Porphyromonas gingivalis adopts intricate and unique molecular mechanisms to survive and persist within the host: a critical update

ABSTRACT Porphyromonas. gingivalis (P. gingivalis) is an obligate, asaccharolytic, gram-negative bacteria commonly associated with increased periodontal and systemic inflammation. P. gingivalis is known to survive and persist within the host tissues as it modulates the entire ecosystem by either engineering its environment or modifying the host’s immune response. It interacts with various host receptors and alters signaling pathways of inflammation, complement system, cell cycle, and apoptosis. P. gingivalis is even known to induce suicidal cell death of the host and other microbes in its vicinity with the emergence of pathobiont species. Recently, new molecular and immunological mechanisms and virulence factors of P. gingivalis that increase its chance of survival and immune evasion within the host have been discovered. Thus, the present paper aims to provide a consolidated update on the new intricate and unique molecular mechanisms and virulence factors of P. gingivalis associated with its survival, persistence, and immune evasion within the host.


Introduction
Periodontitis is a multifactorial immuno-inflammatory disease initiated by the interaction of the host to the pathogenic microorganisms in the oral cavity [1][2][3][4][5]. The host-microbial interaction activates a cascade of signaling pathways that cause release of proinflammatory cytokines and tissue destructive enzymes. The increased inflammatory response in the periodontal tissues alters the composition of the entire microbiome that shifts the gram-positive aerobic cocci to gram-negative anaerobic rods and motile spirochetes [2,[6][7][8][9]. Some of the common gram-negative anaerobic species that predominate the oral biofilm at the later stages of periodontitis include Porphyromonas gingivalis (P. gingivalis), Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), Tanerella forsythia (T. forsythia), Fusobacterium nucleatum (F. nucleatum), Treponema denticola (T. denticola), Camplyobacter rectus (C. rectus), Prevotella intermedia (P. intermedia), etc [3][4][5][6][7][8][9]. Amongst all these pathogens, the role of P. gingivalis in exaggerating periodontal inflammation and dysbiosis is very unique and intricate [10][11][12][13][14][15][16][17] P. gingivalis, formerly known as Bacteroides gingivalis, is a gram-negative non-motile, asaccharolytic, obligate, capnocytophagic, anaerobic rod-shaped bacteria. P. gingivalis is considered as a prime etiological agent in the pathogenesis and progression of periodontal inflammation and alveolar bone loss [2,7,[12][13][14][15]. Approximately, 10%-25% of healthy subjects and 79%-90% of subjects with periodontitis have micro-colonies of P. gingivalis in their oral cavity [16,17]. A positive correlation between the depth of the periodontal pocket and the presence of P. gingivalis has also been established [17]. Even at low abundance, i.e. <0.01% of the total bacterial count, P. gingivalis has the inherent ability to transform the entire microbial community and amplify the disease process [10]. It has been referred to as a 'keystone pathogen' and a 'master of polymicrobial synergy, dysbiosis and immune subversion', as it exploits several sabotage tactics to evade, weaken, or deceive the host's immune system [10][11][12][13][14][15]. P. gingivalis is also known to gain entry into the host cell and systemic circulation to reach distant organ systems [15][16][17][18][19]. Numerous studies have explained and discovered the novel virulence factors and pathogenic mechanisms that help P. gingivalis to survive and persist in the host [12][13][14][15]. However, the knowledge about how P. gingivalis modulates the immune response with activation of newly discovered virulence factors, host proteins, and signaling pathways is limited. No paper to our knowledge has provided a consolidated update on the recent immunological receptors, signaling pathways, and molecular and cellular mechanisms associated with P. gingivalis invasion and persistence within the host tissue. Furthermore, the knowledge about the cellular and molecular mechanisms linked with the interaction of P. gingivalis with other members of the oral microbiome is not fully explored. It is important to understand how P. gingivalis causes of suicidal death of other members of the oral microbiome along with the emergence of pathobionts and virulent commensals in the host.
Therefore the present review aims to provide a critical and consolidated update on the new pathogenic mechanisms and survival strategies of P. gingivalis within host associated with periodontal inflammation and dysbiosis. A thorough understanding of these key virulence factors and pathogenic mechanisms is crucial not only to understand the pathogenesis of periodontal diseases and systemic inflammation but also to develop novel therapeutic modalities that to prevent the onset and progression of periodontal disease.
its growth and survival but also favors the growth and development of other bystanders, commensal, and pathobiont species [29][30][31][32]. Some of the other unique and intricate molecular mechanisms associated with its interaction of other members of the biofilm and host that increases its growth, survival, and persistence can be summarized as follows (Table 1):

2.a. Synergistic interaction of P. gingivalis with other microorganisms and development of pathobiont species
P. gingivalis interacts with other microorganisms to elicit its full range of pathogenicity and virulence factors, as in germ-free conditions, it cannot survive or produce any disease ( Figure 2) [10]. The interspecies synergy of P. gingivalis has been observed with Streptococci mitis (S. mitis), S. gordonii, A.
