Genetic Characteristics and Pathogenic Mechanisms of Periodontal Pathogens

Abstract

Periodontal disease is caused by a group of bacteria that utilize a variety of strategies and molecular mechanisms to evade or overcome host defenses. Recent research has uncovered new evidence illuminating interesting aspects of the virulence of these bacteria and their genomic variability. This paper summarizes some of the strategies utilized by the major species–Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Treponema denticola, and Porphyromonas gingivalis – implicated in the pathogenesis of periodontal disease. Whole-genome sequencing of 14 diverse A. actinomycetemcomitans strains has revealed variations in their genetic content (ranging between 0.4% and 19.5%) and organization. Strikingly, isolates from human periodontal sites showed no genomic changes during persistent colonization. T. forsythia manipulates the cytokine responses of macrophages and monocytes through its surface glycosylation. Studies have revealed that bacterial surface-expressed O-linked glycans modulate T-cell responses during periodontal inflammation. Periodontal pathogens belonging to the “red complex” consortium express neuraminidases, which enables them to scavenge sialic acid from host glycoconjugates. Analysis of recent data has demonstrated that the cleaved sialic acid acts as an important nutrient for bacterial growth and a molecule for the decoration of bacteria surfaces to help evade the host immune attack. In addition, bacterial entry into host cells is also an important prerequisite for the lifestyle of periodontal pathogens such as P. gingivalis. Studies have shown that, after its entry into the cell, this bacterium uses multiple sorting pathways destined for autophagy, lysosomes, or recycling pathways. In addition, P. gingivalis releases outer membrane vesicles which enter cells via endocytosis and cause cellular functional impairment.

Introduction

Pathogenic bacteria use a variety of strategies and molecular mechanisms to evade host defenses and to enhance their penetration into and colonization of host tissues. Some periodontal pathogens, including Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Treponema denticola, and Porphyromonas gingivalis, have developed sophisticated strategies for host evasion and virulence, such as molecular mimicry of host tissues to avoid immune detection, modulation of the host immune response, and entry into host cells to escape immune surveillance. Recent research has shed further light on some of the properties and mechanisms that contribute to the virulence of these key species. Periodontal bacteria exploit a vast number of mechanisms to interact with human tissues during patho-genesis (Berezow and Darveau, 2011; Hernández et al., 2011; Hajishengallis and Lamont, 2012; Teles et al., 2013). Among them, this paper focuses on four topics.

A. actinomycetemcomitans has long been implicated in periodontitis, but is also found in healthy mouths as part of the commensal flora (Haffajee and Socransky, 1994). Such variations in pathogenicity may be due to genetic variability between and among different strains. Recent studies, outlined in this paper, have investigated this possibility.

Recent studies on an enigmatic and relatively less-studied organism, T. forsythia, have revealed that this bacterium induces alveolar bone loss in mice by driving TLR2-dependent polarization of Th cells toward the Th2 subset. This effect depends on the bacterium's ability to decorate its surface with unique O-glycan structures that play an immunomodulatory role.

Table.

A. actinomycetemcomitans Strains Included in the Comparative Genomic Analysis

Strain	2nd Straina	Serotype	Race/Ethnicityb	Age (yrs)	Gender	Diagnosisc	Geographic Location
D7S-1	N.A.	a	Af	29	Female	AP	USA
D17P-3	N.A.	a	As	24	Male	AP	USA
H5P1	N.A.	a	As	27	Female	H	USA
HK1651	N.A.	b/JP2	African	N.A.	N.A.	AP	Ghana
ANH9381	N.A.	b	C	50	Male	H	Finland
I23C	S23A	b	C	48	Male	CP	Finland
SCC1398	SCC4092	b	C	25	Female	AP	Finland
SCC2302	AAS4a	c	C	33	Female	G	Finland
D11S-1	N.A.	c	Af	16	Female	AP	USA
D17P-2	N.A.	c	As	24	Male	AP	USA
I63B	N.A.	d	C	49	N.A.	H	Finland
SCC393	A160	e	C	40	Male	CP	Finland
SC1083	N.A.	e	N.A.	N.A.	N.A.	N.A.	N.A.
D18P-1	N.A.	f	As	20	Female	AP	USA

^aA second strain isolated from the same individual; N.A., not available.

