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Citation: Chen, W.A.; Dou, Y.;
Fletcher, H.M.; Boskovic, D.S. Local
and Systemic Effects of
Porphyromonas gingivalis Infection.
Microorganisms 2023,11, 470.
https://doi.org/10.3390/
microorganisms11020470
Academic Editors: Andrea Scribante,
Mario Alovisi and Andrea Butera
Received: 28 December 2022
Revised: 31 January 2023
Accepted: 2 February 2023
Published: 13 February 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
microorganisms
Review
Local and Systemic Effects of Porphyromonas gingivalis Infection
William A. Chen 1, Yuetan Dou 2, Hansel M. Fletcher 2and Danilo S. Boskovic 1,*
1Division of Biochemistry, Department of Basic Sciences, School of Medicine, Loma Linda University,
Loma Linda, CA 92350, USA
2Division of Microbiology and Molecular Genetics, Department of Basic Sciences, School of Medicine,
Loma Linda University, Loma Linda, CA 92350, USA
*Correspondence: dboskovic@llu.edu
Abstract:
Porphyromonas gingivalis, a gram-negative anaerobe, is a leading etiological agent in peri-
odontitis. This infectious pathogen can induce a dysbiotic, proinflammatory state within the oral
cavity by disrupting commensal interactions between the host and oral microbiota. It is advantageous
for P. gingivalis to avoid complete host immunosuppression, as inflammation-induced tissue damage
provides essential nutrients necessary for robust bacterial proliferation. In this context, P. gingivalis
can gain access to the systemic circulation, where it can promote a prothrombotic state. P. gingivalis
expresses a number of virulence factors, which aid this pathogen toward infection of a variety of host
cells, evasion of detection by the host immune system, subversion of the host immune responses, and
activation of several humoral and cellular hemostatic factors.
Keywords:
dentistry; inflammation; neutrophils; periodontitis; periodontology; platelets; Porphyromonas
gingivalis
1. Overview
Porphyromonas gingivalis is a black-pigmented, gram-negative bacterium that primarily
colonizes the subgingival tissues in the oral cavity. This asaccharolytic anaerobe can adapt
to and survive in the low oxygen tension conditions characteristic of periodontal pockets [
1
].
However, growth rates under microaerophilic conditions are not significantly altered from
those at anaerobic conditions, suggesting that P. gingivalis can tolerate microenvironments
with low oxygen [
2
]. Hemin or heme and vitamin K can be used as growth nutrients [
3
,
4
].
However, P. gingivalis also metabolizes amino acids (AAs) and peptides for energy and as a
supply of carbon [5].
Over 700 bacterial species of diverse microbial flora are estimated to inhabit the
oral cavity [
6
]. New culture-independent and culture-dependent molecular techniques
have been developed to help characterize microbial communities. Culture-independent
approaches include techniques such as next-generation sequencing (NGS) technologies,
such as shotgun metagenomics sequencing, allowing researchers to investigate populations
of oral bacteria [
7
]. Deoxyribonucleic acid (DNA) is extracted from the oral microbiome
and fragmented prior to sequencing [
8
]. Then metagenomic analysis helps to highlight
the genomic characteristics and potential functions of oral microbiota [
9
]. Similarly, meta-
transcriptomic analyses of ribonucleic acid (RNA) help to assess gene expression in mixed
bacterial populations of the oral cavity [
10
]. Such techniques have been used to investigate
interactions of P. gingivalis with various other bacterial species and evaluate its effects on
the microbial community within the biofilm environment [11,12].
The new culture-dependent techniques employ a variety of media prior to analysis
using sensitive mass spectrometric and sequencing techniques, such as matrix-assisted
laser desorption ionization–time of flight mass spectrometry (MALDI–TOF MS) and 16S
ribosomal RNA (rRNA) sequencing, to identify bacterial species [
13
,
14
]. Future studies
can involve such culturomic approaches to characterize particular roles of specific bacteria,
Microorganisms 2023,11, 470. https://doi.org/10.3390/microorganisms11020470 https://www.mdpi.com/journal/microorganisms
Microorganisms 2023,11, 470 2 of 27
such as P. gingivalis during the colonization of periodontal tissues and the pathogenesis of
periodontitis [15].
The subgingival biofilm, within periodontal pockets, comprises over 300 different
species [
16
]. In this context, P. gingivalis is considered to be a late colonizer, often co-
aggregating at the top layer with initial and secondary colonizers [
17
,
18
]. While most
oral microbes are seen as commensal, some, including P. gingivalis, are recognized as
opportunistic or keystone pathogens [19].
2. Virulence Factors
The plasma membrane of P. gingivalis acts as a dynamic interface between the oral
pathogen and its local environment. Successful acquisition of nutrients facilitates growth,
while effective tissue colonization ensures bacterial survival. These are highly dependent
on virulence factors expressed or released by P. gingivalis (Table 1).
Table 1. Virulence factors produced by Porphyromonas gingivalis.
Virulence Factor Action References
Capsule Avoid detection by the immune system
Contribute to P. gingivalis invasion of host tissue [20,21]
Fimbriae Contribute to P. gingivalis invasion of host tissue [22]
Major fimbriae Mediate P. gingivalis attachment to host tissue
Induces remodeling of the actin cytoskeleton in gingival epithelial cells [23,24]
Minor fimbriae Triggers cytokine release by macrophages [25,26]
Gingipains
Chemokine and cytokine degradation
Nutrient (heme and peptide) acquisition
Subversion of host immune response
[27–30]
RgpA
Contribute ECM attachment and fimbriae assembly
Cleave ECM proteins and facilitate tissue invasion
Degrade C3, C5a, and RBCs
Bind to C4BP, resulting in decreased C3 and C4 protein function
Cleave and activate FIX, FX, prothrombin, PAR-1, and PAR-4
[31–40]
RgpB
Mediate P. gingivalis binding to ECM proteins
Involved with the assembly of fimbrial proteins
Cleave and activate FIX, FX, PAR-1, and PAR-4
[40–42]
Kgp
Cleave ECM proteins and facilitate tissue invasion
Bind to and degrade hemoglobin for heme acquisition
Cleave C5a receptor
[31,43,44]
C3, complement component 3; C4, complement component 4; C4BP, complement component 4b binding protein;
C5a, complement component 5a; ECM, extracellular matrix; FIX, factor IX; FX, factor X; PAR, protease-activated
receptor; RBCs, red blood cells.
3. Capsule
Gram-negative bacteria, including P. gingivalis, are covered with cell surface macro-
molecules termed capsular polysaccharides (CPS) [
45
]. Bacterial capsules are typically
carbohydrate homo- or heteropolymers, composed of repeating monosaccharide units
covalently bound through glycosidic linkages [
46
]. Carboxyl or phosphate groups may be
attached to the carbon backbone of the monosaccharide residues, contributing negative
charges to CPS [
47
]. The capsule includes mannuronic acid, glucuronic acid, galacturonic
acid, galactose, and 2-acetamido-2-deoxy-d-glucose (N-acetylglucosamine) at a relative
molar ratio of 1.2:1.8:1.0:1.0:2.0, respectively [
48
]. Additionally, the CPS in several gram-
negative pathogens are terminally linked to reducing phospholipids, which anchor the
glycolipids into the outer membrane [49].
Microorganisms 2023,11, 470 3 of 27
4. Capsular Functions
Encapsulation contributes to P. gingivalis pathogenicity by enabling the evasion of de-
tection by the immune cells and increasing the resistance to phagocytosis [
20
]. Furthermore,
human gingival fibroblasts, when challenged with P. gingivalis lacking a capsule, increased
interleukin (IL)-1
β
, IL-6, and IL-8 cytokine production [
50
]. The presence of a capsule may
help to mask immunogenic bacterial agents, such as adhesins or invasins, and prevent
the triggering of some immune responses [
51
]. In addition to this camouflage-like role, it
was suggested that encapsulated P. gingivalis are also more invasive, although there are
some conflicting reports about this capsular effect. The highest degree of bacterial invasion
is observed when human coronary artery endothelial cells are incubated with wild-type
P. gingivalis compared to other P. gingivalis strains [
21
]. However, it was also reported
that mutant non-encapsulated P. gingivalis strains were more efficient at invading gingival
fibroblasts [
52
]. In this context, the gram-negative Neisseria meningitides, associated with
septicemia and meningitis, are known to downregulate capsule synthesis and assembly
during adhesion to target cells [
53
]. A similar mechanism may be utilized during the early
stages of P. gingivalis infection of subgingival tissue, reducing the capsule production to
increase adhesion. Consistent with this, in contrast to P. gingivalis cultured from healthy
sites, P. gingivalis cultured from diseased periodontal sites are highly invasive to KB cells
(a subline of HeLa cells) [
54
]. Overall, however, encapsulated P. gingivalis strains are associ-
ated with increased virulence, implying that capsule expression contributes to bacterial
survival and its ability to impact host immune responses.
