Identification and characterization of fusolisin, the Fusobacterium nucleatum autotransporter serine protease.

Fusobacterium nucleatum is an oral anaerobe associated with periodontal disease, adverse pregnancy outcomes and colorectal carcinoma. A serine endopeptidase of 61-65 kDa capable of damaging host tissue and of inactivating immune effectors was detected previously in F. nucleatum. Here we describe the identification of this serine protease, named fusolisin, in three oral F. nucleatum sub-species. Gel zymogram revealed fusobacterial proteolytic activity with molecular masses ranging from 55-101 kDa. All of the detected proteases were inhibited by the serine protease inhibitor PMSF. analysis revealed that all of the detected proteases are encoded by genes encoding an open reading frame (ORF) with a calculated mass of approximately 115 kDa. Bioinformatics analysis of the identified ORFs demonstrated that they consist of three domains characteristic of autotransporters of the type Va secretion system. Our results suggest that the F. nucleatum fusolisins are derived from a precursor of approximately 115 kDa. After crossing the cytoplasmic membrane and cleavage of the leader sequence, the C-terminal autotransporter domain of the remaining 96-113 kDa protein is embedded in the outer membrane and delivers the N-terminal S8 serine protease passenger domain to the outer cell surface. In most strains the N-terminal catalytic 55-65 kDa domain self cleaves and liberates itself from the autotransporter domain after its transfer across the outer cell membrane. In F. nucleatum ATCC 25586 this autocatalytic activity is less efficient resulting in a full length membrane-anchored serine protease. The mature serine protease was found to cleave after Thr, Gly, Ala and Leu residues at the P1 position. Growth of F. nucleatum in complex medium was inhibited when serine protease inhibitors were used. Additional experiments are needed to determine whether fusolisin might be used as a target for controlling fusobacterial infections.

Wettability versus electrostatic forces in fibronectin and albumin adsorption to titanium surfaces.

OBJECTIVES: Although the enhancement of plasma protein adsorption to titanium ( Ti ) following wetting has been recognized, the relationship between wettability and electrostatic forces has remained unclear. Thus, we have carried out a series of studies to determine the role of wettability and electrostatic forces on protein adsorption. METHODS: Titanium disks with different surfaces were wetted with a range of solutions, two of which contained divalent positive ions ( Ca and Mg ). Unwetted disks served as a control. Subsequently, the wetted disks were subjected to three treatment regimes: (1) incubation in human serum albumin (HSA) or human serum fibronectin (HSF); (2) drying the wetted disks, followed by incubation in HSA or HSF; and (3) following protein adsorption, the Ca originating in the wetting solutions was removed by divalent positive ions chelator treatment (EGTA), and the remaining quantities were assessed. The quantity of the adsorbed proteins was determined by ELISA. RESULTS: It was found that in the case of HSA, adsorption was enhanced by the wettability, the presence of Ca and Mg in the wetting solution, and the existence of rough surfaces. For HSF, the wettability and rough surfaces enhanced adsorption. CONCLUSION: The results demonstrate that in addition to wettability, the composition of the wetting solution affects the protein adsorption. While wetting reduces the time for the HSA and HSF adsorption to reach saturation, the electrostatic forces enhance the amount of HSA adsorption. Thus, the protein adsorption capacity of titanium rough surfaces can be selectively manipulated by changing of the wetting solution.

The adhesion of oral bacteria to modified titanium surfaces: role of plasma proteins and electrostatic forces.

