Pathogenic Features of Porphyromonas Gingivalis Influence Progression of Rheumatoid Arthritis

The posttranslational modification of proteins is an important determinant of protein function. One type of posttranslational modification recently attracting attention is citrullination of peptidyl-arginine residues in protein and peptides catalyzed in mammals by five peptidyl arginine deiminases (PADs) isotypes play an essential role in many physiological processes (Tilvawala et al. 2018). Unfortunately, excessive citrullination associated with inflammatory responses can lead to generation of autoantibodies recognizing citrullinated epitopes. The Porphyromonas gingivalis enzyme peptidyl-arginine deiminase (PPAD) is also responsible for citrullination (Pyrc et al. 2012), and citrullination by enzymes of both human and bacterial origin promotes autoimmune diseases (Koziel et al. 2014; Ciaston et al. 2022; Krutyholowa et al. 2022; Wielento et al. 2022). The molecular mechanism of arginine modification and its role in rheumatoid arthritis (RA) are the subjects of our presentation.

The oral microbiome is a highly diverse and dynamic environment, and bacteria in the buccal cavity participate in the nutrient metabolism of the host. The first bacterial colonies appear immediately after birth, originating from the mother (Struzycka 2014). The biological characteristics of the oral cavity determine which microorganisms will successfully colonize and predominate the buccal microbiome. Each area of the mouth supports an individual microbiota community with specific characteristics. Moreover, there is a dynamic connection between the activity and composition of the oral microflora and the host environment (Marsh et al. 2016). Initially, the oral microbiome of the neonate primarily contains Streptococcus bacteria, such as S. salivarius, S. mitis, and S. oralis (Struzycka 2014). These species are called the “pioneer community”. Other populations colonize the oral cavity after the metabolic activity of the pioneer community modifies it. After tooth eruption, the diversity of the bacterial species in the oral cavity increases, predominantly around the gingival margins of the newly erupted teeth (Marsh et al. 2016). The oral microbiome becomes more diverse with time, with mainly anaerobic and Gram-negative bacteria, including Fusobacterium nucleatum, Prevotella melaninogenica, and Veilonella. The most stable oral microbiome is generally achieved in the young adult. It is represented by the genera Streptococcus, Veilonella, Fusobacterium, Porphyromonas, Prevotella, Treponema, Neisseria, Eubacteria, Haemophilus, Propionibacterium, Lactobacillus, and Leptotrichia (Struzycka 2014).

Both autogenic and allogenic succession are involved in its development to achieve such a diverse microbial community. In autogenic succession, microbial factors are responsible for microbiome development, whereas in allogenic succession, nonmicrobial factors influence the community (Marsh et al. 2016). The developmental process of the climax community in the human oral cavity is quite complex. Over time, the microbiota remains relatively stable. Still, unexpected and substantial disturbances in the local environment can cause severe disorders of the microbial community, which may lead to several diseases. According to an oral microbiome database (www.homd.org), 688 species of bacteria have been identified in the oral microbiome. Moreover, up to 50% of these species are uncultivated. Factors that significantly influence the oral microbiome include temperature, pH, oxidoreductive potential, and saliva’s nutrients, enzymes, and metabolites (Nelson 2011, Malinowska et al. 2017). During the lifetime of an individual, the oral microbiota changes in response to modifications to the oral habitat, the loss or eruption of teeth, lifestyle changes, or changes in the immune system (Marsh et al. 2016). 16S ribosomal RNA (rRNA) sequencing has shown that the oral microbiome was altered, for example, in patients with stomach cancer (Hu et al. 2015).

