Human Memory B Cells in Healthy Gingiva, Gingivitis, and Periodontitis.

The presence of inflammatory infiltrates with B cells, specifically plasma cells, is the hallmark of periodontitis lesions. The composition of these infiltrates in various stages of homeostasis and disease development is not well documented. Human tissue biopsies from sites with gingival health (n = 29), gingivitis (n = 8), and periodontitis (n = 21) as well as gingival tissue after treated periodontitis (n = 6) were obtained and analyzed for their composition of B cell subsets. Ag specificity, Ig secretion, and expression of receptor activator of NF-κB ligand and granzyme B were performed. Although most of the B cell subsets in healthy gingiva and gingivitis tissues were CD19(+)CD27(+)CD38(-) memory B cells, the major B cell component in periodontitis was CD19(+)CD27(+)CD38(+)CD138(+)HLA-DR(low) plasma cells, not plasmablasts. Plasma cell aggregates were observed at the base of the periodontal pocket and scattered throughout the gingiva, especially apically toward the advancing front of the lesion. High expression of CXCL12, a proliferation-inducing ligand, B cell-activating factor, IL-10, IL-6, and IL-21 molecules involved in local B cell responses was detected in both gingivitis and periodontitis tissues. Periodontitis tissue plasma cells mainly secreted IgG specific to periodontal pathogens and also expressed receptor activator of NF-κB ligand, a bone resorption cytokine. Memory B cells resided in the connective tissue subjacent to the junctional epithelium in healthy gingiva. This suggested a role of memory B cells in maintaining periodontal homeostasis.

Comparative analysis of motility and other properties of Treponema denticola strains.

The periodontitis-associated pathogen Treponema denticola is a spirochetal bacterium that swims by rotating its cell body like a corkscrew using periplasmic flagella. We compared physiologic and pathogenic properties, including motility, in four strains of T. denticola. Phase-contrast microscopy showed differential motility between the strains; ATCC 35404 showed the highest motility, followed by ATCC 33521, and the remaining two strains (ATCC 35405 and ATCC 33520) showed the lowest motility. Transmission electron microscopy showed that the low motility strains exhibited extracellular flagellar protrusions resulting from elongated flagella. Treponemal flagellar filaments are composed of three flagellins of FlaB1, FlaB2 and FlaB3. FlaB1 expression was comparable between the strains, whereas FlaB2 expression was lowest in ATCC 35404. FlaB3 expression varied among strains, with ATCC 35405, ATCC 33520, ATCC 33521, and ATCC 35404 showing the highest to lowest expression levels, respectively. Additionally, the low motility strains showed faster electrophoretic mobility of FlaB3, suggesting that posttranslational modifications of these proteins may have varied, because the amino acid sequences of FlaB3 were identical between the strains. These results suggest that inappropriate expression of FlaB2 and FlaB3 caused the unusual elongation of flagella that resulted in decreased motility. Furthermore, the low motility strains grew to higher bacterial density, and showed greater chymotrypsin-like protease activity, and more bacterial cells associated with gingival epithelial cells in comparison with the high motility strains. There may be a relationship between motility and these properties, but the genetic factors underlying this association remain unclear.

Production of 4-hydroxybutyrate from succinate semialdehyde in butyrate biosynthesis in Porphyromonas gingivalis.

BACKGROUND: Despite evidence demonstrating the importance of butyrate-producing bacteria in host health and disease, the characterization of enzymes responsible for butyrate production has not been fully elucidated in the periodontopathogen, Porphyromonas gingivalis. METHODS: LC-MS/MS and colorimetric analyses were employed to enzymatically characterize recombinant PGN_0724 in P. gingivalis as a succinate semialdehyde reductase. The concentration of short chain fatty acids in the culture supernatant of the wild-type bacteria and a mutant strain lacking the PGN_0724 gene were quantified using GC-MS. RESULTS: Incubation of recombinant PGN_0724 with succinate semialdehyde and NADH resulted in the production of 4-hydroxybutyrate as well as consumption of succinate semialdehyde. Double reciprocal plots showed that the reaction catalyzed by the PGN_0724 protein was associated with a ternary complex mechanism. The growth speed and final turbidity of the mutant strain were much lower than those of the wild-type cells. The capacity of the mutant strain to produce butyrate, isobutyrate, and isovalerate was 30%, 15%, and 45%, respectively, of that of the wild-type strain, while the mutant strain produced approximately 3.9-fold more propionate than the wild type. CONCLUSIONS: The pathway responsible for butyrate production is important for the growth of P. gingivalis and appears to be associated with production of the other short chain fatty acids. GENERAL SIGNIFICANCE: The aim of this study was to delineate the mechanisms involved in the production of 4-hydroxybutyrate, which is an intermediate in the biosynthetic pathway for production of butyrate, which is a virulence factor in P. gingivalis.

