Surface-associated Material Research Articles

Surface immobilization strategies for tyrosinase as biocatalyst applicable to polymer network synthesis

ABSTRACTEnzymes have recently attracted increasing attention in material research based on their capacity to catalyze the conversion of polymer-bound moieties for synthesizing polymer networks, particularly bulk hydrogels. In this study, the surface immobilization of a relevant enzyme, mushroom tyrosinase, should be explored using glass as model surface. In a first step, the glass support was functionalized with silanes to introduce either amine or carboxyl groups, as confirmed e.g. by X-ray photoelectron spectroscopy. By applying glutaraldehyde and EDC/NHS chemistry, respectively, surfaces have been activated for subsequent successful coupling of tyrosinase. Via protein hydrolysis and amino acid characterization by HPLC, the quantity of bound tyrosinase was shown to correspond to a full surface coverage. Based on the visualized enzymatic conversion of a test substrate at the glass support, the functionalized surfaces may be explored for surface-associated material synthesis in the future.

Effects of Porphyromonas gingivalis surface-associated material on osteoclast formation

Porphyromonas gingivalis strongly correlates with periodontitis, but the underlying mechanisms causing dentoalveolar bone resorption are not fully understood. As contradictory effects of P. gingivalis on osteoclastogenesis have been reported, this study investigates the effect of P. gingivalis extract on osteoclast formation. Osteoclast formation in mouse bone marrow (MBM) cell cultures and RAW 264.7 cells was stimulated by nuclear factor-κB ligand (RANKL) or parathyroid hormone (PTH). Cells were cultured with and without P. gingivalis surface-associated material and phenotypic characteristics were examined using microscopy, flow cytometry, and RT-PCR. P. gingivalis significantly decreased osteoclast formation and the expression of osteoclast phenotypic markers in PTH-stimulated MBM cultures. Additionally, P. gingivalis inhibited expression of osteoclast differentiation factors and stimulated expression of the mouse macrophage marker F4/80. The presence of P. gingivalis in RANKL-stimulated MBM cultures and RAW 264.7 cells inhibited osteoclastogenesis. Interestingly, a transient exposure with P. gingivalis before PTH stimulation increased osteoclastogenesis in MBM cultures. Flow cytometric analyses of cells transiently exposed to P. gingivalis demonstrated an increased proportion of potential osteoclast precursor cells. We conclude that a transient exposure of MBM cultures to P. gingivalis increases the number of osteoclast precursors and osteoclast formation, whereas a prolonged exposure completely abolishes osteoclastogenesis.

Nitric oxide production by a murine macrophage cell line (RAW264.7 cells) stimulated with Aggregatibacter actinomycetemcomitans surface-associated material

Nitric oxide (NO) may play a crucial role in the pathogenesis of periodontal disease and, hence, the aim of the present study was to test the hypothesis that Aggregatibacter actinomycetemcomitans surface-associated material (SAM) stimulates inducible nitric oxide synthase (iNOS) activity and NO production by the murine macrophage cell line RAW264.7. Cells were stimulated with untreated or heat-treated A. actinomycetemcomitans SAM and with or without pre-treatment with l- N 6-(1-Iminoethyl)-lysine (L-NIL) (an iNOS inhibitor), polymyxin B, interferon-gamma (IFN-γ) and Interleukin-4 (IL-4), IL-10, genistein [a protein tyrosine kinase (PTK) inhibitor], bisindolylmaleimide [a protein kinase C (PKC) inhibitor], bromophenacyl bromide (BPB) [a phospholipase A 2 (PLA2) inhibitor] or wortmannin [phosphatidylinositol 3-kinase (PI-3K) inhibitor]. The iNOS activity and nitrite production in the cell cultures were determined. Untreated but not heat-treated A. actinomycetemcomitans SAM-stimulated both iNOS activity and nitrite production in RAW264.7 cells. l-NIL, IL-4, IL-10, genistein, bisindolylmaleimide, or BPB, suppressed but IFN-γ enhanced both iNOS activity and nitrite production by A. actinomycetemcomitans SAM-stimulated cells. Wortmannin and polymyxin B failed to alter both iNOS activity or nitrite production by A. actinomycetemcomitans SAM treated cells. Therefore, the present study suggests that a heat-sensitive protein constituent(s) of A. actinomycetemcomitans SAM stimulates both iNOS activity and nitrite production by RAW264.7 cells in a cytokine, PTK, PKC, and PLA 2 but not PI-3K-dependent fashion.