The most common pathobiont that favors P. gingivalis' growth and survival is F. alocis. Studies have shown that P. gingivalis co-cultured with F. alocis can modulate the innate immune response of the host as both these species auto-aggregate and express unique gene expression ( Figure 3) [31][32][33]. P. gingivalis-F. alocis is linked with the remodeling of actin and chromatin molecule, activation of autoinducer (AI) associated quorum sensing, the proliferation of the junctional epithelium, and deposition of collagen fibers in the gingival epithelial cells [30][31][32][33]. The co-infection also upregulate the production of extracellular matrix adhesion proteins like actin, vinculin, vimentin, plectin, transgelin, profilin, endoplasmin proteins (filamin B and filamin C), chaperone proteins (HSP90), non-coding RNA, CRISPRs RNA, 'microbial surface componentrecognizing adhesion matrix molecules' (MSCRAMMs), and toxin-antitoxin system proteins [31]. These novel proteins are required for the adherence and colonization of P. gingivalis with other 'Gram-positive bacteria and host tissues', and are directly linked with increased biofilm formation [31][32][33][34][35][36][37][38][39][40]. The increased production of CRISPR-RNA is also associated with triggering the stress response, chaperone formation, and horizontal gene transfer among oral bacteria [41][42][43]. The CRISPRs RNA even helps P. gingivalis to induce genomic rearrangements and intercellular recombination that helps to acquire the useful DNA sequences for its survival, limit transposition of insertion sequences and acquire resistance to foreign RNA and DNA [44][45][46][47][48]. Watanabe et al. (2017) also observed a highly 'expressed transcripts of CRISPR regulatory small non-coding regulatory RNA (sRNA) in the intergenic sequences (IGS) upstream of CRISPR-associated (cas) gene arrays in P. gingivalis that help to limit the genetic exchange within the biofilm and prevent mutation [42,[45][46][47]. Another highly upregulated hypothetical proteins, known as 'HMPREF0389_00967, containing a CHASE3 extracellular sensory domain', has been observed during F. alocis -P. gingivalis co-infection [31,48]. The HMPREF0389_00967 is found to regulate the production of histidine kinase, adenylate cyclases, and other chemotaxis proteins that helps P. gingivalis to evade the process of phagocytosis [31,[45][46][47][48]. F. alocis -P. gingivalis co-infection is also known to regulate other proteins such as 'histone cluster proteins and PPIA-peptidyl-prolyl isomerase, transferrin receptor protein 1 (involved in iron transport), transmembrane emp24 domain-containing protein 10, dynein (involved in vesicular protein trafficking), surfeit 4, and solute carrier family proteins' in the host [48]. The alteration in the production of these proteins indirectly weakens the innate immune response of the host and favors microbial growth within the tissues. P. gingivalis can even acquire and utilize some of these proteins, nucleosides, and nucleobases for its nutrition, growth, maturation, and survival [49].
• Decrease expression of cyclin D at the G1 phase.
P. gingivalis-F. nucleatum-S. gordonii consortia is known to cause extensive alterations in the cell envelope and cell wall protein, especially the outer membrane of P. gingivalis [78]. Kuboniwa et al., 2006, showed that 84 proteins of cell envelope were detected during the co-infection, out of which 40 showed reduced abundance during the three species community formation [78,79]. Some of the common proteins affected during the co-infection include

gingivalis with F. alocis and its effects on biofilm formation and periodontal inflammation:
P. gingivalis interaction with F. alocis can modulate the innate immune response of the host as both these species autoaggregate and express unique genes expression. P. gingivalis-F. alocis remodeling the actin and chromatin molecule, activation of autoinducer (AI) associated quorum sensing, the proliferation of the junctional epithelium, and deposition of collagen fibers in the gingival epithelial cells. The co-infection also upregulates the production of extracellular matrix adhesion proteins, CRISPRs RNA, and toxin-antitoxin system proteins that increase the adherence and colonization of P. gingivalis with other 'Grampositive bacteria and host tissues', and are directly linked with increased biofilm formation. The CRISPR-RNA also helps with triggering the stress response, chaperone formation, and horizontal gene transfer among oral bacteria that favor microbial community development.
proteins regulating thiamine, cobalamin, and pyrimidine synthesis; OmpH proteins, two lipoproteins, MreB protein (a bacterial actin homolog) that play an important role in determining cell shape; and DNA repair proteins. The HmuR, a TonB dependent outer membrane receptor, was up-regulated in the community with an increase in protein synthesis. Some of the other encoded proteins that affected by this interaction include proteins for maintenance of cell wall integrity (murE), intercellular signaling (cbe), regulation of redox state (spxB and msrA), ribosomal proteins and translation elongation and initiation proteins. A decrease in vitamin B1, B 12, Biotin, pyrimidine synthesis was also observed during this co-infection [78,79] A community derived P. gingivalis biofilm also showed a significant reduction in DNA repair proteins along with an upgradation of DNA repair genes [80].
