INTRODUCTION

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Periodontal diseases comprise a group of infections involving the supporting tissues of the teeth. These range in severity from mild and reversible inflammation of the gingiva (gum) to chronic destruction of periodontal tissues (gingiva, periodontal ligament, and alveolar bone) with eventual exfoliation of teeth. From a microbiological standpoint, several features of these diseases are of interest. The bacterial etiology is complex, with a variety of organisms responsible for the initiation and progression of disease. Many, if not all, of these organisms may also be present in periodontally healthy individuals and can exist in commensal harmony with the host. Thus, disease episodes may ensue from a shift in the ecological balance between bacterial and host factors, as a result of, for example, alteration in the absolute or relative numbers of certain organisms, changes in pathogenic potential, or modulation of particular host factors. The local environment imposes a variety of unique constraints upon the constituent microbiota of the supragingival tooth surface and the subgingival crevice (the channel between the tooth root and the gingiva that deepens into a periodontal pocket as disease progresses). Both the calcified hard tissues of the tooth and the epithelial cells of the gingiva are available for colonization. These tissues are exposed to host salivary secretions and gingival crevicular fluid (a serum exudate), both of which contain molecules that interact directly with bacteria and alter prevailing environmental conditions. In addition, successful colonizers of the teeth and subgingival area must coexist with many (over 300) other species of bacteria that inhabit these regions. Study of the pathogenesis of periodontal diseases is thus complicated by the ecological intricacy of the microenvironment.

The classification of the various manifestations of periodontal diseases is continually changing, and it will suffice to mention that diseases range in severity, rate of progression, and number of teeth affected and that different age groups can be susceptible following the eruption of primary teeth. The nature of the pathogenic agents varies among these disease entities, as well as among patients and even between different disease sites within a patient. In general, however, severe forms of the disease in adults are associated with a number of gram-negative anaerobic bacteria. Of this group, most evidence points to a pathogenic role for Porphyromonas (formerly Bacteroides) gingivalis. The presence of this organism, acting either alone or as a mixed infection with other bacteria, and possibly in concert with the absence of beneficial species and certain immunological deficiencies in the host, appears to be essential for disease activity (83, 251). Intensive study of P. gingivalis has revealed a vast array of potential virulence factors that are now being defined at the molecular level. In this article, we review the pathogenic mechanisms of P. gingivalis.

Colonization of the oral cavity requires that the bacteria first enter the mouth and then localize at and attach to the available surfaces. Host factors that tend to resist bacterial colonization include the mechanical shearing forces of tongue movement along with saliva and gingival crevicular fluid flow. Furthermore, specific salivary molecules can aggregate organisms and promote their clearance via the processes of expectoration or swallowing (262). Successful oral colonizers therefore possess a variety of attributes to overcome host protective mechanisms. The sessile plaque biofilm that subsequently accumulates on the hard and soft tissues of the mouth is a dynamic system composed of diverse microbial species. P. gingivalis is usually among the late or secondary colonizers of the oral cavity, requiring antecedent organisms to create the necessary environmental conditions. The means by which antecedent bacteria may facilitate colonization by P. gingivalis include provision of attachment sites for interspecies adherence, supply of growth substrates, and reduction of oxygen tension to the low levels required for growth and survival of this obligate anaerobe (262).

Initial entry of P. gingivalis into the oral cavity is thought to occur by transmission from infected individuals (77). Saliva is considered an important vector for transmission; however, the spouses and children of individuals with P. gingivalis do not always harbor the same genotype (203, 269). Other vectors would therefore also appear to be operational. The clonal diversity of P. gingivalis isolates has been examined by a variety of techniques including ribotyping, restriction endonuclease analysis, restriction fragment length polymorphism, multilocus enzyme electrophoresis, and arbitrary PCR amplification (145, 146, 156, 261). These studies indicate that individuals are colonized by a single (or at least a predominant) genotype, regardless of site of colonization or clinical status. Strains of many different clonal origins, in contrast, are present in different individuals. This supports the concept that P. gingivalis is essentially an opportunistic pathogen, with virulence not being restricted to a particular clonal type.

The oral cavity provides a variety of surfaces to which P. gingivalis can adhere (262) (Fig. 1). There are the mineralized hard tissues of the teeth, along with mucosal surfaces including those of the gingiva, cheek, and tongue. Colonization of surfaces remote from the gingival crevice and periodontal pocket can still have significance for the disease process since, once established, pathogenic organisms may reach the subgingival area by spreading proliferation or by translocation of dislodged progeny (244, 259). Oral surfaces generally are coated with a pellicle composed predominantly of salivary molecules but also potentially including serum-derived molecules from gingival crevicular fluid along with products related to host nutrition and epithelial cell turnover (262). It is the components of these pellicles, in particular various salivary molecules adsorbed on the tooth surface, that commonly function as receptors for bacterial adherence. Furthermore, oral surfaces rapidly become colonized with the early commensal microbiota of the mouth, as a consequence of which organisms such as P. gingivalis usually encounter surfaces rich in antecedent bacteria and their products. P. gingivalis can adhere to many of these early plaque organisms such as oral streptococci (Streptococcus gordonii, S. sanguis, S. oralis, S. mitis, and S. crista) and Actinomyces naeslundii (76, 108, 130, 131, 141, 166). These coadherence interactions tend not to result in the formation of large aggregates in suspension (109, 131, 229), a mechanism that may facilitate colonization by diminishing the probability that large coaggregates of organisms would be lost from the mouth by expectoration or swallowing before reaching a solid support. In addition, P. gingivalis can bind to other, later colonizers such as Fusobacterium nucleatum, Treponema denticola, and Bacteroides forsythus (79, 120, 279). These kinds of interspecies binding interactions may not only favor colonization but also promote nutritional interrelationships and intercellular signaling mechanisms (122). Other parameters of relevance to oral adherence include the presence, in the fluid phase, of saliva- and crevicular fluid-derived molecules that have the potential to bind to P. gingivalis and modulate its adhesion. The importance of such interactions is difficult to assess, however, since the degree of inhibition and enhancement varies according to the salivary source, the strains under investigation, and even the assay system used (131, 163, 166, 279).

