1. Periodontal diseases are caused by complex biofilms containing multiple bacterial species that interact with host tissues. A key group is the "red complex" comprising Porphyromonas gingivalis, Treponema denticola, and Tanerella forsythia. 2. These bacteria employ various virulence factors to adhere to surfaces, acquire nutrients, and evade the host immune response in order to colonize and cause disease. Important factors include fimbriae, proteases, and capsules that aid adhesion and facilitate tissue destruction. 3. P. gingivalis possesses specific fimbriae, proteases, and a capsule that help it adhere, acquire iron through hemol

1. RED COMPLEX ORGANISMS
CONTENTS

2. INTRODUCTION:
Periodontal diseases are multifactorial infections elicited by a complex of
bacterial species that interact with host tissues and cells causing the release of a
broad array of inflammatory cytokines, chemokines, and mediators, some of which
lead to destruction of the periodontal structures.
The trigger for the initiation of disease is the presence of complex microbial
biofilms that colonize the sulcular regions between the tooth surface and the
gingival margin through specific adherence interactions and accumulation due to
architectural changes in the sulcus (i.e. attachment loss and pocket
formation).Smalley 2002
Nearly 700 bacterial taxa, phylotypes and species, which show some
structural organization in the biofilms (Partridge NC 1985), can colonize the oral
cavity of humans, although it remains unclear how this multitude of bacteria
compete, coexist or synergize to initiate this chronic disease process.
Socransky and Haffajee and their colleagues have described improved
methods for examining the association of oral microbial communities with the
change from health to disease. These investigators catalogued and stratified the
microbiota into groups or complexes, representing bacterial consortia that appear
to occur together and that are associated with the biofilms of gingival health,
gingivitis and periodontitis.

3. BIOFILM
Biofilms are composed of microbial cells encased within a matrix of
extracellular polymeric substances such as polysaccharides, proteins and nucleic
acids.
They are invaded initially by streptococcus, actinomyces, capnocytophaga
species. The red complex, which appears later in biofilm development, comprises
species that are considered periodontal pathogens, namely
 Porphyromonas gingivalis
 Treponema denticola
 Tanerella forsythia
presents as a portion of the climax community in the biofilms at sites
expressing progressing periodontitis.

4. Sigmund Socransky, a researcher at the Forsyth Dental Center in Boston,
proposed criteria by which periodontal microorganisms may be judged to be
potential pathogens. According to these criteria, a potential pathogen must:
1. Be associated with disease, as evident by increases in the number of
organisms at diseased sites
2. Be eliminated or decreased in sites that demonstrate clinical resolution
of disease with treatment
3. Demonstrate a host response, in the form of an alteration in the host
cellular or humoral immune response
4. Be capable of causing disease in experimental animal models
5. Demonstrate virulence factors responsible for enabling the
microorganism to cause destruction of the periodontal tissues
STRATEGIES EMPLOYED BY RED COMPLEX ORGANISMS
 Virulence potential
 Adherence
 Sustenance
 Evasion of host response
BACTERIAL VIRULENCE
The term virulence is generally defined as the relative ability of an organism
to cause disease or to interfere with a metabolic or physiological function of its
host. The word derives from the Latin, virulentus (full of poison). Thus,

5. virulence refers to the ability of a microbe to express pathogenicity (e.g.
virulent), which is contrasted with nonpathogenic or avirulent organisms.
The fact that virulence is a complex of host–parasite interactions,
characteristic of certain microbes and not others, has led to an effort to define
virulence in terms of its own attributes. The characteristic endproducts of
bacterial metabolism, the chemical composition of bacterial components, the
ability of the intact bacterium or its parts to overwhelm host defense
mechanisms, its invasiveness, and of course its ability to kill were all used to
characterize and distinguish a virulent microbe from an avirulent one.
As early as 1924, Kolmer attempted to define virulence as a characteristic
comprising at least two intrinsic microbial factors, toxicity and aggressiveness,
or the ability to invade a susceptible host. Kolmer’s definition of virulence,
toxicity and aggressiveness, depended upon the ability of the microbe to
elaborate specific molecules, or toxins.
TOXICITY (or virulence) included both the ability of the microbe to produce
a specific toxin and host damage from end-products of metabolism, the
production of components which elicited inflammatory reactions, and
components which were capable of interfering with host growth and
reproduction (Stewart)
This ability for a microbe to be AGGRESSIVE, or to invade, grow, and
divide within a susceptible host was also an important attribute of virulence.

