Role P.gingivalis in Periodontitis

2.  Introduction
 Taxonomy
 Morphology & Growth Characteristics
 Biochemical properties
 Invasion of the host by P.gingivalis
 Survival strategies of P.gingivalis
 Subgingival lifestyle of P. Gingivalis
 Exit of P. Gingivalis from periodontal cells
 Polymicrobial Synergy and Dysbiosis (PSD) model
 Oral Ecology & Transmission
 Subgingival distribution of P. gingivalis

3.  Periodontitis is a multifactorial disease due to
complex interaction between the host and plaque
bacteria.
 The main anaerobic and gram negative bacteria
implicated as etiological agent for periodontal
disease include P. gingivalis, B.
forsythus,P.intermedia,A.actinomycetmcomitans,T.
denticola,spirocetes,F.nucleatum,C.rectus and E.
corrodens – putative periodontal pathogens
(Loesche.W 1992)

4.  P. Gingivalis produces by far the greatest
proteolytic activity of any periodontal bacterium.
The hallmarks of periodontitis including bleeding
on probing, neutrophil accumulation, attachment
loss and increased crevicular flow all involve
proteolytic events (Travis J 1997)

5.  According to the model of the dental biofilm
complexes, P. gingivalis belongs to the Red
Complex; hence, it is part of the tertiary colonizers
that colonize dental and periodontal tissue when
the biofilm is mature.
(Socransky & Hafajee;2002)
 Therefore, P. gingivalis is also known as Late
Colonizer, and hence is found in close proximity to
and interacts with the juxta- posing gingival tissue.
(Kolenbrander et al., 2011; Zijnge et al., 2011)

6. Family Bacteroidaceae
(Bergey’s manual 1923)
3 Genera
Bacteroides Fusobacterium Leptotrichia
1989
PORPHYROMONAS Prevotella

7. Bacterium melaninogenicum- 1921
Bacterium melaninogenicus- 1939
1970 Holdeman & Moore
Melaninogenicus Intermedius Asaccharolyticus
1982
B.melaninogenicus B. loescheii B. denticola
Prevotella
Melaninogenicus
1988, 1990
Prevotella
Loescheii
1988, 1990
Prevotella
Denticola
1988, 1990

8. Bacterium melaninogenicum- 1921
Bacterium melaninogenicus- 1939
1970 Holdeman & Moore
Melaninogenicus Intermedius Asaccharolyticus
B. intermedius1983
Prevotella
Intermedia
1988, 1990

9. Bacterium melaninogenicum- 1921
Bacterium melaninogenicus- 1939
1970 Holdeman & Moore
Melaninogenicus Intermedius Asaccharolyticus
B. asaccharolyticus-1977
1980
B.Gingivalis
(oral)
B. asaccharolyticus
(non-oral)
Porphyromonas
gingivalis 1988
Porphyromonas
asaccharolyticus
1988
Shah
&Hardie1979 &
Coykendall et
al 1980

11.  Black-pigmented, anaerobic, assaccharolytic, non-
motile Gram-negative species of 0.5 X 1-2 µm.
 As it is assaccharolytic, it does not depend on
carbohydrates as an energy source.
 It gains its metabolic energy by fermenting amino
acids, a property necessary for its survival in deep
periodontal pockets, where sugars are extremely
scarce.

12.  The black pigmentation of P. gingivalis colo- nies
observed in blood agar culture is itself associated
with the aggregation of heme on its cell surface
(Liu et al., 2004; Smalley et al., 2006)
 But when grown in a heme-
limited medium it becomes less
virulent. (McKee et al. 1986).

13.  Low Oxygen tension
 Abundant nitrogenous substrates
 Subgingival ecosystem Ideal environment
 Low Redox potential
 Protoheme and Menaquinones major
electron carriers present within the
periodontal pocket

14.  For survival into the host, P. gingivalis is able to
invade cells and tissues (Yilmaz, 2008), thus
avoiding the immune surveillance.
 It can actively invade gingival epithelial cells, where
it can maintain viability and replicate. (Belton et al.,
1999; Tribble et al., 2006)
 This invasion is dependent on its major fimbriae,
which bind to ß1 integrin on the surface of host cells
which causes rearrangements of the actin
cytoskeleton to allow internalization.(Yilmaz et al.,
2002, 2003)

15.  It can also invade macrophages, but within these
cells its replication is less active.
(Wang et al., 2007)
 This strategy is maintained by P. gingivalis for
limited exposure to the extracellular environment
and evasion of the immune surveillance.

