PROJECT SUMMARY Significance. The overall goals are to define mechanisms and therapies for the acute respiratory distress syndrome (ARDS) due to septic bacteremia of the opportunistic pathogen, Pseudomonas aeruginosa (PA). These mechanisms remain undefined. Our premise is that blood-borne PA cause ARDS by rapidly internalizing in the lung endothelium. Consequently, there is activation of the gasdermin D mechanism of membrane pore formation. Ca2+ enters the endothelial cytosol through the pores, destabilizing f-actin, and thereby inducing barrier failure – the major cause of ARDS. Approach. We will evaluate the premise in two Specific Aims by means of real-time confocal microscopy (RCM) of live mouse lungs, as well as other general approaches. In SA1, we will determine the effects of LPS transfection of the lung endothelium, a model of PA-associated LPS internalization. In SA2, we will determine lung-endothelial effects of bacteremia, modeled by intravenous PA injection, and extra-pulmonary sepsis by intraperitoneal PA infection. The premise will be evaluated in genetically manipulated mice to evaluate (i) the gasdermin D and other hypotheses of membrane pore formation; (ii) mechanisms of pore repair by the ESCRT- III system; (iii) the role of pore-induced Ca2+ and f-actin mechanisms in barrier failure; (iv) effects of PA internalization in the lung endothelium; and (v) the efficacy of endothelial actin enhancement by cell- permeable proteins as effective therapy against ARDS due to PA peritonitis induced. Impact. Our studies will for the first time, reveal the importance of lung-endothelial pore formation as a mechanism of the endothelial barrier failure that underlies sepsis-induced ARDS due to extra-pulmonary infection by PA. The endothelial internalization of PA will be understood as the critical mechanism in this pathology. The dynamics and mechanisms of endothelial pore formation will be revealed for the first time. Molecular strategies directed against endothelial pore formation, therefore barrier failure, will be evaluated as therapies for ARDS due to PA sepsis. Outstandingly novel understanding will be achieved in mechanisms and therapies of sepsis-induced ARDS due to PA-induced pore formation.