Mfa1 Fimbriae Research Articles

Structural and antigenic characterization of a novel genotype of Mfa1 fimbriae in Porphyromonas gingivalis

ABSTRACT Background Mfa1 fimbriae of the periodontal pathogen Porphyromonas gingivalis are responsible for biofilm formation and comprise five proteins: Mfa1–5. Two major genotypes, mfa170 and mfa153, encode major fimbrillin. The mfa170 genotype is further divided into the mfa170A and mfa170B subtypes. The properties of the novel mfa170B remain unclear. Methods Fimbriae were purified from P. gingivalis strains JI-1 (mfa170A), 1439 (mfa170B), and Ando (mfa153), and their components and their structures were analyzed. Protein expression and variability in the antigenic specificity of fimbrillins were compared using Coomassie staining and western blotting using polyclonal antibodies against Mfa170A, Mfa170B, and Mfa153 proteins. Cell surface expression levels of fimbriae were analyzed by filtration enzyme-linked immunosorbent assays. Results The composition and structure of the purified Mfa1 fimbriae of 1439 was similar to that of JI-1. However, each Mfa1 protein of differential subtype/genotype was specifically detected by western blotting. Mfa170B fimbriae were expressed in several strains such as 1439, JKG9, B42, 1436, and Kyudai-3. Differential protein expression and antigenic heterogeneities were detected in Mfa2–5 between strains. Conclusion Mfa1 fimbriae from the mfa170A and mfa170B genotypes indicated an antigenic differencesuggesting the mfa170B, is to be utilized for the novel classification of P. gingivalis.

Porphyromonas gingivalis Fimbriae Induce Osteoclastogenesis via Toll-like Receptors in RAW264 Cells

The effect of Mfa1 fimbriae of Porphyromonas gingivalis on the progression of bone resorption remains unclear, especially compared with another fimbriae, FimA. We investigated the effect of Mfa1 on osteoclastogenesis together with FimA. We also investigated the role of Toll-like receptors (TLRs) in Mfa1 recognition during osteoclast differentiation. Receptor activator of nuclear factor κβ ligand (RANKL)-prestimulated RAW264 cells were used to examine the effects of purified Mfa1 fimbriae. The number of osteoclasts was examined by tartrate-resistant acid phosphate (TRAP) staining, osteoclast activation was investigated by bone resorption assays, and gene expression of differentiation markers was examined by quantitative real-time PCR. Transfection of Tlr2 and Tlr4 siRNAs into RAW264 cells was also employed and their role in Mfa1 recognition was investigated. Mfa1 effectively induced the formation of TRAP-positive multinucleated cells and activated osteoclasts. Mfa1 also increased gene expression of Acp5, Mmp9, and Ctsk in RANKL-prestimulated RAW264 cells compared with the control. The osteoclastogenesis induced by Mfa1 was significantly decreased in cells transfected with Tlr2 or Tlr4 siRNAs compared with control siRNA. Our results revealed the role of Mfa1 fimbriae in osteoclastogenesis that may contribute to the partial elucidation of the mechanisms of periodontal disease progression and the development of new therapeutic strategies.

A Tale of Two Fimbriae: How Invasion of Dendritic Cells by Porphyromonas gingivalis Disrupts DC Maturation and Depolarizes the T-Cell-Mediated Immune Response.

Porphyromonas gingivalis (P. gingivalis) is a unique pathogen implicated in severe forms of periodontitis (PD), a disease that affects around 50% of the US population. P. gingivalis is equipped with a plethora of virulence factors that it uses to exploit its environment and survive. These include distinct fimbrial adhesins that enable it to bind to other microbes, colonize inflamed tissues, acquire nutrients, and invade cells of the stroma and immune system. Most notable for this review is its ability to invade dendritic cells (DCs), which bridge the innate and adaptive immune systems. This invasion process is tightly linked to the bridging functions of resultant DCs, in that it can disable (or stimulate) the maturation function of DCs and cytokines that are secreted. Maturation molecules (e.g., MHCII, CD80/CD86, CD40) and inflammatory cytokines (e.g., IL-1b, TNFa, IL-6) are essential signals for antigen presentation and for proliferation of effector T-cells such as Th17 cells. In this regard, the ability of P. gingivalis to coordinately regulate its expression of major (fimA) and minor (mfa-1) fimbriae under different environmental influences becomes highly relevant. This review will, therefore, focus on the immunoregulatory role of P. gingivalis fimbriae in the invasion of DCs, intracellular signaling, and functional outcomes such as alveolar bone loss and immune senescence.

