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Periodontal diseases are semi-ubiquitous and caused by chronic, plaque-induced inflammation. The 55- kDa immunodominant RagB outer membrane protein of Porphyromonas gingivalis, a keystone periodontal pathogen, has been proposed to facilitate nutrient transport. However, potential interactions between RagB and the innate response have not been examined. We determined that RagB exposure led to the differential and dose-related expression of multiple genes encoding proinflammatory mediators [interleukin-1α ( IL-1α), IL-1β, IL-6, IL-8 and CCL2; all P < 0.05] in primary human monocytes and to the secretion of tumor necrosis factor and IL-8, but not interferon-γ or IL-12. RagB was shown to be a Toll-like receptor 2 ( TLR2) and TLR4 agonist that activated signal transducer and activator of transcription 4 and nuclear factor-κB signaling, as determined by a combination of blocking antibodies, pharmaceutical inhibitors and gene silencing. In keeping, a Δ ragB mutant similarly exhibited reduced inflammatory capacity, which was rescued by ragB complementation. These results suggest that RagB elicits a major pro-inflammatory response in primary human monocytes and, therefore, could play an important role in the etiology of periodontitis and systemic sequelae. [ABSTRACT FROM AUTHOR]

The fimbriae of Porphyromonas gingivalis, the causative agent of periodontitis, have been implicated in various aspects of pathogenicity, such as colonization, adhesion and aggregation. Porphyromonas gingivalis ATCC 33277 has two adhesins comprised of the FimA and Mfa1 fimbriae. We characterized the PGN0289 (Mfa3) protein, which is one of the three accessory proteins of Mfa1 fimbriae in P. gingivalis. The Mfa3 protein was present in two different sizes, 40 and 43 kDa, in the cell. The 43-kDa and 40-kDa Mfa3 were detected largely in the inner membrane and the outer membrane, respectively. Purified Mfa1 fimbriae contained the 40-kDa Mfa3 alone. Furthermore, the 40-kDa Mfa3 started with the Ala44 residue of the deduced amino acid sequence, indicating that the N-terminal region of the nascent protein expressed from the mfa3 gene is processed in the transport step from the inner membrane into fimbriae. Immuno-electron microscopy revealed that Mfa3 localized at the tip of the fimbrial shaft. Interestingly, deletion of the mfa3 gene resulted in the absence of other accessory proteins, PGN0290 and PGN0291, in the purified Mfa1 fimbriae, suggesting that Mfa3 is required for integration of PGN0290 and PGN0291 into fimbriae. A double mutant of mfa3 and fimA genes (phenotype Mfa1 plus, FimA minus) showed increased auto-aggregation and biofilm formation similar to a double mutant of mfa1 and fimA genes (phenotype Mfa1-, FimA-). These findings suggest that the tip protein Mfa3 of the Mfa1 fimbriae may function in the integration of accessory proteins and in the colonization of P. gingivalis. [ABSTRACT FROM AUTHOR]

The periodontal pathogen Porphyromonas gingivalis generally expresses two distinct fimbriae, FimA and Mfa1, which play a role in biofilm formation. The fimA gene that encodes FimA fimbrilin is polymorphic, and polymerase chain reaction analysis has identified six genotypes called types I-V and Ib. We found recently that fimbriae exhibit antigenic heterogeneity among the genotypes. In the present study, we analysed the fimA DNA sequences of 84 strains of P. gingivalis and characterized the antigenicity of FimA fimbriae. Strains analysed here comprised 10, 16, 29, 13, 10 and 6 strains of types I, Ib, II, III, IV and V, respectively. DNA sequencing revealed that type Ib does not represent a single cluster and that type II sequences are remarkably diverse. In contrast, the fimA sequences of the other types were relatively homogeneous. Antigenicity was investigated using antisera elicited by pure FimA fimbriae of types I-V. Antigenicity correlated generally with the respective genotype. Type Ib strains were recognized by type I antisera. However, some strains showed cross-reactivity, especially, many type II strains reacted with type III antisera. The levels of fimbrial expression were highly variable, and expression was positively correlated with ability of biofilm formation on a saliva-coated plate. Further, two strains without FimA and Mfa1 fimbriae expressed fimbrial structures, suggesting that the strains produce other types of fimbriae. [ABSTRACT FROM AUTHOR]

