Porphyromonas Gingivalis Virulence Factors and their Role in Undermining Antimicrobial Defenses and Host Cell Death Programs in the Pathobiology of Chronic Periodontal Disease

The outer membrane of Gram-negative bacteria is composed of lipopolysaccharide (LPS), which consists of lipid A, a core oligosaccharide, and a heterogeneous O-antigen polysaccharide. Lipid A is the endotoxin’s toxic and pro-inflammatory component, highly conserved among Gram-negative bacteria. It comprises two glucosamine molecules acylated with fatty acids and linked by β(1–6) D-glycosidic bonds (Darveau et al. 2004). However, species-specific variations in lipid A structure influence its immunogenicity, primarily through differences in acylation levels and attached fatty acids.

In Pg, lipid A acylation is influenced by environmental conditions, particularly hemin availability, which serves as a growth factor. Under hemin-rich conditions, Pg predominantly expresses a tetra-acylated LPS variant (LPS1435/1449), while hemin scarcity favors the penta-acylated form (LPS1690) (Fig. 1). The penta-acylated variant, due to its higher affinity to TLR4, triggers a significantly stronger immune response than the tetra-acylated form, which induces only a minimal reaction in host cells (Al-Qutub et al. 2006; Qiu et al. 2021). Studies using human peripheral blood mononuclear cells (PBMCs) have shown that synthetic penta-acylated Pg lipid A from strain 381 strongly induces IL-6 expression compared to tri-acylated lipid A (Sawada et al. 2007). However, both Pg lipid A forms elicit weaker inflammatory responses than Escherichia coli lipid A in mouse macrophages (Sawada et al. 2007).

The ability of Pg to modulate its LPS structure is crucial for its survival within the periodontal environment. The tetra-acylated form suppresses inflammation, enabling Pg to evade immune surveillance and establish infection, whereas the penta-acylated form promotes local inflammation, providing nutrients and growth factors for inflammophilic pathobionts (Herath et al. 2013).

LPS interacts with host cells through Toll-like receptor 4 (TLR4), a pattern recognition receptor (PRR). TLR4 activation triggers the NF-κB signaling pathway, regulating the secretion of pro-inflammatory cytokines such as tumor necrosis factor α (TNFα), IL-6, and chemokines like MCP1 (monocyte chemoattractant protein 1). Initially, Pg LPS was thought to activate both TLR4 and TLR2, unlike typical Gram-negative bacteria such as E. coli. However, recent findings indicate that previous studies used insufficiently purified Pg LPS, which contained bacterial lipoproteins capable of activating TLR2 (Nativel et al. 2017). Highly purified Pg LPS interacts exclusively with TLR4 (Ogawa et al. 2007). Despite this, many researchers continue to use Pg LPS purified by standard methods, which can activate both TLR4 and TLR2.

Fimbriae are thin, filamentous structures composed of non-covalently polymerized proteins anchored in the bacterial cell wall. They play a key role in bacterial adhesion, motility, biofilm formation, and host cell invasion (Hamada et al. 1998). In oral bacteria, fimbriae-mediated adhesion to the acquired pellicle on the tooth surface is a crucial initial step in plaque bio-film formation (Xu et al. 2020). Pg fimbriae also enable coaggregation with other bacterial species, supporting biofilm stability and impeding clearance by the host immune system (Maeda et al. 2004).

Pg produces two types of fimbriae: long fimbriae (composed of the FimA subunit) and short fimbriae (composed of the Mfa1 subunit) (Enersen et al. 2013). Long fimbriae primarily facilitate adhesion and invasion of host cells, particularly human epithelial cells (Nakagawa et al. 2002). Based on genetic variations in the fimA gene encoding FimA, six fimbrial types have been identified: I, Ib, II, III, IV, and V (Fujiwara et al. 1993). Studies on plaque samples from periodontitis patients indicate that Pg strains expressing type II fimbriae are most prevalent and have the highest potential for epithelial cell invasion (Nakagawa et al. 2002). Short fimbriae also enable the adhesion to the other bacteria species, such as Streptococcus gordonii, through the interaction with its surface protein SspA/B, supporting biofilm formation. Additionally, they interact directly with immune cells receptors, binding to dendritic cell specific ICAM-3 grabbing nonintegrin (DC-SIGN), facilitating the cell invasion (Lee et al. 2018).

