Louisiana Research Day Program Book 2025

Biomedical Research: Section 1

Biomedical Research: Section 1

Jedidiah Lim 1 ; David Kang 1 ; Victoria Lucas 1 ; Samreen Shah 1 ; Sherine Thomas 1 ; Carlie Christ 1 ; Meredith Gwin, PhD 2 ; Samir Gautam, MD, PhD 2 ; Melissa Lipsmeyer, PhD 1 ; Rebekah Morrow, PhD 3 ; K. Adam Morrow, PhD 1 ; Sarah Voth, PhD 1 1 Cell Biology and Physiology, VCOM-Louisiana; 2 Pulmonary, Critical Care, and Sleep Medicine, Yale School of Medicine; 3 Microbiology and Immunology, VCOM-Louisiana 12 PSEUDOMONAS INDUCED ARDS PROMOTES INTRAPULMONARY MICROTHROMBUS FORMATION AND ACUTE LUNG INJURY SECONDARY TO CYSTATIN-C DEPLETION

Zachary Dickey, OMS-IV; Annie Pham, OMS-III; Krishna Patel, BS; Melissa Lipsmeyer, PhD; Kasia Michalak, MSc; Troy Stevens, PhD; Rebekah Morrow, PhD; K. Adam Morrow, PhD; Sarah Voth, PhD Dept of Cell Biology and Physiology, VCOM; Dept of Physiology & Cell Biology, USA COM; Dept of Microbiology & Immunology, VCOM 13 PSEUDOMONAS AERUGINOSA TOXIN ExoY INHIBITS SIRT1-MEDIATED AUTOPHAGY TO PROMOTE PULMONOGENIC TAUOPATHY

Rationale: Acute respiratory distress syndrome (ARDS) is a critical respiratory condition caused by the accumulation of fluid in the lungs which impairs gas exchange and ultimately leads to respiratory failure. ARDS is commonly caused by infections from microorganisms, most notably Pseudomonas aeruginosa. Strains responsible for acute infection utilize a type III secretion system (T3SS) to inject toxic exoenzymes directly into host cells. Approximately 90% of clinical strains expressed involve exoenzyme Y (ExoY). Intoxicated cells are afflicted with tau hyperphosphorylation, cytoskeletal collapse, and the release of pathogenic tau into the extracellular space. In the absence of ExoY elicited release of cytotoxic tau, endothelial amyloids including amyloid-beta (A β ) are cytoprotective, antimicrobial, and stabilize the endothelial cell microtubule network to establish barrier integrity within the lungs. Cytotoxic tau disrupts the innate function of endothelial amyloids, suppresses antimicrobicity, and collapses the microtubule barrier in the lungs, leading to the dissemination of cytotoxic tau and beta-amyloid into the circulation of other peripheral organs causing end-organ damage. Cystatin C (CysC), a ubiquitous

cysteine protease inhibitor, maintains amyloid homeostasis. In critically ill patients, blood and urine proteomics indicate that serum CysC is markedly depleted through ARDS-induced inflammatory proteinuria, yet no cause, remedy, or mechanism of this critical care phenomenon has been identified. In these studies, we investigated whether ExoY-competent P. aeruginosa infection induces intrapulmonary microthrombus formation and acute lung injury with concomitant loss of serum CysC. Methods: Male and female wildtype C57BL/6J mice (10-12 weeks old) were infected intratracheally with either vehicle or 1 x 105 colony forming units (CFU) of bacteria in 40 µl of PBS. P. aeruginosa strain ExoY+ (secretes only ExoY in host cells) was used for infections. Weight, urine, and PT/INR were collected daily, and survival was tracked. At 48 hours post-infection, surviving mice were sacrificed. Urine was collected via cystocentesis, blood was collected via cardiac puncture, and the brain, kidneys, heart, and lungs were fixed for histological analysis (H&E and Congo Red). Bronchoalveolar lavage fluid was collected, protein concentration measured, and cell

populations determined via Coulter Counter. CysC levels of the blood and urine were measured via ELISA and immunoblotting. Amyloid and tau levels were assessed via immunoblotting and thioflavin T.

Rationale: Survivors of nosocomial pneumonia often suffer an acute-to-chronic disease transition. Despite resolution of active infection, secondary end-organ damage promotes chronic illness and decreased longevity. Accumulating evidence implicates prion like cytotoxic tau elicited from infected lung capillary endothelium as a notable contributor to this phenomenon. Common nosocomial Pseudomonas aeruginosa utilizes a type III secretion system (T3SS) during acute lung infection. Roughly 90% of clinical isolates express the T3SS toxin Exoenzyme Y (ExoY). ExoY induces unregulated cNMP production, tau hyperphosphorylation, cytoskeletal collapse, antimicrobial amyloid loss-of-function, and the generation of prion-like cytotoxic tau. However, the mechanisms responsible remain unclear. Sirtuin 1 (SIRT1) deacetylase is intricately linked to cellular bioenergetics including autophagy, a fundamental means of clearing dysregulated protein. Here, we tested the hypothesis that ExoY intoxication constrains SIRT1-mediated autophagy to promote the production and release of cytotoxic tau.

cells were serum starved and infected with isogenic strains of P. aeruginosa at an MOI of 20:1 in HBSS with/without the clinical SIRT1 agonist SRT2104. Strains included a mutant incapable of generating cNMPs (ExoYK81M) in host cytosol and a mutant that injects only cNMP-competent ExoY (ExoY+) into host cells. At 4 hours post-infection, whole-cell lysates, RNA, and supernatants were collected. Supernatants were filter-sterilized, boiled, and iced. Lysates were immunoblotted for SIRT1, Tau (TNT-1 antibody), and LC3, and supernatants probed for tau. Transcript levels of SIRT1, Tau, and LC3 were measured using RT-qPCR and immunofluorescence studies determined the cellular localization of SIRT1, Tau, and LC3. Antimicrobial studies (e.g., Kirby Bauer) and cytotoxicity assays (e.g., resazurin) were utilized to determine the downstream impact of cell supernatants. Results: ExoY intoxication significantly diminished protein and transcript levels of SIRT1 while abolishing LC3 in PMVECs as compared controls. The supernatant-to-lysate ratio of tau protein in ExoY+ infected cells was more than double that of controls. ExoY-intoxicated

cells treated with SRT2104 exhibited complete rescue of LC3 and SIRT1 levels, robust LC3-I to LC3-II conversion, and tau was ablated in the supernatant fraction. Immunofluorescence revealed SRT2104 treatment of ExoY-intoxicated cells promotes tau co-localization with LC3 equivalent to vehicle. Supernatants from ExoY-intoxicated cells were cytotoxic whereas SRT2104 treatment produced ExoY-generated cell supernatants with antimicrobicity equivalent to controls.

Results: TBD

Conclusions: TBD

Methods: Pulmonary microvascular endothelial

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2025 Research Recognition Day