title: Expression of Porphyromonas gingivalis gingipain antigen Hgp44 domain on surface of Lactococcus lactis.
abstract: Porphyromonas gingivalis, a pathogen involved in the development of chronic periodontitis, has a number of major virulence factors, among which are its surface cysteine protease gingipains. The purpose of this study was to investigate the feasibility of inducing protective antibodies against P. gingivalis by means of immunization with recombinant Lactococcus lactis expressing the 44-kDa gingipain adhesion/hemagglutinin domain (Hgp44). Part of the Hgp44 sequence encoding the first 314 amino acid residues, residues 188-251, and residues 354-393 was amplified and inserted into shuttle plasmid pSGANC332, with the resulting chimeric plasmids designated as pISTY210, pCOL, and pSHGRP44A, respectively. After confirming the clone sequences, expression of recombinant proteins was investigated by immunoblot. The results revealed that while pISTY210 and pCOL both expressed the Hgp44 antigen on the surface of L. lactis, the level of expression was quite low. To enhance expression of the protein on the surface of the cells, cysteine residues were changed to serine residues by site-directed mutagenesis. Replacement of 3 out of 5 cysteine residues (pISTY213) significantly increased expression of the recombinant protein on the surface of the bacteria. Interestingly, replacement of the 4th cysteine residue (pISTY215) reduced antigenicity of the recombinant protein. These results indicate that expression of Hgp44 on the surface of L. lactis cells requires the replacement of several key cysteine residues, and that L. lactis expressing this antigen could be a promising candidate for immunization against P. gingivalis-induced periodontitis.
authors: Department of Microbiology, Tokyo Dental College.
authors: Isato
authors: I
authors: Yamamoto
authors: Kazuyuki
authors: K
authors: Ishihara
authors: Kyotaro
authors: K
authors: Muramatsu
authors: Yoshiyuki
authors: Y
authors: Wada
authors: Mayumi
authors: M
authors: Kiwaki
authors: Akira
authors: A
authors: Kushiro
authors: Katsuji
authors: K
authors: Okuda
mesh_terms: D018829:Adhesins, Bacterial / Q000276:immunology*; D000907:Antibodies, Bacterial / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology*; D000954:Antigens, Surface / Q000276:immunology; D001426:Bacterial Proteins / Q000276:immunology*; D001428:Bacterial Vaccines; D003545:Cysteine / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D005240:Feasibility Studies; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins / Q000276:immunology*; D006801:Humans; D007114:Immunization; D015151:Immunoblotting; D013294:Lactococcus lactis / Q000276:immunology*; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D016966:Porphyromonas gingivalis / Q000276:immunology*; D011994:Recombinant Proteins; D012694:Serine / Q000235:genetics
chemical_list: D018829:Adhesins, Bacterial; D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D000954:Antigens, Surface; D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins; D011994:Recombinant Proteins; D012694:Serine; D003546:Cysteine Endopeptidases; D003545:Cysteine