Isolation and characterization of a minor fimbria from Porphyromonas gingivalis.

We have discovered two distinctly different fimbriae expressed by the same Porphyromonas gingivalis strain. The construction of a fimA mutant of P. gingivalis ATCC 33277 has previously been reported by N. Hamada et al. (Infect. Immun. 62:1696-1704, 1994). Expression of fimbriae on the surface of the fimA mutant and the wild-type strain, ATCC 33277, were investigated by electron microscopy. The wild-type strain produced long fimbrial structures extending from the cell surface, whereas those structures were not observed on the fimA mutant. However, short fimbrial structures were seen on the surface of the fimA mutant. The short fimbrial protein was purified from the fimA mutant by selective protein precipitation and chromatography on DEAE Sepharose CL-6B. We have found that the second fimbrial structure of P. gingivalis ATCC 33277 is distinct from the 41-kDa (43-kDa) major fimbrial protein (FimA). We provisionally call this protein minor fimbriae. The molecular mass of the minor fimbriae is 67 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions after boiling at 100 degrees C. The component shows a ladder-like pattern at 80 degrees C under nonreducing conditions, suggesting a tendency to aggregate or polymerize. In immunoblotting analysis, anti-minor fimbria serum reacted with both the 100 degrees C- and the 80 degrees C-treated minor fimbriae. The anti-minor fimbria serum also reacts with the same-molecular-size fimbrial preparation from the wild-type strain. Immunogold electron microscopy showed that the anti-minor fimbria serum bound to the minor fimbria on the cell surface of the wild-type strain. This is the first report on the identification of the minor fimbria produced by P. gingivalis. These results suggest that the minor fimbriae appearing on the fimA mutant strain are produced together with numerous long major fimbriae on the wild-type strain. Moreover, the minor fimbriae are different in size and antigenicity from the earlier-reported FimA, a major 41-kDa fimbrial component of P. gingivalis.     

Functional differences among FimA variants of Porphyromonas gingivalis and their effects on adhesion to and invasion of human epithelial cells.

Fimbriae of Porphyromonas gingivalis, a periodontopathogen, play an important role in its adhesion to and invasion of host cells. The fimA genes encoding fimbrillin (FimA), a subunit protein of fimbriae, have been classified into five types, types I to V, based on nucleotide sequences. We previously reported that P. gingivalis with type II fimA was strongly associated with adult periodontitis. In the present study, we compared the abilities of recombinant FimA (rFimA) types I to V to adhere to and invade human gingival fibroblasts (HGF) and a human epithelial cell line (HEp-2 cells) by using rFimA-conjugated microspheres (rFimA-MS). There were no significant differences in the abilities of the rFimA-MS to adhere to HGF; however, the adhesion of type II rFimA-MS to HEp-2 cells was significantly greater than those of other types of rFimA-MS. We also observed that type II rFimA-MS invaded epithelial cells and accumulated around the nuclei. These adhesion and invasion characteristics were eliminated by the addition of antibodies to type II rFimA and alpha5beta1-integrin. In contrast, Arg-Gly-Asp-Ser peptide and a synthetic peptide of proline-rich protein C had negligible inhibitory effects. Furthermore, P. gingivalis strain HW24D1 with type II fimA adhered to cells and invaded them more than strains with other fimA genotypes. These results suggest that type II FimA can bind to epithelial cells most efficiently through specific host receptors.     

Distribution and molecular characterization of Porphyromonas gingivalis carrying a new type of fimA gene