Porphyromonas gingivalis FimA and Mfa1 fimbriae: Current insights on localization, function, biogenesis, and genotype.

In general, the periodontal pathogen Porphyromonas gingivalis expresses distinct FimA and Mfa1 fimbriae. Each of these consists of five FimA–E and five Mfa1–5 proteins encoded by the fim and mfa gene clusters, respectively. The main shaft portion comprises FimA and Mfa1, whereas FimB and Mfa2 are localized on the basal portion and function as anchors and elongation terminators. FimC–E and Mfa3–5 participate in the assembly of an accessory protein complex on the tips of each fimbria. Hence, they serve as ligands for the receptors on host cells and other oral bacterial species. The crystal structures of FimA and Mfa1 fimbrial proteins were recently elucidated and new insights into the localization, function, and biogenesis of these proteins have been reported. Several studies indicated a correlation between P. gingivalis pathogenicity and the fimA genotype but not the mfa1 genotype. We recently revealed polymorphisms of all genes in the fim and mfa gene clusters. Intriguingly, mfa5 occurred in numerous different forms and underwent duplication. Detailed structural and functional knowledge of the fimbrial proteins in the context of the entire filament could facilitate the development of innovative therapeutic strategies for structure-based drug design.

Porphyromonas gingivalis Mfa1 Induces Chemokine and Cell Adhesion Molecules in Mouse Gingival Fibroblasts via Toll-Like Receptors.

Porphyromonas gingivalis Mfa1 fimbriae are thought to act as adhesion factors and to direct periodontal tissue destruction but their immunomodulatory actions are poorly understood. Here, we investigated the effect of Mfa1 stimulation on the immune and metabolic mechanisms of gingival fibroblasts from periodontal connective tissue. We also determined the role of Toll-like receptor (TLR) 2 and TLR4 in Mfa1 recognition. Mfa1 increased the expression of genes encoding chemokine (C-X-C motif) ligand (CXCL) 1, CXCL3, intercellular adhesion molecule (ICAM) 1 and Selectin endothelium (E) in gingival fibroblasts, but did not have a significant effect on genes that regulate metabolism. Mfa1-stimulated up-regulation of genes was significantly suppressed in Tlr4 siRNA-transfected cells compared with that in control siRNA-transfected cells, which indicates that recognition by TLR4 is essential for immunomodulation by Mfa1. Additionally, suppression of Tlr2 expression partially attenuated the stimulatory effect of Mfa1. Overall, these results help explain the involvement of P. gingivalis Mfa1 fimbriae in the progression of periodontal disease.

Role of the RprY response regulator in P. gingivalis community development and virulence.

Porphyromonas gingivalis expresses a limited number of two-component systems, including RprY, an orphan response regulator which lacks a cognate sensor kinase. In this study, we examined cross-phosphorylation of RprY on tyrosine residues and its importance for RprY function. We show that RprY reacts with phosphotyrosine antibodies, and found that the tyrosine (Y) residue at position 41 is predicted to be solvent accessible. Loss of RprY increased the level of heterotypic community development with Streptococcus gordonii, and the community-suppressive function of RprY required Y41. Expression of the Mfa1 fimbrial adhesin was increased in the rprY mutant and in the mutant complemented with rprY containing a Y41F mutation. In a microscale thermophoresis assay, recombinant RprY protein bound to the promoter region of mfa1, and binding was diminished with RprY containing the Y41F substitution. RprY was required for virulence of P. gingivalis in a murine model of alveolar bone loss. Transcriptional profiling indicated that RprY can control the expression of genes encoding the type IX secretion system (T9SS) machinery and virulence factors secreted through the T9SS, including the gingipain proteases and peptidylarginine deiminase (PPAD). Collectively, these results establish the RprY response regulator as a component of the tyrosine phosphorylation regulon in P. gingivalis, which can independently control heterotypic community development through the Mfa1 fimbriae and virulence through the T9SS.