View larger version (66K): [in this window] [in a new window]   FIG. 1.   Multiple adhesive interactions of P. gingivalis. Adherence mechanisms may vary according to substrate, and adherence may be multimodal. P. gingivalis releases membrane vesicles containing functional adhesins that may also bind to the substrates shown. Compiled from references 57, 76, 79, 108, 120, 130, 131, 163, 166, and 279.

Perhaps as an evolutionary adaptation to the diversity of available substrates, P. gingivalis displays a variety of distinct adhesive interactions that are associated with both fimbriae and outer membrane proteins (98, 132, 153). Such multimodal adherence mechanisms may not only improve the likelihood of attachment occurring but also increase the avidity of binding to individual substrates. In addition, in some cases, adherence to host cells can be a prelude to the modulation of eucaryotic intracellular signal transduction pathways. Thus, the specificity or strength of the adherence interaction or the sequence in which different adhesins engage their receptors may be important in the manipulation of the biological activity of the host cell by P. gingivalis.

Fimbriae. Ultrastructural examination has revealed the presence of peritrichious fimbriae, 0.3 to 3.0 µm long and ca. 5 nm wide, on most strains of P. gingivalis (191, 244). The major class of fimbriae are composed of a fimbrillin monomer subunit that varies in size between 41 and 49 kDa depending on the strain (135). Circular dichroism analysis indicates that the secondary structure contains a high percentage of -sheet and random coil with no detectable -helix (253, 284). The gene encoding fimbrillin (fimA) is present in a single copy in the chromosome and is monocistronic (53). Protein sequence analysis reveals no significant homology to fimbrial proteins from other bacteria, indicating that P. gingivalis fimbriae may represent a unique class of gram-negative fimbriae (53).

View larger version (13K): [in this window] [in a new window]   FIG. 2.   Functional domains of P. gingivalis fimbrillin. Numbers refer to amino acid residues. Abbreviations: AG, immunodominant (IgG binding); B Cell, stimulation of B cells; CK, stimulation of cytokines; CT, chemotaxis; FN, binding to fibronectin; LF, binding to lactoferrin; PRP, binding to salivary PRPs; RBC, binding to erythrocytes; STA, binding to salivary statherin; STREP, binding to Streptococcus oralis; T Cell, stimulation of T cells. The boundaries of the domains are not known precisely. Compiled from references 5, 7, 52, 104, 167, 179-184, 187, 252, and 255.

Hemagglutinins. Hemagglutinin proteins are established virulence factors for a number of bacterial species, and P. gingivalis produces at least five hemagglutinating molecules. When expressed on the bacterial cell surface, hemagglutinins may promote colonization by mediating the binding of bacteria to receptors (usually oligosaccharides) on human cells. Since P. gingivalis utilizes heme for growth, binding of bacterial cells to erythrocytes may also serve a nutritional function (138). Early studies suggested that hemagglutination by P. gingivalis might be fimbria-mediated (191). This concept was subsequently confuted when purified fimbriae were shown not to exhibit hemagglutinating activity (283) and neither fimbriae nor antibodies to fimbriae (105) blocked hemagglutination by P. gingivalis cells. More recent evidence, however, does implicate a role for fimbrillin in hemagglutination, since synthetic peptides based on the sequence of fimbrillin (Fig. 2) clearly possess hemagglutinating activities (180). One current view then is that fimbriae do have hemagglutinating activity (54) but that P. gingivalis cells produce a number of hemagglutinins that are distinct from fimbrial protein synthesis, assembly, and structure.

The cells of the junctional epithelium provide an early barrier to tissue penetration by periodontal pathogens. Recently, it has become apparent that epithelial cells function both as a mechanical barrier to the ingress of organisms and as sensors of microbial infection (111). Epithelial cells thus comprise an interactive interface with subgingival bacteria, generating and transmitting signals between bacteria and the adjacent and underlying immune cells in the periodontal tissues. Many bacterial species that initiate infections at epithelial cell interfaces can internalize within the epithelial cells and subvert the intracellular information-processing events that control this signaling network. The molecular dialogue that occurs between bacteria and epithelial cells (for which the term "cellular microbiology" has recently been coined [40]) is thus important in the ultimate outcome of the encounter between pathogen and host. Although it has been known for some time that P. gingivalis is capable of penetrating gingival tissues (64, 202, 218-220, 258), recognition of the ability of this organism to subvert host intracellular events and localize intracellularly is more recent.

