Characterisation of a novel adherence factor of Streptococcus canis affecting cell attachment and cell regeneration under physiological shear stress

The zoonotic pathogen Streptococcus canis (S. canis) is associated with a wide range of local and systemic infections in humans and animals, including endocarditis, bacteraemia, and necrotizing fasciitis. The molecular mechanisms underlying its pathogenesis remain poorly characterised, posing significant challenges for diagnosis and treatment. This study aimed to identify novel bacterial adhesion factors involved during bloodstream infections, focusing on their role in endothelial colonisation under hemodynamic shear stress. A transposon mutant library of a clinical S. canis isolate was generated and subjected to in vitro infection assays using human umbilical vein endothelial cells (HUVECs) under microfluidic conditions. The microfluidic system was employed to precisely simulate physiological shear forces encountered in the bloodstream, which are critical for bacterial-EC interactions during endocarditis and septicaemia. Following infection rounds, non-adherent bacterial populations were isolated for Transposon-Directed Insertion Site Sequencing (TraDIS). Bioinformatic analysis of TraDIS data enabled a genome-wide comparison between the original mutant library and post-infection bacterial pools, identifying 23 potential virulence factors with roles in adhesion. Among these, a serum opacity factor (SOF)-like protein, predicted via sequence homology, was selected for functional validation. A gene-specific deletion mutant (Δsof) was constructed and tested under static and microfluidic conditions. Infection assays and endothelial cell (EC) wound-healing analyses confirmed the SOF-like protein as a novel adhesion factor critical for EC colonisation. To further investigate tissue-invasive mechanisms, a 3D co-culture model was developed, replicating vascular architecture using HUVECs and smooth muscle cells (SMCs) embedded in a collagen matrix. Confocal laser scanning and electron microscopy revealed that S. canis can breach the endothelial barrier and interact with underlying SMCs, highlighting its capacity for tissue penetration. Additionally, ex vivo infections of human heart valve tissue demonstrated the pathogen’s ability to colonise endocardial surfaces under hemodynamic conditions. This study provides novel insights into S. canis pathogenesis, identifying SOF as a key adhesion factor and advancing methodologies for simulating in vivo infection dynamics. The integration of microfluidics, TraDIS, and advanced imaging techniques offers a framework for future research on bacterial-host interactions in bloodstream infections.