Fimbriae of Porphyromonas gingivalis are filamentous appendages on the cell surface and are thought to be one of the virulence factors. The fimA gene encoding the subunit protein of fimbriae, fimbrillin (FimA), was classified into four typeable variants (types I to IV). We previously examined the distribution of P. gingivalis in terms of fimA genotypes in periodontitis patients using a fimA type-specific PCR assay. However, some patients harbored P. gingivalis with untypeable fimA. In this study, we have cloned a new type (type V) of fimA from dental plaque samples. P. gingivalis with type V fimA was isolated from dental plaque of a periodontitis patient, and the isolate was named HNA-99. The deduced amino acid sequences were compared with those of type I P. gingivalis ATCC 33277, type II strain HW24D1, type III strain 6/26, and type IV strain HG564, and the homologies were found to be 45, 44, 43, and 55%, respectively. Southern blot analysis showed that the clinical isolate HNA-99 possessed P. gingivalis-specific genes sod and kgp. However, in terms of serological specificities, type V FimA showed a difference from other types of FimA. In addition, type V P. gingivalis bacteria were detected in 16.4% (12 of 73) of the P. gingivalis-positive patients with periodontitis by PCR assay using specific primers. Thus, a new type of fimA gene is now established, and the fimA genotyping could be useful in determining the disease-associated genotypes of P. gingivalis involved in the development of adult periodontitis.     

Role of the Amino-Terminal Region of Porphyromonas gingivalis Fimbriae in Adherence to Epithelial Cells

Porphyromonas gingivalis fimbriae elicit many responses in eukaryotic cells, including mitogenicity, cytokine production, epithelial cell invasion, and cellular immune response. Specific domains of the major fimbrial protein (FimA) have been shown to be important in triggering some of these functions. The goal of the present study was to identify the domain(s) of P. gingivalis FimA responsible for specific interaction with human mucosal epithelial cells. Fimbriated P. gingivalis strains have been shown to bind to buccal epithelial cells, whereas nonfimbriated strains bind at low levels or not at all. This and other studies provide evidence that FimA mediates the adherence of P. gingivalis to oral epithelial cells. To determine the specific region(s) of P. gingivalis FimA involved in epithelial cell binding, specific antipeptide antibodies were used to inhibit the binding of iodinated purified fimbriae as well as the binding of P. gingivalis cells to epithelial cells. Antibodies directed against peptides 49 to 68 (VVMANTAGAMELVGKTLAEVK) and 69 to 90 (ALTTELTAENQEAAGLIMTAEP) were found to highly inhibit both the binding of fimbriae and the binding of P. gingivalis cells to epithelial cells. The antibody against FimA peptides 69 to 90 also reacted with P. gingivalis fimbriae in immunogold labeling and immunoblot analysis, thereby indicating that this peptide domain is exposed on the surface of fimbriae. Our results suggest that the amino-terminal domain corresponding to amino acid residues 49 to 90 of the fimbrillin protein is a major epithelial cell binding domain of P. gingivalis fimbriae.     

Differential activation of human gingival epithelial cells and monocytes by Porphyromonas gingivalis fimbriae

Humans develop periodontitis in response to challenge by microbial dental plaque. Inflammation begins after perturbation of gingival epithelial cells by subgingival bacteria interacting through pattern-recognition receptors, including the Toll-like receptors (TLR). Porphyromonas gingivalis is a major periodontopathogen that interacts with epithelial cells through its cell surface fimbriae (FimA), leading to colonization and/or invasion. Previous work by our group has established membrane CD14 as an essential coreceptor for TLR2-mediated activation of transfected cell lines by P. gingivalis FimA. We have shown that gingival epithelial cells express TLR2 but not CD14 on their cell surfaces. We thus speculated that P. gingivalis FimA does not readily activate epithelial innate immune responses but rather functions to promote P. gingivalis colonization in the absence of a vigorous FimA-induced response. This hypothesis was verified by the findings that primary human gingival epithelial cells responded poorly to FimA in terms of interleukin (IL)-6, IL-8, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha responses, in stark contrast to the marked response to other TLR2 agonists (Pam3Cys, FSL-1) that are not strictly dependent on CD14. On the other hand, CD14-expressing human primary monocytes responded with high levels of the same cytokines to both FimA and the control TLR2 agonists. The gingival epithelial cells failed to respond to FimA even in the presence of exogenously added soluble CD14. These data indicate that the gingival epithelial cell hyporesponsiveness to FimA is attributable to the lack of membrane-expressed but not soluble CD14. In conclusion, P. gingivalis FimA differentially activates human monocytes and epithelial cells, perhaps reflecting different tactics used by P. gingivalis when interacting with different host cell types or a host strategy to limit inflammation.     