P. gingivalis has invasive potential, actively internalizing within epithelial cells by mechanisms similar to those used by various enteric pathogens (Fig. 3). Invasion of P. gingivalis has been studied in primary cultures of gingival epithelial cells (129, 133), oral epithelial cell lines (56, 175, 223), and cultures of multilayered pocket epithelium (224). Invasion proceeds more efficiently in primary cell cultures than in transformed cells (56, 129), indicating that transformation-induced changes in cell surface receptors or in intracellular signaling pathways may be detrimental to the invasion process. Attachment of P. gingivalis to primary gingival epithelial cells induces the formation of membrane invaginations that surround and engulf the bacteria, effecting their internalization (129, 133). The bacteria rapidly become located in the cytoplasm without, seemingly, being first constrained within a membrane-bound vacuole (129), although such cytoplasmic vacuoles do appear to be present after invasion of KB cells (175). Rearrangement of the host cell cytoskeleton to accommodate these membrane invaginations involves both microfilament and microtubule activity, a characteristic shared with, among others, Neisseria gonorrhoeae, Haemophilus influenzae, and Citrobacter freundii (129, 177, 212, 256). The individual component of the multimodal adherence of P. gingivalis to epithelial cells that is most relevant to subsequent invasion is fimbria mediated. A fimbria-deficient mutant of P. gingivalis exhibited a significantly greater reduction in invasion compared with adherence (274). Fimbriae binding to their cognate receptor(s) may therefore trigger the activation of eucaryotic proteins involved in signal transduction required for internalization (274). The epithelial cell receptor for fimbriae has not been characterized; however, a 48-kDa surface molecule has been found to bind to fimbriae and is thus a potential candidate (274). P. gingivalis is also capable of secreting a novel set of proteins when in contact with epithelial cells (196). Such secretion is generally indicative of the presence of a type III protein secretion pathway which allows bacteria to translocate proteins directly into host cell cytoplasm. These translocated proteins can then impinge upon eucaryotic signaling pathways. The extent to which the P. gingivalis secreted proteins possess intracellular effector functions remains to be determined. Although the ultimate fate of internal P. gingivalis is uncertain, the bacteria can persist for extended periods within host cells and can replicate intracellularly (129, 148). Intercellular spread of the organisms has not been observed. The environment within epithelial cells may thus represent a nutritionally rich, immunologically privileged site for P. gingivalis, which can then serve as a source for recrudescence of active disease episodes. Alternatively, uptake of P. gingivalis by epithelial cells may represent a mechanism by which the host sequesters this pathogenic organism.

View larger version (15K): [in this window] [in a new window]   FIG. 3.   Model of the currently understood P. gingivalis interactions with gingival epithelial cells. Abbreviations: CM, cytoplasmic membrane; MF, actin microfilaments; MT, tubulin microtubules; NM, nuclear membrane; P, phosphate; ; pathway with potential intermediate steps; , translocation; , release; , reversible association. Compiled from references 47, 55, 106, 129, 175, 196, 223, and 274.

The events that occur within epithelial cells following contact with invasive P. gingivalis are only beginning to be defined (Fig. 3). P. gingivalis induces a transient increase in epithelial cell cytosolic [Ca²⁺], a characteristic shared with Salmonella typhimurium and enteropathogenic E. coli (12, 106, 195). Elevated intracellular [Ca²⁺] results from release of Ca²⁺ from thapsigargin-sensitive intracellular stores; influx of extracellular Ca²⁺ does not appear to be involved (106). Such Ca²⁺ ion fluxes are likely to be important in many signaling events and may converge on calcium-gated ion channels in the cytoplasmic membrane, cytoskeletal remodeling, or nuclear transcription factors (17). Eucaryotic cell-signaling pathways also depend on the pattern of protein phosphorylation (40). P. gingivalis invasion is correlated with the tyrosine phosphorylation of a 43-kDa intracellular eucaryotic protein (223), a size consistent with that of mitogen-activated protein (MAP) kinase, which is a target of phosphorylation by invasive S. typhimurium and Listeria monocytogenes (215, 260). Exploitation of MAP kinase-dependent signaling events may also funnel through cytoskeletal remodeling or nuclear transcription factors (63). Furthermore, recent studies in eucaryotic signaling indicate that the cellular skeleton per se may play a direct role in transmitting regulatory information through the cell rather than simply acting as a passive support structure (151, 152). The usurpation of microfilament and microtubule functions by invasive P. gingivalis may therefore be another means by which normal intracellular information flow is compromised. The collective action of these perturbations of epithelial cell intracellular pathways can have phenotypic effects with immediate relevance to the disease process. Regulation of matrix metalloproteinase (MMP) production by gingival epithelial cells is disrupted following contact with the organism (66). Impaired regulation of MMP activation and production will affect extracellular proteolysis in general and, with particular consequence for tissue integrity, interfere with extracellular matrix repair and reorganization. Invasion of P. gingivalis also has implications for innate host immunity. Secretion of interleukin-8 (IL-8) by gingival epithelial cells is inhibited following P. gingivalis invasion (47). P. gingivalis is also able to antagonize IL-8 secretion following stimulation of epithelial cells by common plaque constituents such as Fusobacterium nucleatum (47). Inhibition of IL-8 accumulation by P. gingivalis at sites of bacterial invasion could have a debilitating effect on innate host defense in the periodontium, where bacterial exposure is constant. The host would no longer be able to detect the presence of bacteria and direct leukocytes for their removal. The ensuing overgrowth of bacteria would then contribute to a burst of disease activity.