Apart from the up-regulation of these genes, P. gingivalis regulates the expression of Zinc finger E-box-binding homeobox (ZEB2), a transcription factor, which controls epithelial-mesenchymal  . Schematic representation of the interaction between P. gingivalis and S. gordonii: P. gingivalis interacts with S. gordonii by utilizing its major (FimA) and minor fimbriae (Mfa1). FimA and Mfa1 of P. gingivalis binds to glyceraldehyde-3phosphate dehydrogenase (GAPDH) and streptococcal SspA/B adhesins (SspA/B termed BAR antigen I/II) of S. gordonii, respectively The Mfa1 interactions with SspB lead to phenotypic changes with subsequent activation of tyrosine phosphorylation signal production (PTK). The increased PTK signaling causes exopolysaccharide formation that causes accretion of P. gingivalis colonies. The FimA also interacts to the α5β1-integrin receptors on gingival epithelial to facilitate bacterial entry. transition and inflammatory responses within the periodontal tissues [80,81]. P. gingivalis mediated ZEB2 regulation occurs through pathways involving β-catenin and Forkhead box protein O1 (FOXO1). S. gordonii can antagonize ZEB2 expression, and thereby control P. gingivalis mediated inflammation response. Furthermore, P. gingivalis and S. gordonii coinfection is even found to enhance the expression of Protein Tyrosine Phosphatase (Ltp1) and AI-2 in the periodontal tissues [60][61][62][63][64]69,77,[81][82][83][84][85][86][87][88][89][90][91]. The increased Ltp1 expression subsequently dephosphorylates P. gingivalis tyrosine kinase 1 (Ptk1) receptors and increases the transcription of Complementarity Determining Region (CDrR) to promote colony formation [68,82,83,91] (Figure 4). S. gordonii also inhibits the production of CdhR (Community development and hemin Regulator), a negative regulator of Mfa1, and increases the co-adhesion and coaggregation of P. gingivalis with other microorganisms [60][61][62]84,85]. However, once the colony has been established, it dampens the micro-colony development by utilizing the Ltp1 receptors and reducing Mfa1 expression [90]. The Ltp1 phosphatase activity is also known to down-regulate the transcription of luxS and several other genes involved in exopolysaccharide synthesis and transport of nutrients [90] Ltp1 also promotes the transcription of the hmu operon involved in haem uptake and dephosphorylation of the gingipain proteases. The increased CDrR expression even help P. gingivalis to acquire nutrients such as peptides and heam from its environment at the times of crisis [92]. Since P. gingivalis is unable to synthesize its porphyrin, it relies on exogenous porphyrin and heam biosynthesis intermediates, from host sources and other bacteria. S. gordonii helps P. gingivalis to acquire heam by promoting met-Hemoglobin formation in the presence of Oxy-Hemoglobin. The heme-binding protein receptor (Hmu-Y) of P. gingivalis' captures and extracts the Iron III-Protoporphyrin IX using the Met-Hemoglobin obtained from S. gordonii [89][90][91][92][93][94][95][96][97]. Hmu-Y along with its cognate outer-membrane receptor, HmuR, aid in the transportation of the heam molecule through the outer membrane of P. gingivalis. This process of acquiring heam by P. gingivalis, also dependent upon the protein TonB, which helps to transduce energy for the passage of heam and other ligands into the periplasm. Furthermore, the Hmu-Y receptors help P. gingivalis to increase the extracellular polymeric substances (EPS) production that mediates cohesion among the microbial community and prevents injury from any external physical forces [90][91][92][93][94][95][96][97]. Under the ironlimited conditions, P. gingivalis expresses a haemophore-like protein, HusA, to mediate the uptake of essential porphyrin and support its survival within the host [92][93][94]. The Hmu-Y receptor of P. gingivalis can even interact with the Interpain-A (InpA) of P. intermedia and promote community development [90,98,99].
Additionally, since P. gingivalis is devoid of LuxI/ LuxR-based signaling mechanisms, it utilizes the LuxI/LuxR receptors on S. gordonii to enhance the gene expression responsible for the acquisition of hemin, extracellular polysaccharide formation, and accretion into micro-colonies [91][92][93][94]. The LuxS in S. gordonii regulate the levels of the 'Ssp' adhesins and influence its ability to adhere to micro-colonies and autoaggregation in P. gingivalis [50].
Recently, two novel proteins named 'internalin pro-Clptein (InlJ)' along with the 'expression of the short fimbriae and the universal stress protein UspA' have been linked with monospecies P. gingivalis biofilms formation and initial attachment [100][101][102][103][104][105][106]. Furthermore, the loss of several genes has been observed within the P. gingivalis colonies to regulate the growth of the biofilm and promotes its chance of survival. Some of the inhibitors of homotypic biofilm accumulation include Caseinolytic Mitochondrial Matrix Peptidase Chaperone Subunit X (ClpXP) along with ClpC, and GalE (UDP-galactose 4-epimerase) have been found in P. gingivalis microcolonies [99,[103][104][105][106][107]. P. gingivalis is also observed to cause lysine acetylation in colonies, and this is considered as novel mechanism of metabolic regulation, adaptation, and survival during infection [105]. The acetylation of RprY, a response regulator of P gingivalis during the oxidative stress, has been considered a key factor regulating gene expression. Lysine acetylation reduces the promoter DNA binding ability of RprY, which consequently alters the gene expression of the host [105][106][107].
Apart from regulating the genetic expression and release of important proteins, P. gingivalis can even facilitate the growth of other bacterial species by acting as a 'bridging species' [99,108,109]. P. gingivalis facilitates the entry of pathogens into deep periodontal pockets. Studies have proven that the outer membrane vesicle of P. gingivalis (Omp A) facilitate co-aggregation and piggybacking of T. denticola [109][110][111][112]. Furthermore, P. gingivalis promotes the attachment of Lachnoanaerbaculum. saburreum to T. denticola by acting as a 'bridge' and facilitating complex microbial community formation [109][110][111]. The piggybacking of T. denticola and Lachnoanaerbaculum saburreum favors the migration of bacteria to the anaerobic sub-gingival niches and changing the ecology of the entire sub-gingival sulcus. The cooperation and co-existence of P. gingivalis with T. denticola can also increase the production of gingipains (Kgp, RgpA, HagA) and dentilisism from T. denticola [99]. The increased 'gingipain' production inhibits blood coagulation, escalates nutrient accumulation, and in turn favors attachment of P. gingivalis to the other microorganisms, epithelial cells, and fibroblast [113]. P. gingivalis is also known to 'cheat' its community as it retains some of the energy-consuming mechanisms that are discarded by other membranes of the community. This mechanism of retaining certain discarded features helps P. gingivalis to survive in times of crisis and support the growth and functioning of the entire microbial community [15].