Both long and short fimbriae are recognized by TLR2, leading to NF-κB-mediated secretion of pro-inflammatory cytokines such as TNFα, IL-1β, IL-8, and IL-6 (Hajishengallis et al. 2006) (Fig. 2). TLR2 activation requires the co-receptor CD14, commonly found on monocytes and macrophages (Eskan et al. 2007). Additionally, long fimbriae activate the complement system via complement receptor 3 (CR3; CD11b/CD18) on monocytes and macrophages, inducing ERK1/2 phosphorylation (Hajishengallis et al. 2005; Eskan et al. 2007). Long fimbriae may also downregulate pro-inflammatory responses by signaling through CXCR4, activating the protein kinase A (PKA) pathway, and inhibiting TLR2-mediated inflammation (Hajishengallis et al. 2008).

Fig. 2.

Macrophage polarization pathways.

Upon various stimulus through cell surface receptors macrophage can activate M1 or M2 polarization genes. M1 polarization is related with TLRs, and cytokines receptors, such as TNF-R or IFNγ receptor. NF-κB signaling pathway is the main regulator of M1 polarization genes, but the pro-inflammatory phenotype can be also activated through IRF3, MAP kinase or STAT (members 1, 2, 4, 5). Alternatively, in response to anti-inflammatory cytokines recognized by cytokine receptors, PI3K/Akt and STAT (members 3, 6) pathways can be triggered, leading to activation of M2 polarization genes and inhibiting M1 profile (Kerneur et al., 2022; Xia et al., 2023). AP1, activator protein 1; ARG1, arginase 1; BTK, Bruton’s tyrosine kinase; CSFR, colony-stimulating factors receptor; FIZZ1, found in inflammatory zone 1; GM-CSFR, granulocyte-macrophage colony-stimulating factor receptor; IFNγ, interferon gamma; IL-4R, interleukin-4 receptor; IL-10R, interleukin-10 receptor; IRAK1/4, interleukin-1 receptor-associated kinases 1/4; IRF3/9, interferon regulatory factor 3/9; JAK, Janus activated kinases; MAPK, mitogen-activated protein kinase; MHCII, major histocompatibility complex class II; MyD88, myeloid differentiation primary response 88; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; NOS2, nitric oxide synthase 2; PI3K/Akt, phosphatidylinositol 3-kinase/Akt kinase; STAT, signal transducer and activator of transcription; TGFβ, transforming growth factor β; TLR, toll-like receptor; TNFα, tumor necrosis factor alpha; TNF-R, TNF receptor; TRAF6, TNF receptor associated factor 6; TRIF, TIR-domain-containing adapter-inducing interferon-β; Ym1, chitinase-like protein 3 (Chil3); Created in BioRender. https://BioRender.com/v80l298

Gingipains are cysteine proteases essential for Pg survival and pathogenicity (How et al. 2016). They are classified based on substrate specificity: arginine-specific gingipains (Rgp), which cleave Arg-Xaa peptide bonds, and lysine-specific gingipains (Kgp), which cleave Lys-Xaa peptide bonds (How et al. 2016). Gingipains are localized on the bacterial surface and can also be secreted in soluble forms or within outer membrane vesicles (OMVs) (Guo et al. 2010).

These proteases contribute to periodontal tissue destruction by degrading extracellular matrix components, epithelial cell junctions, and adhesion molecules, facilitating bacterial invasion of connective tissues (Katz et al. 2000; Sheets et al. 2005). Gingipains also help Pg evade the immune system by inactivating complement proteins and degrading immune cell receptors (e.g., CD4, CD8, CD14) and pro-inflammatory cytokines (e.g., IL-1β, IL-6, IL-8, TNFα) (Sugawara et al. 2000; Kitamura et al. 2002; Guo et al. 2010).

Beyond its ability to integrate into dysbiotic bio-films and manipulate the host immune response, Pg can invade subepithelial connective tissue via transcytosis. This process occurs in three stages: entry into the host cell, intracellular survival, and exit from the cell, and was demonstrated in epithelial and endothelial cells (Bélanger et al. 2006; Casadevall 2008; de Jongh et al. 2023). Pg can enter cells passively through phagocytosis (e.g., by macrophages) or actively via long fimbriae interactions with β1 integrins on epithelial cells (Yilmaz et al. 2002). Once internalized, Pg resides within a phagosome, which matures into an autophago-some rich in nutrients, enabling prolonged intracellular survival (Bélanger et al. 2006; Leea et al. 2018).

