectin and laminin and thus may have an
important role in binding of T. denticola to host cells. The Msp protein also aggregates
with the chymotrypsin-like protease complex of T. denticola and may play a role in
binding to epithelial cells. The 64-kDa Msp protein has also been shown to have an
important role in the adhesion of T. denticola to gingival fibroblasts.
As well as binding to host cell matrix proteins the 53-kDa Msp protein is toxic to HeLa
cells, epithelial cells and erythrocytes. Although the precise mechanism by which the 53-
kDa Msp protein induces cell death is not known, it has been shown to alter the
membrane potential of artificial membranes and increase the membrane permeability of
HeLa cells, indicating that it may form a pore in the host cell membrane resulting in
unrestricted influx of ions and eventual cell death.
The ability of the Msp protein to bind to host matrix proteins and form pores may,
in part, explain the cytopathic effects of T. denticola on gingival fibroblasts, epithelial
cells, lymphocytes and erythrocytes.
The 53-kDa Msp protein also enhances the inflammatory response by triggering
degranulation of poly- morphonuclear cells and the specific release of collagenase,
gelatinase and MMP-8 and MMP-9

3. Flagellum
A major distinction between T. denticola and the other three periodontal
pathogens (Aa, Pg & Tf) is that T. denticola is motile.
 The axial helical-flagella and the helically-shaped cell cylinder of T. denticola,
like all spirochetes, result in a cork screw-like locomotion, which aids movement in
highly viscous environments like gingival crevicular fluid and the penetration of
gingival epithelial and connective tissue.

The flagellum of T. denticola is composed of a basal body, rod, hook and a

filament. The flagellar filament consists of three core proteins FlaBl, FlaB2 and FlaB3
and a major sheath protein FlaA. Although each of the filament proteins are antigenic,
FlaA and FlaB3 have been shown to be immunodominant antigens. FlaA has been
suggested to have a role in adherence of T. denticola to host cells as it binds fibronectin.

4. Extracellular proteolytic enzymes

The prolyl-phenylalanine specific, chymotrypsin, serine proteinase known as
dentilisin or trepolisin, is the best characterized T. denticola protease. This enzyme
occurs on the cell surface and has been purified as a complex of three proteins, a large
proteinase encoded by the prtp gene and two smaller proteins.

Has ability to degrade –

 Host matrix proteins (dentilisin has been shown to hydrolyse fibrinogen,

transferrin, gelatin, serum albumin, laminin, collagen type IV, IgG and lgA in
vitro)

 Inflammatory proteins (dentilisin has also been suggested to play a role in the
degradation of pro-IL-I β into its bioactive forms and thus stimulate the
inflammatory response).

 Protease regulatory proteins and peptides (Proteinase has also been shown to
degrade bradykinin, substance P and angiotensin I and II, as well as host
protease inhibitors α1-antitrypsin, antichymotrypsin, α2-macroglobulin,
antithrombin III, antiplasmin and cystatin C).

The ability to degrade host matrix proteins, inflammatory and protease regulatory
proteins and peptides may contribute to the uncontrolled degradation of periodontal
tissues and enhance disease progression. T. denticola has been shown to bind to the
ground substance glycosaminoglycan, hyaluronate via the dentilisin complex. The
dentilisin complex has also been shown to bind and induce apoptosis in epithelial cells.
The presence of T. denticola in subgingival plaque samples has been correlated with
trypsin-like proteolytic activity, which in turn has been correlated with clinical
parameters of periodontitis. Trypsin-like activity has also been localized to the cell
surface of T. denticola.

5. Other antigenic proteins

A putative hemolysin,, named cystalysin is suggested to play a central role in

iron acquisition by T. denticola as it is able to agglutinate and lyse erythrocytes and
cause the oxidation and sulphuration of hemoglobin.