Role of fimbriae in Porphyromonas gingivalis invasion of gingival epithelial cells.

Porphyromonas gingivalis is a periodontal pathogen capable of invading primary cultures of normal human gingival epithelial cells (NHGEC). Involvement of P. gingivalis fimbriae in the invasion process was examined. Purified P. gingivalis 33277 fimbriae blocked invasion of this organism into NHGEC in a dose-dependent manner. DPG3, a P. gingivalis fimbria-deficient mutant, was impaired in its invasion capability approximately eightfold compared to its parent, strain 381. However, adherence of the mutant was only 50% reduced compared to the parent. Biotin labeling of NHGEC surface proteins revealed that two fimbriated strains, but not DPG3, bound a 48-kDa NHGEC protein. Adhesin-receptor interactions, such as fimbriae binding to a 48-kDa NHGEC surface receptor, may trigger activation of eukaryotic proteins involved in signal transduction and/or provoke the generation of surface P. gingivalis molecules required for internalization.     

Invasion of epithelial cells and proteolysis of cellular focal adhesion components by distinct types of Porphyromonas gingivalis fimbriae

Porphyromonas gingivalis fimbriae are classified into six types (types I to V and Ib) based on the fimA genes encoding FimA (a subunit of fimbriae), and they play a critical role in bacterial interactions with host tissues. In this study, we compared the efficiencies of P. gingivalis strains with distinct types of fimbriae for invasion of epithelial cells and for degradation of cellular focal adhesion components, paxillin, and focal adhesion kinase (FAK). Six representative strains with the different types of fimbriae were tested, and P. gingivalis with type II fimbriae (type II P. gingivalis) adhered to and invaded epithelial cells at significantly greater levels than the other strains. There were negligible differences in gingipain activities among the six strains; however, type II P. gingivalis apparently degraded intracellular paxillin in association with a loss of phosphorylation 30 min after infection. Degradation was blocked with cytochalasin D or in mutants with fimA disrupted. Paxillin was degraded by the mutant with Lys-gingipain disrupted, and this degradation was prevented by inhibition of Arg-gingipain activity by Nalpha-p-tosyl-l-lysine chloromethyl ketone. FAK was also degraded by type II P. gingivalis. Cellular focal adhesions with green fluorescent protein-paxillin macroaggregates were clearly destroyed, and this was associated with cellular morphological changes and microtubule disassembly. In an in vitro wound closure assay, type II P. gingivalis significantly inhibited cellular migration and proliferation compared to the cellular migration and proliferation observed with the other types. These results suggest that type II P. gingivalis efficiently invades epithelial cells and degrades focal adhesion components with Arg-gingipain, which results in cellular impairment during wound healing and periodontal tissue regeneration.     

Oral immunization with recombinant Streptococcus gordonii expressing porphyromonas gingivalis FimA domains

Porphyromonas gingivalis, a gram-negative anaerobe, is implicated in the etiology of adult periodontitis. P. gingivalis fimbriae are one of several critical surface virulence factors involved in both bacterial adherence and inflammation. P. gingivalis fimbrillin (FimA), the major subunit protein of fimbriae, is considered an important antigen for vaccine development against P. gingivalis-associated periodontitis. We have previously shown that biologically active domains of P. gingivalis fimbrillin can be expressed on the surface of the human commensal bacterium Streptococcus gordonii. In this study, we examined the effects of oral coimmunization of germfree rats with two S. gordonii recombinants expressing N (residues 55 to 145)- and C (residues 226 to 337)-terminal epitopes of P. gingivalis FimA to elicit FimA-specific immune responses. The effectiveness of immunization in protecting against alveolar bone loss following P. gingivalis infection was also evaluated. The results of this study show that the oral delivery of P. gingivalis FimA epitopes via S. gordonii vectors resulted in the induction of FimA-specific serum (immunoglobulin G [IgG] and IgA) and salivary (IgA) antibody responses and that the immune responses were protective against subsequent P. gingivalis-induced alveolar bone loss. These results support the potential usefulness of the S. gordonii vectors expressing P. gingivalis fimbrillin as a mucosal vaccine against adult periodontitis.     

