Background: Fusobacterium necrophorum is a causative agent of Lemierre’s syndrome (LS) which is characterised by thrombophlebitis of the jugular vein and bacteraemia. F. necrophorum is a Gram-negative, anaerobic bacterium known to possess virulence genes such as a haemolysin, filamentous haemagglutinin and leukotoxin, which target host blood components. Ecotin is a serine protease inhibitor that has not previously been characterised in F. necrophorum, but in E.coli has been shown to have a potent anticoagulant effect.
Methods: Next generation and Sanger sequencing were used to confirm the presence of the ecotin gene in the genomes of a collection of F. necrophorum clinical and reference strains. When translated, it was found to be a highly conserved protein made up of 159 amino acids. A plasmid insert was synthesised and ligated into a pET-16b vector. BL21(DE3) chemically competent E. coli cells were used to express the histidine-tagged protein under IPTG conditions and the protein was purified using IMAC sepharose affinity chromatography. Ecotin was added to human plasma kallikrein at concentrations of 0, 12.5, 25, 50 and 100 nM and incubated for 1 hour at room temperature to equilibrate. Kallikrein substrate was then added at concentrations of 0.015, 0.03, 0.06, 0.125, 0.25 and 0.5 mM and fluorescence was monitored every minute for 30 minutes. Ecotin was also added to human neutrophil elastase (HNE) as above and absorbance was monitored every minute for 30 minutes after addition of HNE substrate at concentrations of 0.015 – 0.5 mM.
Results: Results show that F. necrophorum ecotin inhibits human plasma kallikrein and human neutrophil elastase in a dose-dependent manner. Data will also be presented on the anticoagulant effects of ecotin during activated partial thromboplastin time, thrombin time and prothrombin time tests on human donor blood.
Conclusion: F. necrophorum is known to enter the bloodstream and cause a life threatening condition, therefore understanding the virulence mechanisms that it utilises is of great importance. Inhibition of clotting cascade enzymes suggests that ecotin may play a role in regulating coagulation, while the inhibition of neutrophil elastase suggests another role is to protect the organism from host proteases
