PROJECT SUMMARY/ABSTRACT Pathogens responsible for nosocomial pneumonia elicit production of lung endothelial cell amyloids. These amyloids can have antimicrobial properties and be beneficial to the host, or they can have cytotoxic properties and be detrimental to the host. In the latter case, endothelial-derived amyloids may contribute to end organ dysfunction in the aftermath of critical illness. Bacterial virulence factors, such as the Pseudomonas aeruginosa type III secretion system effectors, and most notably exoenzyme Y, convert endothelial amyloids from antimicrobial to cytotoxic species. Amyloid-beta (Aβ) is one of the cytotoxic amyloids generated following endothelial infection. Aβ is released from endothelium where it becomes a transmissible and self-replicating prion cytotoxin. Our preliminary data reveals that γ-secretase activating protein plays a critical role in generating cytotoxic Aβ. We found γ-secretase activating protein expression in endothelial cells using non- biased microarray, RNAseq, methylation and miRNA screens, and confirmed protein expression and function in microvascular endothelium. γ-secretase activating protein deletion prevented formation of the cytotoxic Aβ species. Rather, following γ-secretase activating protein deletion the Pseudomonas aeruginosa- and exoenzyme Y-induced Aβ had antimicrobial properties. Exoenzyme Y is a promiscuous nucleotidyl cyclase that results in protein kinase A activation, which may phosphorylate Aβ necessary to increase its cytotoxic activity. These data suggest production of cytotoxic Aβ is due to two inter-related mechanisms, including an increase in γ-secretase activating protein function and the phosphorylation of Aβ once it is produced. Hence, this proposal tests the hypothesis that exoenzyme Y promotes the production of cytotoxic Aβ, dependent upon γ-secretase activating protein.