5. Fimbriae
5.1. Major Fimbriae
Characteristic of gram-negative bacterial cells, fimbriae are thin protruding appendages
attached to the outer membrane. P. gingivalis expresses two distinct types of fimbriae that
vary in length, classified as major and minor. The long fimbrial polymer is primarily
composed of the repeating fimbrillin (Fim) A subunits [
55
], which are assembled through
a head-to-tail oligomerization [
56
]. Various P. gingivalis strains produce different overall
FimA monomer sizes with some amino-terminus sequence heterogeneity [
57
]. The fimA
gene, encoding the FimA protein, has six recognized gene variants: type I, Ib, II, III, IV,
and V [
58
], and belongs to a cluster of seven fim genes: fimX,pgmA, and fimABCDE [
59
].
FimB regulates the length of FimA-associated fimbriae. A nonsense mutation in fimB leads
to the production of extended fimbriae in P. gingivalis strains ATCC33277 and 381 [
60
].
FimC, FimD, and FimE represent accessory proteins, forming complexes that facilitate the
biosynthesis of long fimbriae [
61
,
62
]. Deficiency of any of the accessory proteins reduces
the attachment of P. gingivalis mutants to extracellular matrix (ECM) protein fibronectin or
to type I collagen [62].
5.2. Minor Fimbriae
Minor fimbrial antigen 1 (Mfa1) is the main structural protein of P. gingivalis short
fimbriae [
63
]. The mfa1 gene is part of a four-gene cluster operon comprising mfa1-4, while
the mfa5 gene appears to be independently transcribed [
64
]. Formation of short fimbriae
involves the polymerization of monomeric Mfa1 subunits via the amino- and carboxy-
terminus domains [
65
]. In addition, short fimbriae also comprise Mfa2-5 protein subunits.
Mfa2 is a dual-function protein regulating the fimbrial length and its anchoring to the outer
membrane. Mfa3 proteins are localized toward the fimbrial tips and work in conjunction
with Mfa4 and Mfa5 to properly assemble the short fimbriae [
66
]. Mfa4 confers a stabilizing
effect during the formation of short fimbriae, while Mfa5 possesses a von Willebrand Factor
(vWF) type A domain that mediates protein–protein interactions with Mfa1 [
67
,
68
]. For
pathogenic bacteria, such as Enterococcus faecalis and Streptococcus agalactiae, the vWF type
A domain containing pilus proteins are known to mediate bacterial cell adhesion to host
tissue [69,70].
Microorganisms 2023,11, 470 4 of 27
5.3. Functions of Fimbriae
P. gingivalis fimbriae serve dual functions during the pathogenesis of periodontitis:
bacterial adhesion and invasion. The long and short fimbriae support bacterial colonization
of gingival tissue and ensure its survival. First, fimbriae enable bacterial adhesion to
host cells. Purified fimbriae from P. gingivalis cells adhere to human gingival cells in a
concentration and time-dependent manner [
23
]. Impaired adhesion to human gingival
fibroblast and epithelial cells is observed in mutant P. gingivalis strains deficient in long
fimbriae [
71
]. Similarly, bacterial adherence to human gingival epithelial cells is inhibited
in a double knockout strain of fimA and mfa1 genes [
72
]. Second, P. gingivalis invasiveness
is dependent on fimbriae. A FimA deficient strain has diminished potential for bacterial
invasion of gingival epithelial and fibroblast cells [
22
,
73
]. The invasion of epithelial cells
can occur through actin-based cytoskeletal rearrangements, mediated by FimA interactions
with surface epithelial β1 integrins [24,74].
Alveolar bone destruction is a hallmark feature of P. gingivalis-induced periodonti-
tis. Gnotobiotic rats exhibit significant bone loss after infection with fimbriae expressing
wild-type P. gingivalis [
75
]. However, such bone loss is mitigated in the rats that are (a) im-
munized with purified fimbrial proteins before exposure to P. gingivalis or (b) infected with
afimA knockout strain [
75
,
76
]. Consistent with this, alveolar bone loss is not increased
in rats infected with a double knockout strain for fimA and mfa1 [
72
]. Moreover, purified
Mfa1 protein increased the release of the cytokines IL-
β
, IL-6, and TNF
α
from isolated
murine peritoneal macrophages, consistent with the promotion of a proinflammatory envi-
ronment [
25
]. Such chronic inflammation then leads to tissue breakdown facilitating the
further bacterial invasion of the subgingival regions [
26
]. In contrast, downregulation of
fimbrial expression reduces P. gingivalis pathogenicity.
6. Gingipains
Gingipains are surface-expressed or secreted cysteine proteases that can cleave a
variety of host proteins in plasma, ECM, and in association with immune cells [
31
,
32
,
77
–
81
].
Their physiologic effects are functional and focused on bacterial survival. Since P. gingivalis
is asaccharolytic, other types of biomolecules are used for its energy and carbon supply
needs. Gingipains cleave host proteins to generate peptides and amino acids, which in turn
supply some of these requirements. Proteolysis of host proteins also facilitates bacterial
invasion and colonization of subgingival tissues. Furthermore, gingipains can undermine
the host immune responses allowing P. gingivalis to evade neutrophil-mediated bacterial
clearance. Overall, gingipains play a pivotal role in exerting bacterial pathogenicity and
catalyzing the degradation of a broad spectrum of host proteins to support the proliferation
and invasion of the periodontium.
Gingipain Structure
P. gingivalis expresses two distinct forms of gingipains, arginine- and lysine-specific,
that hydrolyze peptide bonds at carboxy termini of arginine or lysine residues, respec-
tively [
82
]. This cysteine protease family comprises three related enzymes: a high molecular
mass arginine gingipain A (RgpA), arginine gingipain B (RgpB), and a lysine gingipain
(Kgp). These gingipains are encoded by rpgA,rgpB, and kgp genes, respectively [
83
]. The
gingipain precursors exhibit similar structural features with a signal peptide attached to an
amino-terminus propeptide domain, followed by an arginine- or lysine-specific catalytic
domain [
84
]. Hemagglutinin/adhesin domains are linked to the catalytic domain of RgpA
and Kgp at their respective carboxy-termini. RgpB lacks such domains. The four identified
hemagglutinin/adhesin domains are designated as Hgp15, Hgp17, Hgp27, and Hgp44 [
85
].
Hgp15 and Hgp44 are implicated with hemagglutination and with hemoglobin binding
activity [
27
,
86
]. Hgp17 mediates coaggregation between P. gingivalis and Prevotella interme-
dia, suggesting a contributing role in periodontal biofilm formation [
87
]. Gingipain gene
products are inactive zymogens that require catalytic activation following post-translational
modifications. Prior to export to the outer membrane, processing of the gingipain gene
Microorganisms 2023,11, 470 5 of 27
translation products includes cleavage of the attached signal peptide and propeptide
domains as well as the noncovalent association of functional domains [
88
]. P. gingivalis
gingipains are expressed in three main forms: membrane-bound monomeric proteins,
membrane-bound multimeric complexes, and secreted soluble proteins. Purified RgpA,
RgpB, and Kgp are synthesized as single-chain glycoprotein enzymes and expressed along
the surface of P. gingivalis [
89
–
91
]. RgpA can interact with Kgp through noncovalent bonds
to form large multifunctional complexes [
92
,
93
]. Outer membrane vesicles with gingipains
can also be secreted into the local environment [94].
Some gingipain expression is central in the development of P. gingivalis virulence
during infection of the oral cavity. These cysteine proteases mediate tissue colonization
to establish a niche in the subgingival regions and degrade host proteins to provide es-
sential nutrients for bacterial growth. Furthermore, gingipain-mediated subversion of the
host immune responses promotes the proinflammatory environment that contributes to
pathogenic persistence and dysbiosis of the oral microbiota.
7. Gingipain Functions
Tissue Colonization
During the initial stages of infection, gingipains assist bacterial adhesion to subgin-
gival tissues. Wild-type P. gingivalis adheres to human oral epithelial cells, while strains
deficient in arginine gingipain activity have reduced attachment to these cells [
41
,
95
]. ECM
cell adhesion molecules (CAMs) located on gingival tissue surfaces mediate such cell-cell
interactions. Purified gingipain enzymes were found to bind to CAMs such as fibronectin
and laminin [
96
]. Similarly,
in vitro
binding activity of wild-type P. gingivalis was demon-
strated toward type I collagen, fibronectin, and laminin [
41
]. Reduced binding to these
ECM proteins was observed for P. gingivalis with inactivated rgpA gene. Adherence to
immobilized collagen is also diminished in the rgpA knockout [33].
