Effects of the tea catechin epigallocatechin gallate on Porphyromonas gingivalis biofilms.

AIMS: The aim of this study was to investigate the effects of tea catechin epigallocatechin gallate (EGCg) on established biofilms and biofilm formation by Porphyromonas gingivalis, a major pathogen of periodontal disease. METHODS AND RESULTS: Biofilm cell survival was measured using adenosine triphosphate (ATP) bioluminescence. In the presence of EGCg, the ATP level in cells of established biofilms was significantly decreased compared to the controls (P < 0·0001). Transmission electron microscopy revealed that EGCg damaged the cell membrane and cell wall of P. gingivalis. Confocal laser-scanning microscopy revealed that the proportion of dead cells was higher in biofilms treated with EGCg. Moreover, the effects of subminimal inhibitory concentrations (MICs) of EGCg on P. gingivalis biofilm formation were dose-dependent (P < 0·0001). CONCLUSION: Our results suggest that EGCg destroys established P. gingivalis biofilms and inhibits biofilm formation. SIGNIFICANCE AND IMPACT OF THE STUDY: Development of chemical control agents against oral biofilms is necessary, because oral biofilms can be only removed using mechanical debridement. This article indicates that EGCg may represent a novel antibiofilm agent that prevents infections involving bacterial biofilms such as periodontitis.

Synergistic effects of antibiotics and an N-acyl homoserine lactone analog on Porphyromonas gingivalis biofilms.

AIMS: To investigate the effects of the combined application of an N-acyl homoserine lactone (HSL) analog and antibiotics on biofilms of Porphyromonas gingivalis, a major pathogen of periodontal disease. METHODS AND RESULTS: Antibiotics used were cefuroxime, ofloxacin and minocycline. A flow-cell model was used for biofilm formation. Samples were divided into four groups: control, analog-treated, antibiotic-treated and combined application groups. Biofilm cell survival was determined using adenosine triphosphate (ATP) bioluminescence and confocal laser microscopy (CLSM). In the combined application group, the ATP count in biofilm cells was significantly decreased compared with the antibiotic-treated group (Games-Howell test, P < 0·05). A combination of cefuroxime and the analog was most effective against the P. gingivalis biofilm. CLSM observations revealed that the proportion of dead cells was highest in the combined application group. CONCLUSIONS: The combined application of the N-acyl HSL analog and antibiotics was effective at reducing the viability of P. gingivalis cells in biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: The combined application of the N-acyl HSL analog and antibiotics may be successful for eradicating infections involving bacterial biofilms, such as periodontitis.

Antibiofilm effects of azithromycin and erythromycin on Porphyromonas gingivalis.

Antibiotic resistance of biofilm-grown bacteria contributes to chronic infections, such as marginal and periapical periodontitis, which are strongly associated with Porphyromonas gingivalis. Concurrent azithromycin (AZM) administration and mechanical debridement improve the clinical parameters of periodontal tissue in situ. We examined the in vitro efficacy of AZM against P. gingivalis biofilms. The susceptibilities of adherent P. gingivalis strains 381, HW24D1, 6/26, and W83 to AZM, erythromycin (ERY), ampicillin (AMP), ofloxacin (OFX), and gentamicin (GEN) were investigated using a static model. The optical densities of adherent P. gingivalis cells were significantly decreased by using AZM and ERY at sub-MIC levels compared with those of the controls in all the strains tested, except for the effect of ERY on strain W83. AMP and OFX inhibited P. gingivalis adherent cells at levels over their MICs, and GEN showed no inhibition in the static model. The effects of AZM and ERY against biofilm cells were investigated using a flow cell model. The ATP levels of P. gingivalis biofilms were significantly decreased by AZM at concentrations below the sub-MICs; however, ERY was not effective for inhibition of P. gingivalis biofilm cells at their sub-MICs. Furthermore, decreased density of P. gingivalis biofilms was observed three-dimensionally with sub-MIC AZM, using confocal laser scanning microscopy. These findings suggest that AZM is effective against P. gingivalis biofilms at sub-MIC levels and could have future clinical application for oral biofilm infections, such as chronic marginal and periapical periodontitis.