An important metabolite that contributes regulate the metabolic interactions of P. gingivalis with other accessory pathogens and prevents its attachment to the host is arginine. Studies have shown that arginine deaminase (ArcA) of Streptococcus cristatus and S. intermedius impairs biofilm formation by inhibiting the production of FimA surface proteins [113][114][115][116]. The down-regulation of FimA catalyzes the conversion of arginine to citrulline. Reduced levels of extracellular arginine and/or citrulline accumulation inhibits FimA expression and biofilm formation [24,117]. An in vitro study on a mouse model has even confirmed that colonization of ArcA-expressing S. cristatus followed by P. gingivalis infection decrease the colonization of P. gingivalis [115]. Although the presence of ArcA from S. gordonii is yet to be confirmed, it is well characterized that S. gordonii ArcA has a well-developed arginine deaminase system (ADS), which catalyzes the intracellular conversion of arginine to ammonia and CO2, along with concomitant production of ATP [117]. Therefore, the distinctive response of P. gingivalis to S. gordonii is due to its ability to transform arginine into citrulline in an extracellular manner. Therefore, targeting ArcA surface proteins could form a 'potential anti-biofilm agent to fight P. gingivalis infections' [114][115][116][117][118]. Arginine and its derivatives also affect the interaction of F. nucleatum to other microorganisms in the biofilm [119]. F. nucleatum harbors an adhesin that is inhibited by arginine (RadD) [117,119]. Therefore high concentrations of arginine can inhibit cell-tocell contact (i.e., coaggregation) between F. nucleatum and other bacterial species [120]. Another, novel cross-feeding interaction between S. gordonii and F. nucleatum is that involving ornithine, which is exported by the arginineornithine antiporter ArcD in the ADS of S. gordonii, has been reported [119]. In-vitro studies confirmed that 'deletion of ArcD attenuates the accumulation of F. nucleatum in S. gordonii biofilm, while ornithine supplementation restored the bio-volume of F. nucleatum in mono-species and dual-species biofilms with the S. gordonii DarcD mutant'. This proves that ArcD-exported ornithine supports the growth of F. nucleatum and sustains the development of its biofilm. F. nucleatum is also known to increase the levels of ornithine decarboxylase (ODC), an enzyme responsible for the conversion of ornithine/arginine to putrescine, in the community biofilms formed with S. gordonii [37]. F. nucleatum utilizes the ornithine released by S. gordonii ArcD as a substrate of ODC and enhance the overall community development by mediating cross-feeding of ornithine, and reinforcing coaggregation between S. gordonii and F. nucleatum. These findings suggest that sustained delivery of ornithine from accessory pathogens induce a state of dysbiosis, by sustaining the growth of the entire microbial community [116]. Furthermore, S. gordonii produces a distinct pattern of protein in communities with F. nucleatum or P. gingivalis, especially with the ADS component enzymes ArcA, ArcB (catabolic ornithine carbamoyltransferase), ArcC (carbamate kinase), and ArcD has been observed [61][62][63][64][65][66][67][68]115,116,118]. The levels of ArcA, ArcB, and ArcC of S. gordonii were reduced in community biofilms formed with P. gingivalis as compared to mono-species biofilms. On the other hand, the interaction of S. gordonii with F. nucleatum showed a marked increase in the levels of ArcA, ArcB, and ArcC despite a significant reduction in the level of ArcD in S. gordonii [62][63][64][65]121].
Apart from using the host cell to gain entry, inactivation of cofilin by SerB impairs the process of phagocytosis, chemotaxis, and transendothelial migration of neutrophils [134][135][136][137][138][139][140][141][142][143][144]. It is also known to upregulate P-21 Activated Kinase (PAK) and Rho-Associated-Kinase (ROCK) receptors that indirectly increase pro-inflammatory cytokines production in the host [90][91][92]. SerB dephosphorylates the serine residues of the Nuclear factor-kappa Beta (NF-κB) and inhibits CXCL8 induced IL8 production [15,[93][94][95][96][97][98][154][155][156][157][158]. Reduced IL8 levels impair the process of phagocytosis and allow P. gingivalis to use the epithelial cells as an autophagosome for its growth and survival [96,[149][150][151][152][153][154][155][156][157]. The SerB even remains functionally viable within the cells by interacting with other bacteria proteins. For example, SerB of P. gingivalis binds to the Heat Shock Protein (HSP) and GAPDH (glyceraldehyde 3 phosphate dehydrogenase) of Streptococcus oralis within the periodontal tissues to increase the inflammatory response [80,83,100] P. gingivalis also inhibit, TLRs, E-selectin and intercellular adhesion molecular (ICAM) expression on the endothelial cell surface and binds to the non-chemotactic methionyl peptides receptor to inhibits the neutrophil chemotactic gradient, leukocyte adhesion, and recruitment of neutrophil to the site of inflammation [150][151][152][153][154][155][156][157][158][159][160]. The reduced chemotaxis decreases the rate of Figure 6. Schematic representation Immune response pathways triggered by the activation of TLR2/TLR4/CXCR5/C5aR receptors by gingipains P. gingivalis: The gingipains of P. gingivalis degrade the C5 and C3 from the complement system and degrade them C5a and C3a. The C5a interacts with the receptors on the neutrophils, epithelial cells, and endothelium in the host to Impairs phagocytosis and increase proinflammatory cytokine. C3a also inhibits the caspase 11-dependent non-canonical inflammasome pathway and prevents the apoptosis of the cell and allows P. gingivalis to use the host cell for its growth. The C5aR-TLR2 cross-talk activated by P. gingivalis pili induced degradation of MyD88 in neutrophils. In the absence of MyD88, the co-association of C5aR-TLR2 promotes P. gingivalis infection v activation of the TIRAP-dependent PI3 K signaling pathway. This, in turn, causes an inflammatory cytokine TNF-α along with inhibition of RhoA activation and actin polymerization that impairs the process of the maturation of phagosomes and P. phagocytosis and facilitates the growth of the entire microbial community. The activation of TLR2 is required for bacterial persistence and its deficiency has been linked with inhibition of alveolar bone resorption [161]. Apart from SerB, lipopolysaccharide (LPS), FimA fimbriae, Lipoteichoic acid of P. gingivalis modulate the immunoinflammatory response by inhibiting the production of antimicrobial molecules, complement system, and recruitment of leukocytes and favoring colonization of microorganisms in the periodontal tissues [95][96][97][98]100,151,155,[160][161][162] P. gingivalis release of various antimicrobial peptides (βeta defensins 2), activate the coagulation cascade and the kallikrein/kinin cascade to increase the inflammation in the host [150,160].