Gingipain adhesin domains mediate tissue colonization of P. gingivalis in the subgin-
gival sulcus. The binding of adhesin proteins to epithelial cells can be detected even if
wild-type P. gingivalis is treated with a protease inhibitor, implying that protease functions
of gingipains are not involved [
97
]. However, treatment with an anti-adhesin antibody
blocks interactions of epithelial cells with native RgpA adhesin proteins or with wild-type
P. gingivalis cells [
98
]. Moreover, gingipain adhesin is known to bind to ECM proteins such
as collagen, fibronectin, and laminin [31,99,100].
P. gingivalis expressed fimbriae are heavily implicated as primary virulence factors
initiating bacterial adhesion to host cells. However, gingipain proteolytic activity plays
a role in effective fimbrial function. Post-translational processing of precursor fimbrillin
proteins is crucial for proper fimbrial assembly along the outer membrane [
101
]. Decreased
expression of cell surface fimbriae is observed following the inactivation of both the rgpA
and rgpB [
42
]. Additionally, fimbrial binding to gingival fibroblast cells is increased in
the presence of purified gingipains [
34
]. In this context, pretreatment of fibronectin with
purified gingipains also increases binding to fimbriae [
102
]. This effect can be reduced by
the addition of competing L-arginine residues or peptides with exposed arginine residues.
Together, these observations imply that proteolytic cleavage of ECM proteins by gingipains
potentially unmask cryptic receptors, or cryptitopes, with exposed arginine residues that
mediate fimbrial attachment [
103
]. Thus, gingipains cleave fimbrial precursors during the
production and maturation of fimbriae, as well as modify target CAMs, to enhance bacterial
adhesion with host cells.
8. Tissue Invasion
Following the attachment to cell surfaces, gingipains facilitate the invasion of gingival
tissues. Transmission electron micrographs from invasion assays illustrate internalized P.
gingivalis bacterial cells within oral epithelial cells [
104
]. Similarly,
in vitro
fluorescence
imaging demonstrates P. gingivalis localization within gingival epithelial cells [
105
]. In
contrast, bacterial pretreatment with protease inhibitors significantly reduces such invasion
Microorganisms 2023,11, 470 6 of 27
of epithelial cells by P. gingivalis [
106
]. Wild-type P. gingivalis infiltrates the entirety of
a human oral mucosal tissue model, including internalization within gingival epithelial
cells, as well as penetration into the basement membrane and lamina propria [
107
]. The
triple (rgpA,rgpB,kgp) gingipain knockout strain, however, is primarily localized to the
epithelial surface. Gingival tissue invasion involves cleavage of CAMs and cytoskeletal
remodeling. Increased fibronectin and its fragments are observed in gingival crevicular
fluid (GCF) from patients with periodontitis but not from healthy subjects [
80
]. Similarly,
fibronectin degradation is observed when purified arginine gingipains are incubated with
human gingival fibroblast cells [
108
]. Moreover, purified RgpA, RgpB, or Kgp gingipains
can cleave isolated human serum fibronectin [80].
Purified RgpA and Kgp can digest laminin directly [
31
]. However, the most prevalent
form of collagen in gingival connective tissues, type I, is resistant to degradation by purified
gingipains [
109
,
110
]. Nevertheless, P. gingivalis can cause connective tissue proteolysis
by upregulating activation and downregulating inhibition of matrix metalloproteases
(MMPs) [
111
–
114
]. MMPs are proteolytic enzymes that degrade ECM proteins in a calcium
and zinc-dependent manner [
115
]. These metalloproteases can be released by fibroblasts
from the periodontal ligament and gingival tissue [114,116].
Gingipains can alter the host cell
'
s cytoskeletal responses to facilitate bacterial invasion.
P. gingivalis-mediated infection of gingival epithelial cells triggers significant depolymeriza-
tion of actin and disassembly of microtubules in host cells [
24
]. Such actin degradation is
not observed when a triple gingipain (rgpA,rgpB,kgp) knockout is incubated with human
gingival epithelial cells [
117
]. However, actin cleavage occurs when the gingival epithelial
cells are challenged with a double (rgpA,rgpB) knockout, implying that Kgp is the effective
enzyme. This is confirmed by dose and time-dependent degradation of isolated actin by
purified Kgp [
117
]. In contrast, the cleavage of isolated actin by purified RgpA or RgpB is
not significant.
Epithelial cells in gingival tissues form tight and adherent junctional complexes via
specialized protein–protein interactions [
118
]. These complexes create homotypic cell–cell
adhesions that together constitute a physical barrier to pathogens [
119
]. P. gingivalis is
known to break down the intercellular junctional complexes, thus disrupting this important
epithelial barrier function [
120
]. Incubation of P. gingivalis with Madin-Darby canine kidney
(MDCK) cells results in reduced labeling intensity of occludin and E-cadherin [
121
]. Western
blots confirm this effect with concentration-dependent degradation of junctional proteins.
In this context, gingipains are the likely enzymes capable of degrading epithelial junctional
proteins. All three forms of purified gingipains (RgpA, RgpB, and Kgp) are known to
catalyze the hydrolysis of immunoprecipitated E-cadherin in a concentration-dependent
manner [122].
Gingipain-mediated degradation of epithelial CAMs, as well as hydrolysis of actin
and junctional proteins, provide P. gingivalis with access to underlying connective tissue.
This creates an environment conducive to bacterial colonization of the periodontium, an
important step in the pathogenesis of periodontitis. The compromising of the structural
integrity of gingival epithelium may lead to further tissue destruction, a hallmark feature
of P. gingivalis-induced periodontitis [
123
]. Immunization of a mouse model with purified
RgpA or RgpB, prior to subcutaneous inoculation of P. gingivalis, inhibits abscess formation
and mortality [
124
]. This implies that gingipain immunization reduces the pathogenicity of
P. gingivalis and protects mice against tissue invasion. Similarly, bacterial preincubation
with cysteine proteinase inhibitors prevents the development of necrotic lesions along
the mouse abdomen and protects the mice from death [
125
]. Additionally, death is not
observed in mice infected with low concentrations of the double gingipain knockout (rgpA,
rgpB) strain [
126
]. Even at higher doses, survival rates for mice infected with this double-
knockout strain are significantly higher compared to mice infected with the wild-type strain.
Similarly, infection with a kgp gene knockout strain also improves survivability in mice.
Taken together, either (a) gingipain gene inactivation or (b) pretreatment of P. gingivalis
Microorganisms 2023,11, 470 7 of 27
culture with gingipain inhibitors attenuates bacterial virulence resulting in diminished
protein degradation and reduced tissue invasion.
9. Nutrient Acquisition
Iron is an essential metal required for a wide range of physiological processes, in-
cluding DNA replication/repair, mitochondrial function, myelin synthesis, and red blood
cell (RBC) mediated oxygen transport [
127
–
130
]. For most organisms, iron bioavailability
is a major requirement for cell proliferation and metabolic maintenance [
131
,
132
]. Ver-
tebrates produce several high-affinity iron-binding proteins, such as ferritin, transferrin,
hemoglobin, and hemopexin, for transport, sequestration, and prevention of cytotoxic-
ity [
133
]. Free iron is in a redox-active state, able to support the Fenton reaction and produce
reactive oxygen species (ROS), including free radicals [
134
]. Heme is composed of a por-
phyrin ring, comprising four pyrrole rings linked by methene bridges, with a bound iron at
the center of the structure [
135
]. P. gingivalis is a porphyrin auxotroph lacking the enzymatic
machinery for porphyrin biosynthesis [
5
]. As a result, this oral pathogen depends on heme
uptake from exogenous sources to meet its needs for iron and for porphyrin-linked iron,
both of which are vital nutrients for bacterial growth and proliferation [136].
RBCs represent a major iron reserve in the host, which can serve as an accessible iron
source for the nutritional requirements of P. gingivalis. Gingipains play a central role in iron
acquisition, starting with gingipain-mediated hemagglutination and hemolysis. RgpA and
Kgp contain four hemagglutinin/adhesin domains, which mediate P. gingivalis interactions
with RBCs [
27
]. Purified RgpA or Kgp is capable of inducing agglutination of RBCs,
while RgpB is not [
35
]. The triple gingipain (rgpA,rgpB,kgp) knockout strain is typically
characterized by inhibited RBCs hemagglutination potential, while single RgpA or Kgp
knockout strains support reduced RBC binding [
137
]. Agglutination of RBCs is followed by
the formation of small aggregates, allowing enzymes to slowly lyse bound RBCs, releasing
their hemoglobin. In the presence of protease inhibitors, the hemolysin activity of wild-type
P. gingivalis is suppressed [
138
]. Furthermore, Kgp-deficient strains are characterized by
their significantly reduced hemolytic function [
139
]. However, degradation of RBCs is not
inhibited when the triple gingipain (rgpA,rgpB,kgp) knockout is used, suggesting that
gingipains may not be the primary sources of the hemolytic function [137].