Acyl-CoA reductase PGN_0723 utilizes succinyl-CoA to generate succinate semialdehyde in a butyrate-producing pathway of Porphyromonas gingivalis.

The molecular basis of butyrate production in Porphyromonas gingivalis has not been fully elucidated, even though butyrate, a short chain fatty acid (SCFA), can exert both beneficial and harmful effects on a mammalian host. A database search showed that the amino acid sequence of PGN_0723 protein was 50.6% identical with CoA-dependent succinate semialdehyde dehydrogenase (SSADH) in Clostridium kluyveri. By contrast, the protein has limited identity (19.1%) with CoA-independent SSADH in Escherichia coli. Compared with the wild type, growth speed, and final turbidity were lower in the PGN_0723 deletion strain that was constructed by replacing the PGN_0723 gene with an erythromycin resistance cassette. Gas chromatography mass spectrometry revealed the supernatant concentrations of the SCFAs butyrate, isobutyrate, and isovalerate, but not propionate, in the PGN_0723 deletion strain were also lower than those in the wild type. The wild-type phenotype was restored in a complemented strain. We cloned the PGN_0723 gene, purified the recombinant protein, and computationally constructed its three-dimensional model. A colorimetric assay and liquid chromatography-tandem mass spectrometry analysis demonstrated that the recombinant PGN_0723 produces succinate semialdehyde, which is an intermediate in the P. gingivalis butyrate synthesis pathway, not from succinate but from succinyl-CoA in the presence of NAD(P)H via a ping-pong bi-bi mechanism. Asn110Ala and Cys239Ala mutations resulted in a significant loss of the CoA-dependent PGN_0723 enzymatic activity. The study provides new insights into butyrate production, which constitutes a virulence factor in P. gingivalis.

Separation of novel phosphoproteins of Porphyromonas gingivalis using phosphate-affinity chromatography.

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_0004, PGN_0375, PGN_0500, PGN_0724, PGN_0733 and PGN_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.

Characterization of wheat germ agglutinin lectin-reactive glycosylated OmpA-like proteins derived from Porphyromonas gingivalis.

Glycosylation is one of the common posttranslational modifications in eukaryotes. Recently, glycosylated proteins have also been identified in prokaryotes. A few glycosylated proteins, including gingipains, have been identified in Porphyromonas gingivalis, a major pathogen associated with chronic periodontitis. However, no other glycosylated proteins have been found. The present study identified glycoproteins in P. gingivalis cell lysates by lectin blotting. Whole-cell lysates reacted with concanavalin A (ConA), Lens culinaris agglutinin (LCA), Phaseolus vulgaris erythroagglutinin (PHA-E4), and wheat germ agglutinin (WGA), suggesting the presence of mannose-, N-acetylgalactosamine-, or N-acetylglucosamine (GlcNAc)-modified proteins. Next, glycoproteins were isolated by ConA-, LCA-, PHA-E4-, or WGA-conjugated lectin affinity chromatography although specific proteins were enriched only by the WGA column. Mass spectrometry analysis showed that an OmpA-like, heterotrimeric complex formed by Pgm6 and Pgm7 (Pgm6/7) was the major glycoprotein isolated from P. gingivalis. Deglycosylation experiments and Western blotting with a specific antibody indicated that Pgm6/7 was modified with O-GlcNAc. When whole-cell lysates from P. gingivalis mutant strains with deletions of Pgm6 and Pgm7 were applied to a WGA column, homotrimeric Pgm7, but not Pgm6, was isolated. Heterotrimeric Pgm6/7 had the strongest affinity for fibronectin of all the extracellular proteins tested, whereas homotrimeric Pgm7 showed reduced binding activity. These findings suggest that the heterotrimeric structure is important for the biological activity of glycosylated WGA-binding OmpA-like proteins in P. gingivalis.