NUTRIENT ACQUISITION

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Peptides

Following entry and attachment, oral bacteria must be able to utilize available nutrients in order to thrive in the oral cavity. P. gingivalis is an asaccharolytic organism, dependent on nitrogenous substrates for energy (230). Although sugars such as glucose can be utilized by the organism, these compounds are not converted to metabolic end products but, rather, are used for the biosynthesis of intracellular macromolecules (230, 231, 234). Among the potential nitrogenous substrates available in the mouth, P. gingivalis has only a limited ability to ferment free amino acids, with the possible exception of aspartic acid and asparagine, which can be metabolized through oxaloacetate, malate, and fumarate to yield succinate (231). In contrast, peptides are efficiently utilized for growth (230). Thus, the action of proteolytic enzymes produced by P. gingivalis, along with other bacterially and host-derived proteases, in the protein-rich subgingival milieu would appear to be pivotal to nutrient acquisition by P. gingivalis. As discussed in considerable detail below, P. gingivalis produces multiple proteases that can degrade a number of potentially important substrates in the gingival crevice, including collagen, fibronectin, fibrinogen, laminin, and keratin (98, 153, 265, 266). Although these proteolytic activities are well established, the identity of the enzyme(s) responsible in each case is less clear. Nonetheless, the high level of proteolytic activity and the wide range of appropriate substrate specificities provide P. gingivalis with the necessary molecular tools to compete metabolically in the protein-rich subgingival environment.

P. gingivalis has an obligate iron requirement for growth. However, it appears to lack a siderophore system (22) and utilizes hemin (iron protoporphyrin IX) to satisfy this iron requirement (14, 23, 98, 250). A number of hemin-containing compounds, such as hemoglobin, haptoglobin, myoglobin, hemopexin, methemoglobin, oxyhemoglobin, albumin, lactoperoxidase, catalase, and cytochrome c, can provide hemin following proteolytic processing (14, 23, 67, 98, 250). Although nonhemin iron sources such as ferric, ferrous, and nitrogenous inorganic iron, along with transferrin and lactoferrin, can also support P. gingivalis growth (23, 102), all the iron demands of P. gingivalis can be fulfilled by hemin. Hemin is stored on the cell surface, a feature considered to give rise to the characteristic black-pigmented appearance of P. gingivalis colonies (70). The intact hemin molecule can be transported into the cell in an energy-dependent process regulated by the levels of available hemin (71). Specific interactions between the protoporphyrin IX ring and outer membrane proteins appear to initiate the uptake process (23, 71). A number of potential hemin binding proteins have been described. The work of Bramanti and Holt (24-26) has identified a 26-kDa outer membrane protein that may be involved in both binding and transport of hemin. Fujimura et al. (67, 68) reported a 19-kDa hemoglobulin binding protein, while Smalley et al. (247, 249) and Kim et al. (119) discovered 32 and 30-kDa hemin binding proteins respectively. Karunakaran et al. (113) identified a 48-kDa protein (HemR) with significant homology within the N-terminal region to iron-regulated, TonB-dependent outer membrane receptor proteins IrgA from Vibrio cholerae and BtuB and CirA from E. coli. (In many bacteria, TonB-linked receptors are involved in periplasmic transport of compounds such as colicins, iron, and vitamin B₁₂.) The C-terminal two-thirds of HemR (aa 173 to 419) contains 99% identical residues to a region (aa 575 to 821) of the P. gingivalis PrtT protease, encoded by a gene upstream of hemR. An additional putative TonB-linked and protease-associated protein (Tla) has also been found in P. gingivalis. The tla gene (3) encodes a predicted protein with structural similarity within the N-terminal region to TonB-linked receptors and also contains an internal region of 98% identity to the  domain of RI protease (see below). Mutants inactivated in tla are unable to grow in medium containing low concentrations of hemin and produce significantly lower proteinase activities. The relationship (if any) among the various hemin-binding proteins, the role of TonB in periplasmic translocation of hemin, and the involvement of other hemin-regulated proteins that have not yet been demonstrated to bind hemin (22, 27, 247) remain to be definitively established. Kinetic analyses indicate the existence of more than one hemin-binding mechanism (248, 249) which would allow the participation both of multiple outer membrane proteins and of LPS, another hemin binding molecule (78).

The levels of hemin in the oral cavity are likely to be variable. Bleeding as a result of gingival inflammation will elevate subgingival hemin concentrations and may be one factor that predisposes a site to P. gingivalis accumulation. P. gingivalis has also developed mechanisms that will increase the availability of hemin. Proteolytic activity, perhaps in direct association with hemin-binding proteins, e.g., HemR and Tla (3, 113), will degrade the host hemin-sequestering plasma proteins such as haptoglobulin and albumin (249). Moreover, P. gingivalis produces (at least) two genetically distinct cell- and vesicle-associated hemolysins which will liberate hemoglobulin from erythrocytes (37, 112). Hemin not only is vital for growth but also regulates various virulence-associated activities of P. gingivalis (as discussed below). Furthermore, membrane-bound hemin can scavenge oxygen and help maintain an anaerobic environment (250). More complete elucidation of the complex binding, uptake, and regulatory pathways relating to hemin will provide significant insights into the fundamental nature of the organism.