6. However, the role of aggressiveness (e.g. invasion) in the definition of virulence
does not always fit the range of microbial pathogenic characteristics. For
example, end-products of the metabolism of Staphylococcus aureus and
Streptococcus pyogenes are capable of killing host cells (leukocidins) but do not
produce a toxin contributing to invasion. In contrast, Streptococcus pneumoniae
is highly aggressive but moderately toxic.
Poulin & Combs defined virulence in terms of virulence factors, that is,
components of a microbe which when present harm the host, but when absent
impair this ability.
VIRULENCE FACTORS
Porphyromonas gingivalis Treponema denticola Tanerella
forsythia
Capsule
Outer membrane proteins
Hemin
Lipopolysaccharide
Fimbriae
Proteinase
Aminopeptidase
Collagenase
Cysteine protease-
gingipains
Proteins
Proteinase
Hemin
Cystalysin
c-glutamyltransferase
H2S
Dentilisin
Lipopolysaccharide
Immuno suppressive
protein
Proteinase
Sulphur compounds
Lipoprotein
S layer
FUNCTIONS OF VIRULENCE FACTORS

7.  The ability to induce microbe–host interactions (attachment)
 The ability to invade the host
 The ability to grow in the confines of a host cell
 The ability to evade ⁄interfere with host defenses.
ADHESINS
The periodontal environment consists of two fluid systems-saliva and GCF,
both of which are capable of flushing out the bacteria. Bacterial adhesion
enhances the ability of organisms to stay in the gingival sulcus and multiply.
The presence of adhesion is a two fold affair :
 Host surface(tooth/gingival surface)
 Other microorganisms (early colonizers). The process of binding of
specific pairs of bacteria is called coaggregation
SUSTENINS
The red complex organisms are ready to express their pathogenecity after
adhesion and invasion. As large numbers of organisms multiply within the
tissues, the rate limiting step is the availability of nutrition to the multiplying
bacteria.
These nutritional demands are met by enzymes and molecular determinants
in the organism that have been collectively termed as ‘sustenins’.
EVASINS

8. Evasion of host immune response is an important part of the strategy to
amplify its pathogenic mechanism. The periodontium is endowed with innate
and acquired immune mechanisms to provide defense capabilities against the
invading microorganisms.
Innate Immunity is conferred by the epithelial barrier and the cells of the
innate immune mechanisms, namely dendritic cells, neutrophils, monocytes and
macrophages. Acquired Immunity is provided by T and B lymphocytes.
Periodontopathogens evade host responses by molecular markers
collectively termed as ‘evasins’.
PORPHYROMONAS GINGIVALIS
MORHOLOGY
Porphyromonas gingivalis belongs to the phylum Bacteroides and is a
nonmotile, Gram-negative, rod-shaped, anaerobic, pathogenic bacterium.
CULTURE

9. The subgingival plaque samples were inoculated into 2 ml of brucella
broth and they were then diluted and plated onto Trypticase soy agar
supplemented with 10% defibrinated horse blood , 5-μg/ml hemin, and 0.4-
μl/ml vitamin K1. The plates were incubated in duplicate in an anaerobic
atmosphere for 7 to 10 days or in air plus 10% CO2 for 2 to 4 days. The bacteria
grown were selected on the basis of size, color, shape, and staining.
The anaerobic bacteria were identified by biochemical tests and by gas
chromatographic analysis. Black-pigmented, anaerobic, gram-negative rods
were submitted to a fluorescence test by longwave UV light; absence of
fluorescence was considered a rapid taxonomic test to distinguish between P.
gingivalis and other black-pigmented, anaerobic, gram-negative rods.
(J Clin Microbiol. 1998 )
VIRULENCE FACTORS OF P. GINGIVALIS

10. ADHESINS
Fimbriae
They are long thin filamentous sructures that extend from the outer
membrane in peritrichous fashion.The major fimbriae consisting of fimbrillin
proteins are constructed from a fimbrillin monomer which is encoded by a
single fimA gene.The major P. gingivalis fimbriae participate in almost all
interactions between the bacterium and both host and other bacteria.
Proline-rich proteins, salivary glycoproteins, and statherins, can function
as specific receptors, enhancing this initial interaction. Gibbons and coworkers
reported that often the receptor epitopes involved in this initial attachment are
hidden when the macromoleules are in the salivary fluid phase. These hidden
attachment sites are known as cryptitopes and exposed when they adsorb to
host surfaces, enabling bacterial binding.
Besides the primary function of the fimbriae in the initial attachment to
host surfaces, it stimulates host cell signalling pathways that involve protein
phosphorylation, Ca++ ion fluxes, and rearrangement of actin and other
cytoskeletal structural .This type of activity can modulate the bacteria capable
of attaching and colonizing the oral cavity.
COAGGREGATION