Hemoglobin is a globular heterotetrameric protein comprising two
α
and two
β
polypeptide chains, each of which noncovalently binds to its own single heme prosthetic
group [
140
]. Gingipains can interact with and degrade the hemoglobin protein, releasing the
heme, including its coordinated iron. High binding affinity for hemoglobin was reported
for both RgpA and Kgp, and a somewhat weaker interaction was observed for RgpB [
43
].
Furthermore, purified hemagglutinin/adhesin domains from both RgpA and Kgp have a
high binding affinity for hemoglobin [
100
]. This suggests that the RgpA or Kgp hemag-
glutinin/adhesin domains mediate protein-protein interactions between gingipains and
hemoglobin. Further, the Hgp15, or hemagglutinin/adhesin domain 2, a conserved domain
expressed in both RgpA and Kgp, can interact with the heme group [
141
]. However, unlike
purified RgpA or RgpB, only purified Kgp completely degrades hemoglobin in a time-
dependent manner [
142
]. Nevertheless, RgpA and Kgp appear to form large multimeric
protease complexes, which are effective for heme acquisition [
143
]. Gingipain-associated
hemagglutinin/adhesin domains facilitate the adhesion of the complex to hemoglobin,
localizing target protein within the range of Kgp for rapid degradation. While gingipains
are able to degrade other iron-binding proteins, including transferrin and hemopexin,
hemoglobin remains the preferred source of iron for P. gingivalis [77,142,144].
As an asaccharolytic pathogen, P. gingivalis relies on host proteins to supply its
metabolic energy and nutrient needs. Peptide fragments appear to be the preferred nu-
trient for bacterial growth. Minimal metabolism of free AAs is observed in P. gingivalis
culture media, with none becoming fully depleted [
145
]. This limited utilization of free
AAs may be due to a lack of suitable transport systems [
146
]. Washed P. gingivalis bacterial
cells incubated with peptides, and free AA, preferentially hydrolyze peptides containing
Microorganisms 2023,11, 470 8 of 27
aspartate, glutamate, leucine, and valine [
28
]. In contrast, free AAs tend to be minimally
used [
147
]. Gingipains can facilitate the breakdown of plasma proteins, such as serum
albumin or transferrin, into peptides for use as carbon and nitrogen sources as well as for
metabolic energy. The presence of a protease inhibitor in the P. gingivalis culture medium,
in addition to human serum albumin, inhibits bacterial growth and partially reduces the
ability to degrade the protein [
78
]. Furthermore, the single gene (rgpA or kgp), double
gene (rgpA,rgpB), or triple gene (rgpA,rgpB,kgp) knockout strains are unable to grow in
human serum supplemented with hemin [
137
]. However, bacterial growth is restored for
all mutant strains after the addition of exogenous peptides. Nevertheless, doubling times
are prolonged for the gingipain knockout strains, consistent with a contributing role of
gingipains in the acquisition of nutrients for optimal proliferation [137].
10. Altered Host Immune Responses to P. gingivalis
Neutralizing host immune defenses is necessary for P. gingivalis to successfully colonize
the periodontium and proliferate within the subgingival region. To this end, the bacteria
(a) evade the host immune response and (b) subvert the immune cell-mediated bacterial
clearance. Avoiding detection by the host immune system is pivotal to bacterial survival
within the oral microenvironment. However, P. gingivalis can also manipulate the host
'
s
inflammatory response. Initial infection in the oral cavity induces the production of a
multitude of chemokines and cytokines. Increased IL-1
β
, IL-6, IL-8, and tumor necrosis
factor (TNF)-
α
are released following incubation of wild-type P. gingivalis with cultured
human gingival fibroblast cells, oral epithelial cells, or periodontal ligament cells from
healthy subjects [
148
–
150
]. Mice infected with wild-type P. gingivalis have significantly
higher serum levels of IL-1
β
, IL-6, IL-8, and TNF-
α
, compared to controls or compared
to mice infected with a mutant strain deficient in functional fimbriae [
151
]. Elevated IL-
1
β
, IL-6, IL-8, IL-17, and TNF-
α
are measured in GCF, inflamed gingival tissues, or in
serum obtained from patients with periodontitis [
152
–
155
]. In this context, tissue cytokine
concentrations are dependent on the biopsy distance from the infection site, suggesting that
cytokine expression is influenced by the progression and severity of periodontitis [154].
11. Disruption of Host Inflammatory Responses to P. gingivalis
Inflammation is a physiologic host response to pathogenic infection. Infected cells
secrete proinflammatory mediators that serve as chemotactic signals to recruit immune
effector cells, which mediate bacterial clearance. IL-1
β
is a proinflammatory cytokine
commonly secreted by monocytes or macrophages [
156
]. It is also produced by other cell
types, including gingival epithelial and fibroblast cells [
157
,
158
]. In response to pathogenic
stimuli, this primary cytokine regulates the initial stages of inflammation by inducing IL-6
and IL-8 production via TLR and nuclear factor-κβ (NF-κB) signaling pathways [159,160].
IL-6 is a proinflammatory cytokine secreted by gingival and periodontal ligament
fibroblasts [
161
,
162
]. P. gingivalis-induced IL-6 expression increases bone resorption by
downregulating osteoprotegerin (OPG) [
158
]. OPG is released by osteoblast cells as a
decoy receptor that binds to and inactivates receptor activator of nuclear factor
κβ
ligand
(RANKL) function [
163
]. RANKL binding to the receptor activator of nuclear factor
κβ
(RANK) receptor triggers a signaling cascade that activates mitogen-activated protein
kinase (MAPK) and other transcription factors involved with osteoclast proliferation [
164
].
Gingipain-mediated disruption of the RANKL/OPG ratio upregulates RANKL function
and drives the system to favor osteoclast differentiation, a predominant cell-type responsi-
ble for bone matrix degradation [
165
,
166
]. The resulting alveolar bone loss is commonly
associated with periodontitis [167].
IL-8 is a chemokine secreted by gingival epithelial cells, gingival fibroblasts, and
periodontal ligament fibroblasts [
168
,
169
]. It is a dual-function inflammatory mediator
involved in neutrophil chemotaxis and activation. IL-8 secretion forms a chemoattractant
gradient that functions as a host signaling mechanism to recruit neutrophils to the infection
site [
170
]. Additionally, IL-8 triggers G protein-coupled receptor (GPCR)-phospholipase
Microorganisms 2023,11, 470 9 of 27
C (PLC) signaling pathways to activate neutrophils [
171
]. Mobilization of intracellular
calcium stores contributes to neutrophil shape polarization, adhesion, degranulation, or
oxidative burst [172–174].
TNF-
α
is a pleiotropic proinflammatory cytokine primarily secreted by activated
macrophages [
175
]. It works synergistically with RANKL to upregulate RANK receptor
expression [
176
]. TNF-
α
and RANKL signaling pathways trigger NF-
κ
B activation to
induce transcription of other transcription factors (nuclear factor of activated T-cells, cyto-
plasmic 1 (NFATc1) and c-Fos) and protease genes (cathepsin K and MMP-9), involved in
osteoclastogenesis and bone resorption, respectively [
177
]. Expression of NFATc1 and c-Fos
messenger RNA (mRNA) is increased in skull bones from mice injected with P. gingivalis
lipopolysaccharide (LPS) [
178
]. In the presence of TNF-
α
, human periodontal ligament cells
have increased expression of MMPs [
179
]. Furthermore, bone resorption is significantly
reduced in P. gingivalis-infected cathepsin K knockout mice [180].
As P. gingivalis infection progresses deeper into the periodontium, the release of
chemokines and cytokines may be altered. Membrane-expressed gingipains can exploit the
host inflammatory responses by degrading proinflammatory cytokines. Human gingival
epithelial cells treated with wild-type P. gingivalis elicit the release of IL-1
β
, IL-6, and IL-
8 [
30
]. However, IL-6 and IL-8 are rapidly digested within an hour, while IL-1
β
degradation
occurs over time. Treatment of gingival epithelial cells with the triple gingipain (rgpA,rgpB,
kgp) knockout strain, but not with the double (rgpA,rgpB) knockout, abolishes cytokine
degradation. Similarly, transient increases in IL-1
β
, IL-6, or IL-8 levels are observed upon
incubation of P. gingivalis with human gingival fibroblasts [
181
]. However, treatments with
heat-killed P. gingivalis or with gingipain-deficient strains lead to significantly higher IL-6
and IL-8 secretion from fibroblasts.
