Fibronectin-binding proteins of pilus-free Streptococcus pneumoniae induce fibronectin-triggered pneumococcal invasive endocytosis and ROS-induced PANoptosis of host cells

Streptococcus pneumoniae causes bacterial pneumonia, upper respiratory tract infection, and meningitis. Pilus is an important virulence factor for of pneumococcal adhesion and invasion. However, the adhesive and invasive mechanisms of pilus-free S. pneumoniae remain poorly understood. This study revealed that pilus-free S. pneumoniae and its recombinant fibronectin-binding proteins (FnBPs), rPavA-, rPavB-N- or rPfbA-coated microbeads adhered to and then invaded human bronchial epithelial, alveolar epithelial or vascular endothelial cells (HBECs, HAECs or HVECs) through pneumococcal FnBPs / human cell Fn (hFn)-triggered integrin (ITG)-focal adhesion kinase (FAK)-microfiament (MF) pathway-dependent endocytosis, which was detected by laser scanning confocal microscopy (LSCM) and transmission electron microscopy. The rFnBPs displayed high adhesiveness and strong binding to hFn with KD values of 0.257-5.690×10⁻8 M determined by ELISA and surface plasmon resonance detection. The pneumococcal strain and FnBP-coated microbeads invaded host cells through endocytosis, leading to NADPH oxidase-dependent reactive oxygen species (ROS) elevation. This ROS- elevation triggered cell death via PANoptosis, characterized by necroptosis followed by pyroptosis, as determined by LSCM, MTT assay, flow cytometry and spectrophotometric colorimetry. These findings demonstrated a novel pathogenic mechanism by which pilus-free S. pneumoniae causes host cell damage and death though pneumococcal FnBPs-hFn-mediated ITG-FAK-MF pathway-dependent endocytosis, resulting in NADPH oxidase-dependent ROS-induced PANoptosis.