Molecular cloning and expression of a major surface protein (the 75-kDa protein) of Porphyromonas (Bacteroides) gingivalis in Escherichia coli

Watanabe, Kan-ichi; Takasawa, Toshihide; Yoshimura, Fuminobu; Ozeki, Masami; Kawanami, Masamitsu; Katô, Hiroshi

doi:10.1016/0378-1097(92)90540-5

Cited by 12 publications

(9 citation statements)

References 19 publications

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“…The 75-kDa protein, one of the major immunodominant cell surface proteins of P. gingivalis, seems to be processed to a mature form by cleavage between residues Arg-49 and Ala-50 (16,19,32,35). To determine whether RGP is also involved in this maturation, immunoblot analysis of the 75-kDa protein of the RGP mutants was performed (Fig.…”

Section: Resultsmentioning

confidence: 99%

Involvement of arginine-specific cysteine proteinase (Arg-gingipain) in fimbriation of Porphyromonas gingivalis

et al. 1996

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Arginine-specific cysteine proteinase (Arg-gingipain [RGP]), a major proteinase secreted from the oral anaerobic bacterium Porphyromonas gingivalis, is encoded by two separate genes (rgpA and rgpB) on the P. gingivalis chromosome and widely implicated as an important virulence factor in the pathogenesis of periodontal disease (K. Nakayama, T. Kadowaki, K. Okamoto, and K. Yamamoto, J. Biol. Chem. 270:23619-23626, 1995). In this study, we investigated the role of RGP in the formation of P. gingivalis fimbriae which are thought to mediate adhesion of the organism to the oral surface by use of the rgp mutants. Electron microscopic observation revealed that the rgpA rgpB double (RGP-null) mutant possessed very few fimbriae on the cell surface, whereas the number of fimbriae of the rgpA or rgpB mutant was similar to that of the wild-type parent strain. The rgpB ؉ revertants that were isolated from the double mutant and recovered 20 to 40% of RGP activity of the wild-type parent possessed as many fimbriae as the wild-type parent, indicating that RGP significantly contributes to the fimbriation of P. gingivalis as well as to the degradation of various host proteins, disturbance of host defense mechanisms, and hemagglutination. Immunoblot analysis of cell extracts of these mutants with antifimbrilin antiserum revealed that the rgpA rgpB double mutant produced small amounts of two immunoreactive proteins with molecular masses of 45 and 43 kDa, corresponding to those of the precursor and mature forms of fimbrilin, respectively. The result suggests that RGP may function as a processing proteinase for fimbrilin maturation. In addition, a precursor form of the 75-kDa protein, one of the major outer membrane proteins of P. gingivalis, was accumulated in the rgpA rgpB double mutant but not in the single mutants and the revertants, suggesting an extensive role for RGP in the maturation of some of the cell surface proteins.Porphyromonas (Bacteroides) gingivalis, an oral anaerobic bacterium, has been isolated frequently from subgingival lesions in patients with adult periodontal disease, implying that the microorganism is one of the most important pathogens for the disease (25,31,36). P. gingivalis possesses several potential virulence factors (13). Among them, secretory proteinases have received much attention because they can degrade host tissue and cause inflammation (9, 27). Many proteinases have been isolated from culture supernatants, vesicles, membrane fractions, and cell extracts of P. gingivalis (for a review, see reference 5), but it has recently been reported that the proteinases with trypsin-like activity are derived from either Arggingipain (RGP) or Lys-gingipain (24). In a previous study (7), we reported the isolation of RGP (formerly, argingipain) from the culture supernatant of P. gingivalis and its unusual catalytic features related to periodontopathogenicity of the organism (e.g., the abilities to degrade periodontal tissue components, to evade inactivation by internal proteinase inhibitors such as cystatins and ...

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Section: Resultsmentioning

confidence: 99%

Involvement of arginine-specific cysteine proteinase (Arg-gingipain) in fimbriation of Porphyromonas gingivalis

et al. 1996

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“…In searching for the appropriate antigen, we and others have examined a group of cell surface carbohydrates designated as K-antigens (12,32,59,60), lipopolysaccharides (10,26,43,58,70), and various proteins including fimbriae and fimbrillin (11,20,21,42,64) and the 53-kDa and 67-kDa cell surface proteins (30,71,72), hemagglutinin (37) and cysteine proteases referred to as gingipains or porphypain (5,13,22,34,40,45,48,55,56). Of the P. gingivalis components studied to date, the cysteine proteases have shown the most potential for use as vaccine antigens.…”

Section: Discussionmentioning

confidence: 99%

Functional characteristics of antibodies induced by Arg‐gingipain (HRgpA) and Lys‐gingipain (Kgp) from Porphyromonas gingivalis

