Mitsuo Hayakawa

ROLE OF PORPHYROMONAS GINGIVALIS 40-kDa OUTER MEMBRANE PROTEIN IN THE AGGREGATION OF P. GINGIVALIS VESICLES AND ACTINOMYCES VISCOSUS

Porphyromonas gingivalis, an important pathogen in periodontitis, produces extracellular vesicles that aggregate with Actinomyces viscosus cells. A 40-kDa outer membrane protein (OMP)-coding gene from P. gingivalis was cloned and the protein was found to be localized in these vesicles. The recombinant 40-kDa OMP did not show aggregation activity. However, affinity-purified antibody against the recombinant protein significantly inhibited aggregation of P. gingivalis vesicles with A. viscosus cells. The antibody also inhibited cellular coaggregation of several strains of P. gingivalis with A. viscosus cells, but not with other periodontal pathogens. Moreover, aggregation of A. viscosus cells with P. gingivalis vesicles was inhibited in a dose-dependent manner by pre-treatment of the A. viscosus cells with the recombinant protein. These findings suggest that the 40-kDa OMP may be an important aggregation factor of P. gingivalis.

Glycylprolyl Dipeptidylaminopeptidase from Bacteroides gingivalis

Dipeptidyl aminopeptidase activity was found in the culture medium of Bacteroides gingivalis 381. The enzyme, hydrolyzing glycylprolyl-4-methylcoumaryl-7-amide, was purified 750-fold from culture medium by ammonium sulfate precipitation, Sephadex G-200 gel filtration, and DEAE Bio Gel A column chromatography. The molecular weight, determined by gel filtration, was approximately 160,000. The isoelectric point of the enzyme, estimated by isoelectric focusing using polyacrylamide disk gel electrophoresis, was about pH 6.2. The optimum pH of the enzyme was about 8.0, and the Km value was 0.05 mM. The enzyme activity was strongly inhibited by phenylmethylsulfonylfluoride and diisopropylfluorophosphate. The purified enzyme specifically cleaved glycylprolyl dipeptide from partially digested type I collagen.

A 35-kDa co-aggregation factor is a hemin binding protein in Porphyromonas gingivalis.

It has been known that Porphyromonas gingivalis has an obligate requirement for hemin or selected heme- or Fe-containing compounds for its growth. In addition, the influence of hemin on the expression of several putative virulence factors produced by this bacterium has also been recently documented; however, the mechanisms involved in hemin uptake are poorly defined. We succeeded in cloning the gene coding for the 35-kDa protein, which was specifically expressed in P. gingivalis and seemed to confer colonizing activities. Recently, we have constructed the P. gingivalis 381 mutant defective in the 35-kDa protein by insertion mutagenesis. The beige mutant exhibited little co-aggregation and the virulence was also decreased. Based on these results and homology search analysis, we focused on assessing the hemin bindings and found the heme regulatory motif (HRM) as a hemin direct binding site. The 35-kDa protein did possess the binding ability of selected protoporphyrins involving the hemin. These results demonstrated that 35-kDa protein is one of the hemin binding proteins in P. gingivalis and suggested that hemin binding ability of 35-kDa protein is important for the expression of virulence in P. gingivalis.

Cloning of a Bacteroides gingivalis outer membrane protein gene in Escherichia coli

Gene banks of chromosomal DNA from Bacteroides gingivalis 381 were constructed using the bacteriophage replacement vector lambda L47.1. A clone encoding an outer membrane protein from B. gingivalis was identified by Western blot screening with antiserum raised against the outer membrane fraction of B. gingivalis 381 cells. The DNA insert contained within this phage was cloned into the plasmid vector pACYC184 to create the recombinant plasmid pMD123. An Escherichia coli transformant, MD123, containing pMD123 produced a protein having an apparent molecular weight of 40 kDa. The recombinant protein was purified, and amino acid analysis revealed the recombinant protein to have a relatively high content of hydrophobic amino acids (43.6%). Antiserum against the purified recombinant 40 kDa protein reacted with a polypeptide of similar size in the outer membrane fraction and vesicles of B. gingivalis.

Purification and immunochemical characterization of a recombinant outer membrane protein from Bacteroides gingivalis

1. Bacteroides gingivalis is thought to be one of the most virulent microorganisms in relation to adult periodontitis. A gene clone, MD125, is an Escherichia coli host which produces an outer membrane protein of B. gingivalis. 2. The recombinant outer membrane protein (rOMP) was purified to homogeneity from cell sonicate of MD125 by four chromatographic steps. The molecular weight of the purified rOMP was estimated to be approximately 40 kDa. 3. Immunodiffusion analysis showed that antiserum against whole cells of B. gingivalis reacted not only with B. gingivalis cells but also with other Bacteroides cells. Antiserum against the purified recombinant protein reacted with cells of B. gingivalis, whereas this antiserum did not react with all of the other Bacteroides species tested. 4. These data suggest that the rOMP may be a B. gingivalis-specific antigen and that the purified rOMP will be useful material for serodiagnosis and for the development of a vaccine against B. gingivalis infection.