Additional inflammatory gingipain targets, including chemokine (C-C motif) ligand
(CCL) 2, CCL5, chemokine (C-X-C motif) ligand (CXCL) 1, and CXCL10, are suppressed
following a challenge of human gingival fibroblasts with P. gingivalis [
182
]. In contrast,
chemokine secretion is observed in fibroblast cell cultures after a challenge with heat-killed
P. gingivalis. Kgp is implicated as the primary gingipain involved in disrupting the cytokine
signaling network since minimal effects are observed when epithelial cells are treated
with a Kgp-null strain [
30
]. Taken together, expressed P. gingivalis gingipains promote a
persistent proinflammatory environment within the subgingival region to extract nutrients
from host proteins and evade host immune cells.
12. Subversion of Host Immune Responses by P. gingivalis
Successful colonization of the periodontium is crucial to establishing a subgingival
niche and fostering bacterial proliferation. Subsequently, long-term bacterial survival
requires evasion of the host immune responses to avoid bacterial killing. Impaired im-
mune responses enable a proinflammatory environment that favors persistent infection,
characteristic of chronic periodontitis.
The complement system, integral to the host immune defenses, comprises a group
of plasma proteins that generally contribute to pathogen identification and elimination.
However, wild-type P. gingivalis viability in human serum is not significantly affected by
incubation with functionally competent complement factors [
137
]. Single (rgpA, rgpB, or
kgp) or double (rgpA, rgpB) gingipain gene inactivation significantly reduces P. gingivalis
survival in human serum, suggesting a contributing role for these cysteine proteases
in disruption of the complement bactericidal functions. Gingipains cleave and degrade
complement components C3, C4, and C5 [
183
]. Purified gingipains cleave isolated C3 into
C3a- and C3b-like peptides. C3a is then susceptible to further time-dependent degradation
into nonfunctional fragments. In addition, purified C3 is not efficiently cleaved when
incubated with a RgpA deficient P. gingivalis strain [
36
]. Furthermore, this mutant strain
is associated with significantly higher levels of bound C3 protein, and the addition of a
protease inhibitor increases C3 binding to wild-type P. gingivalis. Uptake of the RgpA
deficient strain by polymorphonuclear leukocytes (PMNs) is also significantly improved
Microorganisms 2023,11, 470 10 of 27
in the presence of complement factors [
36
]. However, PMN-mediated uptake of the wild-
type strain is observed only at higher complement levels, consistent with the anticipated
degradation of complement factors.
C3a is a proinflammatory anaphylatoxin mediating the degranulation of eosinophils
and chemotaxis of mast cells [
184
,
185
]. The
α
-chains of C3, C4, and C5 can be proteolyt-
ically activated by low concentrations of purified RgpA, RgpB, and Kgp [
32
]. However,
at higher levels, all three gingipains further cleave C3, C4, and C5 into nonfunctional
fragments. Pretreatment of human serum with wild-type P. gingivalis improves the survival
of subsequently added Escherichia coli [
32
]. Similarly, pretreatment of human serum with
purified RgpA, RgpB, or Kgp increases the E. coli viability in a concentration-dependent
manner. The reduction of the bactericidal effect of human serum is most and least efficiently
achieved by RgpA and Kgp, respectively [32].
C4b-binding protein (C4BP) is a circulating inhibitor of the complement system [
186
].
C4BP inhibits the assembly of C3 convertase while also serving as a cofactor for proteolytic
inactivation of C3b and C4b [
186
,
187
]. The C1 complex, a multimeric protease complex
comprising one C1q molecule as well as two C1r and two C1s molecules, cleaves C2 and C4
into C2a and C2b as well as C4a and C4b, respectively [
188
]. The C2a and C4b fragments
interact to form the C3 convertase complex (C4b2a) of the classical pathway [
188
]. In turn,
the C3 convertase cleaves C3 to produce C3a and C3b fragments. Of these, the C3b serves
as an opsonizing agent targeting pathogens for phagocytosis [
189
]. Furthermore, C3b also
mediates the cleavage of C5 into C5a and C5b, allowing C5b to interact with C6-9 to form
a membrane attack complex [
188
]. This complex creates a pore through the targeted cell
membrane resulting in bacterial lysis. Surface-bound C3b on pathogens can also interact
with factor B, a serine protease, to create an alternate C3 convertase complex (C3bBb) from
the alternative pathway [
190
]. This C3bBb complex can cleave C3 to amplify the initial
complement activation signal.
C4BP can interact with the hemagglutinin/adhesin domains of RgpA gingipain [
191
].
In addition, a two-fold increase in surface deposition of C9 on P. gingavlis occurs following
bacterial incubation with human serum deficient in C4BP. Conversely, exogenously added
C4BP reduces C9 surface accumulation on P. gingivalis to levels seen with normal human
serum. The binding of Haemophilus influenzae to C4BP does not affect the cofactor function
of the complement system inhibitor [
192
]. Rather, in the presence of C4BP, reduced C3b
accumulation is observed on the pathogen surface, consistent with ongoing degradation of
C3b and C4b [
192
]. Similarly, RgpA-mediated binding to C4BP may provide P. gingivalis
with an evasion mechanism that attenuates host immune defenses by downregulating
C3b-mediated opsonization and phagocytosis.
13. Gingipains Attenuate Neutrophil Function
The release of chemokines and cytokines at an infection site leads to the recruitment of
neutrophils, which serve as the host’s first line of defense against pathogenic bacteria [
193
].
P. gingivalis can impair bacterial clearance by degrading proinflammatory mediators in-
volved with neutrophil recruitment. Reduced levels of secreted IL-8 are observed in human
gingival epithelial cells following incubation with P. gingivalis [
194
]. In addition, when
oral epithelial cells are challenged with P. gingivalis, the expected IL-8-mediated neutrophil
transmigration through the epithelial layer becomes attenuated [195].
Surface-expressed gingipains can mediate P. gingivalis virulence by disrupting neu-
trophil recruitment and neutrophil functions at the infection site. The release of TNF-
α
and several interleukins contributes toward neutrophil recruitment. PMN responses are
reduced in mice deficient in TNF receptors following wild-type P. gingivalis infection [
196
].
Moreover, TNF-
α
digestion is observed to be dose and time-dependent when murine
fibroblasts are treated with purified RgpA or RgpB [
197
]. Similarly, purified RgpA or Kgp
degrades recombinant IL-8(77), a mature 77 AA cytokine isoform, in a time-dependent
manner [198].
Microorganisms 2023,11, 470 11 of 27
Oral inoculation of P. gingivalis in mice elicits a proinflammatory response with the
release of several cytokines, including IL-17 [
199
]. However, IL-17, which is involved with
neutrophil recruitment, is susceptible to Kgp-mediated cleavage in a concentration and
time-dependent manner [
200
,
201
]. Furthermore, neutrophil accumulation in the maxillary
gingival tissue is comparable in the IL-17 receptor-deficient mice, whether infected with
wild-type P. gingivalis or sham infected [
202
]. Similarly, after another infection, the gingival
tissues from IL-17 receptor knockout mice have reduced neutrophil infiltration, comparable
to sham-infected IL-17 receptor-deficient mice. However, following a third inoculation,
wild-type mice infected with wild-type P. gingivalis have significant neutrophil infiltration
into the gingival epithelium and adjacent connective tissues compared to sham-infected
mice [
202
]. This implies that P. gingivalis tends to suppress the IL-17-mediated signaling
and the subsequent neutrophil infiltration.
Taken together, gingipains help P. gingivalis to exploit host inflammatory and neutrophil
responses. The bacterial infection triggers neutrophil recruitment via chemokine/cytokine
release. P. gingivalis expressed gingipains degrade inflammatory mediators to attenuate
neutrophil functions and promote a chronic proinflammatory environment. In turn, the
inflammation-mediated breakdown of gingival tissue provides essential nutrients for
bacterial growth.
The glycosylated transmembrane glycoprotein CD99 is expressed on the surface of
gingival epithelial, gingival fibroblast, and endothelial cells [
203
,
204
]. CD99 has a role in
neutrophil transmigration between endothelial cells [
205
]. Gingipains impair neutrophil
adhesion to host cells by interacting with and cleaving CD99. Treatment of human um-
bilical vein endothelial cells (HUVECs) with an agonistic anti-CD99 monoclonal antibody
(mAb) leads to increased expression of CAMs, including endothelial leukocyte adhesion
molecule-1 (ELAM-1), vascular cell adhesion molecule-1 (VCAM-1), and intercellular ad-
hesion molecule-1 (ICAM-1) [
204
]. In contrast, treatment of HUVECs with purified RgpA
or Kgp digests CD99 and reduces CAM expression in a dose and time-dependent manner.