OBJECTIVES: Modifications of titanium (Ti) implant surfaces have a significant effect on early biofilm formation and the outcome of implant procedures. The aim of this study was to examine the role of plasma proteins and electrostatic forces in the adhesion mechanism of oral bacteria to modified Ti surfaces. MATERIALS AND METHODS: Ti discs with three different types of surface modifications, machined, acid-etched, and acid-etched and blasted, were examined for adhesion of oral bacteria: Streptococcus mutans, Porphyromonas gingivalis, and Fusobacterium nucleatum. Following pretreatment of the Ti with ion rich solutions or coating by human serum albumin or fibronectin, bacterial adhesion was examined by scanning electron microscopy and assessed quantitatively by DNA analysis. Ti coating by proteins as well as bacterial adhesion and their interrelationships were further investigated through confocal scanning laser microscopy. RESULTS: Acid-etched and blasted Ti surfaces exhibited significantly higher amounts of bacteria adhesion than the other two surfaces. Calcium was found to serve as a bridging agent in the adhesion process of S. mutans and F. nucleatum to Ti surfaces. Although albumin coating of the Ti reduced the adhesion of S. mutans to all surfaces, it had no influence on the adhesion of P. gingivalis or F. nucleatum. Coating the Ti with fibronectin enhanced P. gingivalis and F. nucleatum adhesion. CONCLUSIONS: Bacterial adhesion to Ti surfaces is roughness-dependent, and the adhesion mechanism is influenced by ions and proteins of the initial coating derived from the blood.

The antibacterial activity of LL-37 against Treponema denticola is dentilisin protease independent and facilitated by the major outer sheath protein virulence factor.

Host defense peptides are innate immune effectors that possess both bactericidal activities and immunomodulatory functions. Deficiency in the human host defense peptide LL-37 has previously been correlated with severe periodontal disease. Treponema denticola is an oral anaerobic spirochete closely associated with the pathogenesis of periodontal disease. The T. denticola major surface protein (MSP), involved in adhesion and cytotoxicity, and the dentilisin serine protease are key virulence factors of this organism. In this study, we examined the interactions between LL-37 and T. denticola. The three T. denticola strains tested were susceptible to LL-37. Dentilisin was found to inactivate LL-37 by cleaving it at the Lys, Phe, Gln, and Val residues. However, dentilisin deletion did not increase the susceptibility of T. denticola to LL-37. Furthermore, dentilisin activity was found to be inhibited by human saliva. In contrast, a deficiency of the T. denticola MSP increased resistance to LL-37. The MSP-deficient mutant bound less fluorescently labeled LL-37 than the wild-type strain. MSP demonstrated specific, dose-dependent LL-37 binding. In conclusion, though capable of LL-37 inactivation, dentilisin does not protect T. denticola from LL-37. Rather, the rapid, MSP-mediated binding of LL-37 to the treponemal outer sheath precedes cleavage by dentilisin. Moreover, in vivo, saliva inhibits dentilisin, thus preventing LL-37 restriction and ensuring its bactericidal and immunoregulatory activities.

Involvement of Toll-like receptors 2 and 4 in the innate immune response to Treponema denticola and its outer sheath components.

Treponema denticola is considered an important oral pathogen in the development and progression of periodontal diseases. In the present study, the mechanisms of recognition and activation of murine macrophages by T. denticola and its major outer sheath protein (MSP) and lipooligosaccharide (LOS or glycolipid) were investigated. T. denticola cells and the MSP induced innate immune responses through TLR2-MyD88, whereas LOS induced a macrophage response through TLR4-MyD88. The presence of gamma interferon (IFN-gamma), or of high numbers of T. denticola, circumvented the requirement for TLR2 for the macrophage response to T. denticola, although the response was still dependent on MyD88. In contrast, synergy with IFN-gamma did not alter the TLR dependence of the response to the T. denticola surface components LOS and MSP, despite enhanced sensitivity. These data suggest that although there is flexibility in the requirements for recognition of T. denticola cells (TLR2 dependent or independent), MyD88 is a requirement for the downstream signaling events that lead to inflammation. We also demonstrate that both outer sheath molecules LOS and MSP induce macrophage tolerance to further stimulation with enterobacterial lipopolysaccharide. Tolerance induced by T. denticola components during mixed infections may represent a general mechanism through which bacteria evade clearance.

Degradation of collagen-guided tissue regeneration membranes by proteolytic enzymes of Porphyromonas gingivalis and its inhibition by antibacterial agents.