16.  Once P. gingivalis has invaded intracellularly,
there are no signs of apoptosis or necrosis
(Nakhjiri et al., 2001).
 It can then actively secrete an ATP-hydrolysing
enzyme, thus suppressing ATP- dependent
apoptosis (Yilmaz et al., 2008) and allowing its
survival in host cells.
 Also it can travel from cell to cell, through actin
cytoskeleton bridges without causing cell death,
and spread while avoiding immune surveillance
(Yilmaz et al., 2006)

17.  P. gingivalis is not an aggressor of the
inflammatory response, but rather an opportunist
that can cross-talk with the host and subvert its
defence mechanisms.
 Thus P. gingivalis prolongs its survival and
becomes established in the periodontal pocket.
(Hajishengallis et al., 2011)

18.  It preferentially deregulates innate immunity, which
may in turn disable adaptive immunity.
For examples –
a) its capacity to degrade human defensins.
(Carlisle et al., 2009)
b) its resistance to oxidative burst-killing by
polymorphonuclear neutrophils (PMNs).
(Mydel et al., 2006).
c) its ability to inhibit ‘at will’ the production of
crucial proinflammatory cytokines.
(Bostanci et al., 2007a, b)

19.  Though P. gingivalis has the capacity to stim- ulate
interleukin (IL)-8 [ IL-8 is a potent chemoattractant and
activator of polymorphonuclear leukocytes] production
by epithelial cells (Kusumoto et al., 2004), it can also
inhibit IL-8 production, resulting in hindered PMN
chemotaxis, a phenomenon known as ‘Chemokine
Paralysis’ (Darveau et al., 1998).
 Also by inhibiting IL-12 production by macrophages, it
prevents cytotoxic T-cell activation which inhibits
macrophage bacteriocidal activity and therefore
prevents bacterial clearance
(Hajishengallis et al., 2007).

20.  P.gingivalis can suppress the complement system
activation by degradation of C3 and capturing of
C4b-binging protein, but also by synergizing with
C5a via exploiting toll-like receptor (TLR)-2
signalling.
(Wang et al., 2010)

22.  Within this region, there are three distinct
microenvironments for P.gingivalis in the
subgingival crevice:
a) the complex sessile community on the root
surface
b) the fluid phase of the gingival crevicular fluid
(GCF)
c) in and on the gingival epithelial cells that line the
crevice.

24.  While oral epithelial cells can harbor several
species of oral bacteria simultaneously it is within
the close confines of the multispecies biofilm on
tooth surfaces that interbacterial communication
becomes most relevant.
 As a strict anaerobe, P. gingivalis relies on
antecedent colonizers such as streptococci and
Fusobacterium nucleatum to reduce the oxygen
tension and also provide metabolic support .
Coadhesion among these organisms facilitates
nutritional and signaling interactions.

26. Following cellular entry, P. gingivalis organisms are initially localized within endocytic
vacuoles (early endosomes). Thereafter, some are routed to late endosomes, then
subsequently sorted to lysosomes for degradation. Other bacteria likely promote their
own entry into the autophagic pathway by bacterial escape from endosomes and
sorted to autolysosomes formed by the fusion of autophagosomes with lysosomes for
degradation. A large number of intracellular P. gingivalis organisms escape from these
lytic compartments via endocytosis and autophagy pathways by hijacking a fast
recycling pathway to exit from primarily infected host cells into intercellular space and
then enter new host cells, thus enabling further penetration of host tissues in a trans-
cellular manner.
Exit of P.
Gingivalis
from
periodontal
cells

27.  Bacteria that influence the pathogenicity of the
entire community are keystone pathogens and
best- documented example of which is P.
gingivalis.
 According to this model, physiologically compatible
organisms assemble into heterotypic communities
which exist in a controlled immunoinflammatory
state.

28.  The microbial constituents of the communities can
vary among individuals, among sites, and over
time.
 Colonization by keystone pathogens such as P.
gingivalis elevates the virulence of the entire
community following interactive communication
with accessory pathogens.
 Initially, host immune surveillance is impaired and
the dysbiotic community increases in number.
Subsequently, the community proactively induces
inflammation to sustain itself with derived nutrients,
which will also shape a modified ‘inflammophilic’
community

29.  The action of pathobionts in the community, in
addition to other pathogens, eventually leads to
destruction of periodontal tissues.
 The PSD model reconciles a number of features
of periodontal disease that were discordant with
earlier concepts of pathogenicity. These include:
a) the variable microbiota at disease sites, even
within the same patient
b) the presence of pathogens in the absence of
disease

30. c) the episodic nature of the disease
d) the failure of P. gingivalis to cause periodontitis in
the absence of the commensal microbiota.
 The concept of keystone pathogens in a PSD
model of periodontal disease have a profound
impact on the development of therapeutic options
for periodontal disease.

31.  Targeting P. gingivalis directly is no longer strategy
of choice,as it is difficult to completely eliminate the
organism and P. gingivalis is effective keystone
pathogen at low levels of abundance.
 The ability of P. gingivalis to survive inside
epithelial cells also hinders elimination as
intracellular P. gingivalis are protected from
antibiotics and can serve as a source for
recurrence of infection.

32.  Elevating numbers of organisms that normally
constrain P. gingivalis and reducing those that are
synergistic with P. gingivalis would foster
commensalism and prevent the transition to a
pathogenic community.
 By antagonizing complement pathways in the
gingival tissues could lock the host in a mode that
is non-responsive to the subversive activities of P.
gingivalis, and potentially to other keystone
pathogens.