Transformation and expression of a cloned fimA gene in Porphyromonas gingivalis

The Porphyromonas gingivalis fimbria is an important virulence factor involved in the adherence and colonization of the organism in the oral cavity. In this study, we transformed this organism with a gene, fimA381, encoding the fimbrial subunit of P. gingivalis 381 (fimbrillin) by using the host-vector system that we developed previously and examined expression of the cloned fimA381 gene. The recombinant plasmid pYHF2 was constructed by ligating a fragment containing the fimA381 gene into the plasmid vector pYH420 and transformed into the restriction-deficient P. gingivalis host YH522. pYHF2 was autonomously maintained in YH522 cells, and the fimbrillin polypeptide (recombinant fimbrillin) was fully expressed. The molecular mass of the recombinant fimbrillin was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as 41 kDa, which was identical to that of the native fimbrillin of strain 381. The amino acid sequences of the 20 amino-terminal residues of the recombinant fimbrillin and the native fimbrillin of the strain 381 were identical. In addition, characteristic long and thin fimbrial structures (recombinant fimbriae) that were distinguishable from the host's native fimbriae when examined by immunogold electron microscopy were observed around the cell surface of the transformants containing the fimA381 gene. These results suggested that transformation of fimA gene from a different strain of P. gingivalis followed by accumulation of the mature fimbrial subunit protein was sufficient for production of fimbrial structures that were observable by electron microscopy.     

A role for fimbriae in Porphyromonas gingivalis invasion of oral epithelial cells.

Isogenic mutants of Porphyromonas gingivalis which differ in the expression of fimbriae were used to examine the contribution of fimbriae in invasion of a human oral epithelial cell line (KB). At a multiplicity of infection of 100, the wild-type P. gingivalis strains 33277, 381, and A7436 exhibited adherence efficiencies of 5.5, 0.11, and 5.0%, respectively, and invasion efficiencies of 0.15, 0.03, and 0.10%, respectively. However, adherence to and invasion of KB cells was not detected with the P. gingivalis fimA mutants, DPG3 and MPG1. Adherence of P. gingivalis wild-type strains to KB cells was completely inhibited by the addition of hyperimmune sera raised to the major fimbriae. Examination by electron microscopy of invasion of epithelial cells by the P. gingivalis wild-type strain 381 revealed microvillus-like extensions around adherent bacteria; this was not observed with P. gingivalis fim mutants. Taken together, these results indicate that the P. gingivalis major fimbriae are required for adherence to and invasion of oral epithelial cells.     

Expression of type 1 fimbriae and mannose-sensitive hemagglutinin by recombinant plasmids.

Deletions within the cloned genes (fimA) encoding the type 1 major fimbrial subunits of two isolates of Klebsiella pneumoniae resulted in a nonfimbriate but hemagglutinating phenotype after transformation of Escherichia coli HB101 or ORN103. Phenotypic expression of type 1 fimbriae could be restored by transformation with plasmids containing the fimA genes of the fimbrial gene clusters from different strains. The surface fimbriae expressed were serologically identical to those of the polymerized product of the introduced fimA gene. The fimA gene products of Salmonella typhimurium and Serratia marcescens could utilize the accessory fimbrial genes of K. pneumoniae to produce surface-associated, hemagglutinating fimbriae. The relatedness of the type 1 fimbrial gene clusters from multiple isolates of members of the family Enterobacteriaceae was examined by DNA hybridization techniques. These analyses demonstrated little nucleotide sequence agreement among distinct genera of the enteric bacteria.     

Porphyromonas gingivalis genes isolated by screening for epithelial cell attachment.

Porphyromonas gingivalis is associated with chronic and severe periodontitis in adults. P. gingivalis and the other periodontal pathogens colonize and interact with gingival epithelial cells, but the genes and molecular mechanisms involved are unknown. To dissect the first steps in these interactions, a P. gingivalis expression library was screened for clones which bound human oral epithelial cells. Insert DNA from the recombinant clones did not contain homology to the P. gingivalis fimA gene, encoding fimbrillin, the subunit protein of fimbriae, but showed various degrees of homology to certain cysteine protease-hemagglutinin genes. The DNA sequence of one insert revealed three putative open reading frames which appeared to be in an operon. The relationship between P. gingivalis attachment to epithelial cells and the activities identified by the screen is discussed.     