Purification of Native Mfa1 Fimbriae from Porphyromonas gingivalis.

Fimbriae of the periodontal pathogen Porphyromonas gingivalis mediate its colonization through associations with other bacteria and host tissues. P. gingivalis generally expresses two distinct fimbrial types, FimA and Mfa1. In P. gingivalis ATCC 33277, FimA fimbriae are present as long filaments easily detached from cells, whereas Mfa1 fimbriae are short filaments compactly bound to the cell surface. Because of this unique characteristic, FimA fimbriae have been selectively and easily isolated from the bacterial cell surface through mechanical shearing such as by pipetting and stirring. However, P. gingivalis ATCC 33277 harbors a mutation in the gene encode the fimbrial length regulator, FimB, and thus produces unusually long FimA fimbriae length. Hence, mechanical shearing to remove FimA is potentially applicable only for this type strain. Here we present protocols to purify intact Mfa1 fimbriae from a fimA-deficient mutant strain. Mfa1 fimbriae are purified from cell lysates, using a French pressure cell and through ion-exchange chromatography. The purity of Mfa1 fimbriae can be confirmed through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and electron microscopy.

Phylogenetic diversity in fim and mfa gene clusters between Porphyromonas gingivalis and Porphyromonas gulae, as a potential cause of host specificity

ABSTRACT Background Periodontopathic bacteria Porphyromonas gingivalis in humans and Porphyromonas gulae in animals are phylogenetically close and commonly have FimA and Mfa1 fimbriae. However, little is known about how fimA and mfa1 are phylogenetically different between P. gingivalis and P. gulae. Here, we examined phylogenetic diversity in their fim and mfa gene clusters. Methods Twenty P. gulae strains were isolated from the periodontal pocket of 20 dogs. For their genomic information, along with 64 P. gingivalis and 11 P. gulae genomes, phylogenetic relationship between the genotypes of fimA and mfa1 was examined. Variability of amino acid sequences was examined in the three-dimensional structure of FimA. The distance between strains was calculated for fim and mfa genes. Results Some fimA genotypes in P. gulae were close to particular types in P. gingivalis. Two types of mfa1 were classified as 70-kDa and 53-kDa protein-coding mfa1. The variable amino acid positions were primarily at the outer part of FimA. The genes encoding the structural proteins and the main component were similarly distant from the reference strain in P. gingivalis, but not in P. gulae. Conclusions The differences in the gene clusters between P. gingivalis and P. gulae may result in their host specificity.

Disruption of Immune Homeostasis in Human Dendritic Cells via Regulation of Autophagy and Apoptosis by Porphyromonas gingivalis.

As fundamental processes of immune homeostasis, autophagy, and apoptosis must be maintained to mitigate risk of chronic inflammation and autoimmune diseases. Periodontitis is a chronic inflammatory disease characterized by oral microbial dysbiosis, and dysregulation of dendritic cell (DC) and T cell responses. The aim of this study was to elucidate the underlying mechanisms by which the oral microbe Porphyromonas gingivalis (P. gingivalis) manipulates dendritic cell signaling to perturb both autophagy and apoptosis. Using a combination of Western blotting, flow cytometry, qRT-PCR and immunofluorescence analysis, we show a pivotal role for the minor (Mfa1) fimbriae of P. gingivalis in nuclear/cytoplasmic shuttling of Akt and FOXO1 in human monocyte-derived DCs. Mfa1-induced Akt nuclear localization and activation ultimately induced mTOR. Activation of the Akt/mTOR axis downregulated intracellular LC3II, also known as Atg8, required for autophagosome formation and maturation. Use of allosteric panAkt inhibitor MK2206 and mTOR inhibitor rapamycin confirmed the role of Akt/mTOR signaling in autophagy inhibition by P. gingivalis in DCs. Interestingly, this pathway was also linked to induction of the anti-apoptotic protein Bcl2, decreased caspase-3 cleavage and decreased expression of pro-apoptotic proteins Bax and Bim, thus promoting longevity of host DCs. Addition of ABT-199 peptide to disrupt the interaction of antiapoptotic Bcl2 and its proapoptotic partners BAK/BAX restored apoptotic death to P. gingivalis-infected DC cells. In summary, we have identified the underlying mechanism by which P. gingivalis promotes its own survival and that of its host DCs.

