Soluble adenylyl cyclases generate a cAMP signal that breaks down microtubules leading to endothelial cell barrier disruption. The P. aeruginosa type 3 secretion system effector exoenzyme Y is one such soluble cyclase. However, we have discovered that this enzyme is a promiscuous nucleotidyl cyclase capable of generating both purine and pyrimidine (i.e. non-canonical) cyclic nucleotide monophosphates, including cGMP, cUMP, and cAMP. The cAMP signal, and to a lesser extent the cGMP signal, activates protein kinase A, which phosphorylates tau leading to microtubule breakdown. Phosphorylated tau accumulates inside the endothelium for several hours as a high molecular weight, or oligomeric, form, and is then released from the cell into the supernatant in vitro, and bronchoalveolar lavage and blood in vivo. Preliminary data suggest cellular release of high molecular weight tau is stimulated by cUMP. Our previously published work and recent preliminary studies indicate high molecular weight tau retrieved from cellular supernatant fractions is heat stable, protease resistant, insoluble in certain detergents, insensitive to RNase and DNase treatments, and can be resolved in a 30-50% ammonium sulfate fraction by column chromatography. It can be sedimented by centrifugation with an angular momentum of 1.14 x 1012. This high molecular weight tau is transmissible between cells, leading to inter-endothelial cell gap formation, increased permeability and cytotoxicity; pulmonary microvascular endothelial cells are especially sensitive to this injury. Its cytotoxicity is inactivated by phenol extraction, by post-translational modification with diethylpyrocabonate, and with hexafluoro-2- propanol, all characteristics of amyloid proteins. We have searched for a means to prevent the transmissible cytotoxicity as an anti-inflammatory therapy. We have recently discovered that prion protein is expressed in lung endothelium. Whereas prion antibody treatment initiates a pro-survival signal and prevents tau-induced hyperpermeability and cytotoxicity, prion protein genetic deletion increases endothelial sensitivity to the high molecular weight tau. Hence, this proposal tests the hypothesis that ExoY generates purine and pyrimidine cyclic nucleotides in endothelium and induces release of high molecular weight tau capable of generating transmissible hyperpermeability and cytotoxicity, an effect prevented by antibody ligation of prion protein.

Public Health Relevance

Endothelial cells line blood vessels and form a barrier that separates blood from tissue. In infection, this endothelial cell barrier is disrupted leading to the accumulation of fluids outside of the blood vessels, which compromises tissue function. Here, we resolve a novel mechanism of endothelial cell injury that perpetuates lung fluid accumulation, and we identify a new target for treatment of this important clinical problem.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
2P01HL066299-16A1
Application #
9416709
Study Section
Heart, Lung, and Blood Initial Review Group (HLBP)
Program Officer
Xiao, Lei
Project Start
Project End
Budget Start
2017-09-01
Budget End
2018-08-31
Support Year
16
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of South Alabama
Department
Type
DUNS #
172750234
City
Mobile
State
AL
Country
United States
Zip Code
36688
Khozhukhar, Natalya; Spadafora, Domenico; Rodriguez, Yelitza et al. (2018) Elimination of Mitochondrial DNA from Mammalian Cells. Curr Protoc Cell Biol 78:20.11.1-20.11.14
Leavesley, Silas J; Sweat, Brenner; Abbott, Caitlyn et al. (2018) A theoretical-experimental methodology for assessing the sensitivity of biomedical spectral imaging platforms, assays, and analysis methods. J Biophotonics 11:
Lin, Mike T; Balczon, Ron; Pittet, Jean-Francois et al. (2018) Nosocomial Pneumonia Elicits an Endothelial Proteinopathy: Evidence for a Source of Neurotoxic Amyloids in Critically Ill Patients. Am J Respir Crit Care Med :
Parker, James C (2018) Mitochondrial damage pathways in ventilator induced lung injury (VILI): an update. J Lung Health Dis 2:18-22
Balczon, Ron; Morrow, K Adam; Zhou, Chun et al. (2017) Pseudomonas aeruginosa infection liberates transmissible, cytotoxic prion amyloids. FASEB J 31:2785-2796
Shokolenko, Inna N; Alexeyev, Mikhail F (2017) Mitochondrial transcription in mammalian cells. Front Biosci (Landmark Ed) 22:835-853
Morrow, K Adam; Frank, Dara W; Balczon, Ron et al. (2017) The Pseudomonas aeruginosa Exoenzyme Y: A Promiscuous Nucleotidyl Cyclase Edema Factor and Virulence Determinant. Handb Exp Pharmacol 238:67-85
Blair, Leslie A; Haven, April K; Bauer, Natalie N (2016) Circulating microparticles in severe pulmonary arterial hypertension increase intercellular adhesion molecule-1 expression selectively in pulmonary artery endothelium. Respir Res 17:133
Spadafora, Domenico; Kozhukhar, Natalia; Alexeyev, Mikhail F (2016) Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number. PLoS One 11:e0152705
Jian, Ming-Yuan; Liu, Yanping; Li, Qian et al. (2016) N-cadherin coordinates AMP kinase-mediated lung vascular repair. Am J Physiol Lung Cell Mol Physiol 310:L71-85

Showing the most recent 10 out of 122 publications