In monocytes and macrophages, the interaction of Porphyromonas gingivalis with Toll-like receptor 2 ( TLR2) leads to the activation of a MyD88-dependent antimicrobial pathway and a phosphatidylinositol-3 kinase ( PI3K) -dependent pro-adhesive pathway, which activates the β2-integrin complement receptor 3 ( CR3). By means of its fimbriae, P. gingivalis binds CXC-chemokine receptor 4 ( CXCR4) and induces crosstalk with TLR2 that inhibits the MyD88-dependent antimicrobial pathway. In this paper, we investigated the impact of the P. gingivalis- CXCR4 interaction on the pro-adhesive pathway. Using human monocytes, mouse macrophages, or receptor-transfected cell lines, we showed that the binding of P. gingivalis fimbriae to CXCR4 induces CR3 activation via PI3K, albeit in a TLR2-independent manner. An isogenic strain of P. gingivalis expressing mutant fimbriae that do not interact with CXCR4 failed to efficiently activate CR3, leading to enhanced susceptibility to killing in vivo compared with the wild-type organism. This in vivo observation is consistent with previous findings that activated CR3 mediates safe entry of P. gingivalis into macrophages. Taken together with our previous work, these results indicate that the interaction of P. gingivalis with CXCR4 leads to inhibition of antimicrobial responses and enhancement of pro-adhesive responses, thereby maximizing its adaptive fitness in the mammalian host. [ABSTRACT FROM AUTHOR]

Porphyromonas gingivalis has been implicated as a major pathogen associated with chronic periodontitis. To extend our knowledge of post-translational protein glycosylation in P. gingivalis, a proteomic analysis involving two-dimensional polyacrylamide gel electrophoresis combined with carbohydrate staining and mass spectrometry was performed. Four novel glycoproteins, PGN0743, PGN0876, PGN1513 and PGN0729, in P. gingivalis ATCC 33277 were identified. These four identified glycoproteins possess a range of biochemical activities and cellular localization. PGN0743 contains a sequence motif identifying it as a FKBP-type cis-trans isomerase, which has activity usually associated with chaperone functions. PGN0876 and PGN1513 contain tetratricopeptide repeat domains that mediate protein-protein interactions. PGN0729 encodes the outer membrane protein 41 precursor, which was previously identified as Pgm6, and is homologous to the OmpA protein in Escherichia coli. Several different types of glycoprotein were identified, suggesting that P. gingivalis possesses a general mechanism for protein glycosylation. PGN0743-deficient and PGN0876-deficient mutants were constructed to examine the role(s) of the two identified glycoproteins. Both mutants showed a decreased growth rate under nutrient-limited conditions and reduced biofilm formation activity. These results suggest that the novel glycoproteins PGN0743 and PGN0876 play an important role in the growth and colonization of P. gingivalis. [ABSTRACT FROM AUTHOR]

Summary Tannerella forsythia, a gram-negative fusiform rod, is implicated in several types of oral anaerobic infections. Most gram-negative bacteria have OmpA-like proteins that are homologous to the OmpA protein in Escherichia coli. We identified an OmpA-like protein in T. forsythia encoded by the tf1331 gene as one of the major proteins by mass spectrometric analysis. Two-dimensional, diagonal electrophoresis showed that the OmpA-like protein formed a dimeric or trimeric structure via intermolecular disulfide bonds. A biotin labeling experiment revealed that a portion of the protein was exposed on the cell surface, even though T. forsythia possesses an S-layer at the outermost cell surface. Using a tf1331-deletion mutant, we showed that the OmpA-like protein affected cell morphology. The length of the mutant cell was reduced almost by half. Cell swelling was observed in more than 40% of the mutant cells. Moreover, the mutant exhibited decreased adhesion to fibronectin, retarded autoaggregation, and reduced cell surface hydrophobicity. These results suggest that the OmpA-like protein in T. forsythia plays an important role in cellular integrity and adhesive function. [ABSTRACT FROM AUTHOR]