Tannerella forsythia (formerly Bacteroides forsythus) antigens

T. forsythia is a gram-negative, anaeroble, saccharolyric fusiform bacterium.
Very little is known about the antigens and virulence factors of T. forsythia compared
with the other major periodontal pathogens, P. gingivalis, A. actinomycetemcomitans
and T. denticola. This has mainly been the result of the difficulty in reliably cultivating
T. forsythias. However, the finding that T. forsythia could be grown together with
another bacterium such as F. nucleatum or Streptococcus sanguis revealed that it
requires erogenous N-acetylmuramic acid for growth. Inclusion of N-acetylmurarnic
acid in conventional media now allows routine cultivation of T. forsythia in batch culture

1. Lipopolysaccharide
Vasel et al (1996) indicated that T. forsythia LPS may be similar to P. gingivalis
LPS. Firoozkoohi et al (1997) analysed T. forsythia and showed that LPS was a typical S-
form exhibiting long ladders with multiple steps. The same authors also showed that the
O-polysaccharide side chains of T. forsythia LPS are of similar length to those of
Bacteroides species.

2. Extracellular proteolytic enzymes

Several studies have shown that T. forsythia produces cell surface proteolytic enzymes
These include –
  Trypsin-like serine protease
 Hemolytic cysteine-protease (PrtH)
T. forsythia trypsin-like activity, together with trypsin-like activities of T. denticola and
P. gingivalis in subgingival plaque samples have been correlated with clinical parameters
of periodontitis
Interestingly, unlike P. gingivalis and T. denticola, T. forsythia has been reported
to be unable to proteolytically degrade a variety of host protease inhibitors and lactoferrin
The trypsin-like activity has been suggested to play a role in binding of T. forsythia to
erythrocytes, PMNs and fibroblasts.

3. Other antigenic outer membrane proteins

T. forsythia produces a protein that has been suggested to induce apoptosis
especially in lymphocytes by the formation of membrane pores. A GroEL-like protein is
also produced by T. forsythia which may have similar effects on the immune response as
in Aa
A major surface antigen of T. forsythia is Bsp A. Bsp A contains 14 leucin-rich
repeat (LRR) motifs that are believed to be involved in protein-protein or lipid-protein
interactions. Bsp A also has been shown to stimulate proinflammatory cytokine
production from mononuclear cells via interaction with CD14 and Toll-like receptor 4.

Porphyromonas gingivalis antigens

1. Capsule
Considered an important virulence factor by many authors. Some strains have
either a thin layer or are devoid of it.
Chemical composition differs between strains. Mansheim & Kasper (1977) found
galactose, glucose and galactosamine. Okuda et al (1987) found rhamnose, glucose,
galactose, mannose and methyl pentose.
Biological Function -
 Highly encapsulated strains exhibit decreased autoagglutination, lower bouyant
densities and were more hydrophilic than the less encapsulated strains
 Increased encapsulation was correlated with increased resistance to phagocytosis,
serum resistance and decreased induction of PMN chemiluminiscence.

2. Outer Membrane Proteins

Structure–
Complex multilayered structure
 The inner, cytoplasmic membrane
 A thin, peptidoglycan
 Assymetrical, outer membrane
The outer membrane contains complex LPS,lipoproteins and peripheral & transport
proteins, Porin proteins, short fimbriae. Made of atleast 20 proteins.
Outer membrane proteins & coaggregation –
 Pg may play an important role in the formation and maintenance of the
periodontal biofilm
 The benchmark report of Gibbons and Nygaard (1970) demonstrated that there
were specific interactions bw members of the oral microbiota and for the first
time revealed that bacteria do attach to both hard and soft surfaces as well as to
each other.
 In Pg, this interaction with selected G+ & G- bacteria was mediated by specific
outer membrane proteins in the whole cell-associated outer membrane or in the
outer membrane vesicles

3. Lipopolysaccharide
Chemical Composition -
 Neutral sugars – Rhamnose, mannose, galactose and glucose
 Glucosamine and galactosamine represent the amino sugars
 Fatty acids
 Phosphorus
Biological Properties –
Endotoxic activity is confined to lipid A, while the immunological activity is
contained within the O antigen.
  Ogawa et al (1994) demonstrated induction of IL-1 ra, IL-6, IL-8, IFN-γ, GM-
CSF secretion by cultured mononuclear cells
 Bramati et al (1989) and Sismey Durrant & Hopps (1991) –PGE2
 Tamura et al (1992) and Takada et al (1991) – IL-1β and IL-8
The preponderence of evidence indicates that LPS,especially the Lipid A, is capable of
stimulating the host inflammatory response directly via the induction of host derived
cytokine production.