33.  P. gingivalis is usually found in –
- Periodontal pockets (highest frequency)
(Asikainen et al 1997)
- Dorsum of the tongue (van Winkelhoff et al 1986)
- Saliva (van Winkelhoff et al 1986)
- Pharynx (van steenberg et al 1993)
- Supragingival plaque and oral mucosal surfaces
(Muller et al 1993)

34.  Vertical transmission:
Tuite-McDonnell et al 1997 – Intrafamilial
transmission
 Horizontal transmission:
Siblings-
Petit et al 1993 Identical genotype
Saarela et al 1996
Spouses –
Asikainen et al 1996:- 30-75%

35. a) Distribution pattern within individuals
 Subgingivally P.gingivalis is more widely
distributed.
 The noted distribution patterns may be related to
the efficacy of antibody response especially IgG
response against P.gingivalis whereas elevated
antibody titers to P.gingivalis are not capable of
controlling it which may lead to generalized
periodontal destruction. (Lamster I; 1998)

36.  P.gingivalis, being infrequent in children, is
probably acquired later, or, in order to grow, it
requires a specific environment that is not
generally available in young individuals.
 However, if the environmental requirements are
met, the host response is not able to limit
P.gingivalis infection to a few sites, but the infection
spreads in the dentition leading to generalized
periodontal destruction. This generalized form of
periodontitis predominates in older age groups.

37. b) Association with signs of periodontal disease
 P.gingivalis from subgingival samples is related to
periodontal inflammation, increased probing depth,
poor oral hygiene and attachment loss. Thus,
compared with A. actinomycetemcomitans, the
association of P.gingivalis with periodontal disease
parameters is more rectilinear.
(Ashimoto A; 1996, Beck JD;1992,
Dahlen G; 1992, Gmur R;1989)

38. c) Proportions of flora, balance-imbalance
 In subgingival samples, the proportion of
P.gingivalis reflects the relationship among the
pathogen, the accompaning microbiota and the
host.
 At periodontally healthy sites P.gingivalis is
present, in very low proportions. This suggests that
the growth of these species is under control in
health and that there is a balance among the
microbiota, and a homeostasis in the microbial
ecosystem.

39.  Conversely, high proportions of P.gingivalis can be
interpreted as uncontrolled growth and an imbalance
among microbiota due to breakdown of the
homeostasis in the ecosystem.
 In the study of Torkko & Asikainen1996, The difference
between A. actinomycetemcomitans and P.gingivalis
may indicate that, in most infected adults, P.gingivalis
multiplies subgingivally more effectively than does A.
actinomycetemcomitans. Thereby, P.gingivalis reaches
exceptionally high levels at the expense of other
competing organisms.
 This agrees with the suggestion that the host re-
sponse cannot control P.gingivalis infection.

40.  Route of infection from person to person Saliva,
mucosal contact and inanimate objects

44.  Non-oxidative microbial killing relies on the
contents of three types of cytoplasmic granules,
namely: azurophilic (primary) granules, specific
(secondary) granules, and gelatinase granules.

45.  Neutrophil activation triggers granule fusion with
phagosomes.
 These granules deliver antimicrobial proteins and
peptides, such as azurocidin, cathelicidin, α-
defensins, lysozyme, lactoferrin, elastase, and
cathepsin G, that disrupt bacterial cell envelope,
destroy peptydoglican, degrade proteolytic
bacterial virulence factors, or sequester iron.
(Soehnlein, 2009)

46.  Beside this antimicrobial arsenal, PMNs can
additionally form Neutrophil Extracellular Traps
(NETs), which are composed of decondensed
nuclear or mitochondrial DNA associated with
antibacterial (granule) enzymes, peptides, and
histones.
 These extracellular structures are designed to
disable invading pathogens and elicit pro-
inflammatory responses.
(White P. C. et al., 2016)

47.  PMNs have the shortest lifespan of all immune
cells, i.e., around 24 hours.
 Normally, neutrophils circulate in the blood for 6–
12h and then home to the bone marrow, spleen or
liver where they undergo apoptosis.
 Subsequently, they are phagocytosed by Kupffer
cells in the liver or by red pulp macrophages in the
spleen.
(Summers et al., 2010; Vier et al., 2016)

48.  This short life-span of neutrophils is tightly
controlled by apoptosis, which is a form of
programmed cell death.
 After successful phagocytosis of invading bacteria,
neutrophils undergo apoptosis, which is called
phagocytosis-induced cell death (PICD).

49. During an infection with pathogens, for example E. coli,
lipopolisaccharide enhances the secretion of chemotactic IL-8 and
stimulates the upregulation of E- or P-selectins expression on
gingival endothelial cells (GECs). Selectins facilitate neutrophils
adhesion during transmigration as they interact with PSGL-1
expressed on PMNs.

50. The presence of microbes and their particles activates the complement cascade.
C3a and C5a are anaphylatoxins with a strong chemotactic and pro-inflammatory
potential. IgG and IgM antibodies or C3b recognize bacterial antigens and opsonize
invading pathogens thus facilitating bacterial phagocytosis. LPS activates the TLR4
signaling pathway in recruited neutrophils, eliciting strong inflammatory responses
designed to inactivate the pathogen. Inflammatory responses include the production
of reactive oxygen species, secretion of pro-inflammatory cytokines and
antimicrobial enzymes or peptides, such as cathepsin G, elastase, cathelicidins or
defensins. After a successful bacterial clearance, neutrophils undergo apoptosis, an
essential process triggering the resolution of inflammation.

