[Virulence of outer membrane proteins of periodontopathogenic bacteria].
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Aggregatibacter actinomycetemcomitans↗
Biomedical subjects
Hitoshi Komatsuzawa
Publications and source records attributed to Hitoshi Komatsuzawa.
An N-terminal segment of the active component of the bacterial genotoxin cytolethal distending toxin B (CDTB) directs CDTB into the nucleus.
Cytolethal distending toxin (CDT), produced by Actinobacillus actinomycetemcomitans, is a putative virulence factor in the pathogenesis of periodontal diseases. It is a cell cycle specific inhibitor at the G2/M transition. CDTB, one of the subunits of the CDT holotoxin, is implicated in a genotoxic role after entering the target cells, whereby chromosomal damage induces checkpoint phosphorylation cascades. CDTB microinjected into the cytoplasm was shown to localize in the nucleus and induce chromatin collapse. To investigate the molecular mechanism involved in nuclear transport of CDTB, we used transient expression and microinjection of a CDTB-green fluorescent protein (GFP) fusion protein. After microinjection, His-tagged CDTB-GFP entered the nucleus in 3-4 h. Leptomycin B did not increase the speed of entry of the fusion protein, suggesting that the relatively slow entry of the fusion protein is not due to the CRM1-dependent nuclear export of the protein. Nuclear localization of the CDTBGFP was temperature-dependent. An in vitro transport assay demonstrated that the nuclear localization of CDTB is mediated by active transport. An assay using transient expression of a series of truncated CDTB-GFP fusion proteins revealed that residues 48-124 constitute the minimum region involved in nuclear transport of CDTB. A domain swapping experiment of the region involved in nuclear transport of CDTB with an SV40 T nuclear localization signal indicated that CDTB is composed of two domains, an N-terminal domain for nuclear transport and a C-terminal active domain. Our results strongly suggest that nuclear localization of CDTB is required for the holotoxin to induce cytodistension and cell cycle block. This is the first demonstration that a bacterial toxin possessing a unique domain for nuclear transport is transferred to the animal cell nucleus by active transport.
Active Transport, Cell Nucleus↗
Macrophage inflammatory protein-3alpha and beta-defensin-2 stimulate dentin sialophosphoprotein gene expression in human pulp cells.
Macrophage inflammatory protein (MIP)-3alpha and beta-defensin (BD)-2 have antimicrobial activity and chemotactic activity for immature dendritic cells, natural killer cells, and memory T cells. However, it remains unknown if the widespread effects of these peptides also include an influence on the differentiation of mesenchymal cells. Pulp cells have the capacity to differentiate into odontoblasts and to form dentin. The aim of this study was to determine if inflammatory leukocyte products influence the capacity of pulp cells to differentiate. Dentin sialophosphoprotein (DSPP) is a tooth-specific protein being expressed mostly by odontoblast cells. In the present study, we investigated effects of MIP-3alpha and BD-2 on the DSPP and osteopontin (OPN) gene expression in cultures of human pulp-derived fibroblastic cells (HP cells). HP cells expressed mRNA for the CC chemokine receptor (CCR) 6 to which both MIP-3alpha and BD-2 can bind. Real-time PCR showed that MIP-3alpha and BD-2 significantly increased DSPP mRNA levels, although BD-2 increased DSPP mRNA levels less than MIP-3alpha. MIP-3alpha and BD-2 increased OPN mRNA levels very slightly. MIP-3alpha and BD-2 possessed antibacterial activity against Streptococcus mutans and Lactobacillus casei, which are involved in caries, although the antibacterial activity of MIP-3alpha was lower than that of BD-2. These findings suggest the MIP-3alpha and BD-2 have the ability to stimulate odontoblast differentiation in addition to their more traditional role in inflammation and have potential in the removal of bacteria in infected soft dentin and pulp tissues.
Cell Differentiation↗
Enhancement of alkaline phosphatase synthesis in pulp cells co-cultured with epithelial cells derived from lower rabbit incisors.