Lack of a surface layer in Tannerella forsythia mutants deficient in the type IX secretion system.

Tannerella forsythia, a Gram-negative anaerobic bacterium, is an important pathogen in periodontal disease. This bacterium possesses genes encoding all known components of the type IX secretion system (T9SS). T. forsythia mutants deficient in genes orthologous to the T9SS-encoding genes porK, porT and sov were constructed. All porK, porT and sov single mutants lacked the surface layer (S-layer) and expressed less-glycosylated versions of the S-layer glycoproteins TfsA and TfsB. In addition, these mutants exhibited decreased haemagglutination and increased biofilm formation. Comparison of the proteins secreted by the porK and WT strains revealed that the secretion of several proteins containing C-terminal domain (CTD)-like sequences is dependent on the porK gene. These results indicate that the T9SS is functional in T. forsythia and contributes to the translocation of CTD proteins to the cell surface or into the extracellular milieu.

The surface layer of Tannerella forsythia contributes to serum resistance and oral bacterial coaggregation.

Tannerella forsythia is an anaerobic, Gram-negative bacterium involved in the so-called "red complex," which is associated with severe and chronic periodontitis. The surface layer (S-layer) of T. forsythia is composed of cell surface glycoproteins, such as TfsA and TfsB, and is known to play a role in adhesion/invasion and suppression of proinflammatory cytokine expression. Here we investigated the association of this S-layer with serum resistance and coaggregation with other oral bacteria. The growth of the S-layer-deficient mutant in a bacterial medium containing more than 20% non-heat-inactivated calf serum (CS) or more than 40% non-heat-inactivated human serum was significantly suppressed relative to that of the wild type (WT). Next, we used confocal microscopy to perform quantitative analysis on the effect of serum. The survival ratio of the mutant exposed to 100% non-heat-inactivated CS (76% survival) was significantly lower than that of the WT (97% survival). Furthermore, significant C3b deposition was observed in the mutant but not in the WT. In a coaggregation assay, the mutant showed reduced coaggregation with Streptococcus sanguinis, Streptococcus salivarius, and Porphyromonas gingivalis but strong coaggregation with Fusobacterium nucleatum. These results indicated that the S-layer of T. forsythia plays multiple roles in virulence and may be associated with periodontitis.

E-selectin mediates Porphyromonas gingivalis adherence to human endothelial cells.

Porphyromonas gingivalis, a major periodontal pathogen, may contribute to atherogenesis and other inflammatory cardiovascular diseases. However, little is known about interactions between P. gingivalis and endothelial cells. E-selectin is a membrane protein on endothelial cells that initiates recruitment of leukocytes to inflamed tissue, and it may also play a role in pathogen attachment. In the present study, we examined the role of E-selectin in P. gingivalis adherence to endothelial cells. Human umbilical vein endothelial cells (HUVECs) were stimulated with tumor necrosis factor alpha (TNF-α) to induce E-selectin expression. Adherence of P. gingivalis to HUVECs was measured by fluorescence microscopy. TNF-α increased adherence of wild-type P. gingivalis to HUVECs. Antibodies to E-selectin and sialyl Lewis X suppressed P. gingivalis adherence to stimulated HUVECs. P. gingivalis mutants lacking OmpA-like proteins Pgm6 and -7 had reduced adherence to stimulated HUVECs, but fimbria-deficient mutants were not affected. E-selectin-mediated P. gingivalis adherence activated endothelial exocytosis. These results suggest that the interaction between host E-selectin and pathogen Pgm6/7 mediates P. gingivalis adherence to endothelial cells and may trigger vascular inflammation.

Characterization of iNOS(+) Neutrophil-like ring cell in tumor-bearing mice.