51.  However, in P. gingivalis infection, neutrophils are
unable to phagocyte this periodontopathogen present
within the huge biofilm structure.
 During phagocytosis of something too big to be
ingested, a process so-called “Frustrated
Phagocytosis,” they generate reactive oxygen species
and secrete enzymes in order to destroy the
pathogen.
 Unfortunately, these secreted weapons concomitantly
contribute to the inflammatory destruction of gingival
tissues and alveolar bone (Scott and Krauss, 2012).

52.  P. gingivalis vesicles are between 50 and 250 nm
in diameter but are predominantly around 50 nm
Vesicles from P.gingivalis showing single membrane
surrounding spherical structures.

53.  Bacterial vesicles originate from outer membrane
blebbing and contain mostly outer membrane lipids
including LPS, outer membrane proteins and some
periplasmic and inner membrane components.

54. Why does P. gingivalis produce
vesicles, and what functional
advantage does vesiculation
confer upon the bacteria against
harmful environmental factors?

55.  In an earlier study of P. gingivalis vesicles, Grenier
et al. 1995 demonstrated that vesicles interacted
with chlorhexidine and, as a result, this interaction
protected the organism against antibacterial
treatment by acting as a decoy.
 It was further identified vesicle LPS as the major
component involved in the binding to
chlorhexidine.

56.  It was also reported that human β-defensin-3 bound
specifically to hemagglutinin B of P. gingivalis .
 Human β-defensins produced by gingival epithelia are
known for their activity against oral bacteria including
P. gingivalis .
 Hemagglutinin B is a major protein detected in P.
gingivalis vesicles.
 It was found that secretion of vesicles may reduce
sensitivity of the bacteria to human β-defensin and
thus facilitate survival of P. gingivalis in the oral
environment.

57.  Outer membrane proteins including fimbrial
proteins and gingipains are necessary for
controlling autoaggregation, microcolony
morphology and biovolume, or those processes
required for maturation of P. gingivalis biofilms.
 Moreover, the lack of cell-associated vesicles was
recently linked with a decrease in the
autoaggregation of P. gingivalis, which provided
evidence of an enhanced function of the outer
membrane mediated by vesicles .

58. Porphyromonas gingivalis vesicle and its functions
The formation of outer membrane vesicles is through the blebbing and
pinching-off of the OM. Arrows show OM, PG and IM, respectively.
Bacterial cell-free vesicles may function as decoys neutralizing
antimicrobial agents, interacting with other oral bacteria, host cells
and inducing host immune responses. IM: Inner membrane; OM:
Outer membrane; PG: Peptidoglycan.

59.  Fimbriae, also called pili, are proteinaceous
appendages on the bacterial outer surface, which
promote both adhesion to and invasion of the host
cells (Enersen et al., 2013).
 Two types of fimbriae exist, the 41-KDa (major)
determined (Yoshimura et al., in 1984), and the
67-KDa (minor) found (Hamada et al., 1996).

60.  The Major fimbriae has long appendices
measuring approximately 0.3 to 1.6 micra long, is
comprised of subunits of a protein called
Fimbriline, which is encoded by a gene
denominated fimA of which only one copy exists in
the P. gingivalis chromosome.
 Minor fimbriae are comprised of minor fimbria
protein subunits (Mfa1) encoded by the Mfa1 gene;
these fimbriae measure from 3.5 to 6.5
nanometers long, significantly shorter than the
major fimbriae.
(Amano A;2004, Nishiyama S;2007, Enersen
M;2008)

61.  FimA is the gene that encodes the fimbriline
subunits. Until now, six fimA genotypes have been
found (I, Ib, II, III, IV, V) based on their nucleotide
sequence.
(Inaba H;2008, Teixeira S.;2009)

62.  The fimA I genotypes are less aggressive; they are
associated to the first stages of the infection like
colonization, invasion, and subversion of the
immune response, besides lacking a capsule,
which makes them less virulent bacterial strains
with low invasion capacity in tissues.
 fimA II and fimA IV genotypes are considered pro-
inflammatory genotypes, which exhibit a more
aggressive phenotype with capacity to cause
damage to the tissue;

63.  Generally fimA II and fimA IV , these genotypes
are encapsulated and this gives them an
advantage in terms of invasion, survival within the
host, and resistance, which is related to the
chronicity of the infection.
(Amano A;2000)

64.  The fimA gene is found in a gene cluster that
encode for regulatory factors or fimbriline
accessory proteins.
 Downstream from fimA there are four genes
denominated fimB, fimC, fimD, and fimE.

65.  Recently, it was described that fimC,fimD, and
fimE products play an important role in fimbriae, in
that if they are suppressed in
in vitro experiments, the ability of P. gingivalis to
adhere to eukaryotic and prokaryotic cells, which
affects the capacity of P. gingivalis fimbriae to
colonize tissues, affecting autoaggregation.
Nishiyama S;2007

66.  Nagano et al., in 2010, conducted a study with the
fimB gene.
 He concluded that this gene regulates fimA length
and expression, which is important for colonization
and adhesion of the microorganism.
 Thus he generated the hypothesis that the fimB
gene would be implied in producing an element
responsible for the anchoring of fimbriae to the
bacterial outer membrane.

67.  Serine phosphatase is an enzyme secreted by P.
gingivalis that modulates inflammatory responses,
as it was shown to inhibit IL-8 production by
gingival epithelial cells through the
dephosphorylation.
 This subsequently abolished PMNs recruitment
and gave P. gingivalis enough time for colonization
of periodontal pockets

68.  Such insidious manipulation of cell signaling
protected this periodontopathogen and bystander
bacteria from neutrophil killing during the initial
stages of infection.