^bAf, African American; As, Asian-American; C, Caucasian.

^cA P, aggressive periodontitis; H, periodontally healthy; C P, chronic periodontitis; G, gingivitis.

In another kind of surface modification, “red complex” bacteria have been shown to utilize neuraminidases to scavenge host sialic acid for use as a decorating molecule. This tactic helps the pathogens to evade host immune defenses.

Bacterial invasion of host cells has been well documented and is thought to be an important capability for the successful pathogen. The mechanisms by which P. gingivalis and its products gain entry into host periodontal cells, their possible intra-cellular destinations, and the effects of this bacterial occupation are discussed in the following.

Comparative Genomics of A. Actinomycetemcomitans

Gram-negative A. actinomycetemcomitans is a facultative bacillus phylogenetically related to a number of mucosal pathogens and commensal bacteria in Pasteurellaceae. The organism is considered one of the three or four primary periodontal pathogens and a major cause of localized aggressive periodontitis (Haffajee and Socransky, 1994). However, it is also present as a component of the oral microbiota in periodontally healthy individuals (Slots and Ting, 1999). This implies strain-to-strain variation in their virulence potential. Such variation could be attributed to differences in the gene content and organization in the genome of individual strains (Hacker and Carniel, 2001). It was therefore hypothesized that the A. actinomycetemcomitans genome consists of a core gene pool (i.e., genes shared by all strains) and an accessory gene pool (i.e., genes shared by some but not all strains) similar to many other bacterial species (Hacker and Carniel, 2001). The accessory genes, in particular those organized as genomic islands, may encode novel virulence determinants acquired by A. actinomycetemcomitans via horizontal gene transfer (HGT). Once identified, the accessory genes and genomic islands may be examined further for their roles in the pathogenesis of periodontitis.

This hypothesis was tested in a series of studies that involved a large set of A. actinomycetemcomitans strains (Table) (Kittichotirat et al., 2010, 2011; Sun et al., 2013). First, whole-genome sequencing (WGS) was performed with the 454 platform of 14 clonally, geographically, and clinically diverse strains of A. actinomycetem-comitans (Kittichotirat et al., 2011). The sequencing results were subject to gene identification and annotation by standard protocols. The results showed that the pangenome of A. actinomycetemcomitans is composed of 3,301 protein-coding genes, which included 2,034 core genes and 1,267 accessory genes (Fig. 1). Approximately three-quarters of the accessory genes were classified as poorly defined by Cluster of Orthologous Groups. The pangenome of A. actinomycetemcomitans was open, with16 new genes expected for each future additional strain sequenced. The variation in gene content between and among strains was 0.4-19.5% of their genomes. In total, 171 genomic islands were identified among the 14 strains and accounted for 61% of accessory genes found among 14 A. actinomycetemcomitans strains. The functions of the genomic islands of A. actinomycetemcomitans are largely unknown. However, the utility of comparative genomic analysis for identifying virulence determinants is exemplified in the case of cytolethal distending toxins cdtABC (Mayer et al., 1999). Here the analysis clearly identified a large genomic island carrying cdtABC in the A. actinomy-cetemcomitans strain D7S-1 but not strain SC1083. Such an insertion/deletion relationship between and among strains defines the boundaries of genomic islands, identifies their gene content, and permits testing of the phenotypes associated with the islands.

Figure 1.

The size of the A. actinomycetemcomitans pangenome and the range of the strain-to-strain variation in the gene content of their genomes.

In addition to variations in the gene content of the genome, A. actinomy-cetemcomitans strains exhibit remarkable differences in the arrangement of genes in their genomes (Kittichotirat et al., 2010). For example, the difference in the genomic arrangement (calculated as reversal distances) (Tesler, 2002; Darling et al., 2004) between A. actinomycetemcomitans serotype a strain D7S-1 and serotype b strain HK1651 was much greater than 14 such intra-species comparisons of 7 randomly chosen bacterial species (Kittichotirat et al., 2010). Such variations in gene position could have an impact on gene expression profiles or bacterial growth, due to the interaction between bacterial cellular processes and chromosomes (Rocha, 2008). With detailed genomic information available, one could design a specific hypothesis to test the relationship between gene position and expression in A. actinomycetemcomitans.

