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A5725 - Pseudomonas Aeruginosa’s Exoenzyme Y Intoxication Reversibly Converts Antimicrobial Endothelial Amyloids into Amyloid Prions
Author Block: S. B. Voth, R. Balczon, C. M. Francis, T. Stevens; University of South Alabama, Mobile, AL, United States.
RATIONALE: Pseudomonas aeruginosa is the prevalent bacterial pathogen responsible for nosocomial pneumonia. This opportunistic bacterium utilizes a type III secretion system (T3SS) to directly inject exoenzymes into the host cell. T3SS-dependent intoxication of pulmonary microvascular endothelial cells (PMVECs) is sufficient to significantly attenuate endothelial amyloid antimicrobial activity and promote the release of transmissible cytotoxic amyloid oligomer species that comprise a form of prion disease. Cytotoxic oligomeric amyloid prions are found in patient plasma, cerebrospinal fluid, and bronchoalveolar lavage fluid post-infection, and are capable of suppressing learning and memory. Furthermore, the T3SS effector exoenzyme Y (ExoY) intoxication of PMVECs generates cytotoxic tau amyloid oligomers and is also found in ~90% of clinical isolates. Albeit, the effects of ExoY activity upon the antimicrobicity of endothelial amyloids is unknown. Here, we tested the hypothesis that ExoY intoxication of pulmonary endothelium is sufficient to abrogate endothelial amyloid antimicrobicity. METHODS: PMVECs were infected with bacteria at a multiplicity of infection of 20:1 in Hank’s balanced salt solution. P. aeruginosa strains PA01 (ExoY, ExoS, ExoT), ExoY+ (ExoY), and ExoYK81M (catalytically inactive ExoY) were utilized. Supernatants derived from bacterial infection were collected at 4 (PA01) and 7 (ExoY+ and ExoYK81M) hours, respectively. Following collection, supernatants were filter-sterilized, and the self-replicative potential of putative amyloid prions was assessed through passaging experiments. Supernatant cytotoxicity was measured through supernatant treatment of naïve PMVECs. The antimicrobicity of supernatants was determined by Kirby-Bauer assay, bacterial colony forming units post-treatment, and antimicrobial aggregation effected by supernatant treatment of bacterial lawns plated on Congo red agar. Endothelial gap formation and antimicrobial clearing of bacterial lawns were quantified using custom macros written in ImageJ (NIH). RESULTS: ExoYK81M derived supernatant was negligibly cytotoxic and antimicrobial activity was reduced. On the other hand, supernatants obtained through the infection of PMVECs with ExoY competent strains were profoundly cytotoxic while antimicrobial activity was consistently abolished. Intriguingly, immunodepletion of tau amyloid oligomers entirely rescued antimicrobial activity while abrogating cytotoxicity in supernatants derived from PMVEC infection with ExoY-expressing strains of P. aeruginosa. CONCLUSION: These findings indicate that ExoY activity within the pulmonary endothelium eliminates the antimicrobicity of infection-induced endothelial amyloids through the generation of cytotoxic tau amyloid prions. Most importantly, tau oligomer neutralization restores the innate antimicrobial functionality of endothelial amyloids while eradicating tau amyloid prion pathogenesis. These data also suggest that tau oligomer neutralization may have therapeutic potential in the context of nosocomial pneumonia secondary to P. aeruginosa infection.