P. gingivalis LPS also activates the TLR 2-CXC chemokine receptor 4 (CXCR4) and Complement receptors (C5aR) on the neutrophil surface [150][151][152][153]170]. The Arg-specific gingipains (RgpB and HRgpA) co-activate and mediate the cross-talk between complement receptor (C5aR), TLR2, and CXCR4 [157]. TLR2-C5aR activations increase the ubiquitin via E3 ubiquitin ligase Smurf1 dependent proteasomal degradation and ubiquitination of The atypical LPS of P. gingivalis interacts with the TLR2/TLR4/CXCR5/C5aR receptors on the host cells to activate Phosphoinositide 3-kinase (PI3 K), cyclic AMP and PKA. The C5a-C5aR activation mediated by P. gingivalis gingipain-degradation of C5 synergistically enhances the production of cAMP. The combination of pili and CXCR4 helped maximize cAMP production via C5a-TLR2 cross-talk. The continuous increase in cAMP activated PKA to reduce macrophage-forming NO and destroying the bactericidal function and possible therapeutic targets. The increased cyclic AMP inhibits the release of nitric oxide that further impairs the process of transendothelial migration, chemotaxis, and phagocytosis. Arg-specific gingipains and SerB protein of P. gingivalis alters the process of actin polymerization. The altered actin affects the process of phagocytosis along with promoting the development of the pathogenic species that can indirectly increase the inflammatory process in the periodontal tissues [Abbreviation: CR-complement receptors; TLR-Toll-like receptors; CXCR4: C-X-C chemokine receptor type 4; cAMP: cyclic adenosine monophosphate; iNOS: inducible nitric oxide synthase; Mal: MyD88 adapter-like; p38MAPK: mitogen-activated protein kinase p38; PKA: protein kinase A; PI3 K: phosphoinositide-3-kinase; RhoA: Ras homolog gene family, member A] Myeloid differentiation primary response gene 88 (MyD 88). The inhibition of MyD 88 receptor blocks the signals from the TLR2-PI3 K pathway and activate IL1 receptors with increased production of IL 1 [95,100,110,[156][157][158][159]. The crosstalk between C5aR with TLR2 activates the PI3 K signaling pathway and indirectly increases the inflammatory response [36,126,153]. The PI3 K activation prevents phagocytosis, inhibits RhoA activation, and actin polymerization. The inhibition of TLR2-PI3 K pathway even impairs the process of phagocytosis by inhibiting the RhoA GTPase dependent actin polymerization [95][96][97][98][100][101][102][161][162][163]. The inhibition of LR2/ MyD88 signaling causes the death of infected neutrophils and blocks the process of phagocytosis in the host [29,96]. P. gingivalis can also inhibit all the components of the complement system (classical, alternative and lectin pathways) and degrades the byproducts of the complement system [139,163] P. gingivalis inhibits the Mannose-Binding Lectin (MBL), Ficolins (FCN), C3, C3b, and C4 components of the complement system and prevent the formation of the 'Membrane Attack Complex' (MAC) [161,[163][164][165][166][167][168] The inhibition of MAC and other components of the complement system favors P. gingivalis to evade the complement-mediated phagocytosis and survive within the host [139][140][141][142][143]. Moreover, P. gingivalis utilizes its gingipain to entrap the circulating C4b-binding protein and defend itself from being phagocytized by the molecules of the complement system [109,141]. The Arg specific gingipains even possess C5 convertase like properties that increase the concentration of C5a in the gingival crevicular fluid [108]. The C5a binds to the C5a receptor (C5aR) on the leukocytes and impair leukocyte killing capacity [151,170]. The cross-talk between upon P. gingivalis with TLR2-CXCR4 receptors causes a sustained release of cAMP [171]. The increase in cAMP production weakens the macrophages induced nitric oxide synthase [iNOS] dependent phagocytosis and facilitate the growth of the entire microbial community (Figure 7) [20,114,126]. The increased c-AMP production along with a decrease in the IL10 levels reduces the production of nitric oxide (NO) from the endothelial cells. P. gingivalis also evade the nitric oxide synthetase expression by modifying the structure of the Lipid A moiety of its LPS that indirectly inhibit TLR4 activation [15,21]. Additionally, RgpB and HRgpA gingipains of P. gingivalis along with karilysin enzyme secreted from Tannerella forsythia and InpA molecules from P. intermedia activate the release of C5a from C5 and degrade the central component C3 and Immunoglobulin G [172]. This synergistic interaction of gingipains with Interpain A and karilysin weakens the host immune response and allows persistence of the microbe within the host [90].
