Cell Invasion and Pyruvate Oxidase-Derived H2O2 Are Critical for Streptococcus pneumoniae-Mediated Cardiomyocyte Killing
ABSTRACTStreptococcus pneumoniae(the pneumococcus) is the leading cause of community-acquired pneumonia and is now recognized to be a direct contributor to adverse acute cardiac events. During invasive pneumococcal disease,S. pneumoniaecan gain access to the myocardium, kill cardiomyocytes, and form bacterium-filled “microlesions” causing considerable acute and long-lasting cardiac damage. While the molecular mechanisms responsible for bacterial translocation into the heart have been elucidated, the initial interactions of heart-invadedS. pneumoniaewith cardiomyocytes remain unclear. In this study, we used a model of low multiplicity ofS. pneumoniaeinfection with HL-1 mouse cardiomyocytes to investigate these early events. Using adhesion/invasion assays and immunofluorescent and transmission electron microscopy, we showed thatS. pneumoniaerapidly adhered to and invaded cardiomyocytes. What is more, pneumococci existed as intravacuolar bacteria or escaped into the cytoplasm. Pulse-chase assays with BrdU confirmed intracellular replication of pneumococci within HL-1 cells. Using endocytosis inhibitors, bacterial isogenic mutants, and neutralizing antibodies against host proteins recognized byS. pneumoniaeadhesins, we showed thatS. pneumoniaeuptake by cardiomyocytes is not through the well-studied canonical interactions identified for vascular endothelial cells. Indeed,S. pneumoniaeinvasion of HL-1 cells occurred through clathrin-mediated endocytosis (CME) and independently of choline binding protein A (CbpA)/laminin receptor, CbpA/polymeric immunoglobulin receptor, or cell wall phosphorylcholine/platelet-activating factor receptor. Subsequently, we determined that pneumolysin and streptococcal pyruvate oxidase-derived H2O2production were required for cardiomyocyte killing. Finally, we showed that this cytotoxicity could be abrogated using CME inhibitors or antioxidants, attesting to intracellular replication ofS. pneumoniaeas a key first step in pneumococcal pathogenesis within the heart.