Identifying novel virulence factors in canine infective endocarditis caused by Streptococcus canis using transposon directed insertion sequencing

Question

Infective endocarditis (IE) is a bacterial infection of the endocardium. Streptococcus canis (SC) was found to be involved in almost a quarter of canine IE cases. SC colonizes the skin and mucosae of asymptomatic cats and dogs, and is associated with both superficial and severe infections. Cases of SC causing severe infection or IE in humans have been described after bite or scratch injuries from dogs or cats. The overall prognosis of dogs with IE is poor. The goals of the study are identification and verification of novel virulence factors in SC. This will give new options for vaccine development or treatment of infective endocarditis by for example antibody-antibiotic conjugates where it is essential to have a known target.

Methods

In this study, we use a random transposon mutant library (TraDIS) to discover virulence factors in the adhesion and invasion of SC to endothelial cells. This input library was made by transposon directed insertion-site sequencing, starting from a clinical endocarditis SC strain. After infection of HUVEC cells in vitro, the mutants that did not adhere or invade were sequenced, and this output pool was compared to the input library. This infection was done both statically and under flow conditions to mimic the behaviour of endothelial cells on different locations of the heart valve. By comparing output and input pool, potential virulence factors could be identified. As a selection assay for biofilm formation, a biofilm will be formed on a fibrin matrix under flow conditions to create a pool with mutants that are unable or able to participate in this formation. After sequence analysis we will confirm these factors by mutagenesis.

Results

Genes that are relevant for the pathogenesis of SC in IE could be identified using the TraDIS method. Within these potential virulence factors, some have homologues in other streptococcal species, but for many others the function is unknown. This method tells us which of these are worth investigating.

Conclusions

TraDIS is a great sequencing technique for screening the whole SC genome on a specific function, like in this case infection of endothelial cells and biofilm formation.