RgpA appears to be the more efficient protease in this context. PMN binding to HUVECs
is reduced in the presence of purified RgpA [
204
]. However, PMN adhesion is improved
following HUVEC treatment with RgpA and an agonist anti-CD99 mAb or following
treatment with both a proteinase inhibitor treated RgpA and the mAb agonist. Thus, the
addition of gingipains preincubated with a proteinase inhibitor and treatment of HUVECs
with the agonist anti-CD99 mAb reduces CD99 proteolysis and increases CAM expres-
sion [
204
]. Together, these observations imply that gingipain-mediated CD99 hydrolysis,
along with reduced CAM expression, disrupts the recruitment of leukocytes.
Complement C5 is proteolytically activated to form C5a and C5b fragments. Gingi-
pains are able to cleave C5, releasing C5a, which can serve as an anaphylatoxin and
contribute to neutrophil chemotaxis [
183
]. This proinflammatory mediator establishes
a chemoattractant gradient to direct migrating neutrophils toward sites of bacterial in-
fection [
206
,
207
]. At low gingipain concentrations, purified C5a tends to be resistant to
further degradation by purified RgpA, RgpB, or Kgp [
32
]. Increasing gingipain concentra-
tion, however, promotes further digestion of purified C5a until nonfunctional fragments
are produced.
P. gingivalis subverts neutrophil antimicrobial functions by manipulating the comple-
ment system and the TLR signaling pathway. Cleavage of C5 by P. gingivalis gingipains
produces endogenous C5a, which interacts with the C5a receptor (C5aR), to inhibit toll-like
receptor (TLR) 2 mediated induction of IL-12 [
208
]. IL-12 is a cytokine implicated in the
recruitment of neutrophils for phagocytic bacterial clearance [
209
]. However, if wild-type
mice are injected with a gingipain deficient P. gingivalis strain, exogenously added C5a
promotes bacterial survival by reducing neutrophil-mediated phagocytosis [
199
]. Con-
versely, bacterial viability is significantly reduced if P. gingivalis-infected wild-type mice are
treated with a C5aR antagonist or an anti-TLR2 mAb [
199
]. In addition, lower bacterial cell
counts are measured in C5aR or TLR2 knockout mice infected with P. gingivalis. Similarly,
bacterial killing is increased when isolated neutrophils are challenged with P. gingivalis
Microorganisms 2023,11, 470 12 of 27
in the presence of a C5aR antagonist or with an anti-TLR2 mAb [
199
]. Furthermore, the
total viable oral bacterial counts tend to be reduced in C5aR knockout mice infected with
wild-type P. gingivalis [
210
]. Clearly, the communication between C5aR and TLR2 receptors
plays a protective role in P. gingivalis survival.
Gingipain-mediated C5aR cleavage can be observed when isolated human neutrophils
are incubated with purified Kgp [
44
]. Prior pretreatment of Kgp with proteinase inhibitors
suppresses the C5aR degradation. Such C5aR digestion diminishes calcium flux and
MPO release, implying Kgp-mediated functional inactivation of neutrophils. Together,
gingipain-mediated C5aR degradation represents an alternative form of P. gingivalis viru-
lence, attenuating neutrophil function and enhancing bacterial survival. Suppression of the
neutrophil antimicrobial function may further lead to the overt growth of other common
bacterial species in oral biofilm. Consequently, this can remodel the host commensal micro-
biota into a dysbiotic state favoring proinflammatory conditions and further aggravating
periodontitis's pathogenesis.
Myeloid differentiation primary response 88 (MyD88) is a downstream adaptor pro-
tein that mediates TLR2 signaling for the induction of proinflammatory cytokines and
for the host inflammatory response [
211
]. This suggests that MyD88 may not contribute
to P. gingivalis evasion of neutrophils. On the contrary, P. gingivalis viability is higher in
MyD88 knockout mice [
199
]. In addition, the bacterial killing of P. gingivalis via phagocyto-
sis is MyD88 dependent [
212
]. However, time-dependent degradation of the MyD88 protein
is observed when isolated neutrophils are incubated with P. gingivalis [
190
]. Moreover,
this P. gingivalis-mediated digestion of MyD88 is reduced after treatment with a C5aR
antagonist or with an anti-TLR2 mAb [
199
]. This implies that P. gingivalis survival involves
the exploitation of the C5aR-TLR2 receptor crosstalk and inactivation of the MyD88 protein.
14. P. gingivalis Induced Periodontitis
Periodontitis is a proinflammatory state mediated by a pathogenic infection within
subgingival tissues. The pathophysiology of periodontitis includes a chronic inflammatory
environment that may ultimately progress to a breakdown of gingival tissues, including
periodontal ligament and destruction of supporting structures for teeth [
213
]. Gradual
loss of gingival epithelial attachment to the tooth enamel surface creates deep periodontal
pockets that enable further accumulation of biofilm [214].
Removal of the oral biofilm is key to reducing the tissue destruction associated with
periodontitis. Scaling and root planing (SRP) remains the gold standard for non-surgical
therapy in patients [
215
]. However, bacterial re-colonization continues to be a limitation of
SRP treatment [
216
]. Other therapeutic approaches are being investigated, including the
use of biotics (prebiotics, probiotics, paraprobiotics, lysates, and post-biotics) and various
natural compounds [
217
,
218
]. Further investigations into such adjunct therapies may
provide additional options for controlling microbial biofilm characteristics and modifying
clinical outcomes of oral infections, including those with P. gingivalis.
Contributing factors to the formation of oral biofilm include increased opportunistic
bacterial colonization, periodontitis, dental prosthetics, poor oral hygiene, and smok-
ing [
219
–
221
]. Specifically, implants may be associated with increased biofilm formation,
inflammation, and periodontitis [
222
]. In such circumstances, supportive periodontal
therapy and preventative oral hygiene practice can enhance the success rate of dental
prostheses [
223
–
225
]. Additionally, in order to control microbial growth, new approaches
are being explored, including a variety of nanotechnologies [226–228].
Biofilm formation comprises cell-to-cell interactions among multiple bacterial species.
Early colonizers of the tooth surface include gram-positive anaerobic bacteria such as
Actinomyces (A. oris), Streptococcus (S. gordonii and S. mutans), or Veillonella (V. denticariosi
and V. parvula) [
229
,
230
]. The abundance of Actinomyces sp. and Streptococcus sp., based
on meta-transcriptome analyses of human supragingival dental biofilm, was 3–12% and
12–19%, respectively [
231
]. Following their attachment to the pellicle-coated surface of
teeth, initial colonizers can facilitate interactions with late colonizers, such as P. gingivalis.
Microorganisms 2023,11, 470 13 of 27
The surface-expressed polypeptides of S. gordonii, Streptococcal surface protein A (SspA),
and SspB interact with Mfa1, a protein component of the P. gingivalis fimbriae [
232
,
233
].
Mfa1 binds to the SspB adherence region (BAR), a discrete region on SspB [234].
Secondary colonizers, such as Prevotella intermedia,Aggregatibacter actinomycetemcomi-
tans, and Fusobacterium nucleatum, can interact with early colonizers [
235
]. Subsequently,
late colonizers can bind to these secondary colonizers. High interactions between F. nuclea-
tum and P. gingivalis are observed in coaggregation assays [
236
]. P. gingivalis demonstrated
reduced integration into biofilms formed by mutant F. nucleatum strains deficient in the
outer membrane proteins, fibroblast activation protein 2 (Fap2), and arginine (R)-inhibitable
adhesin (RadD) [236].
Interactions between P. gingivalis and F. nucleatum involve a galactoside moiety and a
lectin-like adhesin (FomA), respectively [
237
–
239
]. Furthermore, the CPS and LPS isolated
from P. gingivalis PK 1924 (serotype K5) can bind to F. nucleatum [
240
]. Consequently, the role
of these ‘bridge’ bacteria is to mediate the coaggregation of early and late colonizers [
241
].
Together, dental biofilm encompasses a diverse bacterial community of over 300 species [
16
].
Keystone pathogens, such as P. gingivalis, transform the biofilm microbiota into a dysbiotic
community, which undermines the host immune response and exploits the inflammatory
responses to infection. Biofilm buildup propagates persistent chemokine and cytokine
production, which is associated with bacterially induced-inflammation of gingival tis-
sues [
242
]. Ultimately, the diseased pathological state of the periodontium is characterized
by irreversible tissue destruction and alveolar bone loss.