Inside the host cell, Pg prevents phagosome-lysosome fusion, evading degradation by lysosomal enzymes (Bélanger et al. 2006). Infected cells exhibit altered metabolic activity, favoring amino acids such as asparagine/aspartate and glutamine/glutamate, preferentially utilized by Pg in energy metabolism (Takahashi et al. 2000).

Pathogen can exit host cells through three mechanisms: inducing programmed cell death, lysing the host cell, or escaping via the plasma membrane without causing damage. Studies suggest that Pg exploits the endocytic recycling pathway involving Rab11 and RalA transferrin receptors to facilitate its exit (Takeuchi et al. 2011).

Transcytosis enables Pg to spread within periodontal tissues and complicates treatment, as the bacterium can evade immune defenses and persist intracellularly, making it more resistant to antibiotic therapy.

Pg type strains ATCC 33277 and W83 are the most frequently used in both in vitro and in vivo research on the microbial and molecular basis of the pathobiology of PD. On the genetic level, differences between these strains mainly stem from the extensive rearrangement within their genomes of similar size (Chen et al. 2017). The same is true for other strains, including clinical isolates, whose genomes are completely sequenced and available in the NCBI database (Murugaiyan et al. 2024).

Based on model in vivo studies using mice, the ATCC 33277 strain is considered a non-invasive strain (Murugaiyan et al. 2024). Similar strains are found in periodontally healthy individuals and those with PD (Murugaiyan et al. 2024). The laboratory ATCC 33277 possesses long fimbriae of type I, which are abundantly present on its surface (Fujiwara et al. 1993). These fimbriae are particularly long compared to other Pg strains, enhancing the bacteria’s ability to adhere (Nagano et al. 2012). Additionally, ATCC 33277 also produces short Mfa1 fimbriae. The reduced invasiveness of ATCC 33277 is partly due to the lack of an external polysaccharide capsule (Singh et al. 2011; Sharaf and Hijazi 2023). The absence of the capsule makes the bacteria more susceptible to bactericidal mechanisms employed by the host, which decreases the bacterium’s ability to survive in the bloodstream, a necessary stage for the systemic spread of bacteria to other organs (Singh et al. 2011).

Pg strains with gene arrangement resembling the W83 strain are found more frequently in individuals with PD than in healthy controls (Murugaiyan et al. 2024). Interestingly, the laboratory W83 strain has no long fimbriae despite the presence of the gene encoding FimA type IV (Fujiwara et al. 1993). The lack of fimbriae results from impaired transcription of the fim operon (fimABCDE), mainly due to the inactive histidine kinase FimS, which is essential for the activation of the translational factor for the fim operon (Nishikawa and Duncan 2010). Furthermore, the laboratory W83 strain does not express short Mfa1 fimbriae due to an insertion of a transposon in the promoter sequence of the mfa1 operon (Nagano et al. 2012). Therefore, the Pg W83 strain is often described as having a non-fimbriated phenotype and, thus, a reduced ability to form biofilms (Ho et al. 2017). Nevertheless, it invades host cells due to a small number of FimA fimbriae on the surface (Nishikawa and Duncan 2010). W83 also possesses a polysaccharide capsule of serotype K1, which limits the phagocytosis of this strain by phagocytic cells (Sharaf and Hijazi 2023). Additionally, the capsule reduces the activation of proinflammatory responses in the host’s cells, facilitating bacterial survival in the periodontal tissue (Singh et al. 2011). Despite both strains secreting gingipains, W83 exhibits higher proteolytic activity, which contributes to its increased virulence (measured by invasiveness) due to mechanisms that evade the host’s immune response, which are dependent on the proteolytic activity of gingipains (Seers et al. 2021).

