Cell Invasion and Pyruvate Oxidase-Derived HO Are Critical for Streptococcus pneumoniae-Mediated Cardiomyocyte Killing.

(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, can 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-invaded with cardiomyocytes remain unclear. In this study, we used a model of low multiplicity of infection with HL-1 mouse cardiomyocytes to investigate these early events. Using adhesion/invasion assays and immunofluorescent and transmission electron microscopy, we showed that rapidly 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 by adhesins, we showed that uptake by cardiomyocytes is not through the well-studied canonical interactions identified for vascular endothelial cells. Indeed, invasion 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 HO production were required for cardiomyocyte killing. Finally, we showed that this cytotoxicity could be abrogated using CME inhibitors or antioxidants, attesting to intracellular replication of as a key first step in pneumococcal pathogenesis within the heart.