VCOM Louisiana Research Day Program

Biomedical Research

Erika Lytle, 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 07 EXOY IMPAIRS TAK1-MEDIATED ENDOTHELIAL AMYLOID INNATE DEFENSE

Background: Pseudomonas aeruginosa is a Gram-negative opportunist and the predominant agent responsible for nosocomial pneumonia. Survivors of nosocomial pneumonia suffer from increased rates of disability, morbidity, and mortality post-discharge. Non-virulent P. aeruginosa lung infection elicits endothelial derived cytoprotective amyloid species including broad-spectrum antimicrobials. However, virulent P. aeruginosa utilizes a type III secretion system (T3SS) to inject toxins directly into the cytoplasm of the target cell which inhibits the efficacy of innate endothelial-derived amyloids. T3SS effector exoenzyme Y (ExoY) is a nucleotidyl cyclase found in roughly 90% of clinical strains. In lung capillary endothelial cells, ExoY generates unconstrained cyclic nucleotides cGMP and cUMP. Further, ExoY has been reported to suppress the innate immune response via inhibition of transforming growth factor- β -activating kinase 1(TAK1). Lung microvascular endothelial cells intoxicated with ExoY also release virulent tau cytotoxins that propagate injury and are transmissible among species indicative of a prion strain. Hypothesis: We hypothesize that ExoY intoxication obstructs TAK1 to suppress endothelial amyloid antimicrobicity and promote production of pathogenic tau.

Methods: Control and tau-/- pulmonary microvascular endothelial cells (PMVECs) were infected with isogenic mutants of P. aeruginosa at an MOI of 20:1 both with and without inhibition of TAK1 via potent and selective (5Z)- 7-oxozeanol over a range of concentrations [4 nM, 8 nM (IC50), 16 nM]. Isogenic mutants include ∆PcrV (incompetent T3SS needle), ∆UT (patent needle but genetic deletion of canonical T3SS toxins), ExoY K81M (ExoY incapable of cyclase activity), and ExoY + (expresses and injects ExoY only). Exogenous caged cyclic nucleotide analogs (cGMP-AM and cUMP-AM) will be utilized in the absence of infection to ascertain whether cytosolic increases in these cNMPs alone are sufficient to inhibit TAK1. Amyloids were monitored via immunoblotting with validated antibodies against recombinant controls, thioflavin T fluorescence, and Congo Red birefringence. TAK1, NF- ƙ B, and cytokine levels were measured via immunoblotting and RT-qPCR, respectively. Cytokines and TAK1 activity were measured with ELISA. Amyloid and tau function (antimicrobial vs. pathogenic) will be ascertained through standard antimicrobial and cytotoxicity assays. Results: The release of lung endothelial-derived innate amyloids and cytokines were decreased in direct proportion to the inhibition of TAK1

in a dose-dependent manner. Probing of non virulent infection-derived cell supernatants for antimicrobial amyloid oligomers decreased concordantly with pharmacologic inhibition. Likewise, thioflavin T studies revealed a dose dependent decrease in the release of amyloid species. ExoY intoxication alone inhibited TAK1 activity, transcript, and expression. Moreover, pharmacologic inhibition of TAK1 was sufficient to induce pathogenic tau production and inhibit antimicrobial amyloid release in fidelity with ExoY intoxication. Conclusion: Taken together, these data indicate that T3SS nucleotidyl cyclase effector ExoY inhibits TAK1 to impair host defense and promote a virulent tauopathy secondary to P. aeruginosa infection.

19 2023 Via Research Recognition Day