69. Model of chemokine paralysis. Under homeostatic conditions, oral bacteria are
kept at bay by steady recruitment of neutrophils following a gradient of IL-8
production by the gingival epithelium. P. gingivalis can manipulate the IL-8
gradient by secreting SerB, an enzyme that dephos- phorylates the p65 subunit of
NF-kB thereby inhibiting translocation into the nucleus and preventing IL-8
transcription. The result is chemokine paralysis that disrupts the recruitment of
neutrophils into the junctional epithelium and control of the outgrowth of oral
bacteria.

70. Manipulation strategies of
neutrophils and the immune
system by P. gingivalis at the
initial phase of infection.
During the initial phase of an
infection with the keystone
pathogen P. gingivalis (Pg) secretes
serine phosphatase (SerB),
inhibiting IL-8 production. At the
same time, a tetra-acylated lipid A
variant of P. gingivalis LPS
suppresses the expression of L- and
P-selectins on gingival epithelial
cells. These manipulation strategies
hinder neutrophil recruitment, giving
periodontal pathogens enough time
for colonization of periodontal
pockets.

71.  The invasion of microorganisms into mammalian
hosts is initially sensed by phagocytic cells
through their receptors, known as pattern-
recognition receptors (PRRs), to activate the
innate immune response, which is the first line of
host defense against pathogens.
 Each PRR can recognize a group of microbial
components having a similar structural pattern,
termed the pathogen- associated molecular
pattern (PAMP), and a limited number of PRRs are
enough for surveillance of almost all microbial
pathogens.

72.  The toll-like receptor (TLR) family is a
representative PRR family, and different members
in this family react with specific PAMPs.
 For defense against Gram-negative infections in
general, LPS is the most suitable target PAMP for
mammalian host cells.

74.  Lipopolysaccharide (LPS), also known as
endotoxin, is a fundamental structural element of
the cell envelope of gram- negative bacteria.
 LPS is composed of three elements, namely O-
antigen, core polysaccharide, and lipidA.
 The Lipid A is responsible for the toxicity of Gram-
negative bacteria

75. On the other hand, LPS recognized by TLR4 is found only in Gram-
negative bacteria as a cell wall component. A hydrophobic membrane
anchor portion of LPS termed lipid A, but not the polysaccharide
portion, is responsible for stimulation of TLR4 signaling.

76.  When considering the heterogeneous acylation
patterns of P. gingivalis lipid A, two variants are
predominant, namely tetra-acylated and penta-
acylated.
 Strikingly, these two variants were described to
induce opposing host responses.
 The penta- acylated lipid A activate TLR4 (Toll-like
receptor 4) signaling, whereas tetra-acylated lipid
A variant had antagonistic effects against TLR4.

77.  In particular, when bacteria were grown in low
hemin conditions, LPS consisted of
phosphorylated, penta-acylated lipid A structure,
which exerted weak LPS agonistic effects.
 Whereas, during high hemin availability, mimicking
an inflammatory condition, phosphorylated penta-
acylated lipid A was converted into non-
phosphorylated tetra-acylated lipid A, and
exhibited an antagonistic activity (Al-Qutub et al.,
2006).

78.  Therefore, P. gingivalis can exert opposing
effects on the expression of this adhesion
molecule expressed on endothelial cells, which
impairs neutrophil transmigration during
inflammation.
 The P. gingivalis-derived LPS is also a potent
activator of TLR2 (Darveau et al., 2004).
 Activation of TLR2 signaling had a protective
effect not only toward P. gingivalis, but also
toward bystander bacteria, otherwise susceptible
to bacterial killing.

79.  Neutrophil apoptosis and subsequent uptake by
macrophages, referred to as Efferocytosis, is
among the key processes leading to the resolution
of inflammation.
 Therefore, neutrophil resistance to cell death and
diminished efferocytosis may additionally
contribute to the chronic inflammation and
collateral tissue damage observed in periodontal
disease.
(Hiroi et al., 1998; Zaric et al., 2010)

80.  P. gingivalis expresses a non-haem iron protein
called Rubrerythrin (Rbr) in order to avoid oxidative
stress and gain protection from reactive oxygen
and nitrogen species.
 Rbr-positive P. gingivalis was shown to be more
resistant to neutrophil killing, which enabled
colonization of the periodontal tissues.

81.  Moreover, this virulence factor increased the
mobilization and activation of neutrophils which
was necessary for the establishment of
inflammation and contributed to greater tissue
damage in vivo (Mydel et al., 2006).

82.  Another enzyme unique to P. gingivalis, which
converts proteins Arg residues, usually present
at the carboxy terminus, into citrulline (McGraw
et al., 1999).

83.  C5a citrullination reduced the chemotactic and
proinflammatory potential of this anaphylatoxin
.
(Maresz et al., 2013; Bielecka et al., 2014;
Koziel et al., 2014a).
 Therefore, modifications of host proteins by
PPAD represent another manipulative strategy
designed to protect P. gingivalis and bystander
bacteria from neutrophil-mediated killing.