Evidence for Calcifying Nanoparticles in Gingival Crevicular Fluid and Dental Calculus in Periodontitis

Calcifying nanoparticles (CNPs), also known as nanobacteria, can produce carbonate apatite on their cell walls and initiate pathologic calcification. The objective of this study was to determine whether CNPs are present in the gingival crevicular fluid (GCF) from subjects with periodontal disease and whether they can induce the pathologic calcification of primary cultured human gingival epithelial cells. GCF and dental calculus samples were collected from 10 subjects with gingivitis and 10 subjects with chronic periodontitis. CNPs in GCF and calculus filtrates were detected with nanocapture enzyme-linked immunosorbent assay kits. The CNPs in cultures of dental calculus filtrates were also identified using immunofluorescence staining, transmission electron microscopy (TEM), and chemical analysis. Pathologic changes in the CNP-treated gingival epithelial cells were observed with TEM, alizarin red staining, and disk-scanning confocal microscopy. CNPs were found in GCF samples from two subjects with chronic periodontitis. Based on chemical analysis, the surface-associated material from CNPs isolated and cultured from calculus has a composition similar to dental calculus. The pathologic calcification of CNP-treated gingival epithelial cells was also observed. Self-replicating calcifying nanoparticles can be cultured and identified from dental calculus. This raises the issue of whether CNPs contribute to the pathogenesis of periodontitis.

Structural and Biological Characterization of a Capsular Polysaccharide Produced by Staphylococcus haemolyticus

The DNA sequence of the genome of Staphylococcus haemolyticus JCSC1435 revealed a putative capsule operon composed of 13 genes in tandem. The first seven genes (capABCDEFG(Sh)) showed > or = 57% similarity with the Staphylococcus aureus cap5 or cap8 locus. However, the capHIJKLM(Sh) genes are unique to S. haemolyticus and include genes encoding a putative flippase, an aminotransferase, two glycosyltransferases, and a transcriptional regulator. Capsule-like material was readily apparent by immunoelectron microscopy on bacteria harvested in the postexponential phase of growth. Electron micrographs of a JCSC1435 mutant with a deleted cap region lacked the capsule-like material. Both strains produced small amounts of surface-associated material that reacted with antibodies to polyglutamic acid. S. haemolyticus cap genes were amplified from four of seven clinical isolates of S. haemolyticus from humans, and three of these strains produced a serologically cross-reactive capsular polysaccharide. In vitro assays demonstrated that the acapsular mutant strain showed greater biofilm formation but was more susceptible to complement-mediated opsonophagocytic killing than the parent strain. Structural characterization of capsule purified from S. haemolyticus strain JCSC1435 showed a trisaccharide repeating unit: -3-alpha-L-FucNAc-3-(2-NAc-4-N-Asp-2,4,6-trideoxy-beta-D-Glc)-4-alpha-D-GlcNAc-. This structure is unique among staphylococcal polysaccharides in that its composition includes a trideoxy sugar residue with aspartic acid as an N-acyl substituent.

