Studies on the Physical Properties of the Pseudomonas aeruginosa‐induced Endothelial Prion Amyloid Cytotoxin(s)

Abstract

RationalePseudomonas aeruginosa lung infection targets capillary endothelium and induces the production of prion amyloid cytotoxin(s), including a high molecular weight tau. These amyloid proteins are heat stable, RNase and DNase insensitive, and protease resistant. They are self‐replicating and transmissible among naïve cells. The physical nature of these amyloid cytotoxins remains incompletely resolved. Here, we examined the amyloid cytotoxin's angular velocity, and determined its degree of protease sensitivity and responsiveness to inactivation by post‐translational modification.MethodsA P. aeruginosa strain, PA103, was used to infect pulmonary microvascular endothelial cells (PMVECs) for 4 hours, at which time cell supernatant was collected, centrifuged (4,500 × g for 10 minutes at room temperature), and filter sterilized (0.22 μm filter). Supernatant was divided into aliquots for centrifugation and sedimentation experiments, for protease treatment, and for post‐translational modification using diethylpyrocarbonate (DEPC). Centrifugation experiments were conducted at 150,000 × g (4°C) over a 24‐hour time course. Supernatant was exposed to proteinase K (100 and 500 μg/mL) digestion for 24 hours. DEPC (1, 10 and 20 mM) was incubated with supernatant for 30 minutes at room temperature for post‐translational modification of histidine residues. After these independent treatments, supernatant was added to PMVECs and gap formation and cytotoxicity (e.g. LDH release assay) determined. We used a custom plugin for ImageJ (NIH) software to automatically quantify intercellular gaps in images. The ratio of black to white pixels within each image was then measured to determine cell gap area.ResultsSupernatant samples from 0 (e.g. control), 1, 2, 4, 8, 16 and 24 hours of centrifugation were placed on PMVECs, and gap formation and cytotoxicity were measured. 0–4 hours centrifugation did not reduce cytotoxicity, whereas 8–24 hours centrifugation nearly eliminated cytotoxicity. Calculation of the angular velocity revealed the endothelial‐derived cytotoxin is small and/or less dense (1.14 × 1012) than the scrapie agent (3 × 1010), consistent with identification of a novel amyloid cytotoxin. The endothelial cytotoxin was extremely protease insensitive, as 24‐hour treatment with proteinase K was insufficient to eliminate its cytotoxicity. However, treatment with DEPC (10 mM), which post‐translationally modifies histidine side chains, eliminated cytotoxicity of the endothelial amyloid cytotoxin, similar to its effect on the scrapie agent.ConclusionP. aeruginosa infection of pulmonary endothelial cells causes production of a novel prion amyloid cytotoxin that is protease resistant and sensitive to post‐translational modification using DEPC.Support or Funding InformationNational Institutes of Health HL66299 and HL60024, American Heart Association 16POST27250139