11. P. gingivalis is capable of coaggregating with Actinomyces naeslundii,
Actinomyces viscosus, Streptococcus gordonii, and S. mitis. Each of these
coaggregation process requires activation of different surface molecule.
Glyceraldehyde-3-phosphate dehydrogenase of S. oralis is critical for
coaggregation with the fimbriae of P. gingivalis. Binding of P. gingivalis to A.
naeslundii has been associated with the presence of a 40 kDa outer membrane
protein of P. gingivalis, resulting in coaggregation of these species
Fusobacterium nucleatum is an important bridging microorganism in the
subgingival microbiota as it is capable of binding to both gram positive and
gram negative organisms. It appears that galactose can specifically inhibit the
binding process (Metzger Z). F. nucleatum possesses a galactose-binding
adhesin site that mediates its coaggregation activities with P. gingivalis, as well
as other oral species. F. nucleatum provides an anaerobic environment
facilitaing the survival of obligate anaerobic bacteria by these interbacterial
interactions.
Umemoto et alexamined coaggregation between Treponema medium and
P. gingivalis, suggesting that the gingipains of P. gingivalis were critical for this
binding. They found a 37 kDa surface protein of T. medium that bound to the
fimbriae of P. gingivalis.
Vesicles from P. gingivalis can aggregate numerous oral bacteria such as
Streptococcus spp., F. nucleatum and A. naeslundii. These vesicles also have

12. the ability to bridge non-aggregating species such as S. aureus with
Streptococcus spp. and certain types of C. albicans.
SUSTENINS
a)CAPSULE
The presence of a capsule in P.gingivalis has been considered an
important anti-phagocytic virulence factor.
Chemical composition
Mansheim & Kasper deter- mined that the capsule of P. gingivalis
contained galactose, glucose and glucosamine, whereas Okuda et al.
determined that the sugar composition of a similar strain was composed of
rhamnose, glucose, galactose, mannose and methylpentose. Schifferle et al
found that it did not contain galactose and was rich in amino sugars. The
capsule of the strain contained galactosamine, glucosamine, galactosaminuronic
acid
Biological function.
. The highly encapsulated P gingivalis strains exhibit decreased
autoagglutination, and were more hydrophilic than the less encapsulated strains
Increased encapsulation was also correlated with increased resistance to
phagocytosis, serum resistance, and decreased induction of polymorphonuclear
leukocyte. The decreased tendency for the highly encapsulated strains to be

13. phagocytized has been pro posed to be due to the increased hydrophilicity of the
strains and their decreased ability to activate the alternative complement
pathway.
Schifferle et aldemonstrated that the P. gingivalis lipo- polysaccharide
alone was responsible for activating the alternative complement pathway and
not the capsule. These authors hypothesized that the thick capsule functioned to
physically mask the lipopolysaccharide, and therefore the complement cascade
could not be activated. The invading bacteria were therefore protected from
opsonization and phagocytosis.

14. b) PROTEINASE
The proteinases of P. gingivalis are considered to be important virulence
factors in the progression of periodontal disease. The proteinases from P.
gingivalis have been shown to degrade types I and IV collagen [major
components of periodontal connective tissue and extracellular matrix proteins
[e.g. fibrinogen, laminin ].
The important proteases of P.gingivalis are the cysteine-proteases that
havebeen given the name gingipains. Gingipains are classified into arginine and
lysine gingipains that are generated by the rgpA and rgpB genes. They cleave
the proteins at the arginine and lysine residues.
They increase gingival vascular permeability in periodontitis sites
resulting in increased gingival fluid flow.They inhibit the generation of oxygen
radicals from activated leukocytes (Yilmaz et al)
Gingipains have the ability to beakdown collagen I, III, V and other
matrix molecules like fibronectin, gelatine and laminin.Theyactivate -
kallikerin/kinin system and coagulation cascade.
COLLAGENASE
The clinical hallmark of periodontal disease is the destruction of the peri-
odontal connective tissue during inflammation. Since collagen (type I) is
resistant to destruction by the majority of the mammalian and bacterially
synthesized proteolytic enzymes, the periodontal tissue destruction observed

