Cytokine production induced by a 67‐kDa fimbrial protein from Porphyromonas gingivalis

Abstract

Fimbriae have been reported to play an important role in the adherence of Porphyromonas gingivalis to oral surfaces and possibly in triggering host responses. P. gingivalis ATCC 33277 has two distinctly different fimbriae expressed on the cell surface. The 67-kDa fimbriae differ in size and antigenicity from the earlier reported FimA, a major 41-kDa fimbrial component of P. gingivalis. Expression of the 67-kDa fimbriae on the cell surface of a fimA mutant was investigated by electron microscopy. The 67-kDa fimbrial protein was purified from the fimA mutant by sonication, precipitation, and chromatography on a DEAE Sepharose CL-6B column. The N-terminal amino acid sequence of the 67-kDa fimbrillin was distinct from that of the 41-kDa fimbrillin. Moreover, we have found that the 67-kDa fimbrial protein from P. gingivalis ATCC 33277 induced IL-1alpha, IL-beta, IL-6 and TNFalpha cytokine expression in mouse peritoneal macrophages. These results suggest that P. gingivalis 67-kDa fimbriae may play a part in the inflammatory response during the development of periodontal diseases.

The Native 67-Kilodalton Minor Fimbria of Porphyromonas gingivalis Is a Novel Glycoprotein with DC-SIGN-Targeting Motifs

“…Protein purification was continued until minor fimbria samples showed no contaminating bands (i.e., for LPS or other contaminants) by Coomassie staining and silver staining (Fig. 1B) (20). Absence of endotoxin was further confirmed by LAL assay (Lonza).…”

“…Fractions were analyzed by 12% SDS-PAGE and silver staining (Bio-Rad) to ensure purity and quantified by Bradford assay. Fimbria preparations underwent further screening to confirm lack of LPS contamination via silver staining (20). Samples were then analyzed by tandem MS (MS/MS) to verify identity and to ensure no other protein contaminants were present.…”

“…The isogenic, major fimbria-deficient mutant DPG3, which expresses only the minor fimbria (Pg min ϩ /maj Ϫ ), was maintained anaerobically (10% H 2 , 10% CO 2 , 80% N 2 ) in a Forma Scientific anaerobic system glove box, model 1025/1029, at 37°C in Difco anaerobe broth MIC. Erythromycin (5 g/ml) was added according to the selection requirements of the strain (11,20,54,68). Fimbriae were purified as described by Davey et al (11).…”

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

“…In addition to their distinct physical properties, and their individual streptococcal receptor specificity, the long and short fimbriae have other contrasting biological properties. The short fimbriae are more active in promoting bone resorption in rats and elicit a secreted cytokine profile from macrophages distinct from that of the long fimbriae (9,10). Adherence of FimA and Mfa1 mutants to epithelial cells is also dissimilar (38).…”

“…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).…”