Polymicrobial synergy within oral biofilm promotes invasion of dendritic cells and survival of consortia members

Years of human microbiome research have confirmed that microbes rarely live or function alone, favoring diverse communities. Yet most experimental host-pathogen studies employ single species models of infection. Here, the influence of three-species oral microbial consortium on growth, virulence, invasion and persistence in dendritic cells (DCs) was examined experimentally in human monocyte-derived dendritic cells (DCs) and in patients with periodontitis (PD). Cooperative biofilm formation by Streptococcus gordonii, Fusobacterium nucleatum and Porphyromonas gingivalis was documented in vitro using growth models and scanning electron microscopy. Analysis of growth rates by species-specific 16s rRNA probes revealed distinct, early advantages to consortium growth for S. gordonii and F. nucleatum with P. gingivalis, while P. gingivalis upregulated its short mfa1 fimbriae, leading to increased invasion of DCs. F. nucleatum was only taken up by DCs when in consortium with P. gingivalis. Mature consortium regressed DC maturation upon uptake, as determined by flow cytometry. Analysis of dental plaques of PD and healthy subjects by 16s rRNA confirmed oral colonization with consortium members, but DC hematogenous spread was limited to P. gingivalis and F. nucleatum. Expression of P. gingivalis mfa1 fimbriae was increased in dental plaques and hematogenous DCs of PD patients. P. gingivalis in the consortium correlated with an adverse clinical response in the gingiva of PD subjects. In conclusion, we have identified polymicrobial synergy in a three-species oral consortium that may have negative consequences for the host, including microbial dissemination and adverse peripheral inflammatory responses.

Maturation of the Mfa1 Fimbriae in the Oral Pathogen Porphyromonas gingivalis.

The Mfa1 fimbriae of the periodontal pathogen Porphyromonas gingivalis are involved in adhesion, including binding to synergistic species in oral biofilms. Mfa1 fimbriae are comprised of 5 proteins: the structural component Mfa1, the anchor Mfa2, and Mfa3-5 which constitute the fimbrial tip complex. Interactions among the Mfa proteins and the polymerization mechanism for Mfa1 are poorly understood. Here we show that Mfa3 can bind to Mfa1, 2, 4, and 5 in vitro, and may function as an adaptor protein interlinking other fimbrial subunits. Polymerization of Mfa1 is independent of Mfa3-5 and requires proteolytic processing mediated by the RgpA/B arginine gingipains of P. gingivalis. Both the N- and C- terminal regions of Mfa1 are necessary for polymerization; however, potential β-strand disrupting amino acid substitutions in these regions do not impair Mfa1 polymerization. In contrast, substitution of hydrophobic amino acids with charged residues in either the N- or C- terminal domains yielded Mfa1 proteins that failed to polymerize. Collectively, these results indicate that Mfa3 serves as an adaptor protein between Mfa1 and other accessory fimbrial proteins. Mfa1 fimbrial polymerization is dependent on hydrophobicity in both the N- and C-terminal regions, indicative of an assembly mechanism involving the terminal regions forming a hydrophobic binding interface between Mfa1 subunits.

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.

Mfa4, an Accessory Protein of Mfa1 Fimbriae, Modulates Fimbrial Biogenesis, Cell Auto-Aggregation, and Biofilm Formation in Porphyromonas gingivalis.

Porphyromonas gingivalis, a gram-negative obligate anaerobic bacterium, is considered to be a key pathogen in periodontal disease. The bacterium expresses Mfa1 fimbriae, which are composed of polymers of Mfa1. The minor accessory components Mfa3, Mfa4, and Mfa5 are incorporated into these fimbriae. In this study, we characterized Mfa4 using genetically modified strains. Deficiency in the mfa4 gene decreased, but did not eliminate, expression of Mfa1 fimbriae. However, Mfa3 and Mfa5 were not incorporated because of defects in posttranslational processing and leakage into the culture supernatant, respectively. Furthermore, the mfa4-deficient mutant had an increased tendency to auto-aggregate and form biofilms, reminiscent of a mutant completely lacking Mfa1. Notably, complementation of mfa4 restored expression of structurally intact and functional Mfa1 fimbriae. Taken together, these results indicate that the accessory proteins Mfa3, Mfa4, and Mfa5 are necessary for assembly of Mfa1 fimbriae and regulation of auto-aggregation and biofilm formation of P. gingivalis. In addition, we found that Mfa3 and Mfa4 are processed to maturity by the same RgpA/B protease that processes Mfa1 subunits prior to polymerization.