Keratinocytes form the oral epithelium, functioning as a protective barrier against environmental insults such as pathogens, chemicals, and physical trauma (Groeger and Meyle 2019). Thanks to transmembrane proteins, keratinocytes are interconnected, which provides the integrity of the epithelial layer. The epithelial integrity depends on transmembrane molecular complexes, which form gap junctions (GJ), tight junctions (TJ), and adherens junctions (AJ). Several studies demonstrated that during Pg infection, the structure and functionality of these junctions are altered. Upon Pg challenge, several genes coding TJ proteins, such as claudin-1, claudin-4, and occludin, are upregulated in keratinocytes (Guo et al. 2018). On the other hand, Pg LPS stimulates the increased expression of claudin-1, claudin-15, and ZO-1 (zonula occludens-1), but decreased expression of occludin, claudin-4, and JAM- (junctional adhesion molecule)-A expression (Guo et al. 2018). Another study has demonstrated that both Pg and Pg LPS treatment of human oral keratinocytes (HOK-16B and OKF6) led to down-regulation of ZO, E-cadherin, claudins, and occludin, as well as GRHL2 (grainyhead-like 2), a regulator of the junction proteins (Chen et al. 2019). Tight junction integrity can also be directly disturbed by gingipains, which degrade members of the JAM family proteins: JAM1 and CXADR (coxsackievirus and adenovirus receptor) (Takeuchi et al. 2019; Takeuchi et al. 2021). Interestingly, gingipains do not affect claudin-1, claudin-4, occludin, ZO-1, or E-cadherin (Takeuchi et al. 2019). The critical role of gingipains in destroying the cell connection was demonstrated in studies comparing wild-type Pg with a gingipain-deficient mutant (Andrian et al. 2004). These studies showed that the wild-type strain had a greater ability to penetrate the gingival tissues deeply than the mutant lacking gingipains.

The permeabilization of the gingival epithelium allows penetration of pro-inflammatory agents, such as LPS, dextran, proteoglycan, and gingipains, into the subepithelial tissue (Takeuchi et al. 2019; Takeuchi et al. 2021; Takeuchi et al. 2022. Application of a microtissue 3D model consisting of human gingival epithelial cells and human oral fibroblasts demonstrated the Pg capacity to overcome the epithelial barrier and reach fibro-blasts, leading to disorganization of tissue structure and fibroblast death (Bugueno et al. 2018). Gingipains were also shown to disturb AJ by proteolysis of N-cadherin, VE-cadherin, and β-integrin, resulting in detachment of the epithelial cells from extracellular matrix proteins (Hintermann et al. 2002). The ability of Pg to disturb keratinocyte (HOK-16) adhesion was much more potent compared to E. coli or A. actinomycetemcomitans: preincubation of cells with Pg at a multiplicity of infection (MOI) 1,000, corresponding to 1,000 bacteria per eukaryotic cell, reduced the adhesion capacity to laminin-5 by 50%, which could not be explained by the cell death (Hintermann et al. 2002). Additionally, Pg triggered selective proteolysis of cell-cell contact structural components in a strain-dependent manner. While ATCC 33277 and 381 were very effective, W50 almost did not affect the integrity of junctional proteins (Hintermann et al. 2002). These results suggest the ability of Pg and its virulence factors to modulate the cell-cell connections in the oral epithelium through activating defense mechanisms and destroying the protective barrier.

The role of keratinocytes goes beyond their structural functions: they are involved in the immune response, regulating it and participating directly in the fight against pathogens. Various types of TLRs are present on the surface of oral epithelial cells (Beklen et al. 2008). During acute or persistent gingival inflammation, the expression level of TLR2 and TLR4, the primary receptors engaged in Pg recognition, increases (Uehara et al. 2007; Beklen et al. 2008; Groeger and Meyle 2019; Chen et al. 2021). Interestingly, when the inflammation enters the chronic stage, characteristic of PD, TLR4 expression decreases in comparison to acute gingivitis. Such subversion may serve as a mechanism for preventing the excessive inflammatory response (Groeger and Meyle 2019). Keratinocytes are sensitive to Pg LPS treatment, which enhances the expression of pro-inflammatory factors such as IL-6, IL-8, TNFα, and IFNγ (Kim et al. 2018). They also respond differently to Pg LPS in different acylation forms. In studies using human oral keratinocytes (HOKs), penta-acylated Pg LPS increased the expression of LPS-binding protein (LBP), involved in the cell’s reaction to LPS, while tetra-acylated Pg LPS did not affect the expression levels of this molecule (Ding et al. 2013).