Previous studies have shown that whole cells of several periodontal pathogenic bacteria including Porphyromonas gingivalis may degrade the clinically used regeneration membranes Biomend Extend and Bio-Gide. Fractionation of P. gingivalis cells revealed that cell membrane-associated proteases are responsible for the in vitro degradation of the collagen membranes. In the present study, the specific role of extracellular vesicles and the purified Arg-gingipain enzyme of P. gingivalis in the degradation of three differently cross-linked collagen membranes (Ossix; Bio-Gide and Biomend Extend) was examined. In addition, the inhibitory effect of antibacterial agents and antibiotics used in local periodontal therapy on the enzymatic degradation was evaluated. The data presented show that while all tested collagen membranes, are prone to lysis by oral bacterial proteases, cross-linked membranes are more resistant to proteolysis. Furthermore, therapeutical concentrations of the antibacterial and antibiotic agents chlorhexidine, cetylpyridiniumchloride, minocycline and doxycycline were found to partially inhibit the enzymatic breakdown of the membranes, while metronidazole had no such effect. These results suggest that the presence of P. gingivalis cells, extracellular vesicles and enzymes in the vicinity of regeneration membranes in the periodontium, may change their physical structure and therefore alter their biological properties. Furthermore, the use of cross-linked collagen membranes and antibacterial agents may significantly inhibit this proteolytic process.

Coaggregation of Treponema denticola with Porphyromonas gingivalis and Fusobacterium nucleatum is mediated by the major outer sheath protein of Treponema denticola.

Coagreggation of Treponema denticola with either Porphyromonas gingivalis or Fusobacterium nucleatum was characterized and the role of the major outer sheath protein (MSP) in the coaggregation process of these bacteria was evaluated. The MSP of T. denticola was found to be able to bind to P. gingivalis and F. nucleatum cells and this binding could be inhibited by MSP in a concentration-dependent manner. While sodium dodecyl sulfate polyacrylamide gel electrophoresis and Periodic acid-Schiff (PAS) staining of MSP revealed that it is a glycoprotein, monosaccharide analysis showed that MSP contains: Glc (44.4), Gal (20.4%) GlcN (1.3%), GalN (31.6%) and Fuc (9.2%). Peptide N-glycosidase F deglycosylation of MSP was found to inhibit its binding to F. nucleatum but not to P. gingivalis cells. Sugar-binding studies showed that the requirements for the binding of both T. denticola and MSP to F. nucleatum cells are similar to those of the F. nucleatum galactose-binding lectin. These data suggest that MSP acts as an adhesin during the coaggregation process of T. denticola with P. gingivalis and F. nucleatum through its protein and carbohydrate moieties, respectively.

Adsorption of human plasma proteins to modified titanium surfaces.

OBJECTIVES: The aim of this study was to examine the effect of modified titanium (Ti) surfaces on the initial events of plasma proteins adsorption. MATERIALS AND METHODS: 'Ti disks' with three types of surface modifications were compared: machined, acid-etched and acid-etched and blasted. Physical and chemical characterizations of the surfaces were performed via scanning electron microscopy (SEM), atomic force microscopy (AFM) used for analysis of surface topography, characterization of the titanium oxide (TiO2) layer was carried out by X-ray photoelectron spectroscopy (XPS) and characterization of surface energy by the determination of contact angles. Evaluation of plasma proteins' adsorption to the treated Ti surfaces was performed by mass spectrometry, confocal laser scanning microscopy and XPS. Quantitative proteins' assessment was carried out by enzyme-linked immunosorbent assay. RESULTS: SEM images revealed major differences in the topography of the examined surfaces. Acid-etched and blasted Ti surfaces were found to have higher roughness values and a thicker TiO2 layer as compared with acid-etched and machined surfaces. Moreover, acid-etched and blasted surfaces showed high surface area differentiation, pointing to a high increase in the three-dimensional (3D) surface area over the 2D surface area compared with the other surfaces. Adsorption of plasma proteins to the acid-etched and blasted Ti surfaces was both qualitatively and quantitatively more intense compared with the machined and acid-etched surfaces. This was shown for each examined protein, total proteins and by the removal degree of the protein coat. CONCLUSIONS: The preferential adsorption of plasma proteins to the acid-etched and blasted Ti surfaces may be explained by its topographical characteristics and by the increase of the 3D surface area of this modified surface.