Introduction: The fimA-encoded fimbriae of the periodontal pathogen Porphyromonas gingivalis display genetic diversity. Type I fimbriated P. gingivalis (Pg-I) has been most widely studied at the molecular level, whereas Pg-II is the most frequent isolate from severe periodontitis. Methods: To investigate virulence differences between Types I and II fimbriae, we examined strains 33277 (Pg-I) and OMZ314 (Pg-II), reciprocal swap mutants (i.e. expressing the heterologous fimbrial type), and their respective FimA-deficient derivatives. These organisms were tested in a mouse periodontitis model and in interactions with mouse macrophages, a cell type that plays important roles in chronic infections. Results: Strain 33277 induced significantly more periodontal bone loss than OMZ314 and substitution of Type II fimbriae with Type I in OMZ314 resulted in a more virulent strain than the parent organism. However, the presence of Type II fimbriae was associated with increased proinflammatory and invasive activities in macrophages. Conclusion: The inverse relationship between proinflammatory potential and ability to cause experimental periodontitis may suggest that an aggressive phenotype could provoke a host response that would compromise the persistence of the pathogen. [ABSTRACT FROM AUTHOR]

Background and Objective: Research on Porphyromonas gingivalis, a periodontopathogen, has provided a tremendous amount of information over the last 20 years, which may exceed in part than that on other closely related members in terms of phylogenetic as well as proteomic criteria, including Bacteroides fragilis and B. thetaiotaomicron as major anaerobic, opportunistic pathogens in the medical field. In this minireview, we focused on recent research findings concerning surface components such as outer membrane proteins and fimbriae, of P. gingivalis. Material and Methods: Elucidation of the surface components in P. gingivalis was especially difficult because outer membrane proteins are tightly bound to lipopolysaccharide and they are resistant to dissociation and separation from each other, even during sodium dodecyl sulfate–polyacrylamide gel electrophoresis, unless samples are appropriately heated. In addition, P. gingivalis is asaccharolytic and therefore a potent proteolytic bacterium, another factor causing difficulty in research. The study of the surface components was carefully carried out considering these unique features in P. gingivalis when compared with other gram-negative bacteria, including Escherichia coli and Pseudomonas aeruginosa. Results: Separation of outer membrane proteins, and characterization of OmpA-like proteins and RagAB as major proteins, is described herein. Our recent findings on FimA and Mfa1 fimbriae, two unique appendages in this organism, and on their regulation of expression are also described briefly. Conclusion: Surface components of P. gingivalis somehow have contact with host tissues and cells because of the outermost cell elements. Therefore, such bacterial components are potentially important in the occurrence of periodontal diseases. [ABSTRACT FROM AUTHOR]

Introduction: Porphyromonas gingivalis is one of the most important bacteria in the progression of chronic periodontal disease. We hypothesized that the major outer membrane proteins Pgm6/7, which are homologous to the OmpA protein in Escherichia coli, might contribute to the stabilization of the cell surface. In this study, the effects of Pgm6/7 on the cell surface were examined morphologically. Methods: Deletion mutants of Pgm6/7 (Δ 694, Δ 695 and Δ 695–694) were constructed using the polymerase chain reaction-based overlap extension method. Wild-type ATCC 33277 and Pgm6/7 mutants were grown under anaerobic conditions. Whole cells and thin sections of fixed cells were stained and examined by transmission electron microscopy. Results: Compared with the wild-type, numerous vesicles released from cells were observed in each deletion mutant. The outer membrane appeared wavy and irregular. Increased numbers of vesicles were confirmed after their preparation from the culture supernatant. Total gingipain activity in vesicles was increased five- to 10-fold in the deletion mutants. Conclusion: This report provides further evidence that Pgm6/7 proteins in P. gingivalis play an important role in the maintenance of bacterial outer membrane integrity. [ABSTRACT FROM AUTHOR]