Stimulated epithelial cells produce various factors, among which the most important are β-defensins (Uehara et al. 2007). β-defensins can directly disrupt microbial cell membranes, leading to pathogen neutralization and induce the release of pro-inflammatory cytokines and chemokines, serving as chemotactic factors for immune cell recruitment (Van Kilsdonk et al. 2017). The recognition of bacteria by keratinocytes also occurs through intracellular PRRs, nucleotide-binding oligomerization domain receptors (NODs), which are cytosolic proteins recognizing peptidoglycan of the bacterial wall. During PD, the expression of NOD1, which is responsible mainly for recognizing Gram-negative bacteria peptidoglycan, is notably elevated in the periodontal tissue (Chen et al. 2021). The activation of NOD1 induces the release of pro-inflammatory factors, such as IL-6 and IL-8 (CXCL8), as well as β-defensins (Groeger and Meyle 2019). IL-8 is one of the key regulators of the influx of immune cells into the gingiva. IL-8 is present in both healthy and diseased tissues, but its levels increase in the presence of pathogens (Ertugrul et al. 2013). The primary function of IL-8, which is recognized by CXCR1 and CXCR2 receptors on polymorphonuclear cells (PMNs), is to induce the influx of neutrophils to the site of inflammation (Sahingur and Yeudall 2015). Unrestricted influx of immune cells leads to increased cytokine production and progressive degradation of structural components of the periodontal tissue. Although keratinocytes are an essential source of IL-8, they also secrete CCL2 (chemokine (C-C motif) ligand 2), CCL5, CXCL10 (chemokine (C-X-C motif) ligand 10), CCL17, and CCL20, which orchestrate the influx of monocytes and T cells (Schutyser et al. 2003).

Human gingival fibroblasts (hGFs) make up approximately 65% of the cells in the gingival tissue (Häkkinen et al. 2014). As mesenchymal cells dominant in connective tissue, they produce extracellular matrix components and maintain the structural integrity of the tissue (Häkkinen et al. 2014). hGFs are also characterized by a high regenerative capacity as the main collagen producer for the extracellular matrix (Roman-Malo et al. 2019). In addition to structural functions, hGFs are involved in the inflammatory response (Wielento et al. 2023). As a source of pro- and anti-inflammatory factors, they influence immune cells by either promoting their activation or attenuating the inflammatory response. Gingival fibroblasts, along with keratinocytes and macrophages, are an essential source of IL-8 (Ertugrul et al. 2013).

GFs’ response to Pg infection occurs primarily through the TLR2 receptor interacting with fimbriae, while sensing LPS plays a far less important role in their stimulation (Schuster et al. 2024). Nevertheless, similarly to keratinocytes, the hGFs response to pentaacylated LPS, but not tetra-acylated LPS, results in the increased secretion of the pro-inflammatory cytokine interleukin (IL)-6 and chemokine IL-8 (Herath et al. 2011). Gingival fibroblasts are also an important source of matrix metalloproteinases (MMPs), especially MMP-1, MMP-3, MMP-8, and MMP-9 (Yucel-Lindberg and Båge 2013). MMPs are responsible for extracellular matrix remodeling through the degradation of its components, including collagen. The activity of secreted MMPs is controlled by tissue inhibitors of MMPs (TIMPs), which prevent excessive matrix destruction (Yucel-Lindberg and Båge 2013). During Pg infection, the balance is disrupted in favor of MMP proteolytic activity due to enhanced expression of MMP-1, MMP-2, and MMP-3, as well as the decreased expression of TIMP-2 in GFs (Bozkurt et al. 2017). In this way, GFs contribute to excessive extracellular matrix degradation, propagating the progression of periodontal disease. Upon Pg infection, fibroblasts also enhance the expression of receptor activator of NF-κB ligand (RANKL), OPG, and PGE2, together with cytokines IL-1β, TNFα, and IL-6, which collectively are involved in the upregulation of osteoclastogenesis (Belibasakis et al. 2007). The synergistic interaction of pro-inflammatory cytokines intensifies the inflammatory response and bone destruction. GFs are also a source of reactive oxygen species (ROS). The respiratory burst is one of the strategies for combating pathogens, but at the same time, it contributes to tissue damage. Activation of the respiratory burst is regulated by the PI3K/Akt/NF-κB signaling pathway and is dependent on the activation of TLR2 and TLR4 (Vo et al. 2021).

A recent study showed that the pro-inflammatory activity of macrophages infected with Pg is reduced in the presence of fibroblasts through post-transcriptional regulation of TNFα activity (Tzach-Nahman et al. 2017). Therefore, fibroblasts may serve as a component of anti-inflammatory control for macrophages stimulated by periodontal pathogens. Reducing fibroblasts’ inflammatory activity could lead to an overall decrease in inflammation in the periodontium by inhibiting the influx of immune cells and limiting the hyperactive inflammatory response.

