Purification and characterization of a 65‐kilodalton diisopropylfluorophosphate‐binding protein in the outer membrane of Fusobacterium nucleatum Fev1

Abstract

Abstract— A 65‐kilodalton protein was identified in the outer membrane of Fusobacterium nucleatum Fevl by SDS‐PAGE. The relative amount of the protein varied during growth, being greatest in stationary phase. The protein was radio‐labeled by [125I]‐lactoperoxidase treatment of live cells and was only partially cxtractable with 2% sodium dodecylsulfatc (SDS) at room temperature, and therefore assumed to be both exposed to the cell surface and peptidoglycan associated. In intact cells the protein bound diisopropylfluorophosphate (DFP), indicating that it may be a serine protease. DFP‐binding activity depended apparently on proper localization of the proteins in the membrane. Several methods were tried in attempts to purify the 65‐kilodalton protein; the method that gave best results was preparative SDS‐polyacrylamide gel electrophoresis. The isoelectric point was determined to about pH 5. Amino acid composition analysis showed that the 65‐kilodalton protein possesses an excess of hydrophilic over hydrophobic residues, polarity index 52%. The N‐terminal end of the protein was apparently blocked.

Identification and Characterization of Fusolisin, the Fusobacterium nucleatum Autotransporter Serine Protease

“…The theoretic isoelectric point of the 55 kDa derivative of Fsp49256 is 5. This calculated isoelectric point is in agreement with that (pH 5) determined for the 65 kDa diisopropylfluorophosphate-binding outer membrane protein of F. nucleatum Fev1 [37]. Mass spectrometry analysis and identification of the high (99 kDa) and the low (55 kDa) molecular weight F. nucleatum serine proteases partially purified from strains ATCC 25586 and ATCC 49256 demonstrated (Fig.…”

“…To date, the only detected endopeptidase activity in F. nucleatum was that of an un-identified serine protease with a molecular weight of 61–65 kDa [36], [37], [38], [39]. In the present work this protease, now named fusolisin, has been identified and characterized at the genetic level.…”

“…Previous studies reported a fusobacterial serine protease activity associated with a molecular mass of 65 kDa [36], [37], [38], [39]. This protease was shown to be capable of degrading components of periodontal tissues, and to inactivate host defense effectors [39].…”

Taxonomy, biology, and periodontal aspects of Fusobacterium nucleatum.

“…The lysine cleavage enzyme has been purified and found to have properties much like those of the enzymes of lysine-fermenting clostridia (19). Brokstad and Jensen (43) and Brokstad et al (42) purified and characterized a 65-kDa OMP of F. nucleatum which appeared to be a serine protease that might be involved in the uptake of peptides.…”

Insights into the Natural Defenses of a Coral Reef Fish Against Gill Ectoparasites: Integrated Metabolome and Microbiome Approach

“…Several studies have shown that extracellular microbe proteases can specifically cleave Hb and produce Hb-derived peptides with antimicrobial activities [62][63][64]. Furthermore, some studies on Fusobacterium nucleatum, a human pathogenic Fusobacteria, suggest peptides and amino acids are the main energy source of Fusobacteria [65,66] and several extracellular serine proteases have been isolated from F. nucleatum [67,68]. As CLHbβ-1, CLHbβ-2 and CLHbβ-3 are cleaved after an arginine residue, these peptides are probably produced by a trypsin-like protease, possibly from bacterial origin.…”