Bacteroides forsythus, which has been recognized as a pathogen associated with periodontitis, produces a hemagglutinin. The hemagglutinin was localized in the envelope of B. forsythus. The hemagglutinating activity was inhibited by lactose at concentrations as low as 1 mM, and by L-arginine and L-lysine at concentrations of 100mM. N-Acetylneuraminyllactose (NeuAc-lactose) was at least 100 times more potent an inhibitor than lactose, as it completely inhibited the hemagglutination at concentrations below 10 µM. This is similar to the Helicobacter pylorl hemagglutinin. The hemagglutinin was heat-labile, and resistant to treatment with proteases such as trypsin. A specific antibody raised against one of the S-layer proteins that are major species-specific proteins had no inhibitory effect on the hemmaglutination. These results suggest that the NeuAc-lactose-sensitive adhesin of B. forsythus may play an important role in colonization in the oral cavity. [ABSTRACT FROM AUTHOR]

A total of 188 Porphyromonas gingivalis strains isolated from 13 periodontal pockets of 8 periodontitis patients were investigated by means of restriction fragment length polymorphism (RFLP) analysis using the fimA gene as a pobe. A total of 5 RFLP genotypes were identified, and over half of the isolates belonged to type I. Four of the 8 patients harbored only 1 RFLP genotype of P. gingivalis, and 1 patient harbored only 2 RFLP genotypes in different sites. On the other hand, in 3 other patients, multiple RFLP genotypes were found in single periodontal pockets. Further studies will be required to clarify whether multiple genotypes of P. gingivalis colonized a single periodontal pocket simultaneously or whether a mutation occurred in the fimbrilin gene locus of P. gingivalis. [ABSTRACT FROM AUTHOR]

The fimbriae of Porphyromonas gingivalis, the causative agent of periodontitis, have been implicated in various aspects of pathogenicity, such as colonization, adhesion and aggregation. P. gingivalis ATCC 33277 has two adhesins comprised of the FimA and Mfa1 fimbriae. We characterized the PGN0289 (Mfa3) protein, which is one of the three accessory proteins of Mfa1 fimbriae in P. gingivalis. The Mfa3 protein was present in two different sizes of 40 and 43 kDa in the cell. The 43‐kDa and the 40‐kDa Mfa3 were detected largely in the inner membrane and the outer membrane, respectively. Purified Mfa1 fimbriae contained the 40‐kDa Mfa3 alone. Furthermore, the 40‐kDa Mfa3 started with the Ala44 residue of the deduced amino acid sequence, indicating that N‐terminal region of the nascent protein expressed from the mfa3 gene is processed in the transport step from the inner membrane into fimbriae. Immuno‐electron microscopy revealed that Mfa3 localized at the tip of fimbrial shaft. Interestingly, deletion of the mfa3 gene resulted in absence of other accessory proteins of PGN0290 and PGN0291 in the purified Mfa1 fimbriae, suggesting that Mfa3 is required for integration of PGN0290 and PGN0291 into fimbriae. A double mutant of mfa3 and fimA genes (phenotype Mfa1 plus, FimA minus) showed increased auto‐aggregation and biofilm formation similar to a double mutant of mfa1 and fimA genes (phenotype Mfa1 minus, FimA minus). These findings suggest that the tip protein Mfa3 of the Mfa1 fimbriae may function in the integration of accessory proteins and in the colonization of P. gingivalis.This article is protected by copyright. All rights reserved. [ABSTRACT FROM AUTHOR]

Phosphorylation of serine, threonine and tyrosine is a central mechanism for regulating the structure and function of proteins in both eukaryotes and prokaryotes. However, the action of phosphorylated proteins present in Porphyromonas gingivalis, a major periodontopathogen, is not fully understood. Here, six novel phosphoproteins that possess metabolic activities were identified, namely PGN&#95;0004, PGN&#95;0375, PGN&#95;0500, PGN&#95;0724, PGN&#95;0733 and PGN&#95;0880, having been separated by phosphate-affinity chromatography. The identified proteins were detectable by immunoblotting specific to phosphorylated Ser (P-Ser), P-Thr, and/or P-Tyr. These results imply that novel phosphorylated proteins might play an important role for regulation of metabolism in P. gingivalis., (© 2016 The Societies and John Wiley & Sons Australia, Ltd.)