Finally, the gains and losses of genes during persistent colonization of A. actinomycetemcomitans in human periodontal sites were assessed (Sun et al., 2013). WGS was combined by comparative genomic hybridization with microarray, transcrip-tomic profiling by microarray, and PCR analysis to examine the genomes of four pairs of A. actinomycetemcomitans strains. The two strains in a pair were isolated from the same individual and were confirmed to be clonally identical. The results showed very limited genomic changes. More importantly, none of the genomic islands exhibited gains or losses of genes. Transcriptome profiling showed little or no difference in the paired strains. The relative stability of genomic islands may support the hypothesis that they provide critical functions for the bacteria and therefore become fixed in the genome after horizontal gene transfer.

T. forsythia Surface Glycosylation: a Strategy for Manipulating Host Immunity

T. forsythia – a Significantly overlooked Culprit

T. forsythia is another species that has been implicated as an etiologic agent of periodontitis. This species is often found cohabiting with P. gingivalis and T. denticola in subgingival dental plaque biofilms (Tanner and Izard, 2006). Recent studies have also detected T. forsythia in acute and chronic endodontic infections (Saito et al., 2009; Montagner et al., 2012). Increased numbers of T. forsythia are reported in subgingival plaque samples from post-menopausal obese women (Brennan et al., 2007; Haffajee and Socransky, 2009) and patients with chronic periodontitis and type 2 diabetes (Li et al., 2013). T. forsythia is also linked to systemic disease such as cardiovascular diseases (Figuero et al., 2011; Leishman et al., 2012) and arthritis (Moen et al., 2006; Scher et al., 2012). Interestingly, domestic cats could become carriers (reservoirs) of T. forsythia and transmit the bacterium to their owners (Booij-Vrieling et al., 2010).

T. forsythia has remained an under-investigated organism because of its fastidious growth and recalcitrant nature to genetic manipulation. In spite of these difficulties, several investigations on the role of this species in the etiology of periodontitis have been conducted (Sharma, 2010, 2011; Settem et al., 2012a; Stafford et al., 2012).

Tannerella's Sugary Coat

Pathogens maintain their survival in the host through manipulation of the host immune defenses. Toward that end, microbes sometimes decorate their surfaces with unique polysaccharides (sugars), which modulate host-microbe interactions at the immune interface. T. forsythia possesses a surface (S)-layer (Tanner et al., 1986), visible as a striated “tooth-like” coat on the bacterium's surface, with an approximate thickness of 25 nm (Fig. 2A). It is composed of 2 high-molecular-weight proteins, TfsA (200 kDa) and TfsB (210 kDa), abundantly glycosylated with N- and O-linked sugars (Lee et al., 2006). A genetic operon involved in the glycosylation of these proteins has been identified (Honma et al., 2007), and the wecC gene encoding a homolog of UDP-N-acetylmannosaminuronic acid dehydrogenase associated with this operon was later shown to be involved in the terminal modification of a protein-linked O-glycan core with a tri-saccharide motif consisting of two sub-terminal mannosur-onic acid residues and a terminal pseudaminic acid residue (Posch et al., 2011) (Fig. 2B).

Figure 2.

(A) TEM micrographs of T. forsythia ultrathin sections

(courtesy of Dr. G. Stafford, University of Sheffield, UK). (b) O-glycan core structure linked to a peptide motif in surface proteins. WecC is involved in the synthesis of a tri-saccharide branch (circled) on the O-glycan core (adapted from Posch et al. 2011).

Manipulating Immunity

In vitro studies utilizing mutants lacking the S-layer suggested that the S-layer helps to delay the ability of macrophages and monocytes to mount a cytokine response (Sekot et al., 2011). Subsequent studies showed that the terminal tri-saccharide motif (two sub-terminal mannosuronic acid residues and a terminal pseudaminic acid residue; Fig. 2B) on the O-glycan core linked to surface proteins in Tannerella affects the ability of the bacterium to regulate cytokine secretion in immune cells (Settem et al., 2012b). The ED1 strain lacking this O-glycan motif induced significantly higher amounts of IL-6 and IL-1β secretion in mouse bone-marrow-derived dendritic cells (mBMDCs) and macrophages compared with wild-type T. forsythia cells. Interestingly, IL-23 (Th17-activating cytokine) levels increased in both mouse macrophages and mBMDCs challenged with the ED1 strain compared with the wild-type strain. In addition, the loss of the O-glycan motif resulted in increased processing of bacteria by DCs (Settem et al., 2012b).