Staphylococcus aureus capsular material promotes osteoclast formation

Osteomyelitis, which is most frequently due to infection by Staphylococcus aureus, commonly causes bone destruction. S. aureus is known to secrete a number of surface-associated proteins that are potent stimulators of bone resorption. The precise cellular and humoral mechanisms that mediate this stimulatory effect are uncertain. In this study, we have determined whether osteoclast formation and resorption is directly promoted by surface-associated proteins. Surface-associated material (SAM) obtained from a 24-hour culture of S. aureus was added to cultures of mouse and human monocytes. Human monocyte cultures were incubated in the presence and absence of a soluble receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony stimulating factor (M-CSF). In cultures where M-CSF, RANKL, and SAM were added together, osteoclast formation did not exceed that seen in cultures with M-CSF and RANKL. In keeping with this finding, SAM did not increase osteoclast formation and resorption when mouse monocytes were cocultured with RANKL-expressing osteoblasts. In the absence of RANKL, however, SAM was capable of inducing osteoclast formation in cultures of human monocytes. This finding was evidenced by the generation of vitronectin receptor and tartrate-resistant acid phosphatasepositive multinucleated cells that were capable of lacunar resorption. Inhibitors of RANKL-dependent (RANK:Fc, OPG) and RANKL-independent (anti-TNF-alpha, gp130, IL-8, TGF-beta) osteoclast formation did not inhibit SAM-induced osteoclast formation. SAM did not stimulate mature osteoclast resorption activity. These findings indicate that RANKL, which is present in the circulation as a soluble factor, does not play a role in osteoclast formation in the presence of S. aureus SAM and that S. aureus SAM contains a soluble factor that promotes osteoclast formation by a RANKL-independent mechanism.

The Surface-Associated Protein of Staphylococcus saprophyticus Is a Lipase

Staphylococcus saprophyticus surface-associated protein (Ssp) was the first surface protein described for this organism. Ssp-positive strains display a fuzzy layer of surface-associated material in electron micrographs, whereas Ssp-negative strains appear to be smooth. The physiologic function of Ssp, however, has remained elusive. To clone the associated gene, we determined the N-terminal sequence, as well as an internal amino acid sequence, of the purified protein. We derived two degenerate primers from these peptide sequences, which we used to identify the ssp gene from genomic DNA of S. saprophyticus 7108. The gene was cloned by PCR techniques and was found to be homologous to genes encoding staphylococcal lipases. In keeping with this finding, strains 7108 and 9325, which are Ssp positive, showed lipase activity on tributyrylglycerol agar plates, whereas the Ssp-negative strain CCM883 did not. Association of enzyme activity with the cloned DNA was proven by introducing the gene into Staphylococcus carnosus TM300. When wild-type strain 7108 and an isogenic mutant were analyzed by transmission electron microscopy, strain 7108 exhibited the fuzzy surface layer, whereas the mutant appeared to be smooth. Lipase activity and the surface appendages could be restored by reintroduction of the cloned gene into the mutant. Experiments using immobilized collagen type I did not provide evidence for the involvement of Ssp in adherence to this matrix protein. Our experiments thus provided evidence that Ssp is a surface-associated lipase of S. saprophyticus.

Surface-associated material from Porphyromonas gingivalis stimulates the release of nitric oxide by inducing expression of inducible nitric oxide synthase

The purpose of this study was to examine the effects of surface-associated material (SAM) from Porphyromonas gingivalis, a major cause of inflammatory periodontal disease, on the production of nitric oxide (NO) and expression of inducible nitric oxide synthase (iNOS) in the murine macrophage cell line RAW264.7. We also attempted to throw light on the signaling mechanisms involved in P. gingivalis SAM-induced NO production. SAM from P. gingivalis 381 was obtained by saline extraction. NO production was assayed by measuring the accumulation of nitrite in culture supernatants. Western blot analysis of iNOS and analysis of reverse transcription-polymerase chain reaction (RT-PCR) products were carried out. We found that P. gingivalis SAM can induce iNOS expression and stimulate the release of NO without additional stimuli and demonstrated an important role of the transcription factor NF-κB and microtubule polymerization in NO production. The production of NO required l-arginine, protein tyrosine kinase, and protein kinase C. The ability of P. gingivalis SAM to promote the production of NO may be important in the pathogenesis of inflammatory periodontal disease.

Antigenic properties of the GroEL-like protein of Campylobacter rectus.