Porphyromonas gingivalis has been implicated in both marginal periodontitis and periapical infection. This study examined the major outer membrane proteins, from P. gingivalis, which related to periradicular lesions. Outer membrane protein profiles of P. gingivalis ATCC 33277 and W83 were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and N-terminal amino acid analysis. Most outer membrane proteins, such as RagA, gingipains, and OmpA-like proteins, were found in both strains in a similar distribution pattern; however, the migration positions of Lys-gingipain and RagB were inverted in SDS-PAGE. Western blot analysis showed that RagA, RagB, and OmpA-like proteins were found in all of the P. gingivalis strains tested. The antiserum of W83 against RagB reacted poorly to some strains, such as ATCC 33277. When strains phylogenetically related to P. gingivalis were examined, RagA and OmpA homologs were immunologically detected in several strains. However, none of the RagB homologs were detected in any strain analyzed, suggesting that RagB is unique to P. gingivalis. To examine immunoreactive antigens in P. gingivalis, sera from patients with periradicular lesions were used. More than half of the sera showed strong reactions to P. gingivalis cell components, especially RagB. Our results indicate that a major outer membrane protein, RagB, is a possible virulence factor in periradicular lesions.

Porphyromonas gingivalis is implicated in the occurrence of adult periodontitis. We have previously identified major outer membrane proteins from P. gingivalis, which include representative virulence factors such as gingipains, a 75 kDa major protein, RagA, RagB, and putative porin. Fimbriae, another important virulence factor, exist on the cell surface. In this study, we identified major supernatant proteins. They were fimbrilin, the 75 kDa major protein, gingipains and their adhesin domains. Microscopic examination showed that supernatant proteins formed vesicle-like and fimbrial structures. To learn more about the character of this bacterium, we examined effects of growth temperature on localization and expression of these virulence factors. In general, localization of major virulence factors did not change at the various growth temperatures used. Most of the 75 kDa major protein, RagA, RagB, and putative porin were found in the envelope fraction, not in cell-free culture supernatant. Gingipains were found in both the envelope fraction and supernatant. More than 80% of fimbriae were associated with cells, less than 20% migrated to the supernatant. Most fimbriae existed in the whole cell lysate, although there was a small amount in the envelope fraction. When the growth temperature was increased, expression of fimbriae, gingipains, the 75 kDa major protein, RagA, and RagB decreased. However, temperature had almost no effect on expression of putative porin. The tendency for expression of major virulence factors to decrease at higher temperatures may enable P. gingivalis to survive under hostile conditions.

To elucidate the mechanism of norfloxacin (a fluoroquinolone) resistance of Bacteroides thetaiotaomicron, a member of the B. fragilis group, we isolated transposon-induced mutants sensitive to this agent using Tn4351. Four norfloxacin-sensitive mutants showed reduced levels of resistance, at least, to ethidium bromide. Cloning and sequencing of three chromosomal fragments adjacent to Tn4351 from the mutants revealed that two partial open reading frames (orfs) were disrupted by a transposon. Amino acid sequences of partial orf products had strong homologies to those of Escherichia coli RecB and B. ovatus transketolase. Two mutants carried a recB homolog inserted by Tn4351 together with R751 (cointegration) and by itself (simple transposition) at the amino- and carboxyl-terminal portions, respectively. Since mutations in recB produce E. coli cells sensitive to DNA-damaging treatments by quinolones, it is concluded that decreases of the minimum inhibitory concentrations (MICs) of the agents for B. thetaiotaomicron resulted from disruption of the recB homolog. Another mutant carried a transketolase gene inserted by Tn4351. There is no reasonable explanation why disruption of the transketolase gene caused a decrease of the MIC of norfloxacin for this organism, although Streptococcus pneumoniae RecP related to DNA recombination was reported to be transketolase.

