VCOM Louisiana Research Day Program Book 2024

Biomedical Research: Section 1

Annie Pham, OMS-II; Kiara Pankratz, OMS-II; Zachary Dickey, OMS-III; Elizabeth Kibodeaux, OMS-II; Erika Lytle, OMS-III; Dara W. Frank, PhD; Troy Stevens, PhD; Rebekah Morrow, PhD; K. Adam Morrow, PhD; Sarah Voth, PhD VCOM-Louisiana; Medical College of Wisconsin; University of South Alabama Whiddon College of Medicine 6 EXOY INTOXICATION SUPPRESSES LUNG ENDOTHELIAL TAK1 TO PROMOTE TAUOPATHY

Background: Nosocomial pneumonia is of particular concern in the critical care setting. Acute opportunistic infection of the distal lung promotes edema, hypoxia, and acute respiratory distress syndrome (ARDS). Both nosocomial pneumonia and ARDS are associated with poor outcomes and reduced longevity among survivors post-discharge. After release, many suffer an acute-to-chronic disease transition with marked functional impairments both physically and neurologically. The Gram negative nosocomial pathogen Pseudomonas aeruginosa is the predominant agent responsible for nosocomial pneumonia and ARDS. Over 90% of virulent clinical isolates of P. aeruginosa utilize a syringe-and-needle like type III secretion system (T3SS) to inject the nucleotidyl cyclase toxin exoenzyme Y (ExoY) into the cytosol of target cells. ExoY generates unconstrained cyclic nucleotides with subsequent breach of the lung air-blood barrier and subversion of the host’s innate immune defense. ExoY has been reported to suppress the innate immune response via inhibition of transforming growth factor- β -activating kinase 1 (TAK1) in some cell types. Pulmonary microvascular endothelial cells (PMVECs) intoxicated with ExoY release virulent tau cytotoxins that propagate injury and are transmissible among species indicative of a prion

strain. In our studies, we have sought to clarify the role of TAK1 in the production of ExoY-generated cytotoxic tau. Objective: We hypothesize that ExoY intoxication of PMVECs inhibits TAK1 to promote production of pathogenic tau. Methods: For infection studies we used isogenic strains of P. aeruginosa expressing 1) an incompetent T3SS needle (∆PcrV; cannot form the translocation pore required to facilitate toxin injection), 2) an activity-null mutant unable to generate cyclic nucleotides in the host cell (ExoYK81M), and a mutant that injects only virulent ExoY (ExoY+). Secondary rat pulmonary microvascular endothelial cells (PMVECs) were infected at a multiplicity of infection (MOI) of 20:1 in HBSS and incubated for 5 hours. Vehicle and ‘no cell’ infection wells were used as controls. Infected cells were imaged over time to assess signs of toxicity and endothelial barrier disruption. Whole cell lysates and cell supernatants were collected at 0 and 5 h post infection. Subsequently, lysates and supernatants were probed for TAK1, endothelial tau harboring a phosphorylation-activating domain (PAD) using the TNT1 antibody, and p65 NF-kB. For lysates, protein concentration was determined using a standard BCA assay and 30 µg of protein

was loaded for each sample. Blots of lysates were normalized to GAPDH. Supernatants were precipitated using TCA-DOC, loaded by volume, and normalized to total protein. Cadaveric Alzheimer’s hippocampal lysate and recombinant proteins were used as controls. Results: In lysates, TAK1 and p65 NFkB were elevated above vehicle for control strains ∆PcrV and ExoYK81M infected cells. However, TAK1 levels were abrogated in ExoY intoxicated cells. ExoY+ infection depressed p65 NFkB in PMVECs. TNT1-reactive tau was elevated above vehicle in ExoY intoxicated cell lysates and increased nearly three-fold in the supernatant fraction collected from ExoY+ infected PMVECs. No TNT1 signal was detected in ‘no cell’ infection controls. Activation of upstream sirtuin 1, a known regulator of TAK1 mediated signaling, rescued TAK1 activity in ExoY infected cells and ablated endothelial derived PAD-harboring tau release. Conclusions: These data implicate ExoY in the suppression of the TAK1 immune regulatory protein in lung endothelium. Moreover, our results shed light on the signaling pathway(s) germane to ExoY-induced pathogenesis. Taken together, our findings suggest that ExoY likely inhibits sirtuin 1 to constrain TAK1 modulated innate immunity and generate endothelial derived tau cytotoxins.

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