To ascertain if the surface O-glycans influence the bacterium's pathogenicity in vivo, investigators infected BALB/c mice with the wild-type or ED1 mutant cells by oral gavage and evaluated the bacterial colonization, tissue inflammation, and alveolar bone loss parameters post-infection. Mice infected with the wild-type T. forsythia strain showed higher alveolar bone loss than the ED1-infected mice (Settem et al., 2012b). In addition, the osteoclastic activity correlated with the alveolar bone loss; significantly fewer tartrate-resistant acid phosphatase (TRAP)-positive cells were observed in jawbone sections of mice infected with the medium (sham) or ED1 strain, compared with the wild-type strain. In line with the increased ability of ED1 to induce inflammatory cyto-kines in monocytes/macrophages, this mutant also caused increased infiltration of CD45⁺ lymphocytes in gingival tissues. Moreover, infection with the wild-type strain skewed the CD4+ T-cell differentiation toward Th2, whereas the ED1 infection resulted in skewing toward the Th17 subset. Furthermore, Th17 polarization in the ED1-infected mice paralleled increased neutro-phil infiltration into the mouse gingival tissues. A consequence of the increased neutrophil infiltration was decreased persistence of the ED1 strain compared with that in the wild-type strain, judged by quantitative PCR for T. forsythia DNA in gingival tissues postinfection. Strikingly, under these conditions, the Th17 response was not associated with enhanced alveolar bone osteoclastic activity, in contrast to the role of Th17 in a rheumatoid arthritis setting (Onishi and Gaffen, 2010).

Neuraminidase — a New Player in the Pathogenesis of Periodontitis

Sialic acid is a group of structurally related nine-carbon sugar acids that feature prominently at terminal positions of many eukaryotic surface-exposed glycoconjugates, where they are involved in a wide range of biological processes, including cell-cell interactions and small-molecule-cell recognition (Vimr et al., 2004). Several bacterial pathogens have evolved to utilize sialic acid as decorating molecules to modify their surface-exposed macromolecules, such as lipopolysaccharides (LPS) and polysialic acid (PSA) capsules. Such modifications allow bacterial pathogens to disguise themselves and thus circumvent and/or counteract the host's immune responses (Severi et al., 2007). Bacteria acquire sialic acid either via a de novo biosynthesis pathway or by means of a scavenger pathway that is primarily mediated by neuraminidases. Neuraminidase (also referred to as sialidase) is a family of enzymes that catalyzes the removal of terminal sialic acid from glycoconjugates (Vimr and Lichtensteiger, 2002). Various mucosal pathogens, ranging from Streptococcus pneumoniae in the airway to Vibrio cholerae in the gut, utilize neuraminidases to scavenge host sialic acid. In these bacteria, neuraminidases are often associated with their virulence (Galen et al., 1992; Uchiyama et al., 2009). Neuraminidase activity is also observed in several oral bacteria, including the “red complex” bacteria (Moncla and Braham, 1989). In addition, Gibbons et al. reported that neuraminidase treatment could expose cryptic receptor which in turn increases the attachment of P. gingivalis and other oral pathogens to gingival epithelial cells (Gibbons et al., 1990).

Neuraminidase Activity in the “Red Complex” Bacteria

Similar to other mucosal pathogens, periodontal pathogens such as the “red complex” bacteria possess neuraminidase activity. For instance, T. forsythia encodes two neuraminidases (NanH and SiaH). NanH (TF0035) is a major neuraminidase, and its deletion mutant fails to attach to and invade human gingival epithelial cells (Honma et al., 2011). P. gingivalis encodes at least one homologue (PG0352) of neuraminidase. Recent studies showed that PG0352 is involved in biofilm formation, capsule synthesis (Fig. 3, left panel), serum resistance, and the pathogenicity of P. gingivalis (Aruni et al., 2011; Li et al., 2012). Similar to P. gingivalis, T. denticola also encodes a putative neuraminidase (TDE0471). Experimental evidence has recently shown that TDE0471 is a neuraminidase that removes sialic acid from human serum glycoconjugates, and that it affects nutrient acquisition, complement activation (Fig. 3, right panel), and the virulence of T. denticola (Kurniyati et al., 2013).