BACKGROUND: Myeloid-derived Suppressor Cells (MDSC) have been identified as tumor-induced immature myeloid cells (IMC) with potent immune suppressive activity in cancer. Whereas strict phenotypic classification of MDSC has been challenging due to the highly heterogeneous nature of cell surface marker expression, use of functional markers such as Arginase and inducible nitric oxide synthase (iNOS) may represent a better categorization strategy. In this study we investigated whether iNOS could be utilized as a specific marker for the identification of a more informative homogenous MDSC subset. METHODS: Single-cell suspensions from tumors and other organs were prepared essentially by enzymatic digestion. Flow cytometric analysis was performed on a four-color flow cytometer. Morphology, intracellular structure and localization of iNOS(+) ring cells in the tumor were determined by cytospin analysis, immunofluorescence microscopy and immunohistochemistry, respectively. For functional analysis, iNOS(+) ring subset were sorted and tested in vitro cell culture experiments. Pharmacologic inhibition of iNOS was performed both in vivo and in vitro. RESULTS: The results showed that intracellular iNOS staining distinguished a granular iNOS(+) SSC(hi) CD11b(+) Gr-1(dim) F4/80(+) subset with ring-shaped nuclei (ring cells) among the CD11b(+) Gr-1(+) cell populations found in tumors. The intensity of the ring cell infiltrate correlated with tumor size and these cells constituted the second major tumor-infiltrating leukocyte subset found in established tumors. Although phenotypic analysis demonstrated that ring cells shared characteristics with tumor-associated macrophages (TAM), morphological analysis revealed a neutrophil-like appearance as detected by cytospin and immunofluorescence microscopy analysis. The presence of distinct iNOS filled granule-like structures located next to the cell membrane suggested that iNOS was stored in pre-formed vesicles and available for rapid release upon activation. Tumor biopsies showed large areas with infiltrating ring cells primarily surrounding necrotic areas. Importantly, these cells significantly impaired CD8(+) T-cell proliferation and induced apoptotic death. The intratumoral accumulation and suppressive activity of ring cells could be blocked through pharmacologic inhibition of iNOS, demonstrating the critical role of this enzyme in mediating both the differentiation and the activity of these cells. CONCLUSIONS: In this study, iNOS expression was linked to a homogeneous subset; ring cells with a particular phenotype and immune suppressive function, in a common and well-established murine tumor model; 4T-1. Since the absence of a Gr-1 homolog in humans has made the identification of MDSC much more challenging, use of iNOS as a functional marker of MDSC may also have clinical importance.

Identification of an L-methionine γ-lyase involved in the production of hydrogen sulfide from L-cysteine in Fusobacterium nucleatum subsp. nucleatum ATCC 25586.

Fusobacterium nucleatum produces an abundance of hydrogen sulfide (H(2)S) in the oral cavity that is mediated by several enzymes. The identification and characterization of three distinct enzymes (Fn0625, Fn1055 and Fn1220) in F. nucleatum that catalyse the production of H(2)S from l-cysteine have been reported. In the current study, a novel enzyme involved in the production of H(2)S in F. nucleatum ATCC 25586, whose molecular mass had been estimated to be approximately 130 kDa, was identified by two-dimensional electrophoresis combined with MALDI-TOF MS. The enzyme, Fn1419, has previously been characterized as an l-methionine γ-lyase. SDS-PAGE and gel-filtration chromatography indicated that Fn1419 has a molecular mass of 43 kDa and forms tetramers in solution. Unlike other enzymes associated with H(2)S production in F. nucleatum, the quaternary structure of Fn1419 was not completely disrupted by exposure to SDS. The purified recombinant enzyme exhibited a K(m) of 0.32±0.02 mM and a k(cat) of 0.69±0.01 s(-1). Based on current and published data, the enzymic activity for H(2)S production from l-cysteine in F. nucleatum is ranked as follows: Fn1220>Fn1055>Fn1419>Fn0625. Based on kinetic values and relative mRNA levels of the respective genes, as determined by real-time quantitative PCR, the amount of H(2)S produced by Fn1419 was estimated to be 1.9 % of the total H(2)S produced from l-cysteine in F. nucleatum ATCC 25586. In comparison, Fn1220 appeared to contribute significantly to H(2)S production (87.6 %).

Identification and enzymic analysis of a novel protein associated with production of hydrogen sulfide and L-serine from L-cysteine in Fusobacterium nucleatum subsp. nucleatum ATCC 25586.