Dental papilla mesenchymal cells differentiate into odontoblasts through epithelial-mesenchymal interactions. However, the mechanism by which enamel epithelial cells affect the differentiation of dental mesenchymal cells remains unknown. Alkaline phosphatase (ALPase) is a marker for odontoblast-like differentiation, because odontoblasts show much higher ALPase activity than dental undifferentiated mesenchymal cells. The continuously growing rabbit incisor is a good model for the epithelial-mesenchymal interaction during odontogenesis. In the present study, we isolated and maintained rabbit incisor-derived epithelial cells and rabbit incisor pulp-derived fibroblastic cells, and examined the effect of epithelial cells on ALPase activity in fibroblastic cells. Epithelial cells were stained with anti-cytokeratin 5 and 8 antibodies and showed the expression of tuftelin mRNA. In separate cultures of epithelial cells or fibroblastic cells, ALPase activity and mRNA levels were very low, but were upregulated in co-cultures of epithelial and fibroblastic cells. Histochemical analysis found high ALPase activity in fibroblastic cells close to epithelial cells. These findings suggest that epithelial cells play an important role in promoting ALPase expression in pulp fibroblastic cells. The co-culture system developed here will be useful for examining the role of the epithelial-mesenchymal interaction during odontoblast differentiation.
Alkaline Phosphatase↗
Syntheses of prostaglandin E2 and E-cadherin and gene expression of beta-defensin-2 by human gingival epithelial cells in response to Actinobacillus actinomycetemcomitans.
The interaction between epithelial cells and microorganisms is the most important step in bacterial infections. Actinobacillus actinomycetemcomitans was suggested to play a significant role in the initiation of periodontitis because of its bacteriological characteristics. Prostaglandins (PG) mediate the inflammatory response. Human beta-defensin-2 (hBD-2) is an antimicrobial peptide and contributes to innate immunity. E-cadherin is responsible for an epithelial intercellular junction. In this study, we investigated the syntheses of PGE2 and E-cadherin and the expression of hBD-2 in human gingival epithelial cells (HGEC) following exposure to A. actinomycetemcomitans. The levels of PGE2 and cyclooxygenase-2, which are responsible for an increase in PGE2, were increased depending on bacteria exposure time. hBD-2 mRNA was induced by A. actinomycetemcomitans, while HGEC exposed to A. actinomycetemcomitans showed a decrease in E-cadherin levels. Etodolac, a selective cyclooxygenase-2 inhibitor reinforced the increase in hBD-2 mRNA levels by A. actinomycetemcomitans. Furthermore, the etodolac suppressed the decrease in E-cadherin levels. Thus, endogenous PGE2 is involved in the hBD-2 and E-cadherin responses of HGEC to A. actinomycetemcomitans. These findings suggest that the inflammatory and antimicrobial response of gingival epithelial cells to A. actinomycetemcomitans is involved in the initiation of periodontal inflammation. A. actinomycetemcomitans may destroy the mechanical epithelial barrier by destroying E-cadherin.
Aggregatibacter actinomycetemcomitans↗
Outer membrane protein 100, a versatile virulence factor of Actinobacillus actinomycetemcomitans.
Actinobacillus actinomycetemcomitans (Aa) is one of the pathogenic bacteria involved in periodontal diseases. We have previously identified six major outer membrane proteins (Omps) of Aa Y4. Among them is an Omp with high molecular mass, designated Omp100, which has homology to a variety of virulence factors. Electron microscopic observation indicated that Omp100 is randomly localized on the cell surface of Aa. Aa Y4 has been shown to adhere and invade KB or normal human gingival keratinocytes. Anti-Omp100 antibody inhibited 50% of adhesion and 70% of invasion of Aa Y4 to KB cells. An Omp100 knock-out mutant had a decreased adhesion and invasion efficiency of 60%, compared with that of the wild type. Escherichia coli HB101 expressing Omp100 adhered twofold and invaded 10-fold more than the wild-type E. coli HB101. HB101 expressing Omp100 showed resistance to serum by trapping factor H, an inhibitor for C3b, with Omp100. Omp100 induced inflammatory cytokine responses of interleukin (IL)-8, IL-6 and tumour necrosis factor (TNF)alpha in epithelial cells, and induced IL-1beta and TNFalpha production in mouse macrophages. These results indicate that Omp100 is a versatile virulence factor that may demonstrate potential significance in the onset of periodontal diseases related to Aa.
Actinobacillus Infections↗
Moenomycin-resistance is associated with vancomycin-intermediate susceptibility in Staphylococcus aureus.
