VCOM Louisiana Research Day Program

Biomedical Research

Zachary Dickey, OMS-II 1 ; Rebekah Morrow, PhD 1 ; K. Adam Morrow, PhD 1 ; Chung-Sik Choi, PhD 2 ; Mike Lin, PhD 2 ; Troy Stevens, PhD 2 ; and Sarah Voth, PhD 1 1 Edward Via College of Osteopathic Medicine-Louisiana, Monroe, Louisiana; 2 University of South Alabama – Whiddon College of Medicine, Mobile, Alabama 02 THE ROLE OF SIRT1 IN THE PATHOGENESIS OF PSEUDOMONAS AERUGINOSA TOXIN EXOENZYME Y

Background: Nosocomial pneumonia describes the onset of pneumonia ≥ 48 hours post-admission and commonly arises in the critically ill. Pseudomonas aeruginosa is the most common cause of nosocomial pneumonia, which frequently gives rise to acute respiratory distress syndrome, sepsis, and consequent multiorgan damage. Sequelae often include long-term physical and cognitive impairment as well as increased rates of mortality with less than 50% of patients surviving the first year post discharge. Within the parenchyma of the lung, the alveolar epithelium and capillary endothelium are closely appositioned and joined by a shared basement membrane forming a thin respiratory barrier to facilitate efficient gas exchange. Virulent strains of P. aeruginosa possess a type III secretion system (T3SS) that functions like a needle and syringe to inject toxins directly into the host cytosol. Roughly 90% of P. aeruginosa clinical isolates utilize a T3SS-mediated nucleotidyl cyclase effector, exoenzyme Y (ExoY). ExoY intoxication generates unconstrained increases in cytosolic cyclic nucleotides (cNMP). In the endothelium of the pulmonary capillary (pulmonary microvascular endothelial cells, or PMVECs), ExoY primarily

microtubule collapse, and the production and release of oligomeric tau. However, the other mechanism(s) of ExoY pathogenesis are not well-understood. Normally, autophagy promotes the breakdown and clearance of dysregulated tau. In neurodegenerative disease, autophagic flux is inhibited and induces the accumulation and oligomerization of dysregulated tau in the endocytic pathway. Pathogenic tau oligomers are then exocytosed into the extracellular milieu where they propagate injury in a prion-like manner. The NAD+-dependent deacetylase sirtuin 1 (SIRT1) modulates mitochondrial function, autophagy, and innate immunity making it a likely target for ExoY-mediated inhibitory phosphorylation. Hypothesis: We hypothesize that ExoY intoxication inhibits lung endothelial SIRT1 to constrain autophagy and innate defense while promoting the production and release of pathogenic tau. Methods: To test our hypothesis, PMVECs will be infected with isogenic mutants of P. aeruginosa at an MOI of 20:1 both with and without pharmacologic inhibition/activation of SIRT1 over a range of concentrations. Exogenous caged cyclic nucleotide analogs (cGMP-AM and cUMP-AM) will be utilized in

the absence of infection to ascertain whether cytosolic increases in cNMPs alone are sufficient to inhibit SIRT1. Isogenic P. aeruginosa mutants include ∆PcrV (incompetent T3SS needle), ExoY K81M (ExoY incapable of cyclase activity), and ExoY + (injects ExoY only). Lysates will be collected over time and examined for 1) SIRT1 levels (immunoblot), 2) SIRT1 activation (immunoblot and ELISA), and 3) SIRT1 transcript (RT-qPCR). Cytokine release will be monitored via RT-qPCR and ELISA, autophagic flux will be assessed through probing lysates for LC3-I to LC3-II conversion, and tau release will be monitored via immunoblotting with validated antibodies against recombinant controls, thioflavin T fluorescence, and Congo Red birefringence. Cyclic nucleotides will be measured with ELISA. Results: TBD Conclusions: TBD

generates cGMP and cUMP, suppresses the innate immune response, induces

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