Figure 3.

Neuraminidases affect capsule synthesis of P. gingivalis (left panel) and complement activation and deposition of T. denticola (right panel). (For details, see Li et al., 2012, and Kurniyati et al., 2013.) (left panel) One central slice of a tomographic reconstruction from Pg83 (wild type) and ΔPG352 (a neuraminidase-deficient mutant) is shown in panels (A) and (b), respectively. (C, d) Corresponding surface views of the reconstructions from Pg83 and ΔPG352, respectively. (e) The proteinase K treatment (200 μg/mL for 40 min at 37°C) does not remove the thin CPS layer of the ΔPG352 mutant. (f) Supplementing with Neu5Ac (30 μg/mL) restored the CPS formation in the ΔPG352 mutant. The thickness of the CPS layer in the mutant is about 30 nm, which is similar to that of Pg83 (G). The numbers represent the thickness of CPS. (right panel) Deposition of membrane attack complex (MAC) on T. denticola strains: Td35405 (wild type), Tde471 ^mut (a neuraminidase-deficient mutant), and Tde471^com (a cis-complemented strain). A 10⁶ quantity of bacterial cells was co-incubated with 25% normal human serum (NHS) for 20 min at 37°C. As a control, 10 mM EDTA was added to the reactions to block complement activation. The resultant serum-treated cells were subjected to SDS-PAGE, followed by Coomassie blue staining (A), or probed with 3 different antibodies as labeled: (b) αC3, a polyclonal antibody against C3; (C) αC9, a monoclonal antibody against C9; and (d) αC5-9, a monoclonal antibody against the C5-9 complex. Notably, complement components were detected only in the serum-treated Tde471^mut but not in the serum-treated Td35405 and Tde471^com strains. The complement factors detected by the antibodies and their molecular weights are indicated.

As periodontal pathogens, the ”red complex” bacteria primarily inhabit the gingival crevices bathed in plasma, which contains a high concentration of sialic acid that is bound to a diverse range of glycans. In this unique niche, utilization of host sialic acid by a bacterium would confer on it a survival advantage. In this regard, it is not surprising that all 3 “red complex” bacteria possess neuraminidases. Recent studies have demonstrated that the “red complex” bacteria use neuraminidases to scavenge host sialic acid, which can benefit the pathogens in at least two ways: nutrient acquisition and immune evasion (Aruni et al., 2011; Honma et al., 2011; Li et al., 2012; Kurniyati et al., 2013). Interestingly, these neuraminidases are surfaced-exposed and share similar biochemical features. Thus, the neuraminidases may be an ideal target for the development of new therapeutic agents for the intervention and prevention of periodontitis, e.g., the design of specific inhibitors against neuraminidases.

Potential Effects of Neuraminidases on Innate and Adaptive Immunity

Interestingly, the surfaces of host cells involved in immune systems contain a family of sialic-acid-binding immunoglobulin-like lectins (Siglecs), which play critical roles in regulating the functions of the cells in innate and adaptive immune systems via glycan recognition (Crocker et al., 2007). It is intriguing to consider the potential effects that neuraminidases could impose on innate and adaptive immunity regulated by the Siglecs. Furthermore, sialylation is essential for the anti-inflammatory activity of human plasma immu-noglobulin G (also referred to as intravenous IgG, IVIG), which is often utilized to treat a variety of human hematological and immu-nological disorders. Recent studies have shown that desialylation of IVIG not only depletes its anti-inflammatory activity but also potentially leads to the generation of pathogenic antibodies (Kaneko et al., 2006). It is even more intriguing to consider the potential impact of neuraminidases on IgG in the GCF and their role in the pathogenesis of periodontitis, as well as in the systemic diseases associated with periodontitis.