15. during the temporal events of periodontal disease must be mediated by specific
proteolytic enzymes, especially the collagenases.
Both humans and P gingivalis produce collagenase. P gingivulis
collagenase may participate with host-derived collagenase in the destruction of
gingival collagen.(Mayrand and Grenier et al 1985)
AMINOPEPTIDASE
P.gingivalis is the only member of this periodontopathic microbiota that
exhibits strong dipeptidyl arylaminopeptidase activity. Abiko et al observed that
when it is exposed to type 1 collagen, it cleaves a glycylpropyl dipeptide from
the collagen protein.
Nakamura et al revealed that it contained at least two additional
aminopeptidases, N-CBz-glycyl-argi- nyl peptidase (N-CBz-Gly-Gly-Arg),
which was both cell associated and extracellular, and an extracellular peptidase,
glycyl-prolyl peptidase (Gly-Pro peptidase).
IRON UTILIZATION
The majority of iron is contained in intracellular complexes with
hemoglobin, myoglobin, catalase, and cytochrome c, or stored in ferritin and
hemosiderin
Outer membrane vesicles.

16. Hemolysin
Hemolyse RBC
Hemin
(growth requirement)
END PRODUCTS OF METABOLISM
P. gingivalis preferentially utilizes peptides rather than free amino acids
as a source of energy and cell material. P. gingivalis anaerobically degrades
aspartylaspartate to butyrate, succinate, acetate or glutamylglutamate to
butyrate, propionate, acetate, generally leading to release of NH4 that
neutralizes acids formed by fermentation of amino acids.
P. gingivalis has the physiologic capacity to produce various volatile
sulfur compounds during its metabolism, including H2S and CH3SH. While
these molecules have been directly linked to halitosis, they can also be toxic to
host cells, and are produced at levels that could degrade disulfide bonds
necessary to the functioning of numerous host defense molecules.
EVASINS
Effect on host innate and immune mechanism

17. P.gingivalis alters the secretion and accumulation of selected chemokines
and interferes with peripheral blood mononuclear cells’ secretion of monocyte
chemoattractant protein (MCP)-1 and interleukin-8. The low chemokine levels
might interfere with the ability of the host to recognize these invading bacterial
species and direct polymorphonuclear leukocytes to remove them which result
in the growth of this resident microbiota.
P. gingivalis gingipains are also able to truncate interleukin-8, an
important polymorphonuclear leukocyte chemoattractant. The outer membrane
vesicles shed into the environment of the host are also capable of degrading
interleukin-8, providing significant protection from host defenses. The
gingipains are able to induce phagocytic events, while controlling these events
such that phagocytosis does not occur. Destruction of the neutrophil before it
can act provides the invading bacterium (P. gingivalis and other invading
periodontopathic species) with important proteolytic nutrients for metabolism
and growth. Hence the proteinase(s) of P. gingivalis are considered to be
important virulence factors in the progression of periodontal disease.
The activation of the complement system in response to an impending
infection or invasion by a foreign object (i.e. lipopolysaccharide, protease) is
probably the most effective first line defense activated by a human host.
The gingipains have developed mechanisms for the use of the complement
system for their own benefit. Initially, they attack and degrade C3, the normal
activation of which results in the formation of C3a and C3b (Wingrove JA1992)

18. Both of these molecules function in the initial elimination of an invading
bacterial species from the host. C3b is initially required for particle
opsonization, as well as to form the C5 convertase complex, essential for the
synthesis of C5a, a potent chemotactic factor. Degradation of both C3a and C3b
therefore provides the invading bacterium with a distinct survival advantage.
Effect on blood factors, coagulation and clotting
Imamura et al has reported that the P. gingivalis gingipains are capable of
rapidly activating both factor X and prothrombin. The activation of the blood
coagulation pathway occurs through the proteolytic cascade pathway. While
thrombin plays a major role in hemostasis, it also functions in the enhancement
of vascular permeability and induce leukocyte chemotaxis.
It also activates the osteoblasts to secrete prostaglandin (Partridge 1985).
There is also stimulation of an interleukin-1 activity by macrophages during
activation of the blood clotting system (Jones 1990). Stimulation of both
thrombin and interleukin-1 in the host gingival crevice fluid by
lipopolysaccharide results in an associated bone and tissue destruction.
P.gingivalis activates kallikrein/kinin system. The Arg-gingipains possess
significant vascular permeability actions, inducing this activity via plasma
prekallikrein activation followed by bradykinin release. Ransjo et al have
suggested that bradykinin might be involved in alveolar bone loss by activating
the prostaglandin pathway in periodontal ligament cells as well as in osteoblasts

