VCOM Louisiana Research Day Program Book 2024

Biomedical Research: Section 1

3 THE ROLE OF SER-214 PHOSPHORYLATED TAU IN LUNG ENDOTHELIAL HOST DEFENSE

Grace Corrier, OMS-II 1 *; Kassady Perkinson, OMS-II 1 *; Elizabeth Turnage, OMS-II 1 *; Ron Balczon, PhD 2,3 ; Chung-Sik Choi 3,4 , PhD; Mike T. Lin, PhD 3,4 ; Troy Stevens, PhD 3,4,5 ; Rebekah Morrow, PhD 6 ; K. Adam Morrow, PhD 1 ; Sarah Voth, PhD 1 * *Indicates equal contributions 1 Department of Cell Biology & Physiology, VCOM-Louisiana 2 Department of Biochemistry, University of South Alabama College of Medicine; 3 Center for Lung Biology, University of South Alabama College of Medicine; 4 Department of Physiology & Cell Biology, University of South Alabama College of Medicine; 5 Department of Internal Medicine, University of South Alabama College of Medicine; 6 Department of Microbiology & Immunology, VCOM-Louisiana

Background: Nosocomial pneumonia is a significant concern in the critical care setting. Lung infection promotes the release of pro inflammatory mediators and cytotoxins into both the distal airways and pulmonary circulation. Patients that recover from nosocomial pneumonia often suffer lasting physical and cognitive impairments post-discharge. Less than one third of those discharged will survive the first year following release. Endothelial derived hyperphosphorylated tau has recently been identified as one of the cytotoxins that comprise the inflammatory milieu generated by lung infection. Infection elicited tau is highly cytotoxic, self-propagating, and transmissible, constituting a tauopathy. Pseudomonas aeruginosa expressing a type III secretion system (T3SS) mediated toxin, exoenzyme Y (ExoY), is one of the most common agents of nosocomial pneumonia. ExoY intoxication of PMVECs induces hyperphosphorylation of lung endothelial tau at the Ser-214 residue which promotes increased permeability and a secondary proteinopathy. ExoY has also been reported to suppress the antimicrobial function of cytoprotective lung endothelial amyloid species. However, whether the contribution of endothelial tau and its phosphorylation status

contributes to this phenomenon remains poorly understood. Objective: ExoY generated endothelial tau is necessary to suppress innate amyloid function whereas Ser-214 phosphorylation significantly contributes to the ExoY mediated impairment of the host defense amylome. Methods: Wildtype and various genetically manipulated PMVECs were utilized for our studies including stable tau KO (global tau KO), control (tau KO carrying an empty plasmid), 1N4R (tau KO with plasmid encoding endothelial tau isoform 1N4R only), and S214A (tau KO harboring a plasmid encoding tau 1N4R with a serine-to-alanine point mutation at residue 214; S214A). PMVECs with plasmids were cultured in full media containing G418 for selection. Confluent monolayers of PMVECs were treated with either vehicle, a non-virulent strain of P. aeruginosa unable to inject ExoY into cells (∆PcrV), and an isogenic strain capable of injecting ExoY (ExoY+) alone into cells. PMVECs were infected with a multiplicity of infection of 20:1 in HBSS and incubated for 6 h. Supernatant fractions were collected at 6 h post-infection, filter-sterilized, and centrifuged in concentrators

with a 3 kDa MW cutoff. Concentrated supernatants were standardized to 0.5 mg/ml of protein in HBSS. Samples were then boiled and cold shocked to denature non-amyloid proteins prior to use in assays. Antimicrobicity was measured through Kirby-Bauer disk diffusion, kinetic bactericidal assays, aggregation assays, and Syto9/propidium iodide staining. Tau species were assessed via immunoblotting and amyloid burden was assessed with Thioflavin T, Congo Red birefringence, and immunoblotting. Results: TBD Conclusions: TBD

15 2024 Via Research Recognition Day