Soluble products from Eikenella corrodens induce cell proliferation and expression of interleukin-8 and adhesion molecules in endothelial cells via mitogen-activated protein kinase pathways.

The periodontal vasculature is profoundly affected during the progression of periodontitis, and several specific bacteria are believed to be involved in this inflammatory disease. Eikenella corrodens is one of the common bacteria detected in periodontitis diseased lesions; however, the function of this organism in periodontitis is not well understood. In this study, we investigated the E. corrodens-induced endothelial cell alteration and inflammation process that leads to leukocyte infiltration in inflamed regions. Soluble products from E. corrodens (EcSP) induced the gene expression and protein production of vascular endothelial growth factor in oral epithelial cells and human umbilical vein endothelial cells (HUVEC). Direct stimulation by EcSP also activated endothelial cell proliferation. Moreover, EcSP induced ERK1/2 (p44/42) and p38 mitogen-activated protein kinase (MAPK) phosphorylation within 10-30 min in HUVEC, as demonstrated by Western blot analysis and up-regulated intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), E-selectin and interleukin-8 (IL-8) production demonstrated by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. The specific p38 MAPK inhibitor SB203580 reduced the expression of ICAM-1, VCAM-1 and IL-8, whereas the blockade of p44/42 by MAPK kinase (MEK1) inhibitor, PD98059, inhibited only IL-8 expression. Our results indicate that E. corrodens can trigger a cascade of events that induce inflammatory responses in periodontal tissue via the MAPK cascade and may promote chronic periodontitis without bacteria-cell contact.

Role of amyloid type cross beta-structure in the formation of soluble aggregate and gel in heat-induced ovalbumin.

The heat-induced denaturation curve of ovalbumin followed by the ellipticity at 222 nm in circular dichroism spectra was consistent with that monitored by fluorescence with thioflavin T, which is an indication of amyloid fibril formation, while other proteins such as lysozyme and ovotransferrin did not fluoresce with thioflavin T during heat denaturation. The amount of soluble aggregate formed during heat denaturation was proportional to the increase in fluorescence with thioflavin T. The binding of soluble aggregates with thioflavin T was greatly suppressed in heat-denatured ovalbumin in the presence of thioflavin T. The similar inhibition effect of thioflavin T on the gel formation of heat-induced ovalbumin was observed. These results suggest that the amyloidogenic intermolecular beta-structure is involved in the formation of soluble aggregate and gel of heat-induced ovalbumin.

Relationship between the stability of lysozymes mutated at the inside hydrophobic core and secretion in Saccharomyces cerevisiae.

The relationship between the stability of lysozymes mutated at the inside hydrophobic core and secretion was investigated to understand the optimal secretion of mutant lysozymes in the Saccharomyces cerevisiae. S91T mutant lysozyme increased in the methyl residue inside the core greatly increased the conformational stability. The secretion amount of S91T in S. cerevisiae increased greatly compared with wild-type lysozyme. On the other hand, I55V and T40S/I55V mutant lysozymes decreased in methyl residue inside the core brought about their unstable conformation. The secretion amounts of these unstable mutant lysozymes significantly decreased. In addition, the effect of glycosylation on the secretion of these mutants was investigated. The secretion amounts of glycosylated lysozyme S91T/G49N with stable hydrophobic core greatly increased compared with that of glycosylated lysozyme G49N, while those of mutant I55V/G49N and T40S/I55V/G49N with unstable hydrophobic core greatly decreased. These results indicate that the secretion amounts of mutant lysozymes increase in proportion to the hydrophobic core stabilities and that a similar good correlation was obtained with glycosylated lysozymes.

Effects of calnexin deletion in Saccharomyces cerevisiae on the secretion of glycosylated lysozymes.

