Project Summary Streptococcus pneumoniae causes over 150,000 hospitalizations annually in the U.S., with a mortality rate of 5- 7%, making the disease both a significant health and financial burden. A key virulence factor during S. pneumoniae infection is pneumolysin (PLY), a cholesterol dependent cytolysin (CDC) that causes ion flux in host cells through its ability to form large 400Å pores in host cell membranes. The goal of the proposed research is to elucidate the role of PLY-dependent ion flux in the disruption of the lung epithelium. Intercellular junctions (IJs) are crucial for maintaining lung epithelial integrity and include adherens junctions and tight junctions. Our hypothesis is that PLY- dependent ion flux disrupts adherens junctions (Aim 1) and tight junctions (Aim 2) during S. pneumoniae infection. We will investigate this hypothesis with an air-liquid-interface (ALI) culture system that generates polarized lung epithelial monolayers. In Aim 1 we will determine how PLY-dependent ion flux disrupts adherens junction proteins. To assess PLY-dependent ion flux removal of adherens junction proteins, we will load cells with ion specific fluorescent indicators, infect with PLY-proficient (WT) or PLY-deficient (∆ply) S. pneumoniae strains, and measure changes in fluorescence. We will also perform these infections with ion specific chelators to show chelator efficacy in blocking ion flux. To determine the role of ion flux in adherens junction disruption, we will infect ALI monolayers with WT or ∆ply S. pneumoniae in the presence or absence of ion specific chelators, stain adherens junctions with fluorescent antibodies, image monolayers by confocal microscopy, perform image analysis, and use Prism for statistical analysis. Finally, we will assess if any adherens junction proteins are cleaved as a result of PLY- dependent ion flux and identify the protease responsible using chemical inhibitors and CRISPR-Cas9 gene-editing. Ion flux caused by pore forming toxins