Although the primary function of proteases secreted by asaccharolytic bacteria such as P. gingivalis is to provide peptides for growth, proteases are also involved directly in tissue invasion and destruction by bacteria and in evasion and modulation of host immune defenses. Specific examples of tissue degradation and attenuation of host defense mechanisms include the degradation of extracellular matrix proteins, activation of MMPs, inactivation of plasma proteinase inhibitors, cleavage of cell surface receptors, activation or inactivation of complement factors and cytokines, and activation of the kallikrein-kinin cascade (266) (Table 1). P. gingivalis cells elaborate a number of proteolytic activities that accomplish all these activities. In the realization that periodontitis is a destructive and inflammatory condition, there naturally has been intense research effort directed toward identifying and characterizing the proteinases produced by periodontal pathogens. The current status of over 15 years of work on P. gingivalis proteinases is a somewhat confusing and still emerging story involving multiple genes and multiply processed products of those genes. The confusion is exacerbated by the plethora of strains used, a lack of consensus with respect to genetic nomenclature, incomplete sequence information, and only partial biochemical characterizations of enzymatic activities. Although it is anticipated that the P. gingivalis genomic sequence will begin to clarify matters, functional studies are needed to resolve the complexities of proteinase production and regulation, since these involve extensive posttranslational processing events and probably also translational controls in addition to transcriptional control. Indeed, major advances in understanding of P. gingivalis proteinases have come about recently through close integration of biochemical and genetic studies (18, 200, 210).

A number of proteases produced by P. gingivalis are thiol-dependent enzymes that cleave C-terminal to arginine (Arg-Xaa) or lysine (Lys-Xaa) within protein or peptide substrates and thus were designated, on the basis of substrate specificity, "trypsin-like" enzymes. Biochemically, these enzymes are members of the cysteine-proteinase family, which includes the papains, calpains, streptopains (from Streptococcus), clostripain (Clostridium), various endopeptidases, and the type IV prepilin leader peptidase. In addition to the cysteine proteinase enzymes, P. gingivalis produces serine proteinase activities (81, 96). Enzymes that degrade immunoglobulins, collagen and/or gelatin, complement factors, fibrinogen, and fibronectin can almost always be ascribed to one of these two families.

Arg-X- and Lys-X-specific proteinases. Before the genes encoding cysteine proteinases of P. gingivalis were cloned and sequenced, the proteolytic enzymatic activities that were purified or semipurified from cell surface extracts or culture fluid were given a variety of names. Thus, gingivain (232), gingipain (34), and argingipain (110) all describe Arg-X-specific proteinase activities which are probably identical. It has been proposed (158, 205) that the genes encoding Arg-X-specific enzymes be designated rgp and those encoding Lys-X-specific enzymes be designated kgp, a classification scheme that would certainly simplify the nomenclature.

View larger version (41K): [in this window] [in a new window]   FIG. 4.   P. gingivalis gene products encoding cysteine proteinase activity and/or containing HagA (hemagglutinin)-related sequences. The HagA (GenBank accession no. U41807) precursor comprises 2628 aa and contains four contiguous repeat blocks of 440 to 456 aa each, with >98% identical residues (). Sequences within the other proteins shown that are 90% or more identical to the HagA sequence are also indicated by this pattern. All polypeptides carry a putative leader peptide () at the NH2 terminus. PrpR1 (accession no. X82680) Arg-X-specific proteinase precursor is proteolytically cleaved at the amino acid residues indicated (arrows) to generate  and  species, as described by Rangarajan et al. (210). Culture supernatant proteinase activities are associated with three forms designated RI (), RIA (), and RIB (*), shown below PrpR1 and discussed in the text. Homologous proteins to PrpR1, from a number of different P. gingivalis strains, include PrtR (L26341), Rgp-1 (U15282), RgpA (A55426), Agp (D26470), and CpgR (X85186). The P. gingivalis strains examined all contain a second gene encoding an Arg-X proteinase designated Rgp-2 (U85038), RgpB (D64081), and PrtRII (AF007124). Rgp-2 consists of a discrete N-terminal catalytic domain without the associated C-terminal adhesin region (158). Cell surface-associated Arg-X-specific proteinase activity is found as a multimeric complex (18) consisting of five polypeptides derived from cleavage of the polyprotein (shown below PrtR) and four proteolytic products of the Lys-X-specific proteinase shown below PrtP (U42210), also designated PrtK (U75366) or Kgp (1,723 aa) (D83258 and U54691). The HagD (hemagglutinin) sequence begins at aa 364 within PrtP. The Tla protein (Y07618) is a putative outer membrane-associated receptor that, over 795 aa, has 99% identity to the COOH-terminal half of PrtK (PrtP, Kgp). Sequences with 90% or greater identity to HagD are indicated (). The patterns of regional sequence similarities amongst the various gene products are far more complex than depicted in this figure, which has been simplified for clarity, and the reader is referred to reference 13 for a detailed comparison of sequence identities between HagA, Rgp-1, and PrtP.