Disruption of the calnexin gene in Saccharomyces cerevisiae did not lead to gross effects on the levels of cell growth and secretion of wild-type hen egg white lysozymes (HEWL). To investigate the function of calnexin in relation to the secretion of glycoproteins, we expressed both stable and unstable mutant glycosylated lysozymes in calnexin-disrupted S. cerevisiae. The secreted amounts of stable mutant glycosylated lysozymes (G49N and S91T/G49N) were almost the same in both wild-type and calnexin-disrupted S. cerevisiae. In contrast, the secretion of unstable mutant glycosylated lysozymes (K13D/G49N, C76A/G49N, and D66H/G49N) greatly increased in calnexin-disrupted S. cerevisiae, although their secretion was very low in the wild-type strain. This indicates that calnexin may act in the quality control of glycoproteins. We further investigated the expression level of the mRNA of the molecular chaperones BiP and PDI, which play a major role in the protein folding process in the ER, when glycosylated lysozymes were expressed in wild-type and calnexin-disrupted S. cerevisiae. The mRNA concentrations of BiP and PDI were evidently increased when the glycosylated lysozymes were expressed in calnexin-disrupted S. cerevisiae. This observation indicates that BiP and PDI may be induced by the accumulation of unfolded glycosylated lysozymes due to the deletion of calnexin.

Effect of chemical and genetic attachment of polysaccharides to proteins on the production of IgG and IgE.

To investigate the effect of polysaccharide attachment to proteins on the production of IgG and IgE, the genetic attachment of polysaccharide to lysozymes (G49N and R21T) using the yeast expression system (Saccharomyces cerevisiae AH 22) and the Maillard-type polysaccharide attachment to native lysozyme and soybean P34 protein were attempted. The production of IgG and IgE was investigated by using mice immunized with the protein-polysaccharide conjugates or native proteins. The attachment of polysaccharide to lysozyme using the yeast expression system greatly suppressed the production level of IgG and IgE. The attachment of polysaccharide to native lysozyme and soybean P34 protein using the Maillard-type reaction was also found to be effective in reducing the production level of IgE compared to IgG.

In vivo glycosylation suppresses the aggregation of amyloidogenic hen egg white lysozymes expressed in yeast.

The mutant hen egg white lysozymes Ile55Thr and Asp66His, corresponding to human amyloidogenic mutant lysozymes Ile56Thr and Asp67His, respectively, were secreted in Saccharomyces cerevisiae. The amyloidogenic mutants (I55T and D66H) of hen egg white lysozymes were remarkably less soluble than that of the wild-type protein. To enhance the secretion of these mutants, we constructed the glycosylated amyloidogenic lysozymes (I55T/G49N and D66H/G49N) having the N-glycosylation signal sequence (Asn-X-Ser) by the substitution of glycine with asparagine at position 49. The secretion of these glycosylated mutant proteins is greatly increased in S. cerevisiae, compared with that of non-glycosylated type. Both the glycosylated mutants retained about 40% enzymatic activity when incubated at pH 7.4 for 1 h at the physiological temperature of 37 degrees C whereas the non-glycosylated proteins eventually lost all activity under these conditions. These results suggest that the glycosylated chains could mask the beta-strand of amyloidogenic lysozymes from the intermolecular cross-beta-sheet association, thus improving the solubility of amyloidogenic lysozymes.

Enthalpic destabilization of glycosylated lysozymes constructed by genetic modification.