Bacteria mainly exist in the mouth in the form of biofilms. Most oral biofilms are composed of Grampositive and Gram-negative bacteria, including aerotolerant and anaerobic bacteria (Salyers and Whitt 2003). An oral biofilm is very complex, and its structure is determined by its location in the mouth (e.g., subgingival, supragingival). Gram-positive bacteria are present on most oral surfaces, with Streptococcus and Actinomyces dominant. These species are located at both healthy and diseased sites, and the only difference between them is their prevalence in the oral microbiome of the host. Gram-negative bacteria vary greatly and include obligative, facultatively anaerobic, or even microaerophilic species (Marsh et al. 2016). Some bacterial species are responsible for plaque formation on the surfaces of teeth. The prevalence and diversity of different species change over time, and new species, such as the spirochete Treponema denticola, are now found with Veillonella, Prevotella, or Propionibacterium (Salyers and Whitt 2003) are incorporated into oral biofilms. Gram-positive bacteria, such as Streptococcus, are dominant in the supragingival regions, whereas Gramnegative bacteria, such as P. gingivalis, are more common in the subgingival space (Chalas et al. 2015), He and Shi 2009).

P. gingivalis is one of the main pathogens responsible for periodontal disease (PD). Periodontitis has not been linked directly to the appearance of certain types of bacteria. Still, it is initiated by a dysregulated immune response, which changes the proportions of various bacterial species in the subgingival dental plaque. This leads to the mixed activities of different bacteria, predominantly anaerobic and proteolytic Gram-negative bacteria, causing an inflammatory response in the deeper periodontal sockets, in which species such as Tannerella forsythia, P. gingivalis, and T. denticola are found (Marsh et al. 2016). Meyle and his colleague have provided a list of critical changes that occur during the development of PD. In general, the host provides all the nutrients to the oral microbiota in the gingival crevicular fluid, and the release of various proteins by microbes can trigger the host’s immune response. The growth of species such as Fusobacterium nucleatum is promoted, influencing its environment via its “quorum sensing” ability. This elicits a stronger response from the host immune system, causing gingival inflammation and stimulating the proliferation of P. gingivalis. In susceptible hosts, the dysbiosis of the oral microbiota can trigger an excessive immune response, with the subsequent overproduction of reactive oxygen species, cytokines, and matrix metalloproteinases, which cause severe tissue damage. These are the first steps in the development of PD. The disease’s further progression involves angiogenesis, which, rather than healing the damaged tissue, leads to chronic inflammation (Meyle and Chapple 2015). The detailed mechanism underlying the progression of periodontitis is described in Meyle and Chapple article (2015), and its mechanism is highly complex. Periodontal disease irreversibly destroys the tissue surrounding the teeth, with the simultaneous loss of attachment between the bone and the teeth. Why PD develops predominantly in older adults is still unclear. Like increased levels of P. gingivalis in the oral microbiota, PD can precipitate autoimmune disorders, such as RA (Koziel et al. 2014).

Periodontal disease is the chronic inflammation of the tissues surrounding the teeth, and P. gingivalis is one of the major pathogens recorded in periodontitis. The colonization of the teeth by pathogenic bacteria, such as T. forsythia, T. denticola, and P. gingivalis, causes the subsequent development of the pathogenic dental plaque. Therefore, these species are called the “red complex” because they are strongly associated with severe periodontitis and are thought to initiate the disease. They have a broad array of virulence factors, which may influence systemic severe diseases, such as the autoimmune disorders that develop in patients suffering from periodontitis (Berthelot and Le Goff 2010; Dissick et al. 2010). Recent studies have suggested that P. gingivalis is the keystone pathogen of the red complex, which may cause an imbalance in the microbiota and promote dysbiosis. However, after the disruption of microbial homeostasis, the other two members of the red complex, T. denticola and T. forsythia, accelerate the progression of the disease in concert with other species because periodontitis is a polymicrobial disease (Lamont et al. 2018; Silva and Cascales 2021). Four different groups of bacteria are involved in the etiology of PD, as well as the red complex (Marsh et al. 2016). Therefore, the pathogenesis of periodontitis is highly complex, involving oral bacteria and environmental factors, the host’s genetic predisposition, and the host’s lifestyle. The initiation of PD is still under investigation.

