Microbial and Host Factors that Promote Epithelial Disruption and S. pneumoniae Transit out of the Lung

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Project Summary Streptococcus pneumoniae causes ~900,000 cases of pneumococcal pneumonia annually in the US, with a mortality rate of 5-7%, making this disease a major health and financial burden. S. pneumoniae lung infections can spread to the bloodstream (bacteremia) and lead to severe patient outcomes. The goal of the proposed research is to elucidate the microbial (Aim 1) and host (Aim 2) factors that enable this bacterium to transit from the lung to the blood, an ability that is critical for many respiratory pathogens to cause disseminated infection. An important virulence factor during S. pneumoniae infection is pneumolysin (PLY), a pore forming toxin, which has been implicated in the development of bacteremia. S. pneumoniae infections are also characterized by an excessive immune response mediated primarily by white blood cells called polymorphonuclear cells (PMNs) that can cause host damage and result in lethal infection. Our overall hypothesis is that PLY and PMN migration disrupt the lung epithelium, promoting bacterial transit from the lung into the bloodstream. We will investigate this hypothesis by using an in vitro transepithelial migration assay, which allows us to assess how bacteria transit across the lung epithelium, analogous to bacterial dissemination from the lungs into the bloodstream in vivo. This versatile system models diverse microenvironments, is easy to maintain, and integrates seamlessly with other molecular biology and microbiology techniques. In Aim 1 we will determine how PLY disrupts intercellular junctions of the lung epithelium and how that promotes S. pneumoniae transit out of the lungs independent of PMNs. To assess PLY-mediated removal of intercellular junction proteins, we will infect polarized lung epithelial monolayers with PLY-proficient (WT) or PLY-deficient isogenic bacterial strains, stain intercellular junction proteins with fluorescent antibodies, image the monolayers by confocal microscopy, and use Image J and Prism software to perform quantitative image and statistical analysis, respectively. In parallel, we will quantify S. pneumoniae transit across lung epithelial monolayers to connect PLY-mediated disruptions of intercellular junctions to changes in bacterial migration in the absence of PMNs. In Aim 2 we will identify how PMNs disrupt intercellular junctions of the lung epithelium and how this perturbation promotes S. pneumoniae transit out of the lungs. To evaluate PMN-mediated removal of intercellular junction proteins, we will infect polarized lung epithelial monolayers with WT S. pneumoniae in the presence or absence of PMNs, stain intercellular junction proteins with fluorescent antibodies, image the monolayers by confocal microscopy, and use Image J and Prism software to perform quantitative image and statistical analysis, respectively. In parallel with these experiments, we will measure S. pneumoniae transit across the lung epithelial monolayers to connect PMN-mediated monolayer disruptions with changes in bacterial migration. Collectively, these experiments will explain how microbial and host factors disrupt the lung epithelium, leading to bacterial dissemination, a fundamental process in S. pneumoniae pathogenesis and other lung infections.