The LPS of P. gingivalis also increases the production of thrombospondin-1 (TSP), an extracellular matrix protein secreted by monocytic cells in the periodontal tissues. TSP stimulates the movement of the macrophages and increases the macrophagemediated inflammation in the periodontal tissues [173]. Additionally, P. gingivalis increases the production of IL17 to synergistically enhance the production of TSP1 and plasminogen activator inhibitor type I in the gingival and periodontal tissues. The increased production of TSP1 and plasminogen activator inhibitor type I contribute to the persistence of the microorganisms within the host that leads to the progression and chronicity of periodontal disease [174,175]. P. gingivalis persistence within the gingival epithelial cells is known to induce the production of several microRNAs, such as miR-105 and miR-203 and short-chain fatty acids. The miR-105 and miR-203 can suppress TLR2 function and inhibit the release of Suppressor of Cytokine Signaling 3 (SOCS3) and SOCS6 respectively [148,149]. The long-term cohabitation of P. gingivalis within the host cell favors the establishment of an 'interkingdom' whereby it impairs the important defense mechanisms of the host ( Figure 6) [171,176,177].
Recently, P. gingivalis is found to increase the production of butyrate and propionate and decrease the release of cytokine-induced neutrophil chemoattractant (CINC) 2αβ, another powerful chemoattractant and inhibitor of chemokines production [132,175]. The outer membrane components of P. gingivalis is proven to possess 'porin-like activity' that can depolarize the electrochemical potential on the neutrophil membrane and prevents its migration in response to the chemotactic stimuli [132,171]. A study done by Chen et al. (2011) showed that the outer membrane protein (PG0027) is 'essential for the O-deacylation of LPS, secretion of gingipain to the cell surface, and attachment of P. gingivalis' to host cells [171]. The hemaagglutinins and gingipains released from P. gingivalis help to create a nutrient-rich but oxygen-deficient environment that protects P. gingivalis from the immune response of the host and promotes its survival [22,113]. P. gingivalis along with T. denticola and T. forsythia degrade and inactivate various antimicrobial molecules and enzymes secreted by the neutrophils to survive in the inflammatory milieu [177][178][179][180]. Recently, the gingipains from P. gingivalis are found to degrade the salivary Triggering Receptor Expressed on Myeloid cells 1 (sTREM-1) on the neutrophils and impede the process of phagocytosis and chemotaxis ( Figure 6). P. gingivalis utilize its Arg-gingipain and sTREM1 receptors to create an inflammation rich environment for its acquiring nutrients. However, when the inflammation starts to compromise its existence, P. gingivalis changes from its Arg-gingipain to Lys-gingipain and attenuates the periodontal inflammation. This 'twin regulation' of sTREM 1 by Lys and Arg gingipains is a unique mechanism adopted by P. gingivalis for its survival and persistence [179][180][181][182][183]. Nylund et al., (2017) evaluated the association of the Salivary TREM-1 (sTREM-1)/its Ligand Peptidoglycan Recognition Protein 1 (PGLYRP1) in periodontitis patients with renal diseases and concluded that PGLYRP1 and sTREM-1 are strongly associated with increased proinflammatory cytokine production in patients with periodontitis [183]. Patients with deep and active periodontal pockets have more salivary sTREM-1 and PGLYRP1 concentration as compared to individuals with shallow probing depth. Further research has shown that P. gingivalis modulates sTREM-1 receptor by acting as a decoy receptor for TLR activation and inhibiting neutrophil migration. The simultaneous activation of TLR4 and sTREM1 synergistically increase the production of TNF αlpha, IL1β, and decreased the production of antiinflammatory cytokines like IL10. The TLR-sTREM1 activation is also known to activate various receptors associated with increased production of proinflammatory cytokines such as IRAK-1 (IL1 R-associated kinases), MAPK, p38 MAPK, Jun N-terminal Kinase (JNK), PI3 K, ERK1/2, NF-kB ( Figure 6 & Figure 7) [183]. TREM-1 molecule is known to activate nucleotide-binding oligomerization receptors (NOD1 and NOD2) and indirectly increase the activation of caspase and NF-kB receptors that can exaggerate the production of proinflammatory cytokine in an autocrine manner [184].

