3 years ago

Cell invasion and pyruvate oxidase derived H2O2 are critical for Streptococcus pneumoniae mediated cardiomyocyte killing.

Anukul T Shenoy, Carlos J Orihuela, LaDonna A Patterson, Terry Brissac
Streptococcus pneumoniae (Spn, 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, Spn can gain access to the myocardium, kill cardiomyocytes, and form bacteria-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 Spn with cardiomyocytes remain unclear. Herein, we used a low multiplicity of Spn infection model with HL-1 mouse cardiomyocytes to investigate these early events. Using adhesion/invasion assays, immunofluorescent and transmission electron microscopy, we show that Spn rapidly adhered to and invaded cardiomyocytes. What is more, pneumococci existed as intra-vacuolar 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 Spn adhesins, we show that Spn uptake by cardiomyocytes is not through the well-studied canonical interactions identified for vascular endothelial cells. Indeed, Spn 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 H2O2 production were required for cardiomyocyte killing. Finally, we show that this cytotoxicity could be abrogated using CME inhibitors or antioxidants attesting intracellular replication of Spn as a key first step in pneumococcal pathogenesis within the heart.

Publisher URL: http://doi.org/10.1128/IAI.00569-17

DOI: 10.1128/IAI.00569-17

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