A Major Fimbrilin Variant of Mfa1 Fimbriae in Porphyromonas gingivalis

The periodontal pathogen Porphyromonas gingivalis is known to express 2 distinct types of fimbriae: FimA and Mfa1 fimbriae. However, we previously reported that fimbria-like structures were found in a P. gingivalis strain in which neither FimA nor Mfa1 fimbriae were detected. In this study, we identified a major protein in the bacterial lysates of the strain, which has been reported as the 53-kDa major outer membrane protein of P. gingivalis (53K protein) and subsequently reported as a major fimbrilin of a novel-type fimbria. Sequencing of the chromosomal DNA of the strain showed that the 53k gene (encoding the 53K protein) was located at a locus corresponding to the mfa1 gene (encoding the Mfa1 protein, which is a major fimbrilin of Mfa1 fimbriae) of the ATCC 33277 type strain. However, the 53K and Mfa1 proteins showed a low amino acid sequence homology and different antigenicity. The 53K protein was detected in 34 of 84 (41%) P. gingivalis strains, while the Mfa1 protein was detected in 44% of the strains. No strain expressed both 53K and Mfa1 proteins. Additionally, fimbriae were normally expressed in mutants in which the 53k and mfa1 genes were interchanged. These results indicate that the 53K protein is another major fimbrilin of Mfa1 fimbriae in P. gingivalis.

Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk.

Signaling via pattern recognition receptors (PRRs) expressed on professional antigen presenting cells, such as dendritic cells (DCs), is crucial to the fate of engulfed microbes. Among the many PRRs expressed by DCs are Toll-like receptors (TLRs) and C-type lectins such as DC-SIGN. DC-SIGN is targeted by several major human pathogens for immune-evasion, although its role in intracellular routing of pathogens to autophagosomes is poorly understood. Here we examined the role of DC-SIGN and TLRs in evasion of autophagy and survival of Porphyromonas gingivalis in human monocyte-derived DCs (MoDCs). We employed a panel of P. gingivalis isogenic fimbriae deficient strains with defined defects in Mfa-1 fimbriae, a DC-SIGN ligand, and FimA fimbriae, a TLR2 agonist. Our results show that DC-SIGN dependent uptake of Mfa1+P. gingivalis strains by MoDCs resulted in lower intracellular killing and higher intracellular content of P. gingivalis. Moreover, Mfa1+P. gingivalis was mostly contained within single membrane vesicles, where it survived intracellularly. Survival was decreased by activation of TLR2 and/or autophagy. Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1. In contrast, P. gingivalis uptake through a DC-SIGN independent manner was associated with early endosomal routing through Rab5, increased LC3-II and LAMP-1, as well as the formation of double membrane intracellular phagophores, a characteristic feature of autophagy. These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs.

SspB Peptide Assay Reveals Saliva-Mediated <i>Porphyromonas gingivalis</i> Attachment

Background: Porphyromonas gingivalis is a major periodontal pathogen that binds efficiently to Streptococcus gordonii, which in turn binds to salivary agglutinin (gp340). The SspB of S. gordonii appears to mediate this association. We previously reported that the strepto-coccal SspB peptide analog, designated SspB (390-T400K-402), showed high binding activity with saliva. To understand the three-way interaction among S. gordonii, P. gingivalis and salivary gp340 as a unit, we established a peptide binding assay using SspB (390-T400K-402). Methods: The binding activity of the SspB (390-T400K-402) to P. gingivalis was detected by ELISA. Ninety-six well plates were coated with whole bacterial cell (P. gingivalis strains ATCC 33277, and W83; S. gordonii DL1) in Na2CO3 coating buffer. After blocking, bacterial cells were incubated with saliva or salivary agglutinin peptide (SRCRP2). Biotinylated SspB (390-T400K-402) was applied and incubated with 1:1000 streptoavidin-conjugated alkaline phosphatase. After development, A405 was recorded. Results: P. gingivalis 33277 showed the highest binding activity of the tested bacteria, whereas P. gingivalis W83, which was deficient in Mfa1 fimbriae, exhibited poor binding activity, as did S. gordonii. The binding of SspB (390-T400K-402) peptide in saliva- or SRCRP2-treated P. gingivalis was significantly higher than that in non-treated cells. Conclusion: The SspB (390-T400K-402) peptide binding assay revealed that initial attachment of P. gingivalis to the substrata of S. gordonii may require gp340-mediated SspB-Mfa1 interactions. The assay is available to assess the relationships among SspB, Mfa1 and salivary gp340 as a unit.