Proteins with molecular masses of 50 and 80 kilodaltons encoded by genes downstream from the fimbrilin gene (fimA) are components associated with fimbriae in the oral anaerobe Porphyromonas gingivalis.

Flanking DNA regions of the fimbrilin gene (designated fimA), which encodes the major subunit protein of Porphyromonas (Bacteroides) gingivalis fimbriae, were cloned in several manners from the P. gingivalis chromosome into Escherichia coli by screening with probes derived from a 2.5-kb SacI DNA fragment previously cloned. A total of 10.4 kb of DNA fragments from the P. gingivalis genome was cloned in the pUC plasmid. Expression of the fimA gene and possible flanking genes in the fragments cloned was examined in a pUC plasmid vector system and in a bacteriophage T7 RNA polymerase-promoter expression vector system. The results show that in the pUC plasmid system, a 45-kDa protein, a product of fimA, was only poorly expressed as a precursor of the fimbrilin protein (FimA) and could be detected from cell extracts in Western blotting (immunoblotting) analysis as a sharp band but not in colony immunoblotting analysis. On the other hand, in the T7 RNA polymerase-promoter system, the product of fimA and products of the possible flanking genes responsible for fimbriation were overproduced as thick bands of the 45-kDa protein and as 63-, 50-, and 80-kDa proteins, respectively, in stained electrophoresis gels. All of the recombinant proteins were insoluble and seemed to be expressed as precursors with leader peptides. The 63-kDa, 45-k*Da (a truncated protein of the 50-kDa protein), and 80-kDa proteins were purified after solubilization with sodium dodecyl sulfate. N-terminal amino acid sequences of the 45-k*Da and 80-kDa proteins were analyzed up to the first 35 residues with a gas-phase sequencer. Monospecific antibodies directed to the recombinant proteins, i.e., the 63-kDa, 45-k*Da, and 80-kDa proteins, were raised in rabbits. By using the antibodies, localization of their matured proteins in P. gingivalis was investigated by Western blotting analysis. Immunoblotting analysis suggests that at least the 50- and 80-kDa proteins, encoded by genes downstream from the fimA gene, are minor components associated with fimbriae.     

FimA, a major virulence factor associated with Streptococcus parasanguis endocarditis.

Adherence of microorganisms to damaged heart tissue is a crucial event in the pathogenesis of infective endocarditis. In the present study, we investigated the role of the FimA protein as a potential virulence factor associated with Streptococcus parasanguis endocarditis. FimA is a 36-kDa surface protein that is a recognized adhesin in the oral cavity where it mediates adherence to the salivary pellicle. An insertion mutant and a deletion mutant of S. parasanguis were employed in the rat model of endocarditis to determine the relevance of FimA in endocarditis pathogenesis. Catheterized rats were infected with either the fimA deletion mutant VT929, the fimA insertion mutant VT930, or the isogenic, wild-type S. parasanguis FW213. Rats inoculated with FW213 developed endocarditis more frequently (50.9%) than animals inoculated with either the deletion mutant (2.7%) or the insertion mutant (7.6%) (P < 0.001). A series of in vitro assays were performed to explore the mechanism(s) by which FimA enhanced the infectivity of S. parasanguis. FimA did not inhibit the uptake or the subsequent killing of S. parasanguis by phagocytic granulocytes. Similarly, FimA did not play a role in the adherence to or the aggregation of platelets. Significant differences were noted between FW213 and VT929 (P < 0.05) and FW213 and VT930 (P < 0.001) in their abilities to bind to fibrin monolayers. The mean percent adherence of FW213 to fibrin monolayers (2.1%) was greater than those of VT929 (0.5%) and VT930 (0.12%). Taken together, these results indicate that FimA is a major virulence determinant associated with S. parasanguis endocarditis and further suggest that its role is associated with initial colonization of damaged heart tissue.     