The purpose of this study was to clarify the antigenic properties of the GroEL-like protein of Campylobacter rectus using a specific polyclonal antibody directed to the purified 64-kDa GroEL-like protein (pAb-CrGroEL), a polyclonal antibody directed to the Actinobacillus actinomycetemcomitans GroEL-like protein (pAb-AaGroEL) and a monoclonal antibody against the recombinant human HSP60 (mAb-HuHSP60). In SDS-PAGE/Western immunoblotting analysis, mAb-HuHSP60, pAb-CrGroEL and pAb-AaGroEL were found to react with the GroEL-like protein (64-kDa) present in all C. rectus strains. A 150-kDa protein in C. rectus ATCC 33238 also reacted strongly with pAb-CrGroEL. This 150-kDa protein was found to be present on the surface-associated material of bacterial cells, as determined by transmission electron microscopy and immunogold labelling of cells with pAb-CrGroEL. Analysis of the first 20 N-terminal amino acids of the sequence of the 150-kDa protein revealed a strong homology (80%) with the C. rectus surface layer (S-layer) protein. Investigation of the biochemical nature of antigenic determinants using periodic acid and proteolytic enzymes showed that the C. rectus GroEL-like protein possessed immunodominant epitopes in both peptide and carbohydrate chains, and that the immunoreactive determinants of the 150-kDa protein belonged to carbohydrate. These results suggest that the GroEL-like protein and the S-layer protein of C. rectus may share the same carbohydrate epitopes.

Protective humoral immunity induced by surface-associated material from Actinobacillus actinomycetemcomitans in mice

The aim of the present study was to determine the role of antibodies specific to anti-surface-associated material from Actinobacillus actinomycetemcomitans (anti-SAM-Aa) in an infection induced by this periodontopathogen in mice. When SAM-Aa obtained by saline extraction of A. actinomycetemcomitans Y4 was separated on one-dimensional gel electrophoresis, this constituent contained antigen fragments with molecular weights ranging from 14 000 to 79 000. Immunoblot analysis revealed that increased antigen dose/immunization resulted in increased numbers of antigen epitopes recognized by serum antibodies of the immunized mice. Rapid healing of the primary lesions and high levels of specific IgG antibodies after challenge with live A. actinomycetemcomitans were seen in the immunized mice, especially at the highest-dose level of 100 μg/immunization. Transfer of SAM-Aa-immunized, but not the SAM-Aa–immunized and adsorbed, serum prior to challenge with live bacteria led to rapid healing of the lesions in the recipient mice. Increased phagocytosis of A. actinomycetemcomitans by murine macrophages (RAW264.7 cells) was observed when this periodontopathogen was opsonized by the SAM-Aa-immunized, but not SAM-Aa-immunized and adsorbed, serum. These results suggest that in mice, SAM-Aa antigens may induce protective antibodies by acting, at least, as an opsonin against challenge with live A. actinomycetemcomitans.

Surface-associated protein from Staphylococcus aureus stimulates osteoclastogenesis: possible role in S. aureus-induced bone pathology.

Staphylococcus aureus is the cause of bone destruction in osteomyelitis, bacterial arthritis and orthopaedic implant failure. We have previously shown that gentle saline extraction of S. aureus has revealed the presence of an extremely potent stimulator of osteoclast activation in both the murine calvarial bone resorption assay and the isolated chick osteoclast resorption assay. In order to investigate the mechanism of action of this surface-associated material (SAM), we have investigated its capacity to recruit osteoclasts. The murine bone marrow osteoclast recruitment assay was used. The ability of the recruited cells to resorb dentine slices was also investigated. Results. The SAM from S. aureus dose dependently stimulated tartrate-resistant acid phosphatase (TRAP)-positive osteoclast formation and pit formation on dentine slices. Neutralization of the cytokines tumour necrosis factor alpha and interleukin (IL)-6 totally inhibited, but antagonism of IL-1 only partially blocked, the stimulated maturation of osteoclast-like cells. These findings suggest that bone destruction associated with local infection by S. aureus is due to the stimulation of osteoclast formation induced by the action of the easily solubilized SAM, and could explain the large numbers of osteoclasts found in infarcted bone in osteomyelitis.

Pro-inflammatory and bone resorbing activity of Staphylococcus epidermidis surface-associated material

Pro-inflammatory and bone resorbing activity of Staphylococcus epidermidis surface-associated material

Immune response to Staphylococcus aureus surface-associated material

Immune response to Staphylococcus aureus surface-associated material

Staphylococcus epidermidis produces a cell-associated proteinaceous fraction which causes bone resorption by a prostanoid-independent mechanism: relevance to the treatment of infected orthopaedic implants.