84.  Gingipains are major cysteine proteases, which
are either membrane-bound or secreted and they
account for 85% of the total proteolytic activity of
P. gingivalis (Potempa et al., 1997).
 The Gingipains family includes three related
cysteine proteases that cleave the c- terminal
peptide bonds of arginine and lysine residues.

85.  The homologous Arg- gingipaain (Rgp)A and
(Rgp)B, are the products of two highly related
genes rgpA and rgpB, while the Lys- specific
gingipain, Lys-gingipain (Kgp) is encoded by a
single gene kgp.
 The assacccharolytic nature of P.gingivalis means
that upon entering the host, the bacterium needs
to compete for and exploit available peptides for
growth, or degrade host proteins in order to
produce peptides for carbon, nitrogen and energy
sources in order to survive.

86.  Thus the actions of proteolytic enzymes such as
gingipains has advantage for the survival of the
bacteria over other periodontopathogenic
bacteria.
 The Gingipains are not only capable of degrading
host proteins but also activating host systems and
cells.

87.  In periodontitis there is increase production of
GCF.
 The Arg- and Lys- specific gingipains enhance
GCF production by activating the Kallikrien/ kinin
pathway and enhancing vascular permeability.
 RgpA and –B factors have been shown to activate
prekallikrien, leading to the formation of
bradykinin.

89.  Kgp acts in synergy with the Arg- specific cysteine
proteinases to degrade high-molecular weight
kininogen directly to bradykinin.
( Imanura T; 1995)
 The Arg- specific gingipains are able to activate
prothrombin to thrombin by direct cleavage and
activation of the activating coagulation factors IX
and X.

90.  The activation of thrombin increases GCF
production and enhances leukocyte accumulation
at inflammed periodontitis sites by enhancing
vascular permeability and inducing leukocyte
chemotaxis at sites of periodontal inflammation.
 As BOP is one of the clinical sign of periodontitis
and the ability of Arg-specific cysteine proteinases
to activate anticoagulant protein C.

91.  Thus inhibiting the blood coagulation could
contribute to bleeding tendency of periodontal
sites.
 Opsonization by natural or acquired antibodies is
an important protective feature of innate and
adaptive immune response and facilitates
phagocytosis of invading bacteria.

92.  Gingipain K (Kgp) has the unique ability to cleave
IgG1 and IgG3 at the hinge region, thus
separating the antigen binding Fab fragment from
the effector Fc fragment of immunoglobulins.
 This activity was observed not only in vitro using
isolated IgGs or human plasma, but it was also
detected in vivo in gingival crevicular fluid (GCF).

93.  Also treatment of patient serum samples with Kgp
inhibited P. gingivalis opsonization and
subsequent phagocytosis by neutrophils
(Kobayashi et al., 2001; Guentsch et al., 2011;
Vincents et al., 2011).
 Therefore, lysine-specific gingipain suppresses
IgG-mediated opsonization and P. gingivalis
phagocytosis, which contributes to pathogen
persistence.

94.  Matrix metalloproteinases (MMPs) are proteolytic
enzymes that helps to degrade the extracellular
matrix of host cells.
 The Arg- and Lys – gingipains affect the
expression and activation of host MMPs, including
MMP- 1,2,3,8,9.

95.  Kgp activates the MMP- 1 and 9 while RgpA
activates MMP-3.
 Activated MMPs degrade collagens and are
destructive to extracellular matrix and PDL.
 These enzymes specifically degrade the collagen
fibres attached to the root surface which contribute
to the formation of periodontal pocket.

96.  Another strategy of immune evasion employed by
P. gingivalis is the inactivation of complement
factors.
 This periodontopathogen produces high quantities
of gingipains targeting these fundamental
complement- mediated innate immune responses
(Popadiak et al., 2007).
 In particular, RgpA can precisely cleave C3 and
C5 to produce active C3b and C5a, the latter
being an anaphilatoxin, strongly promoting
neutrophil recruitment to the gingiva (Wingrove et
al., 1992).

97.  Actually, gingipains have been shown to have
complex, “biphasic” effects on the complement
system.
 At low concentrations of gingipains, which mimic
early infection stages, they precisely cleave C3
and C5 and generate active C3a and C5a,
respectively.
 In turn, at high concentrations or at deeper
gingival localization, where the biofilm resides,
they inhibit the complement pathway.

98.  This indicates that at the beginning of bacterial
invasion, gingipains enhance inflammation in order
to increase nutrients supply.
 While at advanced stages of periodontal disease
they inactivate the complement cascade, which is
designed to protect P. gingivalis and bystander
bacteria from the bactericidal activity of the human
serum and neutrophil killing
(Popadiak et al., 2007; Potempa and Pike, 2009).

100. During a later phase of the infection P. gingivalis releases a penta-acylated LPS
variant leading to the increased expression of L- and P-selectins on GECs and
enhanced production of IL-8. This strongly stimulates neutrophil chemotaxis and
transmigration to the site of infection.

101. Moreover, P.g.-derived LPS and fimbriae strongly stimulate neutrophil pro-
inflammatory and anti-bacterial responses, such as the secretion of reactive oxygen
species and pro-inflammatory cytokines, and the production of anti-microbial peptides
and enzymes. An elevated secretion of these anti-bacterial molecules results in
gingival tissue destruction, while many virulence factors secreted by P. gingivalis
protect this periodontopathogen. The keystone pathogen is protected from oxidative
stress, as it expresses ruberythrin (Rbr) protein.