Although neutrophils and macrophages together account for only 12% of all immune cells, with B lymphocytes/plasma cells (60%) and T lymphocytes (17%) constituting the absolute majority in the periodontitis lesion, the regulatory role of macrophage in the progression of PD cannot be ignored (Berglundh et al. 2011; Yin et al. 2022). In the periodontium, the macrophage pool consists of the resident cells or those differentiated from monocytes that have migrated to the site of inflammation from the bloodstream (Sun et al. 2021). Macrophages are functionally diverse cells, with their functional state revealed under varying environmental conditions. Due to the presence of pattern recognition receptors (PRRs) on their surface, primarily TLRs and C-type lectin receptors (CLRs), macrophages can respond to molecular patterns associated with pathogens (PAMPs) and danger signals (DAMPs) in their surroundings (Sun et al. 2021). Macrophages differentiation from circulating monocytes is directed by macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (Ushach and Zlotnik 2016; Sun et al. 2021). Their primary function is the direct elimination of pathogens, antigen presentation, the mobilization and regulation of other immune cells, and maintaining tissue homeostasis. Macrophages are also responsible for the process of efferocytosis – the phagocytic clearance of apoptotic cells, mainly neutrophils, thus preventing their secondary necrosis at the site of inflammation (Greenlee-Wacker 2016). Upon stimulation, macrophages polarize into various active forms, with the most recognized being the M1 (classically polarized type), the M2 (alternatively polarized type), and various intermediate subtypes (Sun et al. 2021) (Fig. 2).

Apoptosis, the most studied form of programmed cell death, is a controlled process that does not trigger an inflammatory response, often referred to as “silent cell death”. Apoptosis can be induced through two main pathways: the intrinsic (mitochondrial) and the extrinsic pathway (Kayagaki et al. 2024) (Fig. 3).

Fig. 3.

Apoptosis pathways and P. gingivalis.

Upon pro-apoptotic signals, dominating BH3-only proteins inhibit anti-apoptotic members of Bcl-2 family (Bcl-2, Mcl-1, Bcl-xL, Bcl-w, Bfl-1), enabling BAX and BAK oligomerization, and mitochondrion permeabilization. After formation of apoptosome with APAF1, cytochrome c, and pro-capsase-9, the caspase cascade is activated, leading to apoptosis. Alternatively, the activation of death receptors leads to caspase-8 activation, and subsequently intrinsic pathway activation or directly activating caspase cascade (Kayagaki et al., 2024). In sentinel cells, Pg can alter the expression of Bfl-1, Bcl-2, APAF1, XIAP, and caspases 3/7 and 9. APAF1, apoptotic peptidase activating factor 1; FADD, Fas-associated death domain; RIPK1, receptor-interacting serine/threonine-protein kinase 1; SMAC, second mitochondria-derived activator of caspases; TRADD, TNFR1 associated death domain protein; XIAP, X-linked inhibitor of apoptosis; Created in BioRender. https://BioRender.com/t45c156

In contrast to apoptosis, pyroptosis is a highly immunogenic and lytic form of cell death (Kayagaki et al. 2024). It is closely linked to the biology of immune cells and serves as a key mechanism of the inflammatory response. Initially described in macrophages, pyroptosis can also occur in dendritic cells, neutrophils, and epithelial cells (Kayagaki et al. 2024). A hallmark of pyroptosis is cell membrane permeabilization caused by incorporating gasdermin (GSDM) polymers into the membrane. This compromised membrane allows inflammatory factors and danger-associated molecular patterns (DAMPs) to leak into the extracellular space. Depending on the activation source, pyroptosis can proceed via either the canonical or the alternative pathway (Fig. 4).

Fig. 4.

Pyroptosis pathways and P. gingivalis.

Upon TLRs stimulation, NF-κB upregulates the expression of ASC, NLRP3, and pro-caspase-1, which subsequently form inflammasome. Caspase-1 activates IL-1β, IL-18, and cleaves GSDMD into pore forming domains, which oligomerize in cell membrane, creating pores. Alternatively, caspase-8 can activate inflammasome, and IL-1β. Intracellular presence of PAMPs, especially LPS, is recognized by caspase-4/5/11, which upon oligomerization can cleave GSDMD and trigger pore formation, and activate IL-18. Pro-apoptotic caspase-3 acts as a GSDMD inhibitor (Kayagaki et al., 2024). Pg can modulate pyroptosis through changes in the activation of NF-κB, caspase-1, 4, and 3, the expression profile of ASC, NLRP3, pro-capsase-1, inflammasome, GSDMD, and secretion of IL-1β and IL-18. ASC, apoptotic speck protein containing a caspase recruitment domain; BTK, Bruton’s tyrosine kinase; FADD, Fas-associated death domain; GSDMD, gasdermin D; GSDMD-N, cleaved N-terminal GSDMD; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; NLRP3, nucleotide-binding domain, leucine-rich repeat-containing family, and pyrin domain-containing-3/caspase recruitment domain; PAPMs, pathogen associated molecular patterns; RIPK1, receptor-interacting serine/threonine-protein kinase 1; TLR, toll-like receptor; TRADD, TNFR1 associated death domain protein; Created in BioRender. https://BioRender.com/l81p084