15. Pathogen Mediated Dysbiosis
During biofilm formation, P. gingivalis, as a late colonizer that adheres to earlier colo-
nizers, is identified as a keystone pathogen implicated in the pathogenesis and progression
of periodontitis [
18
,
243
]. The polymicrobial dysbiosis model implies that a synergistic
equilibrium exists between host gingival tissue and the microbial community [
244
]. Under
physiologic conditions, the oral microbiota comprises heterotypic microbes residing in a
controlled symbiotic environment. Host inflammatory and immune responses regulate ex-
cessive bacterial proliferation and neutralize overt bacterial pathogenicity [
245
]. Ordinarily,
such homeostasis between the host and the commensal microbiota helps to maintain a bal-
anced state of periodontal health [
246
]. However, during periodontitis, infectious microbes,
such as P. gingivalis, disrupt this homeostatic balance and shift the commensal microbial
community to a pathogenic state [
243
]. Even at low abundance, P. gingivalis mediates a
reinforcing cycle of periodontal dysbiosis leading to enhanced bacterial pathogenicity [
247
].
This opportunistic pathogen manipulates host responses, locally attenuating the immune
system while avoiding total immunosuppression [248]. Chronic infection promotes a con-
tinuing proinflammatory environment, including a prominent role of gingipains in the
enhanced destructiveness of P. gingivalis. Inflammation and gingipain-mediated degra-
dation of gingival tissue proteins provide peptides, iron, and other nutrients crucial for
bacterial growth and further progression of infection.
Dysbiotic polymicrobial communities characteristically develop increased reliance
on the nutrients from the serum-like transudate produced during periodontal inflamma-
tion [
249
]. The adoption of a proteolytic phenotype enables all members of this community
to thrive. This is in clear contrast to growth limitations when individual members are
tested in isolation. Gingipain expression from P. gingivalis contributes to the growth of
such a microbial community. However, the expression and release of such proteases in
the periodontal pocket also appear to be coordinated via signaling from other community
members [
249
]. This microbially driven feedforward inflammatory loop implies a symbiotic
enhancement of the overall virulence potential for progressively faster tissue breakdown
and microbial growth [
250
]. Moreover, interactions of P. gingivalis with the host as well
as other microbial surfaces, are further aided by the adhesive properties of fimbriae [
251
].
Consequently, the microbial biofilm becomes difficult to displace, leading to continued
invasion and persistent tissue destruction.
Microorganisms 2023,11, 470 14 of 27
In turn, the host tissues respond by upregulating the expression of various genes,
including ferric ion binding protein, several proto-oncogenes, an ankryn repeat, and a
β
-enolase [
252
]. The host iron-binding protein may compete with the microbial community
for its iron requirements. The overexpression of the ankryn repeat is commonly associated
with various diseases, such as cancer or cardiovascular disorders [
253
,
254
]. Similarly,
β
-enolase has been associated with metabolism in cancer cells [
255
]. Together, these trends
suggest that chronic inflammation may be associated with increased risks for other diseases,
possibly including cancer.
16. P. gingivalis and Coagulation
The pathogenicity of P. gingivalis can potentially extend beyond the oral cavity. Tis-
sue damage from routine oral hygiene practices or dental procedures may facilitate the
entry of the periodontopathogen into the systemic circulation [
256
]. P. gingivalis can trig-
ger the activation of prothrombotic mediators, including platelets, increasing the risk for
thrombosis [
120
]. Concentration-dependent shortening of plasma clotting time is observed
when human plasma is incubated with purified RgpA or RgpB [
37
]. P. gingivalis expressed
gingipains are cysteine proteases, which can activate plasma serine protease coagulation
factors [
120
]. Purified RgpA proteolytically activates factor IX (FIX), factor X (FX), or
prothrombin in a concentration and time-dependent manner [
37
–
39
]. In contrast, purified
RgpB cleavage of inactive zymogens yields minimal to no activated factor IX (FIXa), ac-
tivated factor X (FXa), or thrombin [
37
–
39
]. The addition of phospholipids and calcium
ions, two contributing clotting cofactors, further enhances the RgpA-mediated activation.
Moreover, in the presence of phospholipids and calcium ions, RgpA catalytic efficiency
(k
cat
/K
m
) of FIX activation is comparable to that observed for a physiological activator,
activated factor VII (FVIIa)-tissue factor (TF) complex [
39
]. However, RgpA is less efficient
at FX or prothrombin activation compared to FVIIa-TF or FXa-activated factor V (FVa)
complex, respectively [
37
,
38
]. Several snake venoms contain enzymes known to activate
coagulation factors, including FX or prothrombin [
257
,
258
]. In this context, RgpA-mediated
FX activation is comparable to that of Russell’s viper venom [
37
]. In addition, the prothrom-
bin activation rate by RgpA is higher compared to Notechis scutulus scutulus venom but
lower compared to venom from Oxyuranus scutellatus [
38
]. Taken together, FIX, FX, and
prothrombin activation by RgpA may be contributing factors in thrombin production.
17. The Role of Gingipains in Platelet Function
Bacterial infection is often transient for individuals with a robust immune system.
However, serious thrombotic complications can develop from persistent infection, including
infective endocarditis and sepsis-associated disseminated intravascular coagulation [
259
].
A distinct feature is the bacterially mediated platelet activation leading to the formation
of intravascular thrombi [
260
]. P. gingivalis interaction with platelets can induce platelet
activation and subsequent aggregation [
261
]. Intracellular calcium mobilization is asso-
ciated with platelet activation [
262
]. Consistent with this, if treated with live P. gingivalis,
isolated platelets undergo intracellular calcium mobilization [
261
]. However, this does not
occur if resting platelets are treated with heat-killed bacteria or with the double (rgpA and
rgpB) gingipain knockout mutant. A single (kgp) gingipain mutant did elicit changes in
intracellular calcium levels, however, this was significantly lower compared to platelet
exposure to the wild-type strain. Similarly, platelet aggregation is observed following the
incubation of P. gingivalis with isolated platelets. However, platelet aggregation depends
on the ratio of platelet/bacteria, consistent with the possibility of either a threshold phe-
nomenon or multiple competing platelet interactions. In whole blood, platelet expression
of CD62P, an adhesion molecule expressed on surfaces of activated platelets, increases
following preincubation of high P. gingivalis colony-forming units (CFU) with or without
subsequent ADP stimulation [
263
]. Conversely, there is a trend towards higher CD62P
expression even in response to low P. gingivalis CFU, particularly as preincubation time
Microorganisms 2023,11, 470 15 of 27
is extended. This suggests a dose and a time dependence for the impact of P. gingivalis
preincubation on platelet surface CD62P expression.
High levels of P. gingivalis may promote an excitable state in platelets that results in
rapid activation following subsequent interaction with physiologic agonists. At lower P.
gingivalis levels, platelet responses may be triggered with prolonged preincubation times.
In this context, whole blood from generalized aggressive periodontitis and periodontitis
patients is associated with higher platelet activation [
264
,
265
]. Moreover, robust platelet
aggregation is observed after incubation of P. gingivalis with whole blood from patients
with the peripheral arterial disease (PAD) [266]. Similarly, agonist-dependent increases in
platelet P-selectin expression are observed after systemic P. gingivalis infusion into rats [
267
].
Preincubation of P. gingivalis with whole blood also impacts platelet plug formation under
shear conditions [
268
]. Extending P. gingivalis preincubation times past 7.5 min significantly
reduces the time for platelet plug-mediated aperture occlusion in the Platelet Function
Analyzer (PFA-100). Thus, platelet plug formation time in whole blood is affected both by
the P. gingivalis concentration and by the duration of bacterial preincubation.
Interestingly, a prolongation of the occlusion time can be observed at certain P. gingi-
valis levels below those needed for the occlusion time shortening [
268
]. This is explainable
either (a) by ineffective platelet activation or (b) by alternate platelet activation pathways.
During the bacterial preincubation phase, platelets may become activated in response to
interaction with P. gingivalis. However, the activated platelets may be insufficient to trigger
full platelet aggregation. Consequently, the spent activated platelets become refractory to
platelet plug formation, leading to a prolonged occlusion time. Alternatively, if P. gingivalis
is capable of interacting with multiple platelet activation pathways with characteristic
interaction affinities, then multiple platelet functions could be triggered in a concentration-
dependent manner. As a result, P. gingivalis in whole blood may trigger a variety of
time-dependent processes, some of which are possibly functionally opposing [268].