We surveyed the Porphyromonas gingivalis W83 genome database for homologues of FimS, the first two-component sensor histidine kinase, which could possibly control virulence factors. Including fimS, we found six putative sensor kinase genes in the genome. The gene encoding one of the homologues was cloned from a P. gingivalis plasmid library, sequenced, and analyzed using its mutants. Two gene-disruption mutants were created in strain ATCC 33277 by introducing a drug cassette into the gene. The mutants formed nonpigmented colonies, indicating that they might be defective in proteinase production, a characteristic of this organism. Proteinase activities, measured as arginine- and lysine-specific (Rgp and Kgp gingipains, respectively) activities, of the mutants were almost half those of the parent strain. Unlike the parent and wildtype strains, most of the gingipain activities were detected in the culture supernatant, not in cells, of the mutants. Abnormal production of gingipains was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analyses. These results strongly suggest that this newly-discovered two-component sensor kinase is involved in maturation and proper localization of gingipains to the outer membrane through an unknown mechanism. The gene encoding the sensor histidine kinase was designated gppX, which represents regulation (X) of gingipains and black pigmentation in P. gingivalis.

The outer membrane of Porphyromonas gingivalis, an oral strict anaerobe, was isolated by sucrose density gradient centrifugation. The outer membrane obtained by the differential detergent extraction method, previously reported, showed an essentially similar protein pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), confirming that the latter method is suitable for the study of outer membrane proteins in this organism. N-terminal amino acid sequence analysis revealed that major outer membrane proteins in this organism included Arg-gingipain, Lys-gingipain, RagA (a TonB-linked receptor), and putative porins that were homologous to Escherichia coli OmpA.

Porphyromonas gingivalis, a periodontopathogen, is an oral anaerobic gram-negative bacterium with numerous fimbriae on the cell surface. Fimbriae have been considered to be an important virulence factor in this organism. We analyzed the genomic DNA of transposon-induced, fimbria-deficient mutants derived from ATCC 33277 and found that seven independent mutants had transposon insertions within the same restriction fragment. Cloning and sequencing of the disrupted region from one of the mutants revealed two adjacent open reading frames (ORFs) which seemed to encode a two-component signal transduction system. We also found that six of the mutants had insertions in a gene, fimS, a homologue of the genes encoding sensor kinase, and that the insertion in the remaining one disrupted the gene immediately downstream, fimR, a homologue of the response regulator genes in other bacteria. These findings suggest that this two-component regulatory system is involved in fimbriation of P. gingivalis.

Bacteroides forsythus produces species-specific major proteins with high molecular weights of 270 and 230-kDa (270K and 230K). A specific antibody raised against 270K was used for Western blot analysis and immunoelectron microscopy. Western blot analysis showed that the 270K and 230K proteins were immunologically similar. Immunogold labeling of ultrathin-sectioned bacterial cells and biochemical fractionation revealed that these proteins were localized at the outermost cell surface, not in the cytoplasm. These results suggest that major proteins ubiquitous to this species may form the S-layer.