Nakagawa

Sims

Fan

et al. 2001

Oral Microbiology and Immunology

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Arginine-specific gingipain (HRgpA) and lysine-specific gingipain (Kgp), enzymes produced by Porphyromonas gingivalis, may be candidates for an anti-P. gingivalis vaccine. The purpose of our study was to determine whether HRgpA and Kgp have opsonic target sites and whether these sites are available and accessible on intact P. gingivalis cells. Rabbits were used to generate polyclonal antibodies to both proteins. Animals were immunized and immunoglobulin G (IgG) fractions were isolated from preimmune and immune sera. Functional characteristics of the antibodies were assessed by determining antibody titers by enzyme-linked immunosorbent assay (ELISA), generating Western immunoblots, and measuring antibody enhancement of P. gingivalis opsonization, phagocytosis and killing by polymorphonuclear leukocytes (PMN) of intact cells of strains of P. gingivalis representative of the four serotypes. Strains studied included 33277 (serotype A), A7A1-28 (serotype B), W50 (serotype C) and 381 (serotype D). Both HRgpA and Kgp induced high titers of IgG antibody. Anti-HRgpA and anti-Kgp bound to both HRgpA and Kgp demonstrating a large proportion of shared antigenic epitopes. The two antibodies bound equally well to all four P. gingivalis serotypes with titers ranging from 77 to 205 ELISA units when compared to preimmune IgG set at 1 ELISA unit. The immunoblot patterns of binding of the two antibodies to HRgpA and Kgp and to sonicates of the four P. gingivalis serotypes were virtually identical. Both antibodies detected components in HRgpA at 27, 35 and 45 kDa and in Kgp at 27, 32, 35, 40 and 55 kDa. The antibodies also detected components at or near these same positions in addition to multiple high molecular mass components in the cell sonicates of P. gingivalis. Both proteins induced antibodies that significantly enhanced opsonization as assessed by chemiluminescence, with values ranging from 130 mV to 375 mV for anti-HRgpA IgG and from 240 mV to 475 mV for anti-Kgp IgG. Both antibodies significantly enhanced PMN-mediated bacterial killing of the four P. gingivalis serotypes, although the percentage of killing varied among the serotypes (24-81% for anti-HRgpA and 37-89% for anti-Kgp). Thus, both HRgpA and Kgp express opsonic target sites and induce high titers of antibodies that opsonize and enhance killing of all four serotypes of P. gingivalis. These two proteins appear to be potential candidate antigens for an anti-P. gingivalis vaccine.

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“…Little is known about the morphology of these fimbriae, however, in part because purification is difficult in the presence of the long fimbriae. Nonetheless, it is now well established that the 75-kDa protein, Mfa1 (67 kDa), and PgII (72 kDa) are the same polypeptides, based on their identical N-terminal amino acid sequences and extensive similarity of primary amino acid sequence deduced from the gene sequences (9,26,41). Furthermore, the short fimbriae comprised of Mfa1 are distinct from a third fimbrial type consisting of a 53-kDa protein (1).…”

Section: Vol 73 2005mentioning

confidence: 99%

Short Fimbriae of Porphyromonas gingivalis and Their Role in Coadhesion with Streptococcus gordonii

et al. 2005

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Porphyromonas gingivalis, one of the causative agents of adult periodontitis, attaches and forms biofilms on substrata of Streptococcus gordonii. Coadhesion and biofilm development between these organisms requires the interaction of the short fimbriae of P. gingivalis with the SspB streptococcal surface polypeptide. In this study we investigated the structure and binding activities of the short fimbriae of P. gingivalis. Electron microscopy showed that isolated short fimbriae have an average length of 103 nm and exhibit a helical structure with a pitch of ca. 27 nm. Mfa1, the major protein subunit of the short fimbriae, bound to SspB protein, and this reaction was inhibited by purified recombinant Mfa1 and monospecifc anti-Mfa1 serum in a dose-dependent manner. Complementation of a polar Mfa1 mutant with the mfa1 gene restored the coadhesion phenotype of P. gingivalis. Hence, the Mfa1 structural fimbrial subunit does not require accessory proteins for binding to SspB. Furthermore, the interaction of Mfa1 with SspB is necessary for optimal coadhesion between P. gingivalis and S. gordonii.

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Molecular cloning and expression of a major surface protein (the 75-kDa protein) of Porphyromonas (Bacteroides) gingivalis in Escherichia coli

Cited by 12 publications

References 19 publications

Involvement of arginine-specific cysteine proteinase (Arg-gingipain) in fimbriation of Porphyromonas gingivalis

Involvement of arginine-specific cysteine proteinase (Arg-gingipain) in fimbriation of Porphyromonas gingivalis

Functional characteristics of antibodies induced by Arg‐gingipain (HRgpA) and Lys‐gingipain (Kgp) from Porphyromonas gingivalis

Short Fimbriae of Porphyromonas gingivalis and Their Role in Coadhesion with Streptococcus gordonii

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