A third enzyme that produces hydrogen sulfide from L-cysteine was identified in Fusobacterium nucleatum subsp. nucleatum. The fn1055 gene was cloned from a cosmid library constructed with genomic DNA of F. nucleatum ATCC 25586. Despite the database annotation that the product of fn1055 is a cysteine synthase, reverse-phase HPLC revealed that no L-cysteine was produced in vitro by the purified Fn1055 protein; however, the enzyme did produce L-serine. In addition, a cysteine auxotroph, Escherichia coli NK3, transformed with a plasmid containing the fn1055 gene did not grow without cysteine, which further suggests that Fn1055 does not function as a cysteine synthase. The Michaelis-Menten kinetics (K(m) =0.09 ± 0.001 mM and k(cat) =5.43 ± 0.64 s(-1)) of the purified enzyme showed that the capacity of Fn1055 to produce hydrogen sulfide was between that of two other enzymes, Fn0625 and Fn1220. Incubation of Fn1055 with L-cysteine resulted in the production of hydrogen sulfide, but not of pyruvate, ammonia or lanthionine, which are all byproducts produced in addition to hydrogen sulfide when Fn0625 or Fn1220 is incubated with L-cysteine. Instead, Fn1055 produced L-serine in its reaction with L-cysteine. Fn1055 produces hydrogen sulfide from l-cysteine by a mechanism that is different from that of Fn0625 or Fn1220.

Analysis of immunostimulatory activity of Porphyromonas gingivalis fimbriae conferred by Toll-like receptor 2.

Bacterial fimbriae are an important pathogenic factor. It has been demonstrated that fimbrial protein encoded by fimA gene (FimA fimbriae) of Porphyromonas gingivalis not only contributes to the abilities of bacterial adhesion and invasion to host cells, but also strongly stimulates host innate immune responses. However, FimA fimbriae separated from P. gingivalis ATCC 33277 using a gentle procedure showed very weak proinflammatory activity compared with previous reports. Therefore, in the present study, biological characteristics of FimA fimbriae were further analyzed in terms of proinflammatory activity in macrophages. Macrophages differentiated from THP-1 cells were stimulated with native, heat-denatured, or either proteinase- or lipoprotein lipase-treated FimA fimbriae of P. gingivalis ATCC 33277. Stimulating activities of these FimA fimbriae were evaluated by TNF-alpha-inducing activity in the macrophages. To clarify the mode of action of FimA fimbriae, anti-Toll-like receptor (TLR) 2 blocking antibody was added prior to stimulation. Weak stimulatory activity of native FimA fimbriae was enhanced by heat treatment and low-dose proteinase K treatment. Higher dose of proteinase K treatment abrogated this up-regulation. The activity of treated FimA fimbriae was suppressed by anti-TLR2 antibody, and more substantially by lipoprotein lipase treatment. These results suggest that lipoproteins or lipopeptides associated with FimA fimbriae could at least in part account for signaling via TLR2 and subsequent TNF-alpha production in macrophages.

Porphyromonas gingivalis outer membrane vesicles enter human epithelial cells via an endocytic pathway and are sorted to lysosomal compartments.

Porphyromonas gingivalis, a periodontal pathogen, secretes outer membrane vesicles (MVs) that contain major virulence factors, including major fimbriae and proteases termed gingipains, although it is not confirmed whether MVs enter host cells. In this study, we analyzed the mechanisms involved in the interactions of P. gingivalis MVs with human epithelial cells. Our results showed that MVs swiftly adhered to HeLa and immortalized human gingival epithelial cells in a fimbria-dependent manner and then entered via a lipid raft-dependent endocytic pathway. The intracellular MVs were subsequently routed to early endosome antigen 1-associated compartments and then were sorted to lysosomal compartments within 90 min, suggesting that intracellular MVs were ultimately degraded by the cellular digestive machinery. However, P. gingivalis MVs remained there for over 24 h and significantly induced acidified compartment formation after being taken up by the cellular digestive machinery. In addition, MV entry was shown to be mediated by a novel pathway for transmission of bacterial products into host cells, a Rac1-regulated pinocytic pathway that is independent of caveolin, dynamin, and clathrin. Our findings indicate that P. gingivalis MVs efficiently enter host cells via an endocytic pathway and survive within the endocyte organelles for an extended period, which provides better understanding of the role of MVs in the etiology of periodontitis.

