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A7615 - Nosocomial Lung Infection Induces a Pulmonogenic Prionopathy that Disrupts the Endothelial Barrier of the Brain
Author Block: S. B. Voth, M. Lin, R. Balczon, C. M. Francis, T. Stevens; University of South Alabama, Mobile, AL, United States.
RATIONALE: Pneumonia is the most common healthcare-acquired infection among the critically ill. Following discharge, patients frequently experience increased rates of mortality, end-organ damage, and neurocognitive decline. Opportunistic bacterial pathogens Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and Klebsiella pneumoniae comprise the majority of nosocomial lung infections. Intoxication of pulmonary microvascular endothelial cells (PMVECs) with P. aeruginosa’s exoenzymes Y or U (ExoU) is sufficient to instigate the release of self-propagating cytotoxic amyloid oligomers. Once elicited from the pulmonary endothelium, these amyloid prions may spread hematogenously to organ vascular beds. However, the potential for pulmonogenic amyloid prions to disrupt the blood-brain barrier has not previously been assessed. Here, we tested the hypothesis that infection of PMVECs with P. aeruginosa, S. aureus, or K. pneumoniae is sufficient to produce amyloid prions capable of disrupting the cerebrovascular endothelial barrier. METHODS: Colony forming units were determined via plating experiments for accurate determination of multiplicity of infection (MOI). PMVECs were cultured in media with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and grown to confluence. Prior to infection, PMVEC monolayers were rinsed with Hank’s balanced salt solution (HBSS) and then infected with bacteria at an MOI of 20:1 in HBSS for 4 hrs. (P. aeruginosa strain PA103 - ExoU) or 9 hrs. (S. aureus and K. pneumoniae). The supernatants were collected, centrifuged, and filter-sterilized. The self-replicative potential and transmissibility of putative amyloid oligomer prions from bacterial-derived supernatants were confirmed via passaging experiments. Brain endothelial cells (BECs) were cultured in media with 20% FBS and 1% penicillin/streptomycin and grown to confluence. Confluent BEC monolayers were rinsed with HBSS, treated with clear media and filter-sterilized supernatants (1:3), incubated, and imaged at 0, 10, and 30 hours. A11 and T22 antibodies were used for immunodepletions. Custom macros written in ImageJ (NIH) were used to quantitatively assess gap formation in BEC monolayers post-treatment. RESULTS: Supernatants derived from bacteria-infected PMVECs contained oligomeric amyloid prions that induced significant gap formation in BEC monolayers. Respective supernatant cytotoxicity upon BEC monolayers ranked as follows: K. pneumoniae > S. aureus > P. aeruginosa (ExoU intoxication). The immunodepletion of amyloid oligomers abolished supernatant cytotoxicity. Conversely, antibody-captured amyloid oligomers eluted and applied to naïve BEC monolayers in media was sufficient to recapitulate original cytotoxic supernatant damage morphology. CONCLUSION: Bacterial infection of lung endothelium by common nosocomial pathogens is sufficient to elicit the formation and release of infectious amyloid prions capable of aggressively breaching the cerebrovascular endothelial barrier.