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. 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
[20]. Gradual loss of gingival epithelial attachment to the tooth enamel surface creates deep periodontal pockets that enable further accumulation of biofilm
[21].
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
[22]. However, bacterial re-colonization continues to be a limitation of SRP treatment
[23]. Other therapeutic approaches are being investigated, including the use of biotics (prebiotics, probiotics, paraprobiotics, lysates, and post-biotics) and various natural compounds
[24][25]. 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 smoking
[26][27][28]. Specifically, implants may be associated with increased biofilm formation, inflammation, and periodontitis
[29]. In such circumstances, supportive periodontal therapy and preventative oral hygiene practice can enhance the success rate of dental prostheses
[30][31][32]. Additionally, in order to control microbial growth, new approaches are being explored, including a variety of nanotechnologies
[33][34][35].
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)
[36][37]. 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
[38]. Following their attachment to the pellicle-coated surface of teeth, initial colonizers can facilitate interactions with late colonizers, such as
P. gingivalis. 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
[39][40]. Mfa1 binds to the SspB adherence region (BAR), a discrete region on SspB
[41].
Secondary colonizers, such as
Prevotella intermedia,
Aggregatibacter actinomycetemcomitans, and
Fusobacterium nucleatum, can interact with early colonizers
[42]. Subsequently, late colonizers can bind to these secondary colonizers. High interactions between
F. nucleatum and
P. gingivalis are observed in coaggregation assays
[43].
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)
[43].
Interactions between
P. gingivalis and
F. nucleatum involve a galactoside moiety and a lectin-like adhesin (FomA), respectively
[44][45][46]. Furthermore, the CPS and LPS isolated from
P. gingivalis PK 1924 (serotype K5) can bind to
F. nucleatum [47]. Consequently, the role of these ‘bridge’ bacteria is to mediate the coaggregation of early and late colonizers
[48].
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 tissues
[49]. Ultimately, the diseased pathological state of the periodontium is characterized by irreversible tissue destruction and alveolar bone loss.
3. Pathogen Mediated Dysbiosis
During biofilm formation,
P. gingivalis, as a late colonizer that adheres to earlier colonizers, is identified as a keystone pathogen implicated in the pathogenesis and progression of periodontitis
[18][50]. The polymicrobial dysbiosis model implies that a synergistic equilibrium exists between host gingival tissue and the microbial community
[51]. Under physiologic conditions, the oral microbiota comprises heterotypic microbes residing in a controlled symbiotic environment. Host inflammatory and immune responses regulate excessive bacterial proliferation and neutralize overt bacterial pathogenicity
[52]. Ordinarily, such homeostasis between the host and the commensal microbiota helps to maintain a balanced state of periodontal health
[53]. However, during periodontitis, infectious microbes, such as
P. gingivalis, disrupt this homeostatic balance and shift the commensal microbial community to a pathogenic state
[50]. Even at low abundance,
P. gingivalis mediates a reinforcing cycle of periodontal dysbiosis leading to enhanced bacterial pathogenicity
[54]. This opportunistic pathogen manipulates host responses, locally attenuating the immune system while avoiding total immunosuppression
[55]. Chronic infection promotes a continuing proinflammatory environment, including a prominent role of gingipains in the enhanced destructiveness of
P. gingivalis. Inflammation and gingipain-mediated degradation 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 inflammation
[56]. 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
[56]. This microbially driven feedforward inflammatory loop implies a symbiotic enhancement of the overall virulence potential for progressively faster tissue breakdown and microbial growth
[57]. Moreover, interactions of
P. gingivalis with the host as well as other microbial surfaces, are further aided by the adhesive properties of fimbriae
[58]. Consequently, the microbial biofilm becomes difficult to displace, leading to continued invasion and persistent tissue destruction.
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
[59]. 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
[60][61]. Similarly, β-enolase has been associated with metabolism in cancer cells
[62]. Together, these trends suggest that chronic inflammation may be associated with increased risks for other diseases, possibly including cancer.
