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A7616 - Lung Infection Elicits Endothelial Amyloids with Distinguishable Antimicrobial and Cytotoxic Properties
Author Block: S. B. Voth, R. Balczon, C. M. Francis, J. Audia, T. Stevens; University of South Alabama, Mobile, AL, United States.
RATIONALE: Nosocomial pneumonia induces lung endothelial amyloid production. These amyloid species are cytotoxic, self-replicating, and transmissible. They are insensitive to boiling, proteases, RNAse, and DNAse. Thus, infection-induced cytotoxic endothelial amyloids represent a form of prion disease. Emerging evidence suggests some endothelial amyloids contribute to end-organ dysfunction in the aftermath of critical illness whereas other amyloids have been implicated in the innate immune response to infection. However, mechanisms of host-pathogen interactions underlying production of endothelial amyloids remain unclear. Here, we tested the hypothesis that Pseudomonas aeruginosa type III secretion system (T3SS) effector intoxication of pulmonary endothelium is sufficient to elicit the release of cytotoxic amyloid prions. METHODS: To test this hypothesis, P. aeruginosa strains both with (PA103 and PA01) and without (ΔPcrV) a functional T3SS were used to infect pulmonary microvascular endothelial cells (PMVECs) for 4 hours at an MOI of 20:1 in HBSS. Supernatants were collected, centrifuged, filter-sterilized, and transferred to monolayers of naïve PMVECs. The A11 (anti-amyloid oligomer) and T22 (anti-tau oligomer) antibodies were used to immunodeplete oligomeric species from supernatants. Antibody captured oligomers were eluted using 4 M MgCl2 and then boiled, extensively dialyzed against HBSS, and filter-sterilized. Eluted amyloid oligomers were then applied to naïve PMVECs to assess amyloid-dependent damage. Antimicrobial characteristics were assessed using standard Kirby-Bauer antibiotic sensitivity assays with disk inoculants standardized to a protein concentration of 10 µg. Amyloid constituents of supernatants were visualized via Congo red staining. Endothelial gap formation was quantified using custom macros written in ImageJ (NIH). RESULTS: Supernatant obtained from both PA103- and PA01-infected cells was cytotoxic. Amyloid and tau antibody neutralization significantly abrogated cytotoxicity, and cytotoxicity was restored by adding back eluted amyloid and tau oligomers. In contrast, supernatant obtained from ΔPcrV-infected cells was not cytotoxic. Consequently, we examined the antimicrobicity of infection-induced endothelial amyloids. In contrast to the cytotoxicity assay, ΔPcrV-derived supernatant effected extensive growth inhibition, whereas PA103 derived supernatant had little effect and PA01-derived supernatant had no effect. ΔPcrV supernatant-induced inhibition progressively increased over a 72-hour time course. Supernatant derived from ΔPcrV infection exhibited antimicrobial activity against Pseudomonas spp., methicillin-resistant Staphylococcus aureus, Klebsiella pneumoniae, and Candida albicans. Antimicrobicity of supernatant obtained from ΔPcrV infection was attenuated through amyloid oligomer immunodepletion but increased via tau oligomer immunodepletion. CONCLUSIONS: These data indicate that T3SS effectors promote endothelial amyloid cytotoxicity while, provocatively, suppressing antimicrobicity. This work provides opportunity to enrich novel antimicrobial compounds as therapeutics for antibiotic-resistant organisms.