Cellular Membrane Trafficking of Intracellular P. gingivalis

Intracellular Infection

Bacterial entry into host cells allows pathogens to occupy various niches within the human body, a process required for successful establishment of bacterial infection. Confocal scanning laser microscopy (CSLM) showed the intracellular localization of several periodontal bacteria (Lamont and Yilmaz, 2002), while a method that used in situ hybridization with 16S rRNA gene probes and CSLM detected P. gingivalis, A. actinomycetemcomitans, T. forsythia, and T. denticola within epithelial cells obtained from periodontal pockets, gingival crevices, and buccal mucosa collected from individuals with or without chronic marginal periodontitis (Colombo et al., 2007). Thus, most periodontal pathogens likely have an ability to enter peri-odontal cells.

Intracellular Dynamics of P. gingivalis

P. gingivalis can enter host cells, including epithelial and endothe-lial cells (Amano et al., 2010a). P. gingivalis fimbriae specifically interact with α5β1-integrin of epithelial cells. The bacterium is then captured by cellular pseudopodia, which enables invagination to occur through the endosomal pathway. This entry event requires host cellular dynamin, actin fibers, microtubules, and lipid rafts. Following entry into the cells, it has not been clearly demonstrated how P. gingivalis trafficks within the infected cells. Intracellular P. gingivalis is reportedly localized in various cellular compartments, such as the cytoplasm, endosomes, and autophagosomes, with autophagosome localization reported to occur in endothelial and smooth-muscle cells, but not in gingival epithelial cells (reviewed by Amano et al., 2010a). In addition, the bacterium has not been found in the cytoplasmic spaces of endothelial cells. Thus, the intracellular localization of P. gingivalis might be specific to the type of host cell.

We showed that P. gingivalis was internalized with early endosomes positive for the FYVE domain of EEA1 (an early endosome marker) and transferrin receptor, and about half of the intracellular bacteria are sorted to lytic compartments, including autolysosomes and late endosomes/lysosomes, in immortalized human gingival epithelial cells (Amano and Furuta, 2012) (Fig. 4). Meanwhile, a considerable number of the remaining organisms are sorted to Rab11- and RalA-positive recycling endosomes, which mediate the bacterial exit from infected cells (Takeuchi et al., 2011). Inhibition experiments revealed that bacterial exit is dependent on actin polymerization, lipid rafts, and microtubule assembly. Furthermore, dominant-negative forms and RNAi-knockdown of Rab11, RalA, and exocyst complex subunits (Sec5, Sec6, and Exo84) significantly disturbed the exit of P. gingivalis. It should be noted that intracellular bacteria were sorted to three different destinations within the same cells and that the bacterial exit likely allowed for the further penetration of periodontal tissues. These results imply the existence of unknown mechanism(s) that alternate multiple sorting pathways destined for autophagy, lysosomes, or the recycling pathway.

Figure 4.

Proposed model of P. gingivalis trafficking in immortalized human epithelial cells. P. gingivalis initially localized within an endocytic vacuole (early endosome) after entry. Some bacteria are routed to late endosomes, then subsequently sorted to lysosomes for degradation. Other bacteria likely promote their own entry into the autophagic pathway by bacterial escape from endosomes or fusion of endosomes with autophagosomes, and then are sorted to autolysosomes, which are formed by the fusion of autophagosomes with lysosomes. Some intracellular P. gingivalis organisms seem able to escape from endosomes via the recycling pathway for exocytosis. Subsequently, the bacteria exit from primarily infected host cells into the intercellular space and enter new host cells, which facilitates further penetration of the host tissue in a transcellular manner.

Cellular Impairment by Intracellular P. gingivalis

Cellular integrins mediate specific attachment between a cell and the extracellular matrix, activate the intracellular cytoskeleton network, and elicit various intracellular events, i.e., cellular anchorage, directed migration, wound healing, regeneration, and tissue integrity (Hakkinen et al., 2000). During these processes, the outside-in signaling of integrins, paxillin, and focal adhesion kinase (FAK) play important roles. Intracellular P. gingivalis has been shown to degrade paxillin and FAK, resulting in impaired cellular function during wound-healing and periodontal tissue regeneration (Kato et al., 2007).

