F. necrophorum Ecotin Inhibits Human Plasma Kallikrein and Human Neutrophil Elastase

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

Bioinformatics: a useful tool for the molecular microbiologist?

Following hard on the heels of the human genome project, microbial genome versions have now begun to produce vast amounts of information on the nucleotide sequences of specific microbes. How useful is this information and how can researchers wade through the millions of base pairs of sequence data to find genes or sequences of interest for either diagnostic or therapeutic strategies? In theory, the answer lies with the new specialty of bioinformatics, which covers genomics, proteomics and metabolomics - terms that are more recognisable to many as molecular genetics and biochemistry

Comparison of virulence genes found in draft genomes of F. necrophorum

Fusobacterium necrophorum is a causative agent of persistent sore throat syndrome, tonsillar abscesses and Lemierre’s syndrome (LS) in humans. LS is characterised by thrombophlebitis of the jugular vein and bacteraemia. It is a Gram-negative, anaerobic bacterium which to date has no available reference genome. Draft genomes suggest it to be a single circular chromosome of approximately 2.2Mb. A reference strain of each of the two F. necrophorum subspecies and a clinical isolate from a LS patient were sequenced on a Roche 454 GS-FLX+. Sequence data was assembled using Roche GS Assembler and the resulting contigs annotated using xBASE, Pfam and BLAST. The annotation data was mined for gene products associated with virulence revealing a leukotoxin, haemolysin, filamentous haemagglutinnin, adhesin, hemin receptor, phage genes, CRISPR-associated proteins, ecotin and a putative type V secretion system. Data will be presented on comparative genomics of the three strains, with a focus on putative virulence genes. Tools such as Artemis Comparison Tool and ClustalO were used for sequence alignments and PhyML was used to generate phylogenetic trees. Conserved motifs associated with virulence were also located. Understanding variations at the genomic level may help to explain the increased virulence of some F. necrophorum strains

Bioinformatics: a useful tool for the molecular microbiologist?

Following hard on the heels of the human genome project, microbial genome versions have now begun to produce vast amounts of information on the nucleotide sequences of specific microbes. How useful is this information and how can researchers wade through the millions of base pairs of sequence data to find genes or sequences of interest for either diagnostic or therapeutic strategies? In theory, the answer lies with the new specialty of bioinformatics, which covers genomics, proteomics and metabolomics - terms that are more recognisable to many as molecular genetics and biochemistry

Human papillomavirus: an overview

Human papillomavirus (HPV) comprises a group of heterogeneous viruses containing many genotypes that can be divided into high-risk and low-risk types, depending on their association with disease. Some 70 HPV types that can infect epithelial surfaces differentially have been identified, including a number that have been implicated in the aetiology of cervical cancer, the most common form of malignant tumour in women worldwide

Proteomics: more than just biochemistry

Growth of the so-called 'omics' technologies may appear to be simply a case of giving new, trendy names to boring, old-fashioned technology. After all, who would dispute that genomics is just genetics and molecular biology, glycomics is the study of glycosylation or that metabolomics is simply the study of metabolic pathways? Surely, then, proteomics is just biochemistry. Indeed, students are now told that they are studying proteomics, as there is a tendency to turn off at the mere mention of biochemistry

Vaccination against cervical cancer

Human papillomavirus (HPV)-associated cervical cancer affects almost half a million sexually active females each year worldwide. Recently, however, clinical trials of two HPV vaccines have demonstrated successful results