Molecular mechanisms underlying organ penetration in disseminated pneumococcal infection

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A molecular understanding of the events responsible for Streptococcus pneumoniae (Spn) organ invasion during bacteremia remains elusive although its consequences are devastating. We have closely examined the interaction between capsule and vascular endothelial cells (VEC) and developed the hypothesis that capsule dynamics are complex and impact all stages of Spn translocation across VEC. This includes attachment, receptor mediated endocytosis (RME), resistance to intracellular killing, and trafficking to the basolateral surface for organ invasion. Testing of our hypothesis will reveal the intracellular mechanics of Spn trafficking that drive organ invasion. Our results will impact risk assessment strategies for serotype-based vaccine design. AIM 1. Determine how serotype impacts platelet-activating factor receptor (PAFr) and laminin-receptor initiated adhesion/uptake of Spn. Our preliminary results indicate capsule has serotype-variable antagonistic effects on the bacterium's interaction with host cells that affects RME. We will use a comprehensive panel of isogenic capsule switch mutants of low and high invasive disease capability, specifically focusing on serotypes belonging to the same serogroup, i.e., having single and defined molecular differences in their capsule structure, to determine how specific biochemical features, such as acetylation, affect interactions with both peripheral and cerebral VEC. We will quantify serotype dependence of surface exposure of the Spn adhesins phosphorylcholine (PC) and choline binding protein A (CbpA), its effect on adhesion to their ligands PAFr and LR, respectively, on receptor-initiated signaling responsible for uptake, and in summate, on invasion rate. AIM 2. Determine how serotype influences the path taken by intracellular Spn for translocation vs recycling vs removal. The degradation of cargo taken up by RME in non-phagocytic cells is mediated by the novel LC3-associated endocytic recycling pathway `LANDO'. Thus, LANDO is likely a critical pathway co-opted by the pneumococcus to cross VEC. Using low and highly invasive capsule mutant swaps, we will determine the ability of PAFr and LR to initiate LANDO. We will subsequently determine the impact of capsule and serotype on Spn trafficking across wild type vs LANDO deficient VEC in vitro. We will characterize targeting of Spn into heart and brain of pafr-/-, LANDO deficient, and LR-blocked mice, thereby assessing how capsule modulates vital endocytic processes at the crossroads of bacterial translocation vs death in the lysosome. AIM 3. Determine how capsule shedding impacts bacterial fate within VEC. Whereas pneumococci in the bloodstream are encapsulated when taken up by VEC, our results suggest capsule is shed within the endosome following RME and provides protection from low pH and oxidative stress which are deployed to kill the bacterium. We will determine the status of capsule (shed or unshed) and the importance of shedding on Spn as they cross the VEC. We will determine if serotype impacts the kinetics of capsule shedding by Spn and how capsule type and shedding influences endosome acidification and lysosome fusion.