Coaggregation of Porphyromonas gingivalis and Fusobacterium nucleatum PK 1594 is mediated by capsular polysaccharide and lipopolysaccharide.

Previous reports have shown that coaggregation between Porphyromonas gingivalis and Fusobacterium nucleatum, two important periodontopathogens, is mediated by a galactoside on the surface of P. gingivalis and a lectin on F. nucleatum. In the present study, purified capsular polysaccharide (CPS) and lipopolysaccharide (LPS) of P. gingivalis PK 1924 (serotype K5) were found to be able to bind to F. nucleatum cells and to inhibit binding of F. nucleatum to P. gingivalis serotype K5. Sugar binding studies showed that the requirements for binding of P. gingivalis serotype K5 CPS and LPS to the F. nucleatum lectin are: the presence of a metal divalent ion, an axial free hydroxyl group at position 4 and free equatorial hydroxyl groups at position 3 and 6 of d-galactose. These data suggest that P. gingivalis serotype K5- CPS and LPS act as receptors mediating coaggregation between P. gingivalis and fusobacteria.

Enzymatic degradation of collagen-guided tissue regeneration membranes by periodontal bacteria.

Bacterial infection in the vicinity of guided tissue regeneration barrier membranes was shown to have a negative effect on the clinical outcomes of this increasingly used technique. Several oral and specifically periodontal bacteria were shown to adhere to such membranes in vivo and in vitro with a higher affinity to membranes constructed from collagen. The present study examined the role of periodontal bacteria and their enzymes in the degradation of commercially used collagen membranes. Degradation of two collagen membranes [Biomend (Calcitek, Colla-Tec Inc., Plainsboro, NJ) and Bio-Gide (Geistlich Biomaterials, Wolhousen, Switzerland)] labeled by fluorescein isothiocyanate was examined by measuring soluble fluorescence. Porphyromonas gingivalis, Treponema denticola and Actinobacillus actinomycetemcomitans and their enzymes were evaluated. Collagenase from Clostridium hystolyticum was used as a positive control. While whole cells of P. gingivalis were able to degrade both types of membranes, T. denticola could degrade Bio-Gide membranes only and A. actinomycetemcomitans whole cells could degrade none of the membranes. Fractionation of P. gingivalis cells revealed that cell membrane associated proteases were responsible for the degradation of the two collagen membranes. In T. denticola, the purified major phenylalanine protease was found to be responsible for the degradation of Bio-Gide membranes. These results suggest that proteolytic bacterial enzymes may take part in the degradation of collagen barrier membranes used for guided tissue regeneration.

Actinobacillus actinomycetemcomitans serotype b lipopolysaccharide mediates coaggregation with Fusobacterium nucleatum.

Purified Actinobacillus actinomycetemcomitans serotype b lipopolysaccharide (LPS) was found to be able to bind Fusobacterium nucleatum cells and to inhibit binding of F. nucleatum to A. actinomycetemcomitans serotype b. Sugar binding studies showed that the requirements for binding of A. actinomycetemcomitans serotype b LPS to the F. nucleatum lectin are the presence of a metal divalent ion, an axial free hydroxyl group at position 4, and free equatorial hydroxyl groups at positions 3 and 6 of D-galactose, indicating that the beta-N-acetyl-D-galactosamine in the serotype b LPS trisaccharide repeating unit is the monosaccharide residue recognized by the F. nucleatum lectin. These data strongly suggest that A. actinomycetemcomitans serotype b LPS is one of the receptors responsible for the lactose-inhibitable coaggregation of A. actinomycetemcomitans to fusobacteria.