We have previously isolated a vancomycin-intermediate susceptibility mutant from methicillinresistant Staphylococcus aureus(MRSA) strain COL, and demonstrated the increased glycan-chain length and the decreased moenomycin-susceptibility. To further investigate the relationship between the resistance to vancomycin and to moenomycin, we isolated moenomycin-resistant mutants (4-16 fold higher compared to the parent) from 5 MRSA and 2 methicillin-sensitive S. aureus(MSSA) strains. The MRSA mutants showed a decreased susceptibility to vancomycin (2-4 fold), teicoplanin (2-4 fold) and an increased susceptibility to methicillin (2-8 fold). MSSA strains also showed similar results with those of MRSA strains except that there was no alteration of methicillin susceptibility. Among the mutants, three mutants including two MRSA mutants and one MSSA mutant were analyzed by electron microscopy, and they showed thickened cell walls compared to those of the parents. The glycan-chain length of the peptidoglycan of the mutant was shown to be slightly longer than that of the parent, but the muropeptide profile was very similar. The expression levels of all PBPs were similar to those of the parent. Furthermore, the nucleotide sequences of sgtA, sgtB and pbp2 in the mutant were identical to those of the parent. These results indicate that the moenomycin-resistance is closely associated with vancomycin-intermediate susceptibility in S. aureus.
Anti-Bacterial Agents↗
Staphylococcus aureus susceptibility to innate antimicrobial peptides, beta-defensins and CAP18, expressed by human keratinocytes.
The antimicrobial peptides human beta-defensin-1 (hBD1), hBD2, hBD3, and CAP18 expressed by keratinocytes have been implicated in mediation of the innate defense against bacterial infection. To gain insight into Staphylococcus aureus infection, the susceptibility of S. aureus, including methicillin-resistant S. aureus (MRSA), to these antimicrobial peptides was examined. Based on quantitative PCR, expression of hBD2 mRNA by human keratinocytes was significantly induced by contact with S. aureus, and expression of hBD3 and CAP18 mRNA was slightly induced, while hBD1 mRNA was constitutively expressed irrespective of the presence of S. aureus. Ten clinical S. aureus isolates, including five MRSA isolates, induced various levels of expression of hBD2, hBD3, and CAP18 mRNA by human kertinocytes. The activities of hBD3 and CAP18 against S. aureus were found to be greater than those of hBD1 and hBD2. A total of 44 S. aureus clinical isolates, including 22 MRSA strains, were tested for susceptibility to hBD3 and CAP18. Twelve (55%) and 13 (59%) of the MRSA strains exhibited more than 20% survival in the presence of hBD3 (1 microg/ml) and CAP18 (0.5 microg/ml), respectively. However, only three (13%) and two (9%) of the methicillin-sensitive S. aureus isolates exhibited more than 20% survival with hBD3 and CAP18, respectively, suggesting that MRSA is more resistant to these peptides. A synergistic antimicrobial effect between suboptimal doses of methicillin and either hBD3 or CAP18 was observed with 10 MRSA strains. Furthermore, of several genes associated with methicillin resistance, inactivation of the fmtC gene in MRSA strain COL increased susceptibility to the antimicrobial effect mediated by hBD3 or CAP18.
Amino Acid Sequence↗
Prevalence of cytolethal distending toxin production in periodontopathogenic bacteria.
Cytolethal distending toxin (CDT) is a newly identified virulence factor produced by several pathogenic bacteria implicated in chronic infection. Seventy three strains of periodontopathogenic bacteria were examined for the production of CDT by a HeLa cell bioassay and for the presence of the cdt gene by PCR with degenerative oligonucleotide primers, which were designed based on various regions of the Escherichia coli and Campylobacter cdtB genes, which have been successfully used for the identification and cloning of cdtABC genes from Actinobacillus actinomycetemcomitans Y4 (M. Sugai et al., Infect. Immun. 66:5008-5019, 1998). CDT activity was found in culture supernatants of 40 of 45 tested A. actinomycetemcomintans strains, but the titer of the toxin varied considerably among these strains. PCR experiments indicated the presence of Y4-type cdt sequences in these strains, but the rest of A. actinomycetemcomitans were negative by PCR amplification and also by Southern blot analysis for the cdtABC gene. In the 40 CDT-positive strains, Southern hybridization with HindIII-digested genomic DNA revealed that there are at least 6 restriction fragment length polymorphism types. This suggests that the cdtABC flanking region is highly polymorphic, which may partly explain the variability of the CDT activity in the culture supernatants. The rest of tested strains of periodontopathogenic bacteria did not have detectable CDT production by the HeLa cell assay and for cdtB sequences by PCR analysis under our experimental conditions. These results strongly suggested that CDT is a unique toxin predominantly produced by A. actinomycetemcomitans among periodontopathogenic bacteria.
