Identification of an antigenic protein Pga30 from Porphyromonas gingivalis W50.
Porphyromonas gingivalis is a black-pigmented, gram-negative bacterium that has been implicated as a major pathogen in the development of adult periodontitis. In an approach to identify a P. gingivalis antigen uniquely seroreactive with healthy subjects, we produced a surface and periplasmic extract of P. gingivalis, separated that extract into 36 fractions using anion-exchange chromatography and screened each fraction for reactivity in an enzyme-linked immunosorbent assay (ELISA) using sera from eight periodontitis patients and eight age- and sex-matched healthy controls. All of the diseased subjects harboured subgingival P. gingivalis by DNA probe analysis and exhibited similar seroreactivity profiles to the anion exchange fractions. However, only two of the healthy subjects (C10 and C15) were seroreactive with the fractions. The highest reactivity for all the seropositive subjects was with the same anion-exchange fractions 13-15. The anion exchange fraction (14) with the highest seroreactivity was subjected to gel filtration chromatography, and fraction 22 from this chromatography exhibited the highest reactivity with all the seropositive subjects. However, fraction 27 was found to be uniquely seroreactive with healthy subject C10, as it was not recognized by sera from any of the diseased or the other healthy subjects. This fraction was further purified by reversed-phase high-pressure liquid chromatography and shown to contain a 30-kDa protein as determined by SDS-PAGE. Control subject C10 had no pocket depths greater than 3 mm and no sites that bled on gentle probing; however, P. gingivalis was detected in subgingival plaque samples at a level of 10(5)-10(6) cells per site in two of the ten sites sampled. This subject was also unusual in that he exhibited a seroreactivity profile similar to that of diseased subjects, which was not characteristic of the healthy control subjects. The unique reactivity of the 30-kDa antigen, designated Pga30, with subject C10 serum was confirmed in a Western blot with the purified antigen. N-Terminal sequence analysis of Pga30 produced a single, unambiguous protein sequence confirming the purity of the protein. A search of the database using the N-terminal sequence obtained did not reveal any significant sequence similarity. In conclusion, we have identified a P. gingivalis antigen that was uniquely reactive in an ELISA and Western blot with serum from a subject with no clinical signs of periodontitis who harbored P. gingivalis in subgingival plaque.
Hendtlass
A
A
School of Dental Science, University of Melbourne, 711 Elizabeth Street, Melbourne, 3000, Victoria, Australia.
Dashper
S G
SG
Reynolds
E C
EC
D000328:Adult; D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000097:blood; D000942:Antigens, Bacterial / Q000737:chemistry / Q000302:isolation & purification*; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D015153:Blotting, Western; D016022:Case-Control Studies; D002850:Chromatography, Gel; D003773:Dental Plaque / Q000382:microbiology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006420:Hemeproteins / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D008875:Middle Aged; D008969:Molecular Sequence Data; D010518:Periodontitis / Q000382:microbiology*; D016966:Porphyromonas gingivalis / Q000737:chemistry / Q000276:immunology*
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D006420:Hemeproteins; C416080:IhtB protein, Porphyromonas gingivalis