Purification and characterization of a putative fimbrial protein/receptor of Porphyromonas gingivalis

Abstract

Porphyromonas gingivalis has been implicated as a major aetiological agent in certain forms of periodontal disease. P. gingivalis is a Gram-negative, asaccharolytic bacterium that obtains energy from the fermentation of amino acids derived from the hydrolysis of host protein. Virulence factors of this bacterium include the capsule, fimbrial adhesins, cytotoxins and extracellular hydrolytic enzymes. A 43 kDa fimbrillin from P. gingivalis has been isolated and characterized. However, there is evidence that a second type of fimbria exists on the surface of P. gingivalis. A putative P. gingivalis fimbrial protein from a membrane preparation has been isolated and identified. This protein was shown to be reactive with sera from patients harbouring P. gingivalis. A 28 kDa protein fragment was purified by anion exchange, gel filtration and reversed-phase chromatography. N-terminal sequence analysis of the 28 kDa protein fragment revealed homology to the fimbrial precursor protein of Dichelobacter nodosus. A peptide corresponding to the N-Terminal 26 amino acyl residues of the 28 kDa protein fragment was synthesized and used to raise antibodies to the protein. Western blot analysis after SDS-PAGE of a P. gingivalis membrane preparation using the antibodies raised to the synthetic peptide detected three proteins of 36, 41 and 67 kDa. When protease inhibitors were not included in the extraction procedure only the 36 and 41 kDa bands were detected. It would appear, therefore, that the intact protein has an Mr of 67 kDa and that the 28, 36 and 41 kDa bands represent protein fragments produced by endogenous proteolytic activity. Based on sequence homology, the 67 kDa protein is possibly a sub-unit of a second P. gingivalis fimbrial type or a surface receptor.