Inhibition of apoptosis increased oxidative stress and activation of the inflammasome
P. gingivalis is also known to inhibit the apoptotic pathways of the infected host cell and utilizes the cellular machinery for its survival. The inhibition of apoptosis contributes to the chronicity of the periodontal disease by promoting the growth of other bacterial species (Figure 6) [17,91]. The LPS of P. gingivalis activates caspase 11 dependent noncanonical inflammasome and initiates the process of pyroptosis and lytic cell death within the macrophages [135,[184][185][186][187][188]. P. gingivalis LPS, specifically the O-antigen region and HmuY is known to affect the 'viability and apoptosis of gingival epithelial cells' [54,62,155,[187][188][189][190]. The HmuY protein of P. gingivalis can induce apoptosis in the gingival epithelial cells by increasing FAS Ligand expression and NFκB receptor activation. The Fas ligand, also referred to as CD95 L or CD178, is a type-II transmembrane protein belonging to the TNF family, which initiates the process of apoptosis. A study by Meghill et al., 2019 elucidates the underlying mechanisms by which P. gingivalis manipulates dendritic cell signaling to perturb both autophagy and apoptosis. The results of their study showed that the minor (Mfa1) fimbriae of P. gingivalis induce Akt nuclear localization and activate the Akt/mTOR axis required for autophagosome formation and maturation [190]. P. gingivalis also increase the mitotic cell cycle and suppress apoptosis by inhibiting Janus A Kinase (JAK), Phosphoinositide 3 Kinase (PI3 K), Signal Transducer of Activation (STAT), alpha-serine /threonine-protein kinase (Akt) and purinoceptor (P2X7) pathways (Figure 7) [91] Furthermore, P. gingivalis reduce the renewal capacity of cells and inhibit apoptosis by activating p38, mitogen-activated protein kinase (MAPK), and extracellular-signalregulated kinase (Erk1/2) pathways [184][185][186] The activation of p38, MAPK, and Erk1/2 pathways decrease the expression of cyclin D and inhibit cellular proliferation by arresting the cell cycle at the G1 phase [72,73]. Studies have confirmed the deregulation of apoptosis-related genes, such as Bax, Bcl2, Nlrp3, or Smad2, in the gingival tissues of patients with periodontitis [181,189] P. gingivalis has also confirmed to possess cellspecific modulation of apoptosis receptors and signaling pathways such as Apoptotic protease activating factor (APAF 1), B-cell lymphoma Associated X (Bax1) and Caspase and reduce B-cell lymphoma (BCL 2) in the epithelial cells and fibroblasts [184]. In the epithelial cells, P. gingivalis blocks the epithelial cell death by decreasing the APAF-1 expression, reducing the enzymatic activity of caspase enzyme, and increasing the expression of X-linked inhibitor of apoptosis protein (XIAP). However, in the fibroblast, P. gingivalis stimulates the APAF-1 pathway and reduces XIAP expression, increases caspase expression and apoptosis. The oligomerization of APAF1, induced by its binding to cytochrome C, helps apoptosome formation, a structure that recruits and activates a caspase initiator, caspase 9. The activation of caspase 9 cleaves and activates caspase effector caspase 3 and caspase 7, leading to apoptosis [184]. The increased caspase expression following P. gingivalis infection increases the process of pyroptosis, hypoxia, and exaggerate the inflammatory burden in the periodontal tissues and systemic circulation [190][191][192][193][194][195].
P. gingivalis can even inhibit F. nucleatum induced activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome and modulates the NLRP3 inflammasome cytokine secretion from the macrophages [189]. NLRP3 inflammasome is associated with the release of cytokines (IL1b and IL18), apoptosis, phagocytosis, and pyroptosis. NLRP3 regulates the process of apoptosis in the osteoblasts cells infected with P. gingivalis and A. actinomycetemcomitans [188]. Additionally, P. gingivalis interacts with the CR3 receptors on the macrophages and decreases the IL12p70 induced bacterial clearance by the macrophages [190][191][192][193]. The enzyme sialidase in P. gingivalis has been lately discovered as novel mechanisms to inhibit macrophage activity. Studies have shown that sialidase-deficiency in P. gingivalis can attenuate CR3 activation in macrophages, reduce inhibition of lncRNA GAS5, with less miR-21 and more IL12 production. Inhibition of sialidase in P. gingivalis would render P. gingivalis more easily cleared by macrophages [193]. The compromised macrophage function impairs the process of antigen presentation, lymph node activation, and delays the onset of cellular immunity in the host [189].
Studies have even confirmed that P. gingivalis infection increases the production of 8 hydroxy 2ʹ deoxyguanosine, superoxide anions, matrix metalloproteinases, caspase-3, catalase enzyme in the host cells [192,197]. P. gingivalis protect itself against the extracellular ATP and oxidative stress by releasing a unique enzyme known as nucleotide-diphosphatekinase (NDK) (Figure 1) [147]. NDK cleaves the extracellular ATP molecules and down-regulates the P2X7/pannexin 1 receptor that is activated by autocrine action of ATP, within the host [200]. The NDK from P. gingivalis accumulates in the cytoplasm and is subsequently carried along myosin-9 filaments and actin filaments to the host cell periphery [150]. Upon translocation to the extracellular environment through the formation of the P2X7/pannexin 1 channel, NDK hydrolyzes ATP, and decrease the ATPinduced IL-1β release [92,150,152]. Additionally, the NDK inhibits ATP and increase the reactive oxygen species (ROS) production that helps in bacterial persistence [150][151][152][201][202][203] Furthermore, it has been observed that even though P. gingivalis increase the oxidative stress in the host, it remains unaffected by the ROS due to the release of various endogenous antioxidants, like thiol peroxidase, glutathione, superoxide dismutase, rubrerythrin, in and around its cell surface [18,147,[180][181][182][183]200,204,205]. The endogenously produced antioxidants protect P. gingivalis from the ROS mediated injury and allow it to survive within the host. The increase in the levels of glutathione suppresses the mitochondrial-mediated intrinsic cell death and protects the bacteria against the reactive oxygen species (ROS) that help P. gingivalis to survive within the host [18,147]. P. gingivalis selectively inhibits the macrophage-mediated cytokine production without affecting the production of T cell-mediated chemokines [206] The selective inhibition of the inflammatory process allows it to acquire the essential nutrient derived from the dead bacteria and tissue breakdown products for its growth and provide a competitive advantage to P. gingivalis during the inflammatory process [18,20,119,126,188].