Staphylococcus epidermidis is the most commonly isolated coagulase-negative staphylococcus from the skin, gut and upper respiratory tract. Although it is less virulent than Staphylococcus aureus, it has emerged in recent years as an important causative agent of infections associated with metal implants, prosthetic devices and i.v. lines. We have previously demonstrated that a saline wash of S. aureus contained proteins which had potent bone-resorbing activity in vitro. The purpose of this study was to determine whether gently washing S. epidermidis in saline also released osteolytically active proteins. The so-called surface-associated material (SAM) eluted from S. epidermidis in saline was able to induce osteolysis, and activity was heat and trypsin sensitive, suggesting that the active component was proteinaceous. Fractionation studies have suggested that activity is due to a small number of cationic proteins. This SAM-induced bone resorption was not inhibited by the cyclo-oxygenase inhibitor, indomethacin, or the 5-lipoxygenase inhibitors BWA70C and MK886. However, it was partially inhibited by high concentrations of interleukin-1 receptor antagonist and was completely blocked by a neutralizing antibody to tumour necrosis factor-alpha. Thus, the SAM from this organism is a potent osteolytic agent which differs from that of S. aureus SAM in not being inhibited by cyclo-oxygenase inhibitors. As adjunctive therapy is becoming necessary in infectious diseases, either as a result of the side-effects of antibiotics or their lack of efficacy, consideration should be given to the future treatment of bone infections with staphylococci in the light of the different mechanisms of action of the surface proteins produced by these bacteria.

Pro- and anti-inflammatory activity of surface-associated material from Staphylococcus aureus

Pro- and anti-inflammatory activity of surface-associated material from Staphylococcus aureus

Surface-associated material from the bacterium Actinobacillus actinomycetemcomitans contains a peptide which, in contrast to lipopolysaccharide, directly stimulates fibroblast interleukin-6 gene transcription.

The oral commensal Gram-negative bacterium Actinobacillus actinomycetemcomitans is believed to be the causative organism of localized juvenile periodontitis, a disease in which there is rapid loss of alveolar bone supporting the teeth. Previously, we have reported that gentle saline extraction of this bacterium removed a loosely adherent proteinaceous fraction from the cell surface of the bacterium, which we have termed surface-associated material. This material contained potent bone-resorbing activity. We now report that surface-associated material is also a potent stimulator of cytokines, and in particular, interleukin-6 (IL-6) synthesis, while the lipopolysaccharide from this bacterium is only a weak stimulator of IL-6 synthesis by fibroblasts and monocytes. In contrast to enteric lipopolysaccharide (LPS), which induces fibroblast IL-1, IL-6 and tumour necrosis factor (TNF) alpha synthesis, surface-associated material stimulated gingival fibroblasts to synthesize only IL-6, with no induction of IL-1 or TNF (the normal inducers of IL-6 synthesis). Reverse transcriptase PCR also failed to detect mRNA for IL-1 or TNF in surface-associated-material-stimulated fibroblasts, although both mRNAs were present in Escherichia coli LPS-stimulated cells. Neutralizing antibodies to IL-1 and/or TNF or the natural IL-1 receptor antagonist (IL-1ra) inhibited enteric LPS-induced IL-6 synthesis, but did not inhibit surface-associated-material-induced synthesis. In addition, dexamethasone, which completely suppressed LPS-induced IL-6 synthesis, only inhibited surface-associated-material-induced IL-6 synthesis by 50%. This suggests that the active constituent in the surface-associated material stimulates IL-6 gene transcription by a transcriptional control mechanism distinct to that of E. coli LPS. The IL-6 stimulating activity of the surface-associated material is inhibited by both heat and trypsin, suggesting that it is proteinaceous. The activity has been isolated using anion-exchange, reverse-phase and size-exclusion HPLC. The active moiety is a peptide of molecular mass 2kDa which may be the product of a bacterial short open reading frame.