102. Additionally, the expression of Lys-specific gingipains degrades
immunoglobulins at the hinge region, and, coupled with the activation of the
TLR2 signaling pathway (by LPS), abolishes bacterial opsonization and
phagocytosis. Also, gingipains manipulate anti-bacterial responses by
deregulating the complement cascade and IL-8-mediated neutrophil
chemotaxis.

103. In particular, depending on the concentration and the position of gingipains
within the biofilm, these enzymes can exert opposing effects. C3, C5, and IL-8
are degraded at high gingipain concentration or by gingipains associated with
bacterial cells or vesicles thus inhibiting pro-inflammatory responses and
protecting bacteria from elimination. In contrast, low levels of soluble Arg-
specific gingipains activate C5 and C3 by limited proteolysis that results in the
generation of C5a and C3a anaphylatoxins.

107.  Temperature
- Mean temperature of the gingival sulcus during
health -35°C (30°C to 38°C )
(Socransky SS, Haffajee AD, 1991)
-P. gingivalis when exposed to elevated temperature –
heat shock response
(Lu et al 1994)
- HSP6O and HSP70 family homologs have been
described in P. gingivalis which stimulates bone
resorption, inducing bone destruction & inhibiting
bone formation

108.  pH
As pH increases pH range within gingival sulcus
during health -7.0 to 8.5 (Cimasoni 1983)
Periodontal pockets deepen and host inflammatory
response is induced.
pH increases
Increases in Gram –ve anaerobic colonization

109.  Optimal pH for P. gingivalis - 7.5 (7.5 to 8.5)
(Marsh et al 1994)
 Trypsin-like activity increases with pH.

110.  Oxygen
Oxygen concentration - induce the HSP6O-like
stress protein in P. gingivalis
(Vayssier et al)

111.  Scaling and root planing - temporary decrease
P.gingivalis levels but not capable of eradicating
the organism from subgingival sites.
 Non - resective periodontal surgery - not effective
in removing P. gingivalis

112.  Elimination of periodontal pockets along with
proper oral hygiene provides a more predict- able
suppression of P.gingivalis.
 Systemic antibiotic therapy + scaling and root
planing may not ensure subgingival eradication of
P. Gingivalis.
 Topical antimicrobial therapy - not very useful

113.  Resective Periodontal surgery + systemic antibiotic
therapy + good oral hygiene: promote more
effective control of P.gingivalis.
 Zarkesh et al. (1999) – coating PTFE barrier
membranes with Tetracycline. They permitted less
P. gingivalis colonization and more clinical
attachment gain.

114.  P.gingivalis comprise major pathogens in
infectious implant failure.
 P.gingivalis was found to be associated with the
development of ligature-induced peri-implantitis.
 P.gingivalis can attach to barrier membranes and
thus can penetrate porous barrier membranes
from one side to the other.

115.  P.gingivalis seem to be indigenous only to the
oral cavity, these organisms’ occurrence in
extraoral sites may suggest translocation from
oral to nonoral sites.
 Occasionally been recovered from non - oral
sites.

117.  P.gingivalis is responsible for metastatic infection due
to transient bacteremia.
 Tooth brushing, dental flossing, oral irrigation, scaling
and root planing and surgical procedures among
others give rise, in the presence of accumulated
plaque and gingivitis, to transient bacteremias
(Baumgartner et al, 1977)
 Most common example of it is atheresclerosis and
myocardial infarction.

118.  The oral bacteria such as Streptococcus sanguis
and Porphyromonas gingivalis induce platelet
aggregation, which leads to thrombus formation .
 These organisms have a collagen-like molecule,
the platelet aggregation-associated protein, on
their surface.
 Possibly, antibodies reactive to periodontal
organisms localize in the heart and trigger
complement activation, a series of events leading
to sensitized T cells and heart disease.

119.  Potempa et al. 1999, studied proteolytic enzymes
referred to as gingipains R, which are released in
large quantities from P. gingivalis.
 After entering the circulation, gingipains R can
activate factor X, prothrombin, and protein C.
 It promotes a thrombotic tendency through the
ultimate release of thrombin, subsequent platelet
aggregation, conversion of fibrinogen to fibrin, and
intravascular clot formation.

120.  Second mechanism involved in it is metastatic
injury due to immunological injury.
 In this process could be an exaggerated host
response to a given microbial or LPS challenge,
as re- flected in the release of high levels of
proinflammatory medi- ators such as PGE2,
TNF-a, and IL-1b.

121.  Third mechanism involved metastatic injury from
circulation of oral microbial toxins.
 LPS from periodontal organisms being
transferred to the serum as a result of
bacteremias or bacterial invasion may have a
direct effect on endothelia so that atherosclerosis
is promoted.

122.  LPS may also elicit recruitment of inflammatory
cells into major blood vessels and stimulate
proliferation of vascular smooth muscle,
vascular fatty degeneration, intravascular
coagulation, and blood platelet function.
 These changes are the result of the action of
various biologic mediators, such as PGs, ILs,
and TNF-a on vascular endothelium and
smooth muscle

123.  The brain is immunologically privileged due to a
physical blood brain barrier (BBB) and due to
the absence of a lymphatic system and
therefore displays low levels of molecules that
are critical to antigen presentation.

