Discovery of a new fibronectin-binding surface protein of Streptococcus canis with serum opacification activity through transposon directed insertion-site sequencing.

Infective endocarditis is a rare but severe disease in humans and dogs which can be caused by Streptococcus canis. To understand how S. canis can adhere to and invade the endocardium, we combined a high-throughput approach called transposon directed insertion-site sequencing (TraDIS) with a physiologically relevant endothelial cell infection model that incorporates venous-range shear stress to identify genes encoding factors relevant for bacterial adherence and invasion to vascular human cells under simulation of physiological blood flow conditions. A saturated transposon library of clinical strain IMT49926 was screened in a microfluidic infection assay, enabling genome-wide selection of mutants impaired in endothelial adhesion. Comparative analysis of input and non-adherent output pools revealed several candidate genes, including a fibronectin-binding LPXTG-anchored surface protein with high homology to streptococcal serum opacity factors (SOFs). Our findings identified ScSOF as a multifunctional surface protein that plays an important role in the infection potential of S. canis. It facilitates adhesion to endothelial cells, prevents endothelial wound closure, contributes to the streptococcal surface architecture, binds fibronectin, opacifies serum, and inhibits β-hemolytic activity. These properties make ScSOF a compelling candidate for further, more targeted studies to confirm it as a virulence factor in S. canis that can be used for vaccine development or therapeutic targeting.