Relationship between hemagglutinin and proteinase. Historically, the hemagglutinating activities and adherence of P. gingivalis cells have both been associated with protease activity (80, 99, 173, 204). Inactivation of a cysteine protease gene in P. gingivalis 381 had a direct effect on hemagglutinin activity (280) as well as on the ability of P. gingivalis to bind gram-positive bacteria, oral epithelial cells, and extracellular matrix proteins (263). While some of these effects could be the result of a pleiotropic mutation (as discussed below), the sequences of proteinase genes reveal a possible molecular basis for the association of protease and adhesive activities. It is now evident that the C-terminal coding regions of the genes encoding the RgpA family of cysteine proteinases (equivalent to the  and  regions within PrpR1) encode extensive amino acid blocks that have up to 90% identity to sequences that are also found within the hagA, hagD and hagE genes encoding hemagglutinins (13) (Fig. 4). For example, Rgp1, Agp, PrpR1, and PrtR Arg-X-specific proteinases each contain a 522-aa residue region with 93% identical residues to HagA (89). This region carries a hemoglobin receptor domain (171), localized within the 15-kDa segment of PrtR and PrtP, as shown in Fig. 4. It has not yet been determined if these protease-associated sequences are functional hemagglutinins, in addition to the hemagglutinating activities that have been assigned to HagA, HagB, and HagC. Moreover, it now appears that hagD specifically comprises the sequence encoding the C-terminal region of the Lys-X proteinase, encoded by a single gene in P. gingivalis and variously designated prtK, kpg, and prtP (38) (Fig. 4). As discussed above, the Lys-X proteinase is proposed to be generated by processing of a polyprotein (PrtP is 1,732 aa long) into an active protease (comprising aa 229 to 738 of PrtP) and C-terminally derived hemagglutinin sequences. However, it is not firmly established that posttranslational modifications of the polyproteins account exclusively for the various forms of proteinase and hemagglutinins identified. It is formally possible that translational initiation occurs internally in the primary mRNA transcripts, effectively generating an additional level of control over hemagglutinin production.

Other nonrelated proteinases. Three additional genes (prtT, prtC, and tpr) encoding proteases have been cloned and sequenced from P. gingivalis. These genes are clearly distinguished from the previous protease genes, and they do not contain sequence homologies to hagA. Nevertheless, prtT (from strain ATCC 53977) does possess a C-terminal coding region for hemagglutinin activity (194) that is distinct from the hagA, hagB, hagC, and hagD sequences. The prtT gene is transcribed as a 3.3-kb mRNA (147) and encodes a protein of approximately 99 kDa. This polypeptide contains 31% identical residues (over 401 aa within the N-terminal region) to streptococcal pyrogenic exotoxin B, which is itself related to streptococcal cysteine proteinase (streptopain). The prtT gene is immediately downstream of prtC, which encodes a putative collagenase comprising 417 aa (115). It is not known how this relates, if at all, to the reports of 55-kDa (16, 124) and 44-kDa (110) collagenolytic activities isolated from P. gingivalis. Furthermore, sequences with significant amino acid identities (up to 55%) to PrtC are found within the inferred amino acid sequences of putative proteins from a range of bacteria including Methanobacterium, Methanococcus, Bacillus subtilis, H. influenzae, and E. coli. Lastly, the tpr gene, which appears to be present as a single copy in strains W83, W50, and ATCC 33277 (198, 199), is expressed as a 1.7-kb mRNA (197). The gene encodes a thiol-dependent, papain-related, protease with a molecular mass of approximately 55 kDa that has no significant sequence homology to any of the other proteases (20). Evidence suggests that this proteinase, although not acting on native collagen, may be involved in the later stages of collagen degradation, since it is active in hydrolyzing gelatin and the bacterial collagenase substrate Pz-peptide (197). Another Pz-peptidase has been isolated from P. gingivalis ATCC 33277 (254) and may be distinct from Tpr, based on N-terminal amino acid sequence information.

Concerted activities. To recapitulate, P. gingivalis contains two homologous genes encoding Arg-X-specific proteinases, one gene encoding a Lys-X specific proteinase (Table 2), and three genes encoding a putative collagenase (PrtC), a streptopain-related protease (PrtT), and a Pz-peptidase (Tpr). The activity of these cloned gene products accounts for most of the wide variety of substrates degraded by P. gingivalis (Table 1). However, because the enzyme activities derived from a single gene product are present as several molecular species and are complexed with the products of both homologous genes and heterologous genes, it has been immensely difficult if not impossible to ascertain unequivocally the substrate specificities of individual proteinases "purified" from P. gingivalis (50). For example, a protease activity purified from spent culture medium of P. gingivalis was shown to run as a single 55-kDa band upon gel electrophoresis and was found to hydrolyze collagens type I, III, IV, and V, complement factor C3, fibrinogen, fibronectin, ₁-antitrypsin, ₂-macroglobulin, apotransferrin, and human serum albumin (16). Likewise, the 44-kDa Rgp proteinase purified from P. gingivalis culture fluid was shown to degrade collagens type I and IV and immunoglobulin G (110). However, it is reported that the purified recombinant gingipains (RgpA, RgpB, and Kgp) do not degrade native collagen (205), and so a broad range of substrates degraded by an apparently single enzyme species from P. gingivalis culture fluid may in fact be due to the combined activities of two or more copurified enzyme species. Binding studies with the Arg-X- and Lys-X-specific proteinases Rgp and Kgp, respectively, show that they both bind to and degrade fibrinogen, fibronectin, and laminin (204) and that purified PrtP hydrolyses fibrinogen (38). The combined effects of these activities, in conjunction with the ability of P. gingivalis proteases to activate MMPs (51), would therefore lead to extensive host tissue disruption. In addition, evidence suggests that Arg-X proteinases activate the kallikrein/kinin and complement pathways, disrupt polymorphonuclear leukocyte (PMN) functions (107, 170), inactivate tumor necrosis factor alpha (TNF-) (30), and modify neutrophil elastase (1). These activities, together with the evidence that Kgp (Lys-X) proteinase is not inhibited by plasma ₂-macroglobulin (82), suggest that P. gingivalis proteinases are major virulence factors in the development of periodontal disease (265).