19. Gingipains R and K therefore are important factors in vascular
permeability, and might contribute to gingival crevice fluid production and
edema formation at periodontitis sites infected with P. gingivalis. Thus,
gingipains might function to provide a ready source of low molecular weight
peptides for the growth and reproduction (and virulence) of P. gingivalis and
other members of the periodontopathic microbiota (i.e. T. denticola and T.
forsythia).
Outer membrane vesicles
P.gingivalis form small, spherical structures on the surface of their outer
membrane during growth. These structures, which are released from the outer
membrane proper during growth, are referred to as outer membrane vesicles.
Trapped within these closed sacs are numerous enzymes that occur in the
periplasmic region of the intact cell. These include phospholipase C,
proesterases, alkaline phosphatase, hemolysins, and autolysins(Saito 1994).
These vesicles are able to fuse with the outer membrane of other bacterial
species, into which virulence factors are released, resulting in an impairment of
target cells (Kadowaki 2000). The outer membrane vesicles also contain
lipopolysaccharide, DNA, RNA, and porins. As such, the outer membrane
vesicles may play an antagonistic role in evading the host immune system.
TREPONEMA DENTICOLA
These obligatory anaerobic bacteria have been recognized since van
Leeuwenhoek first observed them almost 350 years ago. A large body of

20. experimental evidence supports the importance of the oral treponemes,
including T. denticola, in the progression of periodontal diseases. During
periods of oral health the number and distribution of these types of bacteria are
low or nearly undetectable. However, during gingivitis and the progression to
periodontitis there is a large increase in the number.
MORPHOLOGY OF TREPONEMA DENTICOLA
 Dark field microscope Spirochetes present a long and slightly helically
coiled (spiral, helical or serpentine-shaped) - this particular morphology
causes a twisting motion which allows to penetrate into dense media
 In particular, family of Spirochaetes are distinguished from other
bacterial phyla by the location of their flagella, axial filaments, which
run lengthwise between the cell wall (peptidoglycan layer) and outer
membrane.
CULTURE OF TREPONEMA DENTICOLA
T. denticola was isolated from human periodontal pockets. All strains
were stored at −78 °C in 15% glycerol medium. For normal cultivation, the
spirochetes were grown in medium which contains 2% heat-inactivated rabbit
serum and 6 mMl-cysteine, in screw-cap tubes. The bacteria were incubated

21. either anaerobically in a Coy anaerobic chamber (5% CO2, 10% H2, and 85%
N2) or aerobically in a 37°C warm room for the times appropriate for the
experimental design. The culture purity was checked by dark-field microscopy
at a magnification of ×400.
ADHESINS
Bacteria-host interactions
T. denticola has been shown to bind to a variety of oral surfaces,
including the tooth surface, to extracellular matrix proteins, including laminin,
fibronectin, and heparin and host cells, such as human gingival fibroblasts.
Binding of T. denticola to fibronectin, a component of the extracellular matrix
of host cells, was shown to occur via the bacterial pole. This polar adherence
likely plays an important role in the localization of many bacteria along the
matrix border, in close proximity to membrane proteins and other molecules.
Li et al have shown that the collagen binding proteins of T. denticola
bind type I, IV, and V collagens, which suggested that they play a role in
adherence and colonization by this microorganism.
Bacterial coaggregation
The protein, LrrA binds human epithelial cells and to T. forsythia,
another member of the red complex. Coaggregation between P. gingivalis and
T. denticola was also mediated by the T. denticola fimbriae-binding protein,
dentilisin.(Hashimoto et al).