Alteration of T cell function with activation of Th17 cell
Another important mechanism by which P. gingivalis impairs the host immune response is by altering the T cell function and activation of Th17 mediated cytokine production [201][202][203][205][206][207][208]. It has been observed that P. gingivalis can induce TLR2dependent IL-10 production that 'leads to inhibition of IFN-γ production by CD4+ and CD8 + T cells [209]. P. gingivalis down-regulates IL12 secretion and increases the release of Interferon-alpha (IFN-alpha) from the CD4 Th1 cells. The decreased levels of IL12 and increase the production of IFN-alpha shifts the immune response from Th1 to Th2 [14]. The outer membrane proteins of P. gingivalis is found to activate the Th1 cells that in turn increase the production of proinflammatory cytokines [210] Furthermore, P. gingivalis interacts with the dendritic cells of the host and favors the production of Th17 related cytokines such as TNFα, IL8, CXCL8, IL17, IL1β, IL6, IL23, IL12p40, IL21, IL3, granulocyte colony-stimulating factor (GCSF), etc. from both immune and non-immune cells of the host [147][148][149]202,203,[206][207][208][209][210]. The increased production of IL1, IL 6, IL 21, IL23, TGF βeta amplifies and stabilizes the Th17 cell differentiation and favors the onset of dysbiosis and periodontal inflammation. The activation of Th17 cells increases the levels of IL1β, IL6, and TNFα in the periodontal tissues, which is associated with increase bone and soft tissue destruction in the periodontal tissues [210]. IL17 activates the RANKL receptor on the osteoblasts and fibroblasts and induce bone resorption by expressing the 'osteoclast activating factor' on the alveolar bone [210]. Furthermore, the arg-gingipain from P. gingivalis is known to modulate and increase the production of proinflammatory cytokines by acting on novel receptors sites like protease-activated receptors [PAR]-2 and soluble triggering receptor expressed on myeloid cells [sTREM-1] [211]. The activation of PAR-1, PAR-2, and PAR-4 induce CD69 and CD25 expression in CD4 + T cells that in turn increase the production of IL17 [211][212][213][214][215]. P. gingivalis also interacts with the NF-κB and RAR-related orphan receptor (ROR) transcription factor on the alveolar bone and increases the production of IL17 mediated bone resorption [213].

P. gingivalis facilitates 'Fratricide and Programmed Cell
Death' of other bacterial species within the biofilm to increase the inflammatory response and promotes its survival [60,216] Fratricide is the pathogenic process by which P. gingivalis kills and obtain DNAs fragments from noncompeting host cells and microbes by inducing their death [216,[218][219][220][221] During the process of programmed cell death, P. gingivalis promotes a fraction of the microbial population to perform a self-sacrificing 'suicide' and discharge its nutrients and extracellular DNA fragments into the environment. The discharged nutrients and extracellular DNA fragments are utilized by P. gingivalis and other members of the microbial community for their growth. The process of 'Fratricide and Programmed Cell Death' is tightly regulated by a pattern of genes along with the presence of an intricate toxin and antitoxin system [216]. For example, when P. gingivalis strains are added to the biofilm containing Streptococcus. mitis and Staphylococcus aureus, cell death and altruistic suicide of microbial colonies take place [60] Some of the essential molecules that are linked with Fratricide and Programmed Cell Death include Choline Binding protein D (CbpD), Competence-induced bacteriocins (CibAB), Autolysin-Encoding Gene (LytA, LytC, LrgAB, LytR, and LytSR), CidABC operon, etc [220]. When P. gingivalis is added to biofilms containing S. mitis, apoptosis-like death with release of DNA fragments and production of transposase that marks the onset of programmed cell death have been observed [60]. P. gingivalis is even known to activate the bacterial apoptosis endonuclease (BapE) enzyme that can fragment the chromosomes by sequentially cleaving the supercoiled DNA. The DNA damage and induced chromosome fragmentation activate the process of apoptosis in the entire microbial community and intensify the process of dysbiosis and inflammation in the host [166][167][168].

Conclusion
P. gingivalis is a potential and highly virulent periodontal pathogen that adopts intricate molecular mechanisms to interact with other members of the microbial community and host to pave its path of invasion, survival, and persistence. It adopts unique molecular and intricate mechanisms to survive and persist within the tissues and exaggerate inflammation and dysbiosis. P. gingivalis modulated the entire oral microbiome and creates an environment that not only favors its growth and survival but also facilitates the growth of many other commensal and pathobionts species. The interbacterial interactions, immune evasion, and tissue invasive properties help P. gingivalis to survive even in adverse environmental conditions and reach a distant organ system. However further research on how P. gingivalis invade the epithelial barrier of different organs and induce organ dysfunction is intriguing and needs more research. It is also necessary to explore if the altered immune response by P. gingivalis considerable enough to acquire other opportunistic infections and systemic diseases. More research is necessary at the genomic level to understand and discover the transcriptomes, nucleotide, and metabolites affected in the host by P. gingivalis with the host and other members of the microbial community upon invasion. It is also necessary to explore if these immunologic mechanisms form a link between P. gingivalis and the development of various cancers. Furthermore, it is also critical to explore if any of these cellular and molecular mechanisms are associated with the development of emerging antimicrobial resistance of periodontal pathogens to conventional antibiotics. There is a need to study the strain-specific variation of P. gingivalis on the modulation of host response and systemic inflammation. The paper is of paramount importance to researchers, oral biologists, microbiologists, immunologists, scientists, and pharmacologists across the globe to initiate further research and develop novel therapeutic modalities that can target the specific mechanism and prevent the onset of oral and systemic inflammation in the host