124.  Thus, during neurodegeneration, the brain relies
mainly on the resident central nervous system
(CNS) cells particcularly Microglial cells to
recognise and mount a response against the
invading pathogens.
 Poole et al. 2013 reported that periodontal
pathogen P. gingivalis components are also
identified in AD subjects.

125.  PD presents after 30 years of age,where as the
late-onsetAD appears later(80+ years onwards)
in life.
 Thus there is sufficient time for an established
chronic periodontal pathogen such as P.gingivalis
to exploit the haematogenous route to access the
brain as illustrated by (Singhrao et al.
2014)

126.  Given the resilience of P. gingivalis in the human
host, even the presence of fewer P. gingivalis in the
brain over at least three decades would be sufficient
to contribute to a low but persistent level of local
inflammation for its own nutritional sustenance and
survival.
 Bacteria and/or their immunogenic components at
the appropriate concentration initiate the classical
innate imm- une signalling pathways via TLR-2 and
TLR-4 mechanisms whereby the release of cytokines
by microglia (IFN-𝛾 and TNF-𝛼) is an inevitable
consequence.

127.  Chronic release of cytokines will eventually
change the permeability at the BBB and reduce
the efflux of A𝛽 from the CNS into the systemic
circulation.
 Under appropriate concentrations of
LPS/peptidoglycan, TLR-2 and TLR-4 signalling,
and release of reactive oxygen/nitrogen species
such as superoxide ions, iNOS/NO, cytokine
secretion and bacterial activation of the
complement system become inseparable.

128.  Together these factors lead to vital neurons being
destroyed and enhanced maintenance of chronic
inflammation with consequences for development
of disease.

129.  Peptidylarginine deiminase (PAD) has been
recently suggested to assume a key role in the
citrullination of arginine residues in host proteins.
 Thus alter their normal folding and creating
conditions for the induction of auto-antibodies
against host cartilage-specific antigens.

130.  P. gingivalis’ ability to activate host matrix
metalloproteases (MMPs) and the organisms’
role in altering cytokine responses have also
been implicated in the protein degradation,
observed in arthritic joints.
(Mangat et al., 2010, Hitchon et al., 2010,
Detert et al., 2010)

131.  The significance of P. gingivalis involvement in
cancer has evolved within the last decade.
 Chronologically initial studies have comprised
mostly epidemiological and clinical association
between oral microbiome, periodontal disease or
tooth loss, with orodigestive cancers including
cancers of the oral cavity, gastrointestinal tract
and pancreas. (Michaud, 2013)

132.  Among all observed cancer relations the most
direct and strong association of P. gingivalis has
been found with oral squamous cell carcinoma (
OSCC).
(Katz et al., 2011)
 P. gingivalis, can invade primary cultures of oral
epithelial cells (OECs), render them resistant to
cell death, and alter the chemokine and cytokine
secretion.

133.  OECs that form the outer lining of the oral
mucosa are the initial targets for colonizing
bacteria, and stand an important arm of the host
innate immune response.
 They show response by secreting antimicrobial,
pro-inflammatory and chemotactic signals, and
modulating apoptosis upon sensing invading
organisms .
(Takeuchi et al., 2013, Yilmaz et al., 2010, Hung
et al., 2013)

134.  Therefore, the ability of P. gingivalis to
colonize in the oral cavity and modulate both
the epithelial cell and systemic responses
potentially places the organism not only in the
category of central contributors in the
multifactorial etiology of the periodontal
disease, but also orodigestive cancers and
other chronic diseases

135.  Attenuated and inactivated bacterial vaccines
 Live bacterial vectors
 Passive immunization
 Purified antigen (subunit) vaccines
 Synthetic antigen vaccines

136.  Attenuated and inactivated bacterial vaccines
- Production of serum antibody, which correlated
withimmune protection from the virulence
properties ofP.gingivalis (Ebersole 1997, Genco
CA, 1992,Kesavalu,1992)
- Active immunization of mice (Baker et al 1997) or
rats(Taubman et al 1983) with P. gingivalis -
ability to alterdisease manifestations of
periodontitis in these animals

137.  Live bacterial vectors
- The hemagglutinin gene of P. gingivalis has been
clonedinto an avirulent strains of S. Typhimurium
(Dusek DM 1995)
- Used to orally immunize mice and resulted in a
systemic and mucosal response to this antigen

138.  Passive immunization
- Booth et al. (1996) produced a murine
monoclonalantibody to P. gingivalis which
prevented recolonization ofdeep pockets in
periodontitis patients.
- Laboratory tests revealed that this antibody
inhibited the hemagglutination of red blood
cells.

139.  Purified antigen (subunit) vaccines
- Bird et al. (1995) used the mouse abscess
model and immunized it with an outer
membrane – induction of protective immunity

140.  Synthetic antigen vaccines
- Requires synthesis of linear & branched polymers
of 3-10amino acids based on known sequences of
microbialantigens.
- Weakly immunogenic.
- Coupled to large proteins antibody response.
- Safe, cheap, easy to store & handle & ideally
suited tospecific targets.

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145. Thank You