22. Since P. Gingivalis is more common in the later stages of periodontitis
along with T. denticola, it is possible that the coaggregation observed functions
to transport P. gingivalis into the deeper regions of the developing periodontal
pocket. These locations might be more conducive to P. gingivalis growth (being
more anaerobic), as well as providing a richer nutritional environment (from
gingival crevice exudates).
PROTEINS
T. denticola produces an array of major outer sheath proteins, several of
which might be important to the interaction of the bacterium with its host.
When a host environment is chronically exposed to a bacterial protein, it
may perturb host physiological processes engaged in collagen synthesis
functioning to maintain connective tissue homeostasis or to heal infected
wounds. (Batista da Silva et al).
In addition to the major outer sheath protein of T. denticola, several other
small polypeptides show the capacity to adhere to fibronectin. One of these
outer membrane proteins, a 70 kDa surface protein, binds both plasminogen and
fibronectin. Thus, the adherence of T. denticola to host cells via major sheath
proteins and other cell surface molecules, as well as the ability of these bacterial
ligands to bind to an array of host cellular receptors and ⁄ or surface structures is
important to host cell interactions and colonization by this pathogen.
PROTEINASE

23. T. denticola possesses several peptidases associated with its outer sheath.
One of these, a prolyl-phenylalanine specific protease appears to be important in
T. denticola virulence. Uitto et al called this protease a chymotrypsin-like
protease with a molecular mass of approximately 100 kDa. When denatured, the
100 kDa native protein was separated into three peptides, with molecular
masses of 72, 43, and 38 kDa .
The 72 kDa protein appears to be similar to a protease domain and is
homologous to the active site of Bacillus subtilus subtilisin. Ishihara et al have
named this protease dentilisin, which is characterized as a 72 kDa serine
protease. Its enzyme quality makes it potentially very important to the
interaction of T. denticola with host cells.
IRON ACQUISITION
T. denticola is capable of interacting with erythrocytes, resulting in
cell lysis and competing with the host for available hemin-derived iron.
Cystalysin, a 46 kDa protein produce hemolysis by 2 mechanisms.Krupka et al
Hemolysis occurs by degradation of cysteine to H2S, which is secreted
from the bacterium, and crosses the red blood cell plasma membrane where it
results in the destabilization of the red blood cell membrane.
Secondly, cystalysin in the presence of cysteine results in the formation
of sulfane–sulphur derivatives. These strong oxidants would interact with the
red blood cell membrane, causing cell damage and death as the result of a
depolarization of the red blood cell plasma membrane and hemoxidation

24. of the iron atom.
T. denticola also synthesizes two low-iron induced outer membrane
proteins, HbpA and HbpB, that bind hemin. These proteins appear to be
necessary for efficient iron utilization.
CHEMOTAXINS
T. denticola is a rapidly motile bacterium that expresses chemotaxis,
sensing and responding to selected chemical gradients to gather
and utilize nutrients. Otteman & Miller have reported that both motility and
chemotaxis play a role in bacterial–host cell interactions. Greene & Stamm
characterized three chemotaxis (che) genes, which they identified as cheA,
cheW, and cheY. Lux et al have reported that these genes are also important for
the penetration of the bacterium into periodontal tissue where they then migrate,
presumably to a host environment more favorable for their growth and
reproduction.
END PODUCTS OF METABOLISM
Elevated levels of T. denticola were identified along with other species in
sulfide-positive compared with sulfide-negative sites. The results suggested that
the sulfide levels in the pockets reflected the proportion of bacteria, whose
metabolism resulted in the production of sulfide as an end-product.
Cystalysin also functions as a crucial physiologic enzyme for growth of T.
denticola.(Chu et al). It metabolizes host sulphur containing molecules (e.g.
cysteine, glutathione) with the release of pyruvate, NH3, and H2S.

25. The pyruvate is used as a energy source for growth. The NH3 would be
predicted to contribute to the generally alkaline nature of the progressing
periodontal lesion, and the H2S can act on both red blood cells, being toxic for
other host cells, and can hydrolyze disulfide bonds of host proteins, i.e.
immunoglobulins, cytokines, chemokines, leading to altered host responses.
Glutathione is a peptide comprising glutamine, glycine, and cysteine.
T. denticola has been shown to produce a c-glutamyltransferase that initially
removes the glutamine from the peptide, resulting in the release of a cysteinyl-
glycine dipeptide. A unique cysteinylglycinase is produced that specifically
attacks the peptide bond, releasing free cysteine, which is subsequently
degraded by cystalysin.
EVASINS
Chemotrypsin like protease
The 72 kDa subtilisin like lipoprotein complexes with two accessory
proteins (PrcA1 and PrcA2) in the outer sheath of T. Denticola forms the
chymotrypsin-like protease complex
It degrades humoral proteins, e.g. basement membrane components (type
IV collagen, laminin, and fibronectin (Grenier D, Uitto VJ 1990) and serum
proteins such as transferrin, fibrinogen, IgG, IgA, and a1-antitrypsin, as well as
bioactive peptides.
Being an enzyme like subtilisin, the dentilisin can function in the
destruction (hydrolysis) of substance P and angiotensin 1. It activates selected