Inhibition of a Porphyromonas gingivalis colonizing factor between Actinomyces viscosus ATCC 19246 by monoclonal antibodies against recombinant 40-kDa outer-membrane protein.

1. Porphyromonas gingivalis, an important pathogen in human periodontal disease, aggregates with Actinomyces viscosus ATCC 19246. 2. Monoclonal antibodies (mAbs) against purified recombinant 40-kDa outer-membrane protein (r40-kDa, OMP) of P. gingivalis 381 inhibited its coaggregation with A. viscosus ATCC 19246 in a dose-dependent manner. 3. Five mAb clones against r40-kDa OMP were selected. The isotype of the five was IgG1. 4. Pg-ompA2 inhibited the coaggregation of several strains of P. gingivalis with A. viscosus ATCC 19246 cells.

Specific antibodies induced by nasally administered 40-kDa outer membrane protein of Porphyromonas gingivalis inhibits coaggregation activity of P. gingivalis

In this study, we have assessed the efficacy of the 40-kDa outer membrane protein (40k-OMP) of Porphyromonas gingivalis as a nasal vaccine for the prevention of adult periodontitis. Mice nasally immunized with 40k-OMP and cholera toxin as mucosal adjuvant displayed significant levels of 40k-OMP-specific serum IgG1, IgG2b and IgA as well as mucosal IgA antibodies (Abs) in saliva and nasal secretions. Ab-forming cell (AFC) analysis confirmed the antibody titers by detecting high numbers of 40k-OMP-specific AFCs in spleen, salivary glands and nasal passages. Because 40k-OMP-specific IgG inhibited coaggregation of P. gingivalis vesicles and S. gordonii, it may be an important tool for the prevention of adult periodontitis.

Sequence analysis of the Porphyromonas gingivalis dipeptidyl peptidase IV gene

We previously constructed a Porphyromonas gingivalis genomic library and isolated the 2.9 kb EcoRV fragment which specified glycylprolyl dipeptidyl aminopeptidase (GPase). Nucleotide sequencing of this fragment identified the single 2169 bp open reading frame which coded for a 723 amino acid protein. The amino acid sequencing of the NH2-terminal domain of the native and recombinant mature enzymes suggested that the protease possessed a 16 amino acid residue signal peptide. The calculated mass of the precursor and mature proteases were 82,018 and 80,235 daltons, respectively. The homology search of this enzyme in registered protein sequences revealed that this enzyme was homologous to dipeptidyl peptidase (DPP) IV from the Flavobacterium meningosepticum and that from eukaryotic cells, including the human, mouse, and rat. Three amino acid residues, Ser-593, Asp-668, and His-700, were identified as a putative catalytic triad, a common feature of eukaryotic serine proteases. In addition, this enzyme showed a broad proteolytic spectrum toward synthetic substrates capable of splitting not only Gly-Pro-derivative but also Ala-Pro, Lys-Pro, and Phe-Pro-derivatives. Therefore, we conclude that this enzyme belongs to DPP IV rather than GPase.

H2O2-derived free radicals treated fibronectin substratum reduces the bone nodule formation of rat calvarial osteoblast

Fibronectin (FN) is involved in various cellular activities such as adhesion, proliferation and migration as a substratum. Since the metabolic turnover of FN is much slower than other cellular components, it may be affected by the oxygen free radicals produced in the aging process. However, the effect of oxygen free radicals on FN as substratum in bone formation has not been well characterized. The objective of this study was to examine the effect on the bone forming activity of osteoblasts using an oxygen free radical treated FN substratum in vitro (H2O2-Cu2+system). SDS-PAGE, Western blotting and immuno-blotting analysis revealed that FN was degradated and/or modified by H2O2-Cu2+ (.OH) treatment. Bone nodule formation per well was examined for total number, total area and area per nodule, which data were then compared between non-coated and FN-coated, and between FN-coated and .OH treated FN-coated. Bone nodule formation in the FN-coated was significantly greater than in the non-coated. Furthermore, bone nodule formation in .OH treated FN-coated was significantly less than that of FN-coated. These findings suggested that FN plays important roles in osteoblast activity and that FN substratum damaged by the oxygen free radicals produced by the aging process may cause decline of bone nodule formation through inhibition of the proliferation, differentiation and calcification processes.

Cloning and nucleotide sequence analysis of the Streptococcus sobrinus gtfU gene that produces a highly branched water-soluble glucan.

Streptococcus sobrinus has four gtf genes, gtfI, gtfS, gtfT, and gtfU, on the chromosome. These genes correspond respectively to the enzymes GTF-I, GTF-S1, GTF-S2, and GTF-S3. An Escherichia coli MD66 clone that contained the S. sobrinus gtfU gene was characterized. Immunological properties showed that the protein produced by the E. coli MD66 clone was similar to S. sobrinus GTF-S1. Biological properties and a linkage analysis of the glucans by 13C NMR spectrometry revealed that the protein produced by the E. coli MD66 clone was GTF-S1.