The canonical pyroptosis pathway is triggered by receptors that recognize pathogen-associated molecular patterns (PAMPs) and DAMPs, such as toxins, double-stranded DNA (dsDNA), proteases, ubiquitin ligases, and bacterial proteins (Kayagaki et al. 2024). The bin ding of these factors to specific receptors induces conformational changes that promote the assembly of an active inflammasome complex within the cytoplasm. The inflammasome consists of intracellular NLR receptors (nucleotide-binding domain and leucine-rich repeat-containing receptors), pro-caspase-1, and the adaptor protein ASC (apoptotic speck protein containing a caspase recruitment domain). The best-characterized inflammasome is NLRP3 (nucleotide-binding domain, leucine-rich repeat-containing family, and pyrin domain-containing-3/caspase recruitment domain) (Shao et al. 2015).

NLRP3 activation is tightly regulated by BTK kinase, which directly binds to the inflammasome complex. BTK phosphorylates tyrosine residues during activation in the NACHT domain of NLRP3, enhancing its interaction with ASC, which is crucial for full inflammasome activation (Bittner et al. 2021). Once assembled, the inflammasome facilitates the auto-activation of pro-caspase-1, which then proteolytically processes the precursors of pro-inflammatory cytokines IL-1β and IL-18 into their active forms (Kayagaki et al. 2024). In addition, activated caspase-1 cleaves gasdermin D (GSDMD), releasing its N-terminal pore-forming domain (PFD) from the C-terminal inhibitory domain. GSDMD polymers embed into the cell membrane to form pores that allow the release of IL-1β and IL-18 into the extracellular space, thereby amplifying inflammation. These GSDMD pores also induce non-selective ion flux, disrupting the electrochemical gradient and leading to cell death. This process is accompanied by the release of cellular contents, including lactate dehydrogenase (LDH), commonly used as a marker of pyroptosis in culture media (Wang et al. 2022).

In the alternative pyroptosis pathway, intracellular lipopolysaccharide (LPS) is the primary signal for inflammasome activation. Intracellular LPS is bind by caspase-4 and -5 (in human cells) or caspase-11 (in murine), which leads to activation and oligomerization of these caspases (Gabarin et al. 2021). Subsequently, they initiate inflammasome assembly and cleave GSDMD, forming membrane pores and the induction of pyroptosis (Gabarin et al. 2021).

Necroptosis is a form of programmed cell death exhibiting apoptosis and necrosis characteristics. This pathway is initiated by receptors such as Fas, TNF, and TRAIL, which, upon binding to their respective ligands, activate receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3) (Dhuriya and Sharma 2018) (Fig. 5). Caspase-8 negatively regulates necroptosis by cleaving and inactivating RIPK1 and CYLD (cylindromatosis) proteins, thereby preventing necroptotic signaling (O’Donnell et al. 2011). Consequently, for necroptosis to proceed, caspase-8 must be inhibited in addition to RIPK3 being expressed in the cell (Dhuriya and Sharma 2018).

Fig. 5.

Necroptosis pathways and P. gingivalis.

The activation of death receptors triggers formation of complex I, which activates NF-κB pathway and pro-survival genes. Upon inhibition of RIPK1 ubiquitination by CYLD, complex IIa is formed. The cleavage of RIPK1 by caspase-8 activates apoptosis pathway. If pro-caspase-8 remains inactive by cFLIP, complex IIb is formed. Subsequently, phosphorylation of RIPK1, and RIPK3, and their oligomerization, together with phosphorylated MLKL leads to necrosome formation. Necrosome translocates to cellular membranes, forming pores, which leads to necroptosis (Dhuriya & Sharma, 2018; Holler et al., 2000; O’Donnell et al., 2011). Pg modulates necroptosis pathways targeting RIPK3, and MLKL. cIAP1/2, cellular inhibitor of apoptosis protein 1/2; CYLD, cylindromatosis; FADD, Fas-associated death domain; MLKL, mixed lineage kinase domain-like; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; RIPK1/3, receptor-interacting serine/threonine-protein kinases 1/3; SMAC, second mitochondria-derived activator of caspases; TRADD, TNFR1 associated death domain protein; TRAF, TNF receptor-associated factor; TRIF, TIR-domain-containing adapter-inducing interferon-β; Created in BioRender. https://BioRender.com/r22c556