Role of Porphyromonas gingivalis protease activity in colonization of oral surfaces.

Cysteine proteases, including Arg-gingipain of Porphyromonas gingivalis, have been implicated as important virulence factors in periodontal diseases. These enzymes are also involved in the hemagglutinating activity of the organisms. In order to determine the role of proteases in the colonization of the gingival margin, we have compared the attachment properties of P. gingivalis 381 with those of its Arg-gingipain-defective mutant, G-102. Interactions with gram-positive bacteria, human oral epithelial cells, extracellular matrix proteins, and type I collagen were evaluated. In all cases, mutant G-102 was deficient in attachment relative to the parental strain. The mutant's defects could be explained, in part, by the weak autoaggregation displayed by the mutant, which appeared to result from altered fimbrial expression. Both Western blot (immunoblot) and Northern (RNA) blot analyses indicated reduced expression of the major 43-kDa fimbrillin subunit in the mutant. These results suggest that Arg-gingipain may play both direct and indirect roles in the colonization of the gingival margin. In addition, fimbriae may play a direct role in interacting with some host surfaces.     

Distribution of Porphyromonas gingivalis strains with fimA genotypes in periodontitis patients

Fimbriae (FimA) of Porphyromonas gingivalis are filamentous components on the cell surface and are thought to play an important role in the colonization and invasion of periodontal tissues. We previously demonstrated that fimA can be classified into four variants (types I to IV) on the basis of the nucleotide sequences of the fimA gene. In the present study, we attempted to detect the four different fimA genes in saliva and plaque samples isolated from patients with periodontitis using the PCR method. Four sets of fimA type-specific primers were designed for the PCR assay. These primers selectively amplified 392-bp (type I), 257-bp (type II), 247-bp (type III), and 251-bp (type IV) DNA fragments of the fimA gene. Positive PCR results were observed with reference strains of P. gingivalis in a type-specific manner. All other laboratory strains of oral and nonoral bacteria gave negative results. The sensitivity of the PCR assay for fimA type-specific detection was between 5 and 50 cells of P. gingivalis. Clinical samples were obtained from saliva and subgingival plaque from deep pockets (>/=4 mm) of 93 patients with periodontitis. Bacterial genomic DNA was isolated from the samples, and the targeted fragments were amplified by PCR. The presence of P. gingivalis was demonstrated in 73 patients (78.5%), and a single fimA gene was detected in most patients. The distribution of the four fimA types among the P. gingivalis-positive patients was as follows: type I, 5.4%; type II, 58.9%; type III, 6. 8%; type IV, 12.3%; types I and II, 6.8%; types II and IV, 2.7%; and untypeable, 6.8%. P. gingivalis with type II fimA was detected more frequently in the deeper pockets, and a significant difference of the occurrence was observed between shallow (4 mm) and deep (>/=8 mm) pockets. These results suggest that P. gingivalis strains that possess type II fimA are significantly more predominant in periodontitis patients, and we speculate that these organisms are involved in the destructive progression of periodontal diseases.     

Bacterial fimbriae activate human peripheral blood monocytes utilizing TLR2, CD14 and CD11a/CD18 as cellular receptors

Bacterial fimbriae are associated with a specific adherence factor, adhesin, in their microbial etiology. Porphyromonas gingivalis, as an anaerobic Gram-negative periodontopathogenic organism, is known to possess fimbriae on its cell surfaces. In this study, we demonstrated that P. gingivalis fimbriae and an active synthetic peptide composed of residues 69 - 73 of thefimbrial subunit protein, ALTTE, induced IL-6 mRNA expression and cytokine production, p38 mitogen-activated protein (MAP) kinase phosphorylation, and NF-kappaB activation in human peripheral blood monocytes. P. gingivalis fimbriae and ALTTE also induced IL-6 production via human Toll-like receptor (TLR) 2, CD14, and CD11a/CD18 (LFA-1) molecules on human monocytes. These results suggest that P. gingivalis fimbriae and these degraded peptides may play an important role in the inflamed gingival and periodontal tissues seen in the development and progression of periodontal diseases.