P. gingivalis is known as the “keystone pathogen” that disturbs the homeostatic system of the human host through its virulence factors, including lipopolysaccharides (LPSs), hemagglutinins, and fimbriae, which allow P. gingivalis to colonize the periodontal pockets. One of the most critical factors leading to PD is the production of extracellular cysteine proteases, such as gingipains. P. gingivalis uses the immune response of its human host for its benefit, i.e. through the activation of complementary pathways. The gingipains of P. gingivalis affect the host proinflammatory signaling pathways by activating proteinase-activated receptor 2 (PAR2) in human neutrophils. This induces connective tissue damage, including the resorption of the alveolar bone (Wegner et al. 2010; Koziel et al. 2014). The primary role of gingipains is the degradation of host proteins, including cytokines and chemokines, causing the deterioration of the host’s immune response and contributing to the transition to dysbiosis. Gingipains are transported through bacterial cell membrane via the general secretory (Sec) pathway. They are then recruited and transported by the type IX secretion system (T9SS) throughout the cell and outer membrane. Gingipains are considered the major virulence factors of P. gingivalis (Silva and Cascales 2021).

P. gingivalis differs from other periodontal microbes in several unique features, including its ability to synthesize peptidyl-arginine deiminase (PPAD). The enzymatic properties of PPAD differ from those of human deiminases in that its activity is optimal at a higher pH and does not require calcium ions. The primary function of PPAD is the citrullination of the C-terminal arginine residues of human proteins (Pyrc et al. 2012). PPAD occurs on the bacterial surface with argininespecific gingipains (cysteine proteases), which cleaves host proteins, exposing C-terminal Arg residues that are then citrullinated by PPAD. The citrullination of surface proteins generally depends on the activity of gingipain proteases. The degradation of a-enolases and fibrinogen by P. gingivalis gingipains generates PPAD-citrullinated peptides. Moreover, the deimination of the C-terminal arginine in epidermal growth factor (EGF) by PPAD can disturb the biological activity of cytokines (Koziel et al. 2014, Olsen et al. 2018).

As the crucial virulence factors of P. gingivalis, gingipains are necessary to assimilate nutrients, such as heme, a growth-limiting agent for bacterial cells. Among pathogenic bacteria, the accumulation of cellsurface heme is unique to P. gingivalis when cultured on a blood-containing medium (Silva and Cascales (2021)). The hus, hmu and iht operons encode several proteins, including HmuY, which is responsible for transporting heme molecules through the bacterial membrane of P. gingivalis (Fig. 1). Proteins that bind heme molecules advantage P. gingivalis during the initial stages of infection. These proteins enhance the pathogenic capacity of the bacterium, leading to dental periodontitis and the subsequent development of RA (Sato et al. 2013; Smiga et al. 2020).

Fig. 1.

The pathogenicity of P. gingivalis is associated with its low abundance and reduced inflammatory potential. One possible explanation for its association with pathogenicity is the structure of lipid A of LPS, which has only one phosphate residue of 4-acyl lipid A moieties, which confers low proinflammatory potential (Olsen and Singhrao 2018). The inhibition of host interleukin 8 (IL8) in the presence of LPS type A and local chemokine paralysis facilitates the colonization of the host mouth tissue by P.gingivalis (Hajishengallis and Lamont 2012).

Apart from the virulence factors mentioned above, P. gingivalis also produces capsular polysaccharides, fimbriae, and outer-membrane vesicles to avoid the immune system of its host. After it is established within the cell, P. gingivalis produces an ATP-hydrolase that contributes to its survival by inactivating the host’s ATP-dependent apoptosis pathway (Silva and Cascales 2021).

PPAD localizes to different sites: as a component of the outer membrane, anchored to LPS-A; on outermembrane vesicles (OMVs); and secreted as a 47-kDa soluble form (type II). It has been reported that 93 clinical P. gingivalis isolates produced OMVs carrying 75-85-kDa type I PPAD modified with LPS-A. It has been suggested that replacing glutamine with lysine at position 373 of PPAD is crucial for intracellular sorting. The delivery of PPAD type I to phagocytic cells by OMVs results in the presentation of citrullinated peptides and the production of anti-citrullinated-protein antibodies (ACPAs). The role of soluble PPAD type II seems to be less critical (Gabarrini et al. 2018, Stobernack et al. 2018).