4. Fimbriae
Arranged in an peritrichous fashion.Comprised of atleast 1000 protein subunits
(fimbrillin subunits). Sex pili not present in the isolated strains thus far.
Atleast 2 distinct fimbriae molecules –
 Major
 Minor

Major fimbriae -
Composed of fimbrillin
F/n –
1. Act in bacterial interactions with host tissue by mediating bacterial adhesion &
colonization in targeted sites
2. Are capable of binding specifically to and activating various host cells, resulting
in release of cytokines, as well as cell adhesion molecules
3. Have been shown to be necessary for bacterial invasion of host cells.

Lee et al (1991) first reported the variation of Fim A proteins and divided a number
of Pg strains into 4 types based on their N-terminal aa sequences. Pg fim A genes have
been further classified into 6 variants (type I to V , and Ib) on the basis of nucleotide
sequence
Nakagawa & coworkers (2000) found a majority of patients were found to carry type II
fim A organisms, followed by type IV. In healthy it was type I (2001)
Minor fimbriae -
Was found in 1996 (Hamada et al) and was shown to be short fimbria-like
appendages in an fim A deficient mutant mfa 1 gene encodes for the subunit protein of
the minor fimbriae (Mfa 1)
It is thought that production of both major &minor fimbriae is required for the
expression of pathogenic traits by Pg
- Amano et al (2004)

4. Proteinases
One of the potentially significant virulence characteristics of Pg is the large number of
hydrolytic, proteolytic and lytic enzymes that are produced by
essentially all known strains
Many of the enzymes are either-
 Exposed at the surface (in the outer membrane) of the bacterium where they are
able to come in contact with the host cells and tissues
 Within the periplasmic space capable of being transported to the cell surface
 In the outer membrane vesicles, which are sloughed from the outer surface during
growth

Serine
Aspartate
Proteinases are of 4 types Thiol
Metalloproteinases

Secretory pattern is type IV

I. Trypsin-like protease (the gingipains)
2 proteases-
 Arg gingipains (Rgp) encoded by rgpA & rgpB
 Lys gingipains (Kgp) encoded by kgp
II. Aminopeptidases
Pg is probably the only periodontoppathic microbiota that exhibits strong
dipeptidyl arylaminopeptidase activity.

III. Caseinases
Hydrolyses the protein, casein. Exists in atleast 3 isoforms called Pase A, B & C.
B & C display a cleavage pattern similar to that of trypsin (ie cleavage of carboxy side of
arginine). Pase A has its preferred cleavage site as the carboxyl side of lysine.

IV. Collagenase
It was almost 35 years ago that Shultz-Haudt & Schrep (1955) demonstrated that a
mixed culture of bacteria isolated from the gingival cavity displayed collagenolytic
activity. This activity was demonstrated to be collagenase from Bacteroides
melanogenicus. Data support the participation of bacterially derived collagenase;
however these data are equivocal.

5. Hemagglutinins
Pg produces at least 5 hemagglutinating molecules. When expressed on the
bacterial cell surface, hemagglutinins may promote colonization by mediating binding of
bacteria to receptors on human cells. Since Pg utilizes heme for growth, binding of
bacterial cells to erythrocytes may also serve a nutritional function. Evidence does
implicate a role for fimbrillin
Hemaggluttin activities expressed by Pg include those complexed with LPS on
the cell surface and a released form designated exohemagglutinin. 4 hemagglutinin genes
from Pg strains have been identified: hag A, hag B, hag C & hag D. Several studies have
demonstrated that the hemagglutination events mediated by Pg might be due to the
combined effects of at least 3 enzymes formed into a large protein complex.