Secondary Lymphoid Organ Homing Phenotype of Human Myeloid Dendritic Cells Disrupted by an Intracellular Oral Pathogen

Several intracellular pathogens, including a key etiological agent of chronic periodontitis, Porphyromonas gingivalis, infect blood myeloid dendritic cells (mDCs). This infection results in pathogen dissemination to distant inflammatory sites (i.e., pathogen trafficking). The alteration in chemokine-chemokine receptor expression that contributes to this pathogen trafficking function, particularly toward sites of neovascularization in humans, is unclear. To investigate this, we utilized human monocyte-derived DCs (MoDCs) and primary endothelial cells in vitro, combined with ex vivo-isolated blood mDCs and serum from chronic periodontitis subjects and healthy controls. Our results, using conditional fimbria mutants of P. gingivalis, show that P. gingivalis infection of MoDCs induces an angiogenic migratory profile. This profile is enhanced by expression of DC-SIGN on MoDCs and minor mfa-1 fimbriae on P. gingivalis and is evidenced by robust upregulation of CXCR4, but not secondary lymphoid organ (SLO)-homing CCR7. This disruption of SLO-homing capacity in response to respective chemokines closely matches surface expression of CXCR4 and CCR7 and is consistent with directed MoDC migration through an endothelial monolayer. Ex vivo-isolated mDCs from the blood of chronic periodontitis subjects, but not healthy controls, expressed a similar migratory profile; moreover, sera from chronic periodontitis subjects expressed elevated levels of CXCL12. Overall, we conclude that P. gingivalis actively "commandeers" DCs by reprogramming the chemokine receptor profile, thus disrupting SLO homing, while driving migration toward inflammatory vascular sites.

Localization and function of the accessory protein Mfa3 in Porphyromonas gingivalis Mfa1 fimbriae

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 Ala(44) 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.

Genetic and antigenic analyses of Porphyromonas gingivalis FimA fimbriae

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.

Arginine deiminase inhibits Porphyromonas gingivalis surface attachment

The oral cavity is host to a complex microbial community whose maintenance depends on an array of cell-to-cell interactions and communication networks, with little known regarding the nature of the signals or mechanisms by which they are sensed and transmitted. Determining the signals that control attachment, biofilm development and outgrowth of oral pathogens is fundamental to understanding pathogenic biofilm development. We have previously identified a secreted arginine deiminase (ADI) produced by Streptococcus intermedius that inhibited biofilm development of the commensal pathogen Porphyromonas gingivalis through downregulation of genes encoding the major (fimA) and minor (mfa1) fimbriae, both of which are required for proper biofilm development. Here we report that this inhibitory effect is dependent on enzymic activity. We have successfully cloned, expressed and defined the conditions to ensure that ADI from S. intermedius is enzymically active. Along with the cloning of the wild-type allele, we have created a catalytic mutant (ADIC399S), in which the resulting protein is not able to catalyse the hydrolysis of l-arginine to l-citrulline. P. gingivalis is insensitive to the ADIC399S catalytic mutant, demonstrating that enzymic activity is required for the effects of ADI on biofilm formation. Biofilm formation is absent under l-arginine-deplete conditions, and can be recovered by the addition of the amino acid. Taken together, the results indicate that arginine is an important signal that directs biofilm formation by this anaerobe. Based on our findings, we postulate that ADI functions to reduce arginine levels and, by a yet to be identified mechanism, signals P. gingivalis to alter biofilm development. ADI release from the streptococcal cell and its cross-genera effects are important findings in understanding the nature of inter-bacterial signalling and biofilm-mediated diseases of the oral cavity.