Citrullination is a complex posttranslational modification that occurs in higher organisms and leads to the deimination of arginine in proteins and peptides in both physiological processes and pathological diseases, including Alzheimer’s disease, RA, and multiple sclerosis. This process is catalyzed by peptidyl arginine deiminases (PADs) found in vertebrates. As mentioned above, P. gingivalis also secretes PAD, designated PPAD (Porphyromonas peptidyl arginine deiminase), which differs from the eukaryotic enzyme. PPAD consists of a planar, cylindrical catalytic domain with a quintuple α/β-propeller architecture and a C-terminal immunoglobulin-like domain (Olsen et al. 2018). The reaction side of PPAD is on one of the cylinder’s bases, which allows it to accommodate arginine from peptide substrates after rearranging the so-called “Michaelis loop, “ which closes the cleft. The close relationship between the guanidinium and carboxylate groups of the substrates explains the activity of PPAD on the arginine at the C-terminus, although not in the case of peptides. The entire catalytic process is based on the cysteine (C)-histidine (H)-asparagine (R) triad and is similar to those of human PAD1-PAD4, with a guanidino group used to modify enzymes (Goulas et al. 2015).

L-Citrulline is a nonprotein amino-acid derivative and one of the animal urea cycle intermediates. Citrullination causes the transition of free arginine into citrulline through its deamination, which involves the replacement of the guanidino group with a ureido group. This causes the removal of the positively charged arginine side chain with the liberation of ammonia. This reaction increases the functional and structural diversity of the proteome. Citrullination is essential in the immune response because PADs are involved in autophagy, apoptosis, and NETosis. In some specific genetic backgrounds, citrullinated proteins behave as autoantigens, inducing antibodies against citrullinated proteins, leading to autoimmune responses and prolonged inflammation, which make them hallmarks of both PD and RA (Koziel et al. 2014, Goulas et al. 2015). A comparison of human-origin PAD and bacterial PPAD is described in detail in several references (Jonsson et al. 2020; Ciesielski et al. 2022; Matuz-Flores et al. 2022; Curran et al. 2023).

PPAD includes a catalytic triad (C³⁵¹-H²³⁶-N²⁹⁷) and a seven-stage citrullination process. The Michaelis loop containing tyrosine (Y²³³) is in the substrate-free state and has an open conformation, which accommodates the reaction side peptides with C-terminal arginine. Electrostatic interactions between the guanidinium group and the side chain stabilize arginine. These groups are arranged in an extended conformation and then oriented appropriately for the catalysis reaction (de Diego et al. 2014). Arginine at residue 152 (R¹⁵²) and R¹⁵⁴ bind to the C-terminal carboxylate of arginine and the carbonyl of the previous peptide bond. The formation of the Michaelis complex also involves a significant regrouping of the Michaelis loop, which blocks the active site. This binds the C-terminal carboxylate to the substrate (de Diego et al. 2013). Further changes in the structure indicate that the H²³⁶ side chain is rotated here. As a result of this process, the plane of the guanidinium group is compressed between H²³⁶Nδ1, C³⁵¹Sγ, and H²³⁶Nε2. This geometry is a determinant of the identification of H²³⁶ as the general acid/base of the mechanism and the Nη1 guanidinium atom as the nitrogen atom, leaving the ammonia product (Shirai et al. 2006).