Direct stimulation of IL-6 synthesis by a peptide isolated from the surface-associated material of the bacterium Actinobacillus actinomycetemcomitans

Direct stimulation of IL-6 synthesis by a peptide isolated from the surface-associated material of the bacterium Actinobacillus actinomycetemcomitans

Characterization of an antiproliferative surface-associated protein from Actinobacillus actinomycetemcomitans which can be neutralized by sera from a proportion of patients with localized juvenile periodontitis.

The gentle agitation of suspensions of Actinobacillus actinomycetemcomitans serotype a, b, or c in saline resulted in the release of a proteinaceous surface-associated material (SAM) which produced a dose-dependent inhibition of tritiated thymidine incorporation by the osteoblast-like cell line MG63 in culture. This cell line was sensitive to low concentrations of SAM (50% inhibitory concentration, 200 ng/ml for serotype c). Immunoglobulin G antibodies to constituents of the SAM were found in the blood of patients with localized juvenile periodontitis (LJP). Sera from 9 of 16 patients with LJP significantly neutralized the antiproliferative activity of the SAM, while sera from 15 controls, with no evidence of periodontal disease, were unable to neutralize this activity. Neutralization was not directly related to the patient's antibody titer to the whole SAM. Characterization of the antiproliferative activity in the SAM demonstrated that it was not cytotoxic and was heat and trypsin sensitive. The active component separated in a well-defined peak in anion-exchange high-performance liquid chromatography (HPLC) which, when further analyzed by size exclusion HPLC, revealed a single active peak, which had an apparent molecular mass of approximately 8 kDa. The lipopolysaccharide from A. actinomycetemcomitans was only weakly active. SAM from Porphyromonas gingivalis W50 and Eikenella corrodens NCTC 10596 did not exhibit any antiproliferative activity with this cell line, even at concentrations as high as 10 micrograms/ml. This study has shown that SAM from A. actinomycetemcomitans contains a potent antiproliferative protein whose activity can be neutralized by antibodies in the sera from some patients with LJP.

Comparison of the osteolytic activity of surface‐associated proteins of bacteria implicated in periodontal disease

To compare the osteolytic activity of surface-associated material (SAM) and lipid A-associated proteins (LAPs) from periodontopathogenic bacteria. Surface-associated material was extracted from the surface and LAPs from the cell walls of a range of periodontopathic bacteria including Actinobacillus actinomycetemcomitans and Eikenella corrodens. These bacterial fractions were assayed to determine their composition and their capacity to induce bone resorption was determined by use of the neonatal murine calvarial bone resorption assay. The SAMs from E. corrodens and A. actinomycetemcomitans demonstrated bone-resorbing capacity at concentrations as low as 1 ng ml-1 which, given the molecular weights of the active components, is in the picomolar range of activity. In contrast, the SAMs from the other three bacteria were significantly less potent and showed a lower efficacy. The LAPs all showed significant, and similar, capacities to induce bone breakdown. This is the first demonstration that LAP from periodontopathic bacteria can stimulate bone degradation. The LAPs from diverse bacteria all produced similar levels of bone-resorbing activity. In contrast, the SAM showed significant differences in potency and in efficacy (maximal stimulation). This may mean that in vivo certain periodontopathic bacteria have significantly more bone-resorbing capacity than others and should be therapeutic targets.

Serum antibody response to surface-associated material from periodontopathogenic bacteria

Saline extracts of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Eikenella corrodens contain surface-associated components of these bacteria. It has been shown that these extracts are potent stimulators of bone resorption in vitro. The possibility that the components of these surface-associated materials (SAM) could contribute to the serum immune response in patients with juvenile or adult onset forms of rapidly progressive periodontitis were investigated by direct binding ELISA. Very high titres of serum IgG antibodies to SAM from A. actinomycetemcomitans were detected in patients with localized juvenile periodontitis (LJP). Patients with adult onset rapidly progressive periodontitis (RPP) had significantly raised antibody levels to SAM from P. gingivalis. Both groups of patients had significantly raised levels of antibodies to SAM from E. corrodens compared with control sera. Thus, not only does solubilized SAM have the capacity to induce bone resorption, but it also contributes to the antigenic load on the immune system in LJP and RPP.