Host adhesive activities and virulence of novel fimbrial proteins of Porphyromonas gingivalis.

The fimbriae of Porphyromonas gingivalis mediate critical roles in host colonization and evasion of innate defenses and comprise polymerized fimbrilin (FimA) associated with quantitatively minor accessory proteins (FimCDE) of unknown function. We now show that P. gingivalis fimbriae lacking FimCDE fail to interact with the CXC-chemokine receptor 4 (CXCR4), and bacteria expressing FimCDE-deficient fimbriae cannot exploit CXCR4 in vivo for promoting their persistence, as the wild-type organism does. Consistent with these loss-of-function experiments, purified FimC and FimD (but not FimE) were shown to interact with CXCR4. However, significantly stronger binding was observed when a combination of all three proteins was allowed to interact with CXCR4. In addition, FimC and FimD bound to fibronectin and type 1 collagen, whereas FimE failed to interact with these matrix proteins. These data and the fact that FimE is required for the association of FimCDE with P. gingivalis fimbriae suggest that FimE may recruit FimC and FimD into a functional complex, rather than directly binding host proteins. Consistent with this notion, FimE was shown to bind both FimC and FimD. In summary, the FimCDE components cooperate and impart critical adhesive and virulence properties to P. gingivalis fimbriae.

Subversion of innate immunity by periodontopathic bacteria via exploitation of complement receptor-3.

The capacity of certain pathogens to exploit innate immune receptors enables them to undermine immune clearance and persist in their host, often causing disease. Here we review subversive interactions of Porphyromonas gingivalis, a major periodontal pathogen, with the complement receptor-3 (CR3; CD11b/CD18) in monocytes/macrophages. Through its cell surface fimbriae, P. gingivalis stimulates Toll-like receptor-2 (TLR2) inside-out signaling which induces the high-affinity conformation of CR3. Although this activates CR3-dependent monocyte adhesion and transendothelial migration, P. gingivalis has co-opted this TLR2 proadhesive pathway for CR3 binding and intracellular entry. In CR3-deficient macrophages, the internalization of P. gingivalis is reduced twofold but its ability to survive intracellularly is reduced 1,000-fold, indicating that CR3 is exploited by the pathogen as a relatively safe portal of entry. The interaction of P. gingivalis fimbriae with CR3 additionally inhibits production of bioactive (p70) interleukin-12, which mediates immune clearance. In vivo blockade of CR3 leads to reduced persistence of P. gingivalis in the mouse host and diminished ability to cause periodontal bone loss, the hallmark of periodontal disease. Strikingly, the ability of P. gingivalis to interact with and exploit CR3 depends upon quantitatively minor components (FimCDE) of its fimbrial structure, which predominantly consists of polymerized fimbrillin (FimA). Indeed, isogenic mutants lacking FimCDE but expressing FimA are dramatically less persistent and virulent than the wildtype organism both in vitro and in vivo. This model of immune evasion through CR3 exploitation by P. gingivalis supports the concept that pathogens evolved to manipulate innate immune function for promoting their adaptive fitness.

Immunocytochemical approach for surface layer proteins of freeze-substituted Tannerella forsythensis by energy-filtering transmission electron microscopy.

Tannerella forsythensis (Bacteroides forsythus), an anaerobic gram-negative potential periodontal pathogens in the progression of periodontitis. IT forsythensis has unique bacterial protein profiles containing major proteins with apparent molecular weight of more than 200-kDa shown by sodium dodecylsulfate-polyacrylamide gel electrophoresis. It is also known to have a typical surface layer (S-layer) consisting of regularly arrayed subunits outside the outer membrane revealed by electron microscopy. On the other hand, electron microscopy showed that the best preservation of structure was obtained when cells were postfixed with OsO4, but this resulted in very low levels of gold particles labeling. Therefore, cells were applied to pieces of filter paper and freeze-substituted by plung-freezing in Liquid propane, substituted in methanol containing 0.5% uranyl acetate, and infiltrated with LR-White resin. We also examined the relation between high molecular weight proteins and S-layer in energy-filtering transmission electron microscopy (EF-TEM) to visualize 3,3'-diaminobenzidene, tetrahydrochloride (DAB) reaction. The three-window method in electron spectroscopic images (ESI) of nitrogen (N) element, reflecting the presence of DAB moieties by the DAB reaction solution, horseradish peroxidase (HRP)-conjugated secondary antibodies instead of immunogold particles obtained by the EF-TEM. The mapping patterns of net N element were restricted to the outermost cell surface.