Bacterial binding phenomena among different bacterial genera or species play an important role in bacterial colonization in a mixed microbiota such as in the human oral cavity. The coaggregation reaction between two gram-negative anaerobes, Treponema medium and Porphyromonas gingivalis, was characterized using fimbria-deficient mutants of P. gingivalis and specific antisera against purified fimbriae and bacterial whole cells. T. medium ATCC 700273 strongly coaggregated with fimbriate P. gingivalis strains ATCC 33277 and 381, but not with afimbriate strains including transposon-induced fimbria-deficient mutants and KDP98 as a fimA-disrupted mutant of P. gingivalis ATCC 33277. In the P. gingivalis-T. medium coaggregation assay, the presence of rabbit antiserum against the purified fimbriae or the whole cells of P. gingivalis ATCC 33277 produced different "aggregates" consisting predominantly of P. gingivalis cells with few spirochetes, but both preimmune serum and the antiserum against the afimbriate KDP98 cells did not inhibit the coaggregation reaction. Heated P. gingivalis cells lost their ability to bind both heated and unheated T. medium cells. This T. medium-P. gingivalis coaggregation reaction was inhibited by a cysteine proteinase inhibitor, leupeptin, and also by arginine and lysine, but not by EDTA or sugars including lactose. A binding assay on nitrocellulose membranes and immunoelectron microscopy demonstrated that a heat-stable 37 kDa surface protein on the T. medium cell attached to the P. gingivalis fimbriae.

A gene upstream from fimA, the gene encoding fimbrilin, on the chromosome of Porphyromonas gingivalis was sequenced and shown to be the gene encoding an outer membrane protein in this organism based on homology and biochemical analyses. Therefore, the gene (formerly ORF5) was designated pgmA, the P. gingivalis outer membrane protein A gene. The gene product, PgmA, was sensitive to protease, and was detected as a 60-kDa protein from wild-type strains with trichloroacetic acid treatment, which was carried out to destroy intrinsic proteases, and from protease-deficient mutants without this treatment prior to electrophoresis. PgmA was indeed present in the membrane fraction. Its nature was determined to be that of outer membrane proteins in gram-negative bacteria based on attempts at differential extraction of inner membrane proteins with detergents. No evidence has been found thus far from functional analyses that this protein is related to fimbrial morphogenesis and functions or to serum resistance of this organism.

The downstream DNA region of the fimbrilin gene (fimA), which encodes the major subunit protein of Porphyromonas gingivalis fimbriae, was fully sequenced. Gene products, expressed from this region in Escherichia coli, were purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their partial amino acid sequences were determined to verify open reading frames (ORFs) found in the region by DNA sequencing. Four ORFs, designated ORF1, ORF2, ORF3 and ORF4, were found in the 5.8-kb PstI fragment downstream from fimA, which was previously cloned and partially characterized by Yoshimura, Takahashi, Hibi, Takasawa, Kato, and Dickinson (Infect. Immun. 61: 5181-5189, 1993). The direction of transcription of all the ORFs was the same as that of fimA. The 50 and 80 kDa encoded proteins, ORF2 and ORF3, respectively, have been reported to be minor components associated with fimbriae. The 15 and 19 kDa proteins, ORF1 and ORF4, respectively, have been expressed in E. coli but not identified in P. gingivalis. However, all the gene products of the ORFs, expressed in E. coli, appeared to contain intact signal peptides based on their N-terminal amino acid sequences.

Inhibition of hemagglutinin (HA) activity in a membrane fraction of Bacteroides gingivalis was examined using various compounds. Leupeptin and anti-pain inhibited the HA activity at nM order. This potency was lost when the aldehyde group of leupeptin was converted to an alcohol moiety. Irreversible protease inhibitors, tosyl-L-lysine chloromethyl ketone (TLCK), p-chloromercuribenzoate (PCMB), and N-ethylmaleimide (NEM) were also inhibitory. From the inhibition experiments, we speculate that the HA possesses protease activity and that the same site of the molecule participates in the erythrocyte binding and the substrate binding.

Sera from a number of patients with periodontal diseases were shown to have specific immunoglobulin G (IgG) antibodies against fimbriae and membrane proteins of Bacteroides gingivalis, a suspected pathogen, by using Western blottin analysis. The sera had a strong tendency to react with fimbriae, or exactly oligomeric structures of fimbriae with a native beta-structure rich-conformation. However, the sera did not react with fimbrilin, a constituent protein of fimbriae, which is denatured by sodium dodecyl sulfate.