6. Other Enzymes
i. Alkaline phosphatase: Enzyme appears to be cell associated and limited to the
periplasmic space. However, some evidence suggests location of enzyme in the
 extra cellular environment. It has been shown to be positively correlated with
periodontal disease activity as well as with alveolar resorption in advanced cases
of human periodontitis. However, the role of alkaline phosphatase in Pg
pathogenesis has not been clearly defined.

ii. Superoxide dismutase (SOD): While anaerobe by definition, it can withstand

significant amounts of oxygen. Therefore Pg is an anaerobe and cannot grow in an
aerobic environment but can tolerate high levels of dissolved oxygen. Therefore,
it must possess the enzymes necessary for detoxifying oxygen radicals. The
enzymes superoxide dismutase, peroxidase, and catalase are capable of providing
such protection. SOD was isolated and purified from Pg. The superoxide is
scavenged by SOD which functions in the conversion of 2 superoxide anions into
hydrogen peroxide and oxygen. Under the catalytic direction of catalase,
hydrogen peroxide is converted to oxygen and water.

iii. Sulfatase, heparinase and chondroitinase: Enzymes which are capable of

degrading gingival glycose amino glycans and proteoglycans.
 CONCLUSION
Each of the periodontal pathogens produces an array of antigens that stimulate
epithelial cells, macrophages, monocytes and fibroblasts to produce proinflammatory
cytokines and induce a Th-1 (inflammatory) cytokine response. However, a number of
studies have shown that some antigens of the pathogenic bacteria also stimulate a Th2
cytokine response which is known to be anti-inflammatory, This Th1 and Th2 response
may be mixed, a possible explanation being that, it reflects the dysregulation of the host
defense in periodontal lesions thus allowing bacteria at the site of infection to evade
immune response.
Proteolytic activity, in particular ‘trypsin-like’ proteolytic activity of subgingival
plaque (produced by all 4 periodontal pathogens), have been positively correlated with
clinical parameters of periodontitis. These enzymes are likely to be involved directly or
indirectly in destruction of host tissue and dysregulation of host defenses by activating
latent host enzymes and degrading host matrix proteins, cellular receptors, proteinase
inhibitors, complement, antibodies and certain cytokines. They are also likely to be
involved in adherence to host cells and other bacteria and therefore in the colonization of
the gingival crevice.
The current treatment of periodontitis is non-specific and centered on the removal
of subgingival plaque by mechanical debridement and the use of antibiotics (systemic and
local). The elucidation of the specific bacterial etiology of periodontitis suggests the
development of specific treatment modality to target site colonization or virulence of Pg,
Tf, Td and Aa is now a more rational approach to treat the disease. This would include
development of a vaccine or multi-species vaccine that is able to target all 4 bacterial
species. Vaccination may also have a therapeutic benefit as although the bacteria may be
more resistant to the adaptive immune response (when present in subgingival plaque as
an undisturbed biofilm) , specific antibodies may still restrict the progression of disease
by blocking the penetration of the major virulence factors into the gingival tissues. Also,
if the antibodies were to be directed against key epitopes involved in function, it may
neutralize their action and also facilitate their removal through opsonisation and
phagocytosis. Specific antibodies may also neutralize key virulence factors associated
with acquisition of essential nutrients, thereby restricting proliferation within the plaque
biofilm.
Thus it is important to use a combined proteomic, genomic and immunological
strategy to identify bacterial antigens of periodontal pathogens and evaluate their
potential as vaccine candidates for the development of a multi-species vaccine for
periodontitis as an adjunct to current periodontal therapies.
 REFERENCES

 Immunoregulation in periodontal disease. Perio 2000, vol 35, 2004

 Textbook of microbiology – Anathnarayan & Paniker

 Contemporary oral microbiology & immunology – Slots

 Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in

periodontal disease. Perio 2000,m vol 20, 1999