Similarly, C³⁵¹Sγ hydrogen bonds to N²⁹⁷Oδ1, which probably extends the nucleophilicity of the catalyst of the sulfur residues. At the beginning of the reaction, C³⁵¹Sγ executes a nucleophilic attack on the flat like the sp2 conformation Cζ atom of the guanidine substrate. This process gives rise to the first tetrahedral reaction intermediate and yields a Cζ atom with an sp3 conformation. Overall, H²³⁶, which first reacts as a general base, strips a proton from Nη1. The last substrate then captures protons from the catalytic thiol group, after which histidine remains without a proton. The tetrahedral intermediate breaks down into a positively charged flat covalent thiouronium compound. This causes ammonia cations to adopt a function after receiving a proton from H²³⁶Nδ1. Ammonia omits the active site through the NH₃-exit/H₂O-entry channel to reach the enzyme surface. The diluent molecule fills the former position of ammonia, leading to the polarization of the aspartic acid (D)²³⁸ and H²³⁶Nδ1 side chains (Goulas et al. 2015). The latter acts as a base and takes a proton from the water molecule, which then executes a nucleophilic attack on the central carbon atom of thiouronium. This produces another neutral intermediate centered on the sp3-like tetrahedral Cζ and diprotonated H²³⁶. The middle substrate then breaks down into a citrullinated product and an intact catalytic mercaptocysteine group, forming hydrogen bonds with N²⁹⁷Oδ1. The repulsion between the carbonyl oxygen of the impartial reaction product and D²³⁸ provides the motive force for the clearance of the substrate from the active-site gap. At the end of the process, a hydroxide formed by the reaction of ammonia with water may provide entrance to the active site via the hydroxide entry channel. This process ultimately leads to the exchange of one of two solvent molecules bound to H²³⁶Nε1. A proton is transferred to hydroxide with the subsequent transfer of histidine and the shift of a proton from Nδ1 to Nε2, which restores the functional monoprotonated condition of H²³⁶. Therefore, the active site is left available to repeat the reactions described above (Goulas et al. 2015).

Data have shown that P. gingivalis infection precedes RA. Moreover, this microbe is one of the most significant factors in maintaining and prolonging the autoimmune inflammatory response that occurs during infection. Therefore, the PPAD enzyme produced by P. gingivalis may have a profound effect on the appearance and progression of RA through protein citrullination, generating neo-epitopes and thus breaching the immunological tolerance of citrullinated proteins (Bielecka et al. 2014). Citrullination also leads to changes in the inter- and intramolecular interactions of proteins containing arginine residues, which are essential for their structure. Therefore, it potentially changes the three-dimensional structures of the modified proteins and their water solubility. Ultimately, this process can trigger a cascade of events leading to RA (Maresz et al. 2013).

There may also be an association between RA and PD based on the similarities in their environmental and genetic risk factors, including smoking and the expression of MHC class II HLA-DRB1 alleles (Berthelot and Le Goff 2010; Koziel et al. 2014). There are also similarities in their initiating mechanisms, with evidence emerging from various studies of an association between RA and PD. A comparison of the whole population and individuals with PD demonstrated an increased risk of developing RA among subjects with PD. Moreover, the course of PD in patients with RA is more severe than in patients without RA, independently of age, sex, smoking history, and ethnicity. Moreover, RA and PD use similar mechanisms insofar as proinflammatory cytokines and inflammatory cells cause the chronic erosion of bones in RA and chronic gum destruction in PD similarly. A recent study suggested that PD is one of the main factors in the induction and maintenance of the inflammatory response by the autoimmune system that occurs in RA (Maresz et al. 2013).

Many studies have shown that RA is caused by the dysregulated response of the immune system by citrullinated proteins. Such proteins are produced under physiological conditions, but the loss of immunotolerance for citrullinated proteins in genetically susceptible individuals initiates the production of autoantibodies against citrullinated proteins (ACPAs), ultimately leading to RA (Smit et al. 2012).

Our studies have indicated that molecular mimicry and autoantibodies were important factors in the etiology of RA. We observed a positive correlation between ACPAs and antibodies directed against Proteus mirabilis LPS O3 or high levels of anti-urease antibodies, which recognized synthetic ureases epitopes (Durlik-Popinska et al. 2020; Konieczna et al. 2020).