26. matrix metalloproteinases, which in the polypeptide state - modulate host
chemokine and cytokine activity.
DENTILISIN
The enzyme dentilisin has the ability to move T. denticola across
basement membranes thereby increasing the permeability of these membranes.
Sorsaet al demonstrated that the dentilisin is capable of acting to direct
proteolytic activation of human procollagenases, and therefore playing a
potentially important role in host enzyme tissue destruction. Since epithelial
cells are the initial barrier that the periodontal microbiota encounters at the
gingival margin, the ability of this member of thered complex as well as other
bacterial species to disrupt this barrier and penetrate into deeper tissue supports
T. denticola as an important member of the periodontopathic microbiota.
Effect on host innate and immune mechanism
When human gingival fibroblasts were exposed to T. denticola the
fibroblasts underwent significant cytoskeletal rearrangement,characterized by
cell rounding, the formation of surface blebs, and detachment from the cell
surface.
T. denticola lipopolysaccharide have been shown to possess considerable
chemokine and cytokine activity. The results demonstrated that the
lipooligosaccharide stimulates osteoclastogenesis by up-regulating osteoclast
differentiation factor and down-regulating osteoprotegerin, and selective matrix

27. metalloprotease up-regulation, supporting a contribution of this microorganism
to local tissue destructive processes.
T. denticola produces an immunosuppressive protein (Sip) that decreases
human lymphocyte proliferation by inducing an arrest at the G1 phase in human
T-cells.(Lee et al)
T. denticola and murine infection models
One of the most useful ways to determine the function of a putative virulence
factor is to mutate ⁄ delete the factor from the bacterium and subsequently
evaluate its growth, colonization, and pathogenetic capabilities. Ishiharaet al
injected dentilisin-defective mutant (T. denticola, K1) into mice resulted in the
inability of the microorganism to produce a subcutaneous abscess, characteristic
of its wild-type parent. Dentilisin, then, can be considered a potential virulence
factor of T. denticola.
Kesavalu et al showed that a polymicrobic T. denticola–P. gingivalis
infection of mice was significantly more pathogenic than infection with either
microorganism alone. More specifically, the addition of T. denticola to the
bacterial challenge mixture enhanced the tissue-destructive properties of
P. gingivalis and significantly decreased the dose of P. gingivalis necessary for
lethal infections.
TANNERELLA FORSYTHIA
The third member of the red complex of Socransky et al is T. forsythia.
The original isolate, identified as a fusiform Bacteroides was first reported in

28. the literature by Tanner et al. B. forsythus was reclassified by Sakamoto et al on
the basis of 16S rDNA analysis. This latter procedure did not reveal B.
forsythus to be a species within the genus Bacteroides which resulted in the
proposal to adopt a new genus, Tannerella, with one species, T. forsythia (T.
forsythensis).
MORPHOLOGY
 Gram –negative obligate anaerobe
 Non-motile
 spindle-shaped
 highly pleomorphic rods
CULTURE OF TANNERELLA FORSYTHIA
Samples were diluted 100 μl of each dilution was plated in media
containing blood agar with N-acetyl muramic acid (NAM) disks (10 mg/l). The
blood agar plates were studied after 5 to 7 days of anaerobic incubation (80%
N2; 10% H2; 10% CO2 at 37°C). Plates were examined for the identification of
T. forsythia based on the morphology of the colony which are tiny and opaque,
pale pink and speckled circular, slightly convex with depressed centre (donut
shape) and by using grams reaction and different standard biochemical tests
(Indole, Catalase and Nitrate reductase test) to confirm the initial
identification.[J Indian SocPeriodontol. 2014]
VIRULENCE FACTORS OF TANNEREELA FORSYTHIA
ADHESINS