Role in virulence. Biochemical and genetic evidence points to an essential but highly complex role for proteinases in P. gingivalis metabolism and virulence. While molecular genetics has demonstrated that P. gingivalis cells express multiple proteinases, the relative roles that these enzymes play in adhesion, nutrition, and virulence are hard to determine because these processes are all proteolytically interrelated and interdependent. A current molecular picture of the P. gingivalis cell surface would include major macromolecular complexes of adhesins (hemagglutinins), proteinases, outer membrane-associated protein receptors, and LPS. The complexing of adhesin domains and proteinases might increase the repertoire of targets for the proteinases. At the same time, the hemagglutinin sequences are highly antigenic and might act to shield essential proteolytic functions from the host immune system. Given this potential complexity of proteinase-associated phenotypes and the number of distinct proteinase genes identified, it is perhaps not surprising that the analysis of gene knockout mutants has in the main simply confirmed that proteinases impinge on multiple metabolic and virulence-related processes. A novel erythromycin resistance determinant cassette, developed to insertionally inactivate the prtH gene in P. gingivalis W83, resulted in a strain deficient in Arg-X proteinase activity, with decreased virulence in a mouse model of infection (61). In addition, the isogenic mutant was less able to degrade complement factor C3, and opsonized mutant cells were taken up in much larger numbers by human PMN than were wild-type cells (226). In separate experiments, a double-knockout rgpA rgpB mutant of P. gingivalis ATCC 33277 that was abrogated in Arg-X proteinase activity showed much less inhibition of leukocyte bactericidal function than did the wild-type strain, as well as having markedly reduced hemagglutinin activity (170). Another isogenic mutant with a mutation in the rgp-1 (Arg-X) protease gene of strain 381 exhibited decreased binding to epithelial cells, gram-positive bacteria, extracellular matrix proteins, and type 1 collagen (263). These experiments all confirm the notion that proteinases are obligatory for P. gingivalis growth and survival in the host. However, since it has been shown that Arg-X-specific proteinase mutants are also deficient in Lys-X-specific proteinase activity, the data do not point to a specific role that any one proteinase might play in pathogenesis. This information will be important if inhibition of proteinase function is to be pursued as a viable method of controlling the growth of and tissue destruction by periodontal pathogens such as P. gingivalis.

Proteinases and fimbriae. A further complicating factor in the study of proteinase function in P. gingivalis is the discovery that the production and activity of the major fimbriae are modulated by proteolytic activity. This control appears to occur on at least three levels: posttranslational modification, transcriptional modulation, and substrate activation. The work of Onoe et al. (193) suggested a role for trypsin-like proteases in the processing of the leader peptide from the fimbrillin precursor. The subsequent observations of Nakayama et al. (172) that the ATCC 33277 rgpA rgpB double-mutant cells possessed very few fimbriae on their surface compared with rgpA or rgpB single-mutant or wild-type cells demonstrated that fimbrial production required the expression of Arg-X and/or Lys-X-specific proteinases. These data suggest that proteinase activity was necessary for processing and maturation of fimbrillin, in addition to its role in modifying other cell surface-associated proteins. Control at the transcriptional level is inferred from the finding that inactivation of the rgp-1 (Arg-X proteinase) gene in P. gingivalis 381 resulted in reduced production of the 43-kDa fimbrillin subunit as well as reduced transcription of fimA (263). Since fimbriae are implicated in a large number of adhesive interactions of P. gingivalis (see above), most notably binding to host and bacterial cells, it would not be possible through simply generating proteinase gene knockout mutants to delineate precisely which adhesive functions are related directly to protease/hemagglutinin activity and which are the result of secondary effects on fimbriation. The third control level, substrate activation, is facilitated by the ability of P. gingivalis to bind to and degrade (perhaps progressively) human plasma fibronectin, along with other host proteins such as laminin, fibrinogen, and collagen. Both the fimbriae and the Arg-X-specific (204) and Lys-X-specific (134) proteinases are associated with these binding and degradative processes. Hydrolysis of fibronectin or other matrix proteins such as collagen by P. gingivalis Arg-X proteinase enhances the binding of fimbriae to these substrates (124). Specifically, it seems that the Arg-X proteinase is able to expose sequences, within host matrix protein molecules, that carry C-terminal Arg residues, thus promoting adhesion of the organism through a fimbria-Arg interaction (123). Proteolytic exposure of cryptic binding domains on host molecules and potentially on epithelial cells and other bacteria (35, 97, 124, 130) could involve host-derived as well as bacterial enzymes. It has been conjectured (35, 97) that this may represent one mechanism by which initial gingivitis progresses to more severe periodontitis. A buildup of plaque bacteria in the gingival sulcus may irritate the tissues and cause increased proteolytic activity in the gingival milieu. The resulting exposure of previously hidden receptors would then enhance colonization by P. gingivalis. Subsequently, P. gingivalis proteases themselves could contribute to further exposure of adherence receptors.