To understand the role of polyglycosylation in protein stability, the thermodynamic changes in the denaturation of various polymannosyl lysozyme mutants (R21T, G49N, R21T/G49N) constructed by genetic modification were analyzed using differential scanning calorimetry (DSC). The denaturation temperature and the enthalpy change for unfolding of the lysozymes were reduced with an increase in the length of the polymannose chain and the number of binding sites to a protein, although the polymannosyl lysozymes revealed apparent heat stability in that no aggregation was observed and the enzymatic activity was conserved under conditions in which the wild-type lysozyme coagulated [S. Nakamura et al., J. Biol. Chem. 268 (1993) 12706-12712]. The reversibility of the denaturation of polymannosyl lysozymes was observed in the DSC curves obtained by reheating after heat denaturation, while it was not observed for the wild-type lysozyme. Based on these results, the polymannosyl lysozyme seems to easily refold due to the excellent reversibility of denaturation, despite the decreases in the enthalpic stabilization due to the strain in the protein molecule by the introduction of a polysaccharide chain.

Bactericidal action of egg yolk phosvitin against Escherichia coliunder thermal stress.

Chicken egg yolk phosvitin showed a remarkable antibacterial effect against Escherichia coli under thermal stress at 50 degrees C. E. coli cells (10(6)/mL) completely disappeared in 1 mL of L-broth coexisting with 0.l mg/mL phosvitin when incubated at 50 degrees C for 20 min, whereas a considerable amount of cells (10(5)/mL) survived at the same thermal stress without phosvitin. Blocking of the chelating effect of phosvitin by the addition of Ca(2+) ion displayed a protective effect against the bactericidal activity at 50 degrees C. In addition, the antibacterial activity of phosvitin was dramatically reduced by treatment with alpha-chymotrypsin, although the chelating effect remained. The surface properties, such as interfacial tension and emulsifying properties of phosvitin, which are an index of the affinity with the outer membrane, were greatly reduced by the alpha-chymotrypsin digestion. This indicates that the alpha-chymotrypsin-digested membrane-penetrating hydrophobic domains at the N- and C-terminal regions play an important role in antibacterial activity. These results suggest that a significant part of the bactericidal activity of phosvitin against E. coli resides in the synergistic effect of the high metal-chelating ability and the high surface activity under the influence of thermal stress.

Lipophilization of lysozyme by short and middle chain fatty acids.

Hen egg white lysozyme was lipophilized with short and middle chain saturated fatty acids (caproic, capric, or myristic acid). The yield, bactericidal properties, and structural properties of lipophilized lysozymes were investigated. The yield of lipophilization of lysozyme greatly increased with the decrease in the chain length of fatty acid. Lipophilization broadened the bactericidal action of lysozyme to Gram-negative bacteria with little loss of enzymatic activity. The bactericidal activity increased in proportion to the number of bound short chain fatty acids. The thermal stability of lipophilized lysozyme decreased in proportion to the chain length and number of bound fatty acids.

Immunological characterization of recombinant soy protein allergen produced by Escherichia coli expression system.

To elucidate the molecular mechanism of the allergenicity of soybean P34 protein recognized as the most allergenic protein in soybean, the protein was expressed in Escherichia coli transformed with a plasmid carrying P34 cDNA. SDS-PAGE pattern showed that the molecular weight of the recombinant P34 was approximately 2 kDa less than that of the native soybean P34. The difference in the molecular mass between these two proteins could be due to the native P34 in soybean being glycosylated at position Asn(170), whereas the recombinant protein generated in E. coli lacks this post-translational modification. Immunoblot analysis showed that both soybean and recombinant P34 proteins cross-reacted not only with polyclonal and monoclonal antibodies produced against P34 and crude soybean protein but also with patients' sera. The results suggest that the recombinant P34 is immunologically reactive, indicating that both proteins have similar epitope structures. Thus, the recombinant P34 produced by the E. coli expression system can be used as a standard allergen for molecular design to reduce the allergenic structure.

Improvement in the yield of lipophilized lysozyme by the combination with Maillard-type glycosylation.

Hen egg white lysozyme was modified using the Maillard-type glycosylation method prior to the lipophilization with palmitic acid. The yield of lipophilized lysozyme significantly increased by the pre-glycosylation of the protein. The lipophilized lysozyme derivative was separated into two main fractions with different level of glycosylation. All fractions showed a strong antimicrobial activity against Gram-negative bacteria, Escherichia coli. The lipophilization of the lysozyme combined with glycosylation is a promising method for potential industrial applications of the lysozyme due to the enhanced antimicrobial activity and the improved yield.