The citrullination of proteins through the deimination of the guanidino group on the side chain of arginine is a posttranslational modification that converts positively charged peptidyl arginine to neutral peptidyl citrulline (Fig. 2). The citrullination reaction is essential in various physiological processes, including the differentiation of the epidermis (citrullination of profilaggrin and keratin), brain development (citrullination of myelin basic protein [MBP]), and the regulation of gene expression (Smit et al. 2012; Maresz et al. 2013).

Figure 2.

PPAD-induced citrullination reduces the activity of the immune system by inactivating EGF or activating the prostaglandin E2 signaling pathways in fibroblasts, which causes alveolar bone loss. The citrullination of the surface proteins of P. gingivalis is also involved in its invasion of host cells and adherence to other bacteria. PPAD also significantly affects the host cell cytokine response to P. gingivalis infection. This enzyme is vital for the expression of IL36 in the epithelial cells of the human gingiva and increases the expression of other cytokines, such as IL8, IL13, CCL20, and CXCL8. Previous studies have shown that IL36 regulates dendritic and T-cell responses and is essential for inflammatory diseases (Goulas et al. 2015).

The bone destruction associated with RA and periodontitis involves similar inflammatory responses. In both cases, an immune system imbalance is linked to the dysregulation of immune cells, including Treg and Th17 cells (Zhou et al. 2021).

The association between PPAD, ACPAs, and the development of RA has been discussed in several comprehensive reviews and experimental studies (Koziel et al. 2014; Tilvawala et al. 2018; Jonsson et al. 2020; Gomez-Banuelos et al. 2022; Matuz-Flores et al. 2022; Zoubi and Gordon 2022). Recent progress in microbiome studies has correlated the oral and gut microbiomes with the progression of RA (Stobernack et al. 2018; Du Teil Espina et al. 2019).

Porphyromonas gingivalis strongly affects the human immune system. There is evidence from many studies that P. gingivalis infections correlate with the development of PD and, subsequently, with the progression of RA. Citrullination, which converts arginine to citrulline, initiates the immune system’s inflammatory response, and the whole process is triggered by the expression of P. gingivalis peptidylarginine deiminase (PPAD). Citrullination occurs under the pathological inflammatory conditions caused by P. gingivalis invasion. It is a natural process involved in cellular apoptosis, necrosis, and NETosis. For instance, histone hypercitrullination is necessary to create neutrophil extracellular traps. These traps are components of the immune system that respond to the disturbance of the homeostasis of the oral microbiota caused by an increase in P. gingivalis, leading to the imbalanced autoimmune responses that result in PD, RA, and other diseases of the immune system (Wang et al. 2009). Therefore, further detailed study of the mechanisms and relationships between the citrullination caused by P. gingivalis and the development of RA and PD is required.

Other proinflammatory factors that must be considered in both RA and PD are lipopolysaccharides of P. gingivalis types of O-, A- and K. Their binding to and activation of toll-like receptors (TLRs 2 and 4) via the NF-κB ligand contribute to peripheral polyarthritis. It has been suggested that the inhibition of the inflammatory potential of oral LPSs reduces the progression of RA, and fluoride is proposed as an inactivator of bacterial LPS (Marcano et al. 2021). The short-chain fatty acids (SCFAs) produced during the anaerobic metabolism of P. gingivalis reportedly attract neutrophils to the gingival pocket through the interaction between SCFAs and free fatty acid receptor 2 (FFAR2) (Dahlstrand et al. 2021). It is suggested that monitoring the level of SCFAs might reduce the proinflammatory potential of the oral microbiome and, consequently, the likelihood of RA. A meta-analysis of 28 of 2050 studies with human subjects revealed a high odds ratio (1.86, 95% confidence interval) for the risk of RA in individuals infected with P. gingivalis (Li et al. 2022). Periodontal disease and RA are multifactorial and interrelated, and P. gingivalis is undisputedly an essential player in both.