4. P. gingivalis and Coagulation
The pathogenicity of
P. gingivalis can potentially extend beyond the oral cavity. Tissue damage from routine oral hygiene practices or dental procedures may facilitate the entry of the periodontopathogen into the systemic circulation
[63].
P. gingivalis can trigger the activation of prothrombotic mediators, including platelets, increasing the risk for thrombosis
[64]. Concentration-dependent shortening of plasma clotting time is observed when human plasma is incubated with purified RgpA or RgpB
[65].
P. gingivalis expressed gingipains are cysteine proteases, which can activate plasma serine protease coagulation factors
[64]. Purified RgpA proteolytically activates factor IX (FIX), factor X (FX), or prothrombin in a concentration and time-dependent manner
[65][66][67]. In contrast, purified RgpB cleavage of inactive zymogens yields minimal to no activated factor IX (FIXa), activated factor X (FXa), or thrombin
[65][66][67]. 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
[67]. However, RgpA is less efficient at FX or prothrombin activation compared to FVIIa-TF or FXa-activated factor V (FVa) complex, respectively
[65][66]. Several snake venoms contain enzymes known to activate coagulation factors, including FX or prothrombin
[68][69]. In this context, RgpA-mediated FX activation is comparable to that of Russell’s viper venom
[65]. In addition, the prothrombin activation rate by RgpA is higher compared to
Notechis scutulus scutulus venom but lower compared to venom from
Oxyuranus scutellatus [66]. Taken together, FIX, FX, and prothrombin activation by RgpA may be contributing factors in thrombin production.
5. 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
[70]. A distinct feature is the bacterially mediated platelet activation leading to the formation of intravascular thrombi
[71].
P. gingivalis interaction with platelets can induce platelet activation and subsequent aggregation
[72]. Intracellular calcium mobilization is associated with platelet activation
[73]. Consistent with this, if treated with live
P. gingivalis, isolated platelets undergo intracellular calcium mobilization
[72]. 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 phenomenon or with 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
[74]. Conversely, there is a trend towards higher CD62P expression even in response to low
P. gingivalis CFU, particularly as preincubation time is extended. This suggests a dose and 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
[75][76]. Moreover, robust platelet aggregation is observed after incubation of
P. gingivalis with whole blood from patients with the peripheral arterial disease (PAD)
[77]. Similarly, agonist-dependent increases in platelet P-selectin expression are observed after systemic
P. gingivalis infusion into rats
[78]. Preincubation of
P. gingivalis with whole blood also impacts platelet plug formation under shear conditions
[79]. 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. gingivalis levels below those needed for the occlusion time shortening
[79]. 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
[79].
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)
[80]. Such interaction is enhanced following ADP-mediated platelet activation
[74]. 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
[74]. 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
[81]. 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
[82].
P. gingivalis expressed gingipains, however, can also cleave and activate PAR-1 and PAR-4
[83]. RgpA is up to six-fold more efficient in activating PAR-4 compared to thrombin
[83]. Thrombin, however, is significantly more efficient at PAR-1 activation compared to either RgpA or RgpB
[83]. 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
[84]. 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
[83]. Similarly, the treatment of platelets with a protease inhibitor completely abolished this effect
[83]. In 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 mediating 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
[85]. 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
[86]. 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
[85]. 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
γRIIa mAb inhibits platelet aggregation
[85]. Similarly, platelet aggregation can be somewhat reduced when PRP is incubated with
P. gingivalis and an anti-glycoprotein (GP) Ibα mAb. However, aggregation of washed platelets, treated with gingipain deficient strains, is restored if anti-
P. gingivalis immunoglobulin G (IgG) is added
[85]. Taken together,
P. gingivalis can induce platelet aggregation independent of gingipains via pathways that involve contributing roles from Fc
γRIIa, IgG, and GPIbα.
6. Conclusions
Porphyromonas gingivalis is a gram-negative anaerobic opportunistic pathogen that infects the subgingival tissues of the oral cavity. It is a late colonizer that disrupts the relationship between the local commensal microbes and the host. Consequently, P. gingivalis is a leading etiological agent in periodontitis. 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.