Isolation and characterization of fimbriae from a sparsely fimbriated strain of Porphyromonas gingivalis.
Porphyromonas gingivalis W50 (ATCC 53978) possesses the gene for fimbriae; however, the surface-expressed fimbriae are sparse and have not been previously isolated and characterized. We purified fimbriae from strain W50 to homogeneity by ammonium sulfate precipitation and reverse-phase high-performance liquid chromatography [H. T. Sojar, N. Hamada, and R. J. Genco, Protein Expr. Purif. 9(1):49-52, 1997]. Negative staining of purified fimbriae viewed by electron microscopy revealed that the fimbriae were identical in diameter to fimbriae of other P. gingivalis strains, such as 2561, but were shorter in length. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, the apparent molecular weight of isolated fimbrillin from strain W50 was found to be identical to that of the fimbrillin molecule of strain 2561. Unlike 2561 fimbriae, W50 fimbriae, under reducing condition, exhibited a monomeric structure on SDS-PAGE at room temperature. However, under nonreduced conditions, even at 100 degrees C, no monomer was observed. In immunoblot analysis as well as immunogold labeling of isolated fimbriae, polyclonal antibodies against 2561 fimbriae, as well as antibodies against peptide I (V-V-M-A-N-T-G-A-M-E-V-G-K-T-L-A-E-V-K-Cys) and peptide J (A-L-T-T-E-L-T-A-E-N-Q-E-A-A-G-L-I-M-T-A-E-P-Cys), reacted. However, antifimbrial antibodies against strain 2561 reacted very weakly compared to anti-peptide I and anti-peptide J. Negative staining of whole W50 cells, as well as immunogold electron microscopy with anti-peptide I and anti-peptide J, showed fimbriae shorter in length and very few in number compared to those of strain 2561. Purified fimbriae showed no hemagglutinating activity. Amino acid composition was very similar to that of previously reported fimbriae of the 2561 strain.
Sojar
H T
HT
Department of Oral Biology, School of Dental Medicine, State University of New York at Buffalo 14214, USA. Hakim_Sojar@sdm.buffalo.edu
Hamada
N
N
Genco
R J
RJ
D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid; D036781:Fimbriae Proteins; D010861:Fimbriae, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D006801:Humans; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology; D016966:Porphyromonas gingivalis / Q000737:chemistry* / Q000235:genetics / Q000472:pathogenicity; D014774:Virulence
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D010446:Peptide Fragments; C063055:fimbrillin; D036781:Fimbriae Proteins