In addition to proteolytic activity, P. gingivalis can degrade the glycosaminoglycans hyaluronate, chondroitin sulfate, and heparin (98). The repertoire of enzymes and metabolites that could be detrimental to the host also includes phospholipase A, which can provide the prostaglandin precursors that could stimulate prostaglandin-mediated bone resorption (29, 75); alkaline and acid phosphatases, which may contribute to alveolar bone breakdown (65, 242); DNase and RNase (217); sialidase (98); volatile sulfur compounds such as hydrogen sulfide, methylmercaptan, and dimethyl disulfide, which are cytotoxic and can inhibit protein synthesis (243, 264); butyrate and propionate, which are cytotoxic for epithelial cells, fibroblasts, and lymphocytes (125, 238, 243); and indole and ammonia, which also exhibit cytotoxicity (243, 270).

By a variety of intricate and interconnected mechanisms, P. gingivalis can contribute to alveolar bone loss by stimulating bone resorption, inducing bone destruction, and inhibiting bone formation (98, 144). P. gingivalis LPS can activate osteoclasts directly and causes the release of prostaglandin E₂ and of the cytokines IL-1 and TNF- from macrophages, monocytes, and fibroblasts (28, 142, 214, 239, 276, 278). These compounds are potent local mediators of bone resorption and, moreover, can inhibit collagen synthesis by osteoblasts and induce the production of host metalloproteases that destroy connective tissue and bone (94, 98). In addition to LPS, other cellular constituents may be involved in bone loss. Heat-stable polysaccharide antigens can stimulate the release of IL-1 from monocytes (168), and saline-extracted proteins can stimulate bone resorption in in vitro models (275). The identities of some of the active P. gingivalis proteins have been revealed. A 24-kDa outer membrane protein has bone-resorptive and -destructive activity in vitro (157), while an outer membrane protein of 75 kDa and a 12-kDa lectin-like protein can stimulate macrophages to secrete IL-1 (221, 272). P. gingivalis fimbriae (fimA gene product) stimulate bone resorption in vitro (116), probably by inducing the expression and production of IL-1 and TNF- from monocytes, macrophages, and gingival fibroblasts (90, 92, 181, 187) and by stimulating the expression of IL-1 and granulocyte-macrophage colony-stimulating factor in bone cells themselves (116). The amino acid sequence LTxxLTxxN, which occurs within fimbrillin aa 61 to 80 and 171 to 185 (Fig. 2), appears to be involved in cytokine stimulation in monocytes (187). Within this domain, the xLTxx sequence may be the minimum essential unit (184). Fimbrial binding, and subsequent stimulation of cytokines and bone resorption can be inhibited by N-acetyl-D-galactosamine (161) or by interaction with fibronectin through the heparin binding and cell attachment domains in the fibronectin structure (117). Fibronectin domains and other receptor analogs therefore have the potential to act as antagonists to neutralize fimbria-mediated bone resorption. The intracellular signalling events that are the target of fimbriae appear to involve eucaryotic cell kinase activity. Tyrosine phosphorylation of several proteins in osteoclasts and macrophages is induced by fimbriae, and inhibition of tyrosine kinase prevents cytokine stimulation and fimbria-mediated bone resorption in vitro (91, 221). The 12-kDa lectin-like surface component also acts via activation of tyrosine kinases within macrophages (222).

The list of P. gingivalis components and products with the capability of compromising tissue integrity is long. Undoubtedly, many of these activities, alone or in combination, are important in vivo. Definitive evidence, however, about the ones that are operational in the disease process is, unfortunately, still lacking.

Of the various immune mechanisms in the periodontal pocket, PMNs appear to play a major role in controlling the overgrowth of periodontal bacteria (49). P. gingivalis impinges on almost all aspects of PMN recruitment and activity. Although there are numerous phenomenological reports of the organism adversely affecting PMNs, this section will concentrate on processes for which functionality has been associated with specific molecules. Neutrophil chemotaxis is inhibited by low-molecular-weight fatty acids, such as succinic acid, that are produced by the organism (19, 216). Succinate may act by reducing the intracellular pH of neutrophils (216). P. gingivalis can also immobilize PMN responses to chemotactic peptides by depolarizing PMN membranes (176). This activity is associated with an outer membrane protein of 31.5 kDa, which may be a porin. Novak and Cohen (176) proposed that depolarization could be caused by translocation of this porin into the PMN membrane, thus producing an ion channel that the PMN would be unable to regulate. Package of the porin in extracellular vesicles could then be a means by which P. gingivalis could act at remote sites to inhibit PMN influx in the periodontal pocket.

As is frequently the case with investigation of bacterial interactions with the host innate immune response, different bacterial components can demonstrate diametrically opposing affects. In the case of P. gingivalis, LPS strongly activates complement, generating the chemotactic product C5a. However, the activity of cell-associated LPS may be tempered by capsular polysaccharide that can physically mask LPS on the cell surf