Platelets are involved in a variety of ways with leukocyte functions, including those of
neutrophils. Platelet-neutrophil interactions are believed to be mediated by an interaction
between platelet P-selectin and neutrophil P-selectin glycoprotein ligand-1 (PSGL-1) [
269
].
Such interaction is enhanced following ADP-mediated platelet activation [
263
]. Platelet-
neutrophil interactions are also enhanced in the presence of P. gingivalis in a preincubation
time-dependent manner. Moreover, bacterial exposure to whole blood can trigger the
neutrophil release of nuclear DNA, also known as neutrophil extracellular traps (NETs).
Such release of NETs in response to P. gingivalis is known to be at least in part dependent
on an interaction between activated platelets and neutrophils [
263
]. This implies that the
interaction of P. gingivalis with the various blood cells does not only potentially alter their
cell-specific functions in response to this pathogen but can also impact their physiologic
cell-cell interactions.
Protease-activated receptors (PARs), members of the GPCR family, are characterized
by a unique activation mechanism. The amino terminus of PARs is cleaved to expose
an auto-activating tethered ligand that triggers intracellular signal transduction via an
internal salt bridge formation [
270
]. Human platelets express two types of PARs, PAR-1,
and PAR-4. These receptors are normally proteolytically activated by the serine protease
thrombin as one of the mechanisms of platelet activation [
271
]. P. gingivalis expressed
gingipains, however, can also cleave and activate PAR-1 and PAR-4 [
40
]. RgpA is up to
six-fold more efficient in activating PAR-4 compared to thrombin [
40
]. Thrombin, however,
is significantly more efficient at PAR-1 activation compared to either RgpA or RgpB [
40
].
The particular activation efficiency of PAR-1 by thrombin is likely due to a hirudin-like
sequence contained within the exodomain of PAR-1, which binds with high affinity to the
anion-binding exosite of thrombin [
272
]. Furthermore, cytosolic calcium levels are increased
following the incubation of isolated platelets with purified RgpA or RgpB. Pretreatment of
platelets with an anti–PAR-1 antibody abrogates this effect, supporting the role of arginine
gingipain dependent PAR-1 cleavage in platelet calcium activities [
40
]. Similarly, the
treatment of platelets with a protease inhibitor completely abolished this effect [
40
]. In
Microorganisms 2023,11, 470 16 of 27
this context, lower levels of RgpA are required to induce platelet aggregation compared to
RgpB, emphasizing its higher efficiency at mediating platelet responses.
However, the proteolytic functions of gingipains are not solely responsible for mediat-
ing platelet aggregation. In the presence of P. gingivalis, platelet aggregation is observed in
platelet-rich plasma (PRP) treated individually or in combination with inhibitors for Rgp
or Kgp [
273
]. This suggests that other bacterial products may also mediate some platelet
aggregating effects. Hgp44, an adhesin domain expressed at the C-termini of RgpA and
Kgp, plays a role in hemagglutination and hemoglobin binding [
27
]. Incubating PRP with
a mutant P. gingivalis strain deficient in adhesin domains only or with a strain deficient
in Rgp, Kgp, and adhesin domains does not induce platelet aggregation [
273
]. However,
platelet aggregating potential is restored when a recombinant Hgp44 is preincubated with
either mutant strain prior to incubation with PRP. Furthermore, incubating PRP with P.
gingivalis in the presence of anti-Fc
γ
RIIa mAb inhibits platelet aggregation [
273
]. Similarly,
platelet aggregation can be somewhat reduced when PRP is incubated with P. gingivalis
and an anti-glycoprotein (GP) Ib
α
mAb. However, aggregation of washed platelets, treated
with gingipain deficient strains, is restored if anti-P. gingivalis immunoglobulin G (IgG) is
added [
273
]. Taken together, P. gingivalis can induce platelet aggregation independent of
gingipains via pathways that involve contributing roles from FcγRIIa, IgG, and GPIbα.
18. Limitations
This review represents the current understanding from a basic science perspective of
the role of P. gingivalis in the pathogenesis of oral inflammatory processes. In addition, the
potential impact on the functioning of certain blood cells, such as platelets and neutrophils,
is also considered, particularly regarding their subsequent roles in increased prothrombotic
risks. However, this review does not represent an encyclopedic compilation of everything
currently known about this pathogen. Similarly, it does not detail the impact of this
pathogen on every cell type that it may come in contact with and possibly infect. Of
particular interest might be the effects of P. gingivalis on the cells and tissues of blood vessel
walls. That would be a worthy topic for a review in its own right.
19. Conclusions
P. gingivalis is an opportunistic pathogen that infects the subgingival tissues of the
oral cavity. Virulence factors, most notably arginine- and lysine-specific gingipains, play a
central role in mediating P. gingivalis pathogenicity. Gingipains are involved with all aspects
of P. gingivalis-induced infection, including nutrient acquisition essential for bacterial
growth, tissue breakdown to facilitate bacterial invasion, and degradation of cytokines
to disrupt the host inflammatory responses. Gingipains also contribute to P. gingivalis-
mediated subversion of host immune responses by cleaving complement proteins and
exploiting the TLR signaling pathway to attenuate neutrophil functions. Ineffective bacterial
clearance reinforces persistent P. gingivalis infection and propagates chronic inflammation,
driving the periodontitis pathophysiology. A growing body of evidence further suggests a
potential circulatory prothrombotic role of P. gingivalis. Platelet activation and aggregation
are observed in the presence of this periodontopathogen. Future studies should include
further characterization of the thrombotic mechanisms triggered by P. gingivalis and its
expressed virulence factors.
Author Contributions:
Conceptualization, writing—original draft preparation, W.A.C.; writing—
review and editing, W.A.C., Y.D., H.M.F. and D.S.B. All authors have read and agreed to the published
version of the manuscript.
Funding: NIH grants to H.M.F.: R-56-DE13664, DE019730, DE022508, DE022724.
Data Availability Statement:
This is a narative review that largely relies on published peer-reviewed
research articles and discussions. Data is presented with citations of supporting source publications.
Conflicts of Interest: The authors declare no conflict of interest.
Microorganisms 2023,11, 470 17 of 27
Abbreviations
AAs: amino acids; BAR, SspB adherence region; C, component; C3bBb, C3 convertase complex;
C4b2a, C3 convertase complex; C4BP, C4b-binding protein; C5aR, C5a receptor; CAMs, cell adhesion
molecules; CCL, chemokine (C-C motif) ligand; CFU, colony-forming unit; CPS, capsular polysac-
charides; CXCL, chemokine (C-X-C motif) ligand; DNA, deoxyribonucleic acid; ECM, extracellular
matrix; ELAM-1, endothelial leukocyte adhesion molecule-1; Fap2, fibroblast activation protein 2;
Fim, fimbrillin; FIX, factor IX; FIXa, activated factor IX; FVa, activated factor V; FVIIa, activated factor
VII; FX, factor X; FXa, activated factor X; GP, glycoprotein; GCF, gingival crevicular fluid; GPCR, G
protein-coupled receptor; HUVECs, human umbilical vein endothelial cells; ICAM-1 intercellular
adhesion molecule-1; IgG immunoglobulin G; IL, interleukin; k
cat
/K
m
, catalytic efficiency; Kgp,
lysine gingipain; LPS, lipopolysaccharide; mAb, monoclonal antibody; MALDI–TOF MS, matrix-
assisted laser desorption ionization–time of flight mass spectrometry; MAPK, mitogen-activated
protein kinase; MDCK, Madin-Darby canine kidney; Mfa, Minor fimbrial antigen; MMPs, matrix
metalloproteinases; mRNA, messenger RNA; MYD88, Myeloid differentiation primary response 88;
N-acetylglucosamine, 2-acetamido-2-deoxy-d-glucose; NFATc1, nuclear factor of activated T-cells, cy-
toplasmic 1; NGS, next generation sequence; OPG, osteoprotegerin; PAD, peripheral arterial disease;
PARs, protease-activated receptors; PLC, phospholipase C; PMNs, polymorphonuclear leukocytes;
PRP, platelet-rich-plasma; PSGL-1, P-selectin glycoprotein ligand-1; RadD, arginine (R)-inhibitable
adhesin; RANK, receptor activator of nuclear factor
κβ
; RANKL, receptor activator of nuclear factor
κβ
ligand; RBCs, red blood cells; RgpA, arginine gingipain A; RgpB, arginine gingipain B; RNA,
ribonucleic acid; ROS, reactive oxygen species; rRNA, ribosomal RNA; SRP, Scaling and root planing;
Ssp, Streptococcal surface protein; TF, tissue factor; TLR, toll-like receptor; TNF, tumor necrosis factor;
VCAM-1, vascular cell adhesion molecule-1; vWF, von Willebrand Factor.
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