Actinobacillus Infections↗
Osteoprotegerin levels increased by interleukin-1beta in human periodontal ligament cells are suppressed through prostaglandin E(2) synthesized de novo.
Osteoprotegerin (OPG) is a novel tumor necrosis factor receptor superfamily that inhibits osteoclast differentiation, activity, and survival. Interleukin-1beta (IL-1beta) increases OPG expression. IL-1beta also increases prostaglandin E(2) (PGE(2)) production and stimulates bone resorption. In the present study, we examined the involvement of PGE(2) in IL-1beta-induced increases in OPG levels in human periodontal ligament cells (HPL cells) in an effort to clarify apparently conflicting IL-1beta actions on bone resorption and understand IL-1beta-induced increases in secretion of OPG and PGE(2) in HPL cells. 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole, a mRNA synthesis inhibitor, partly inhibited the increase in OPG mRNA levels induced by IL-1beta. Cycloheximide, a protein synthesis inhibitor, enhanced the stimulatory effect of IL-1beta. Etodolac, a selective cyclooxygenase-2 inhibitor, suppressed the increase in PGE(2) levels. Furthermore, etodolac reinforced the promotion of OPG expression by IL-1beta at the mRNA and protein levels. PGE(2) added to cultures of HPL cells decreased OPG mRNA levels in a dose- and time- dependent manner. These findings suggest that the increase in OPG levels induced by IL-1beta in HPL cells is suppressed through PGE(2) synthesized de novo.
Base Sequence↗
Clonal association of Staphylococcus aureus causing bullous impetigo and the emergence of new methicillin-resistant clonal groups in Kansai district in Japan.
A molecular epidemiological analysis was performed to reveal the clonal association of Staphylococcus aureus strains isolated from patients with bullous impetigo. Pulsed-field gel electrophoresis with cluster analysis, genetic and phenotypic characterizations, and antimicrobial susceptibility profiling of 88 S. aureus strains isolated from outpatients at 4 hospitals in the Kansai district in Japan were undertaken. Three distinct clonal groups were identified: 2 of them carried the exfoliative toxin (ET) A gene (eta), and the other carried the ETB gene (etb). The former groups represent 2 eta-positive clonal groups that have not been described previously. All the strains in the more dominant eta-positive clonal group and some of the strains in the etb-positive clonal group were methicillin-resistant S. aureus (MRSA) showing borderline-to-moderate resistance to beta-lactams. These MRSA strains appear to be emerging clonal groups that have not been considered in previous epidemiological studies of ET-producing S. aureus in Japan and thus pose a significant threat for future treatment of patients with bullous impetigo and/or staphylococcal scalded-skin syndrome.
Blister↗
Identification of six major outer membrane proteins from Actinobacillus actinomycetemcomitans.
We have identified six major sarcosyl-insoluble outer membrane proteins (Omp) of Actinobacillus actinomycetemcomitans Y4, and designated them as Omp100, Omp64, Omp39, Omp29, Omp18 and Omp16 according to the molecular mass. A similar N-terminal sequence was found in the first 15 amino acid residues of Omp16 and Omp18. The N-terminal sequence of Omp29 matched perfectly with the sequence previously identified. We cloned and determined the DNA sequences of three complete genes encoding Omp100, Omp64 and Omp18/16, and one incomplete gene encoding Omp39. Each Omp revealed homologies with some bacterial virulence factors responsible for adhesion, invasion, serum resistance, or protein antigenicity. Serum from patients with periodontitis suspected to be related to A. actinomycetemcomintans infection strongly reacted with Omp100, Omp29 and Omp16 as did serum from mice immunized with A. actinomycetemcomitans Y4 whole bacteria. These findings suggest that Omps of A. actinomycetemcomitans can be associated with periodontal disease.