Entry Mechanism of outer Membrane vesicles of P. gingivalis

A great many Gram-negative bacteria, including P. gingivalis, have an ability to release outer membrane vesicles (MVs) in an extracellular manner, as noted above (Amano et al., 2010b) (Fig. 5). P. gingivalis MVs retain major components of outer membrane constituents, including LPS, muramic acid, capsule, fimbriae, and proteases termed “gingipains.” Fimbriae mediate bacterial adherence to and entry into host cells (Enersen et al., 2013), while gingipains contribute to the destruction of peri-odontal tissues (Kadowaki et al., 2007). P. gingivalis MVs swiftly entered gingival epithelial cells via lipid rafts through the actin filament assembly, which is dependent on phosphati-dylinositol 3-kinase and Rac1, whereas the clathrin- and dyna-min-dependent pathways were not involved in MV entry. Next, the MVs were routed to early endosomes and subsequently sorted within 90 min to lysosomes, where they remained for more than 24 hr (Furuta et al., 2009b). Although taken up by the cellular digestive machinery, MVs degraded the cellular transferrin receptor and focal adhesion complex proteins, which resulted in depletion of intracellular transferrin and inhibition of cellular migration (Furuta et al., 2009a). These findings suggest that P. gingivalis MVs enter host cells via an endocytosis pathway and that MV-associated gingipains degrade cellular functional molecules, resulting in cellular impairment. This indicates a novel role for P. gingivalis MVs in the etiology of periodontitis.

Figure 5.

Outer membrane vesicles (MVs) secreted from P. gingivalis entering human epithelial cells. Human epithelial cells were incubated with P. gingivalis strain TDC60 for 15 min, and bacterial entry was observed by transmission electron microscopy. Arrowheads indicate MVs on the surface of a P. gingivalis organism of which entry was triggered by cellular membrane ruffling.

Conclusion

Ongoing research is increasing our understanding of the factors that contribute to the pathogenic behavior of some periodontal bacteria, including their genetic characteristics and their interactions with the host environment.

A. actinomycetemcomitans strains are remarkably diverse in gene content and gene organization of their genomes. This diversity can be used to design hypothesis-driven experiments to understand the molecular basis for intra-species variations in the virulence potential of A. actinomycetemcomitans. The results may also serve as an example for the evolution and adaptation of oral bacteria to humans.

Studies have demonstrated that T. forsythia induces alveolar bone loss in mice by driving the TLR2-Th2 inflammatory axis. The pathogenicity of the organism is dependent on its ability to decorate the surface with a unique glycan motif, which plays a vital role in modulating the cytokine responses of antigen-presenting cells to ensure subsequent blockade of the Th17 and induction of Th2 responses. An understanding of how T. forsythia shapes the immunity conducive for its survival, and possibly that of cohabiting pathogens, will provide better insight into the pathobiology of periodontitis and facilitate the future development of glycan-based immunotherapies in the prevention of periodontitis.

Accumulating evidence shows that neuraminidases play critical roles in the physiology and pathogenicity of “red complex” bacteria and may be an important new player in the development and progression of periodontitis. Further research is required to investigate the feasibility and therapeutic potential of agents targeting bacterial neuraminidases.

Analyses of bacterial strategies used by P. gingivalis to determine intercellular persistence, dissemination, and fate are well-advanced, though we still know only a part of the picture. Efficient bacterial entry into host cells is considered to be tightly related to cytotoxicity expressed by periodontal pathogens. However, recent studies showed that human oral epithelial cells harbor large masses of intracellular bacterial consortia, resembling the polymicrobial nature of tooth-surface biofilm (Colombo et al., 2007). In addition, no significant induction of apoptosis or necrosis was observed in epithelial cells heavily invaded by bacteria (Rudney et al., 2005). These new findings may represent a periodontal bacterial intracellular lifestyle characterized by rest and quiet, which allows them to persist in, but not destroy, the host.

These sophisticated strategies of the pathogens contribute to periodontal destruction, which could be further enhanced by the bacterial mutual communication with a complex polymicrobial etiology (Hajishengallis and Lamont, 2012). We must move ahead powerfully toward an in-depth understanding of periodontal pathogenesis and new targets for therapeutic intervention.