Molecular mechanism of the excellent emulsifying properties of phosvitin-galactomannan conjugate.

The emulsifying properties of native and N- and C-terminal-deleted phosvitin (protease digests) were compared after conjugation with galactomannan. The emulsifying properties of Maillard-type phosvitin-galactomannan conjugates were greatly improved, whereas those of the protease-digested phosvitin-galactomannan conjugates were not so dramatically improved. Phosvitin was highly glycosylated with galactomannan, whereas the protease-digested phosvitin conjugate consisting of a highly phosphorylated core peptide fragment was not. The results suggest that both N and C termini of the peptide moiety, digested by protease, were essential for the improvement of emulsifying properties of phosvitin-galactomannan conjugates. In addition, the role of N and C termini as anchors in oil droplets was supported from the comparative studies of native phosvitin, phosvitin-galactomannan conjugates, and protease-digested phosvitin-galactomannan conjugates.

Enhanced secretion of hydrophobic peptide fused lysozyme by the introduction of N-glycosylation signal and the disruption of calnexin gene in Saccharomyces cerevisiae.

The insertion of a hydrophobic pentapeptide (Phe-Phe-Val-Ala-Pro) into the C-terminus in hen egg white lysozyme by genetic modification resulted in an unstable structure which caused little secretion in a yeast expression system, although this modification is useful to enhance bactericidal action to gram-negative bacteria [Ibrahim et al. (1994) J. Biol. Chem. 269, 5059-5063]. To enhance the secretion of the unstable hydrophobic pentapeptide fused lysozymes (H5-Lz), we attempted to introduce the signal sequence (Asn-X-Ser/Thr) of N-linked glycosylation into lysozyme and to suppress the quality control of the unstable mutant in the yeast expression system. The polymannosyl hydrophobic fused lysozyme (H5/G49N-Lz) having the N-glycosylation signal sequence was expressed in the medium at 3.4 times that of unglycosylated lysozyme. Further, the secretion of the unstable mutant lysozyme was done in the Saccharomyces cerevisiae disrupted calnexin gene to avoid the degradation of the unstable mutant by the quality control. Although disruption of the calnexin gene did not lead to gross effects on the levels of growth of S. cerevisiae (W303-1b), the secretion amount of H5/G49N-Lz in calnexin disrupted S. cerevisiae was 2.5 times larger than that in wild type S. cerevisiae. These results suggest that the secretion of unstable glycosylated lysozyme (H5/G49N) was suppressed by the quality control function of calnexin and that the disruption of calnexin is effective to increase the secretion of unstable glycosylated protein.

Cloning, sequencing and expression of an Eikenella corrodens gene encoding a component protein of the lectin-like adhesin complex.

A lectin-like substance (LS), that was isolated from Eikenella corrodens (Ec) 1073, migrated as proteins of about 300 and 45 kDa upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. In this study, we cloned the gene encoding the 45-kDa protein and predicted its structure and function. Based on the N-terminal 23-amino acid (aa) sequence of this protein, we cloned the region for its N-terminus. We cloned the entire gene by means of gene walking using polymerase chain reaction and Southern hybridization. The nucleotide sequences of cloned fragments revealed an open reading frame encoding a polypeptide of 330 aa (M(r), 35748). This ORF displayed high homology to those of porins of Neisseria species. Using the T7-expression system, the 45-kDa protein was produced in E. coli. Our results suggested that the 45-kDa protein of Ec 1073 is a component of the EcLS complex, and that it is the major outer membrane protein.

Molecular analysis of the gene encoding a protein component of the Eikenella corrodens adhesin complex that is close to the carbohydrate recognition domain.