Aggregatibacter actinomycetemcomitans↗
Molecular characterization of an atl null mutant of Staphylococcus aureus.
atl is a gene encoding a bifunctional peptidoglycan hydrolase of Staphylococcus aureus. The gene product of atl is a 138 kDa protein that has an amidase domain and a glucosaminidase domain, and undergoes processing to generate two major peptidoglycan hydrolases, a 51 kDa glucosaminidase and a 62 kDa amidase in culture supernatant. An atl null mutant was isolated by allelic replacement and characterized. The mutant grew in clusters and sedimented when grown in broth culture. Analysis of peptidoglycan prepared from the wild type and the mutant revealed that there were no differences in muropeptide composition or in glycan chain length distribution. On the other hand, the atl mutation resulted in pleiotropic effects on cell surface nature. The mutant cells showed complete inhibition of metabolic turnover of cell wall peptidoglycan and revealed a rough outer cell wall surface. The mutation also decreased the amount of protein non-covalently bound to the cell surface and altered the protein profile, but did not affect proteins covalently associated with the cell wall. Lysis of growing cells treated with otherwise lytic concentration of penicillin G was completely inhibited in the mutant, but that of non-growing cells was not affected by the mutation. The atl mutation did not significantly affect the ability of S. aureus to provoke an acute infection when inoculated intraperitoneally in a mouse sepsis model. These results further support the supposition that atl gene products are involved in cell separation, cell wall turnover and penicillin-induced lysis of the cells.
Animals↗
Increased glycan chain length distribution and decreased susceptibility to moenomycin in a vancomycin-resistant Staphylococcus aureus mutant.
A vancomycin-resistant Staphylococcus aureus mutant, COL-VR1 (MIC, 16 microg/ml), was isolated from methicillin-resistant S. aureus COL by exposure to vancomycin. COL-VR1 also showed decreased susceptibility to teicoplanin (8-fold), methicillin (2-fold), macarbomycin (8-fold), and moenomycin (16-fold). Macarbomycin and moenomycin are thought to directly inhibit transglycosylase activity. Characterization of the mutant revealed a thickened cell wall and suppression of penicillin-induced lysis, although the amounts of the five penicillin-binding proteins (PBPs 1, 2, 3, 4, and 2') and the profiles of peptidoglycan hydrolases were not altered. Analysis of muropeptide profile and glycan chain length distribution by reversed-phase high-pressure liquid chromatography revealed slightly decreased peptide cross-linking and an increased average glycan chain length compared to those of the parent. These results together suggest that a transglycosylase activity was enhanced in the mutant. This may represent a novel mechanism of glycopeptide resistance in S. aureus.
Aminoglycosides↗
Identification of the Staphylococcus aureus etd pathogenicity island which encodes a novel exfoliative toxin, ETD, and EDIN-B.
We identified a novel pathogenicity island in Staphylococcus aureus which contains open reading frames (ORFs) similar to the exfoliative toxin (ET) gene, glutamyl endopeptidase gene, and edin-B gene in tandem and the phage resistance gene, flanked by hsdM, hsdS (restriction and modification system), and IS256. The protein encoded by the ET-like gene showed 40, 59, and 68% amino acid sequence identities with exfoliative toxin A (ETA), exfoliative toxin B (ETB), and Staphylococcus hyicus ETB (ShETB), respectively. When injected into neonatal mice, the recombinant protein derived from the ET-like gene induced exfoliation of the skin with loss of cell-to-cell adhesion in the upper part of the epidermis as observed in histological examinations, just as was found in neonatal mice injected with ETA or ETB. Western blot analysis indicated that the recombinant protein is serologically distinct from ETA and ETB. Therefore, the product encoded by this new ORF is a new ET member produced by S. aureus and is termed ETD. ETD did not induce blisters in 1-day-old chickens. In the skins of mice injected with ETD, cell surface staining of desmoglein 1 (Dsg1), a cadherin type cell-to-cell adhesion molecule in desmosomes, was abolished without affecting that of desmoglein 3 (Dsg3). Furthermore, in vitro incubation of the recombinant extracellular domains of Dsg1 and Dsg3 with the recombinant protein demonstrated that both mouse and human Dsg1, but not Dsg3, were directly cleaved in a dose-dependent manner. These results demonstrate that ETD and ETA induce blister formation by identical pathophysiological mechanisms. Clinical strains positive for edin-B were suggested to be clonally associated, and all edin-B-positive strains tested were positive for etd. Among 18 etd-positive strains, 12 produced ETD extracellularly. Interestingly, these strains are mainly isolated from other sources of infections and not from patients with bullous impetigo or staphylococcal scalded-skin syndrome. This strongly suggests that ETD might play a pathogenic role in a broader spectrum of bacterial infections than previously considered.
Amino Acid Sequence↗