Objectives: profile and identify F. nucleatum proteases.
Methods: SDS-PAGE Zymograms containing fibrinogen were used to profile proteolytic activity in F. nucleatum strains ATCC 49256 and ATCC 25586 which have been sequenced. The proteases were characterized using specific inhibitors and identified by mass spectrograph analysis.
Results: A 55kDa proteolytic band was detected in F. nucleatum ATCC 49256 and a 90 kDa proteolytic band was detected in F. nucleatum ATCC25585. Both proteolytic activities were inhibited by PMSF, a serine protease specific inhibitor. Mass spectrograph analysis of the proteolytic bands followed by sequence analysis of the annotated genomes of both fusobacterial strains, revealed that the presence of peptide sequences homologues to putative serine proteases. Both 55 kDa and 90 kDa proteases are encoded by homologous genes encoding a putative serine protease of 104 kDa.
Conclusion: Our results link for the first time proteolytic activity with specific fusobacterial genes, and suggest that the 55 kDa serine protease is derived from a 104 kDa precursor.
This study was supported the United States-Israel Binational Science Foundation grant number 2005084