29. P. gingivalis and T. forsythia coaggregate with F. nucleatum, suggesting a
process for enhancing colonization in the subgingival biofilm. The lrrA gene of
T. denticola codes for the LrrA protein that binds to a portion of the T. forsythia
leucine-rich repeat protein, BspA, through an N-terminal region, supporting an
additional strategy for colonization and providing some molecular mechanism
for the consortium relationship of these species.
Proteinase
T. forsythia produces an enzymatic peptidase activity that degrades
benzoyl-DL arginine-naphthylamide (BANA), the activity of which appears
related to sites of periodontal tissue destruction. The sialidases are enzymes that
cleave a-ketosidic linkages between sialic acid and the glycosyl residues of host
glycoproteins, glycolipids or colominic acids.
T. forsythia has been reported to express a number of enzymatic and
proteolytic activities that could contribute to its ability to compete effectively in
the complex biofilm of the subgingival sulcus (EbersoleJL, Novak KF)
End products of metabolism
T. forsythia, along with nearly a dozen other species, was elevated in
sulfide-positive versus sulfide-negative sites. The results suggested that the
sulfide levels in the pockets reflected the proportion of bacteria, whose
metabolism resulted in the production of sulfide as an end-product.
EVASINS

30. T. forsythia produces lipoproteins which activate gingival fibroblasts to
produce elevated levels of interleukin-6 and tumor necrosis factor-a. It also
induces nuclear factor kappaB production by fibroblasts.
Arakawa et al examined the effects of cell extracts from T. forsythia to
induce cytolytic activity against HL-60 and other human leukemic cells. The
cytolytic activities, loss of both mitochondrial membrane potential and
membrane integrity, damage to the cell cytoplasmic membrane, DNA ladder
formation, and the activation of caspase-3 were used as apoptotic inducing
indicators.
T. forsythia appears to invade the periodontal pocket along with P. gingivalis
(and T. denticola) and these species could be attacked by the host’s white blood
cells. The apoptotic-inducing activity could result in the elimination of host
immune or preimmune cells; loss of these host immune cells from the
developing periodontal pocket would support bacterial colonization of the
pocket, and the potential rapid progression of the disease.
S LAYER
In the surface of cell envelope of T.forsythia, there is an additional layer
which possess a crystalline protein or glycoprotein -protective covering against
external host or natural environmental forces. It also acts as a molecular sieve
and ion trap, as well as providing an adhesion and surface recognition
mechanism. (Kerosuo et al)
TANNERELLA FORSYTHIA AND THE MURINE ABSCESS MODEL

31. Takemoto et al found that coinfection with P. gingivalis and T. forsythia
induced both large lesions and sometimes death, while monoinfection with
either of the bacterial species induced no lesions. The synergism observed
between P. gingivalis and T. forsythia might be due to the presence of the
significant proteolytic activity of P.gingivalis.
Sabet et al also employed a murine abscess model in their studies of the
T. forsythia S-layer. Mice immunized with either isolated and purified S-layer
or whole cells did not develop any abscesses when challenged with viable T.
forsythia.
ANTIBIOTIC SENSITIVITY
 They are generally sensitive to antibiotics that are active against
anaerobes.
 Most active antibiotics were amoxicillin with clavulanate (100%),
ampicillin (98%), doxycycline (98%), amoxocillin (96%),tetracycline
(90%), and clindamycin (86%).
 Fairly active antibiotics were penicillin (70%) and spiramycin (68%)
 Poorly active antibiotics were erythromycin (54%) and ciprofloxacin
(46%).
 In addition, they are also found to be sensitive to metronidazole
MOLECULAR METHODS TO ANALYZE PERIODONTAL MICROFLORA
 DNA probes
 Whole genomic probe

32.  Checker board DNA
 Randomly cloned probes
 Oligonucleotide probes
 PCR
 Static 96 well microtiter plate assay
 Static 12 well microtiter plate assay
 Flow cell system
CONCLUSION
Microbes have evolved and developed a large repertoire of strategies to
enable a continual interaction with their hosts. Many of the features of these
microbes that enable them to make a transition from a commensal symbiont to
an opportunistic pathoge are quite different from classical virulence
determinants.

33. The molecular details of microbial interactions and ⁄ or synergism in
polymicrobial infections remain poorly understood. Thus, research in molecular
signaling, communication, and virulence in oral biofilms has an opportunity to
forge new ground in the knowledge base of polymicrobial infections, not
dissimilar from contributions made two to three decades earlier in the areas of
specific bacterial adhesins and biology of the mucosal immune system .
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