Structural and functional characterization of shaft, anchor, and tip proteins of the Mfa1 fimbria from the periodontal pathogen Porphyromonas gingivalis.

Very little is known about how fimbriae of Bacteroidetes bacteria are assembled. To shed more light on this process, we solved the crystal structures of the shaft protein Mfa1, the regulatory protein Mfa2, and the tip protein Mfa3 from the periodontal pathogen Porphyromonas gingivalis. Together these build up part of the Mfa1 fimbria and represent three of the five proteins, Mfa1-5, encoded by the mfa1 gene cluster. Mfa1, Mfa2 and Mfa3 have the same overall fold i.e., two ß-sandwich domains. Upon polymerization, the first ß-strand of the shaft or tip protein is removed by indigenous proteases. Although the resulting void is expected to be filled by a donor-strand from another fimbrial protein, the mechanism by which it does so is still not established. In contrast, the first ß-strand in Mfa2, the anchoring protein, is firmly attached by a disulphide bond and is not cleaved. Based on the structural information, we created multiple mutations in P. gingivalis and analysed their effect on fimbrial polymerization and assembly in vivo. Collectively, these data suggest an important role for the C-terminal tail of Mfa1, but not of Mfa3, affecting both polymerization and maturation of downstream fimbrial proteins.

Characterization of RagA and RagB in Porphyromonas gingivalis: study using gene-deletion mutants.

The major outer-membrane proteins RagA and RagB of Porphyromonas gingivalis are considered to form a receptor complex functionally linked to TonB. In this study, P. gingivalis mutants with ragA, ragB or both deleted were constructed from strain W83 as the parent to examine the physiological and pathological functions of RagA and RagB. The double-deletion mutant completely lacked both RagA and RagB, whereas the DeltaragA mutant reduced RagB expression considerably and the DeltaragB mutant produced degraded RagA. Growth of the three mutants in a nutrient-rich medium and synthetic media containing digested protein as a unique nutrient source was similar to that of the parental strain; however, both the DeltaragA and DeltaragAB mutants exhibited very slow growth in a synthetic medium containing undigested, native protein, and the two mutants tended to lose their viability during experiments, although gingipain (protease) activities were unchanged in the mutants. A mouse model showed that the DeltaragB mutant had reduced virulence. Cell-surface labelling with biotin and dextran revealed that both RagA and RagB localized on the outermost cell surface. A cross-linking experiment using wild-type P. gingivalis showed that RagA and RagB were closely associated with each other. Furthermore, co-immunoprecipitation confirmed that RagA and RagB formed a protein-protein complex. These results suggest that physically associated RagA and RagB may stabilize themselves on the cell surface and function as active transporters of large degradation products of protein and in part as a virulence factor.

Novel fimbrilin PGN_1808 in Porphyromonas gingivalis.

Porphyromonas gingivalis, a periodontopathic gram-negative anaerobic bacterium, generally expresses two types of fimbriae, FimA and Mfa1. However, a novel potential fimbrilin, PGN_1808, in P. gingivalis strain ATCC 33277 was recently identified by an in silico structural homology search. In this study, we experimentally examined whether the protein formed a fimbrial structure. Anion-exchange chromatography showed that the elution peak of the protein was not identical to those of the major fimbrilins of FimA and Mfa1, indicating that PGN_1808 is not a component of these fimbriae. Electrophoretic analyses showed that PGN_1808 formed a polymer, although it was detergent and heat labile compared to FimA and Mfa1. Transmission electron microscopy showed filamentous structures (2‒3 nm × 200‒400 nm) on the cell surfaces of a PGN_1808-overexpressing P. gingivalis mutant (deficient in both FimA and Mfa1 fimbriae) and in the PGN_1808 fraction. PGN_1808 was detected in 81 of 84 wild-type strains of P. gingivalis by western blotting, suggesting that the protein is generally present in P. gingivalis.