A monoclonal antibody against a lectin-like substance (LS) of Eikenella corrodens (Ec) was used for screening the Ec DNA library. Three positive clones that carried an identical 12-kb segment were obtained. A 25-kDa protein, which specifically binds to the antibody, was overproduced in all of the Escherichia coli clones. Deletion analysis showed that the gene encoding the 25-kDa protein was located within a 1.2-kb segment. The nucleotide (nt) sequence of this segment contained an open reading frame encoding a protein of 24600 Da. We purified the 25-kDa protein from the cloned E. coli strain. The sequence of the first 10 amino acids(aa) from the N-terminus of the purified 25-kDa protein agreed with that deduced from the nt sequence. Since the monoclonal antibody used in this study inhibits the physiological activity of EcLS, we concluded that the 25-kDa protein is a component of the adhesin complex, which is located near the carbohydrate recognition domain of lectin in EcLS.

The monoamine regulon including syntheses of arylsulfatase and monoamine oxidase in bacteria.

Bacterial cells respond to monoamine compounds, such as tyramine, dopamine, octopamine, or norepinephrine, and induce the syntheses of tyramine oxidase encoded by tynA and monoamine oxidase encoded by maoA. These monoamine compounds also derepress the synthesis of atsA-specified arylsulfatase that is repressed by sulfur compounds. These complex mechanisms of regulons regulated by monoamine and sulfur compounds has been analyzed by cloning and characterization of genes that are involved in the repression and derepression of the synthesis of arylsulfatase. The atsA gene forms an operon with the atsB gene, which encodes an activator of the expression of atsA. The negative regulator gene for arylsulfatase was found to code for dihydrofolate reductase (folA). The maoA gene forms an operon with the maoC gene, which has similarity to a dehydrogenase involved in the tyramine metabolism. The moaF gene encoding a 30-kDa protein, which is induced by tyramine, also forms an operon with the moaE gene. Finally, the moaR gene, which is induced by monoamine, was found to play a central role in the positive regulation of the expression of the monoamine regulon (moa) including the atsBA, maoCA, moaEF, and tyn operons. The moaR expression is subject to autogenous regulation and to cAMP-CRP control. The MoaR protein has a helix-turn-helix motif in its C terminus. Thus, the MoaR protein probably regulates the operons by binding to the regulatory region of the moa regulon.

maoB, a gene that encodes a positive regulator of the monoamine oxidase gene (maoA) in Escherichia coli.

The structural gene for copper- and topa quinone-containing monoamine oxidase (maoA) and an unknown amine oxidase gene have been located at 30.9 min on the Escherichia coli chromosome. Deletion analysis showed that the unknown gene was located within a 1.1-kb cloned fragment adjacent to the maoA gene. The nucleotide sequence of this fragment was determined, and a single open reading frame (maoB) consisting of 903 bp was found. The gene encoded a polypeptide with a predicted molecular mass of 34,619 Da which was correlated with the migration on a sodium dodecyl sulfate-polyacrylamide gel. The predicted amino acid sequence of the MaoB protein was identical to the NH2-terminal amino acid sequence derived by Edman degradation of the protein synthesized under the self-promoter. No homology of the nucleotide sequence of maoB to the sequences of any reported genes was found. However, the amino acid sequence of MaoB showed a high level of homology with respect to the helix-turn-helix motif of the AraC family in its C terminus. The homology search and disruption of maoA on the chromosome led to the conclusion that MaoB is a transcriptional activator of maoA but not an amine oxidase. The consensus sequence of the cyclic AMP-cyclic AMP receptor protein complex binding domain was adjacent to the putative promoter for the maoB gene. By use of lac gene fusions with the maoA and maoB genes, we showed that the maoA gene is regulated by tyramine and MaoB and that the expression of the maoB gene is subject to catabolite repression. Thus, it seems likely that tyramine and the MaoB protein activate the transcription of maoA by binding to the regulatory region of the maoA gene.