RIPK1 interacts with RIPK3 through the receptor homology domain (RHD), leading to the formation of the necrosome complex. This complex subsequently activates the pseudokinase MLKL (mixed lineage kinase domain-like) (O’Donnell et al. 2011). Like in pyroptosis, activated MLKL translocates to the cell membrane, facilitating the accumulation of ion channels transporting Na⁺ and Ca²⁺ or initiating the formation of membrane pores by interacting with phosphoinositol molecules in the membrane. During necroptosis, pro-inflammatory cytokines are produced and released, particularly in response to TNFα pathway activation (O’Donnell et al. 2011).

Each previously described cell death type follows a well-characterized and distinct process. However, while danger-associated molecular patterns (DAMPs) serve as common activators across these pathways, they do not solely dictate which form of cell death will occur. Instead, programmed cell death is the response to many competing signals, with regulatory proteins either promoting or inhibiting different pathways.

Some proteins play roles in multiple cell death mechanisms, while others exert mutually inhibitory effects. For example, caspase-3, a key apoptosis regulator, also negatively regulates pyroptosis by proteolytically cleaving gasdermin D (GSDMD) into a form incapable of pore formation (Dai et al. 2023). Paradoxically, caspase-3 can also trigger pyroptosis by activating gasdermin E (GSDME), implying that pyroptosis may be a sequel of apoptosis (Jiang et al. 2020). Similarly, caspase-8, another critical apoptosis regulator, inhibits necroptosis (Dhuriya and Sharma 2018).

Conversely, RIPK3, a central protein in necroptosis, can directly activate the NLRP3 inflammasome, promoting pyroptosis and the secretion of IL-1β and IL-18 (Bertheloot et al. 2021). These interactions highlight the complex crosstalk between cell death pathways.

Cellular aging influences various pathophysiological processes and has been implicated in aging-related and inflammatory diseases, including atherosclerosis, cardiovascular disorders, type 2 diabetes, and Alzheimer’s disease (Tchkonia et al. 2010; Ritzel et al. 2019). Recent research also suggests a role for cellular senescence in periodontal disease (PD) (Albuquerque-Souza et al. 2022; Rattanaprukskul et al. 2024).

Rattanaprukskul et al. (2024) demonstrated that PD patients exhibit significantly elevated senescence markers, including p16, lipofuscin, β-galactosidase, and SASP. Notably, these senescence markers were present regardless of patient age, indicating that periodontitis-related senescence can occur even in young individuals. Senescent cells were identified in both the epithelium and connective tissue, particularly within fibroblast and macrophage populations derived from PD patient biopsies (Rattanaprukskul et al. 2024).

Interestingly, a case study followed by longitudinal research in a mouse model provided evidence that periodontitis-induced senescence can be triggered via TLR9-dependent signaling (Albuquerque-Souza et al. 2022). Another study revealed that prolonged exposure to F. nucleatum led to increased expression of senescence markers (p16, p21, and β-galactosidase) alongside decreased levels of p14 and p53, impairing gingival epithelial wound healing (Albuquerque-Souza et al. 2024). Additionally, senescence-associated changes in the human periodontal ligament (PDL) have been linked to increased expression of microRNA (miR)-34a, which promotes inflammation and tissue destruction through SASP-related protein secretion (Ikegami et al. 2023).

Emerging evidence also suggests that senescence can be initiated by sub-lethal apoptotic signaling and the release of cellular contents from cells undergoing pro-inflammatory cell death (Zhao et al. 2021). For instance, human gingival fibroblasts (hGFs) exposed to pyroptotic RAW264.7 macrophages displayed increased expression of senescence-associated markers. A similar effect was observed in an in vivo mouse model of hyperglycemia, suggesting that initial macrophage pyroptosis triggered by hyperglycemia can act as a secondary signal, promoting cellular senescence in periodontal tissues (Zhao et al. 2021).

These findings highlight a growing interest in the role of senescence in PD pathogenesis. Given the extensive evidence for the co-occurrence of multiple cell death pathways in PD and their ability to drive cellular senescence, it is likely that both processes are linked to the disease progression.