Abstract The pathogenic bacteria Pseudomonas aeruginosa is a major cause of blinding corneal infections in the USA and worldwide. This includes a recent outbreak of drug resistant P. aeruginosa keratitis in contaminated eyedrops that caused keratitis in 68 patients from 16 US states and resulted in 3 deaths and 8 enucleations. Our earlier studies supported by this grant demonstrated that P. aeruginosa virulence in infected corneas is due to expression of the Type III secretion system, which comprises a needle structure that injects exoenzymes directly into the host cell cytosol. Our recent data show that using either NLRP3-/- mice or the NLRP3 small molecule inhibitor MCC950, P. aeruginosa ExoS ADP ribosyl transferase (ADPRT) selectively licenses NLRP3 inflammasome usage in neutrophils (where macrophages use the NLRC4 inflammasome) for IL-1β processing and secretion, and importantly that NLRP3 is required for effective bacterial killing in infected corneas that can be blocked with the small molecule inhibitor MCC950. Proposed studies in Aim 1 will examine the early events in NLRP3 activation in neutrophils, including a role for NEK7 and HDAC6, which will also be examined during infection. Aim 2 will use an HEK293 based expression system to produce mg levels of NLRP3 that can used to identifying NLRP3 domains and specific arginine residues that are targeted by ExoS ADPRT. Aim 3 will examine cell death pathways in corneal epithelial cells and neutrophils infected with P. aeruginosa expressing the Type II secretion protein ToxA and determine if there is a role for NLRP1 pyroptotic cell death and in corneal infections. Results of these proposed studies will identify novel targets for immune intervention in this blinding infection.