VCOM Louisiana Research Day Program Book 2024

Biomedical Research: Section 1

Zachary Dickey, OMS-III 1 , Kiara Pankratz, OMS-II 1 *, Annie Pham, OMS-II 1 *, Elizabeth Kibodeaux, OMS-II 1 *, Dara W. Frank, PhD 2 , Troy Stevens, PhD 3 , Rebekah Morrow, PhD 1 , K. Adam Morrow, PhD 1 , Sarah Voth, PhD 1 *Indicates equal contributions 1 VCOM-Louisiana; 2 Medical College of Wisconsin; 3 University of South Alabama Whiddon College of Medicine 4 SIRT1 ACTIVATION ABLATES TAU RELEASE DURING EXOY-COMPETENT PSEUDOMONAS AERUGINOSA INFECTION

Background: Pseudomonas aeruginosa is one of the most common agents of nosocomial pneumonia and acute respiratory distress syndrome (ARDS). While mortality rates in ARDS have improved, survivors often face significant chronic physical and cognitive impairment. P. aeruginosa strains responsible for acute infection of the distal airways usually have a syringe-and-needle type III secretion system (T3SS) that delivers a combination of up to 3 of a potential 4 toxins directly into the cytosol of host cells. The T3SS-mediated toxin most frequently utilized in acute lung infection is the nucleotidyl cyclase, exoenzyme Y (ExoY). ExoY produces marked disruption of interendothelial junctions, edema, and suppression of the innate immune response. Moreover, ExoY intoxicated endothelium generates the release of cytotoxic endothelial-derived amyloids including hyperphosphorylated tau. ExoY elicited amyloids propagate damage independently of the primary infection and transmit injury from cell-to-cell and host-to-host thereby constituting a proteinopathy. However, the mechanisms germane to ExoY induced cytotoxic amyloid release remains poorly resolved. The sirtuin 1 (SIRT1) NAD+ dependent deacetylase has been implicated in the regulation of the autophagic degradation of dysregulated amyloidogenic proteins. Thus, activating SIRT1 may present a viable option for alleviating the cytotoxic amyloid burden arising from lung infection. Objective: In this study, we tested whether pharmacologic activation of SIRT1 would reduce the

pathogenic tau burden elicited secondary to ExoY competent infection. Methods: Our studies utilized rat pulmonary microvascular endothelial cells (PMVECs). Bacteria included strains expressing either ExoY (ExoY+) alone or an isogenic activity-null mutant (ExoYK81M). PMVECs were infected at a multiplicity of infection (MOI) of 20:1 in HBSS. To ascertain the potential role of SIRT1, we identified a particularly selective SIRT1 agonist (SRT2104) with negligible affinity for other sirtuin proteins as well as a highly selective SIRT1 inhibitor, EX-527. Infection experiments were conducted with and without SRT2104 and EX-527. Initial experiments were conducted over a range of concentrations for SRT2104 (1, 3, and 6 µM) and EX 527 (5, 10, and 20 µM). Infections were imaged over time to assess signs of toxicity and interendothelial disruption. To determine whether the drug killed the bacteria directly or conferred protection to the cells despite bacterial doubling, aliquots from ExoY+ infections were serially diluted and plated over time. Lysates and supernatants were collected at 0 and 6 h post-infection and probed for pathogenic tau harboring a phosphatase-activating domain (PAD) with the TNT1 antibody, amyloid oligomers (A11 antibody), and SIRT1. Supernatants were also filter-sterilized, boiled, ice shocked, and applied to naïve cells to determine the potential for transmissible cytotoxicity. Results: ExoY intoxication alone was sufficient to significantly impair levels of SIRT1 in whole cell

lysates while amplifying pathogenic tau export into the supernatant fraction. Activation of SIRT1 via treatment with SRT2104 during ExoY infection notably reduced intracellular tau and ablated ExoY generated PAD-bearing tau in the extracellular space. SIRT1 inhibition (EX-527 treatment) during infection with the activity null mutant ExoYK81M was sufficient to increase intra- and extracellular PAD tau as compared to the untreated control. Tau was not observed in ‘cell free’ infection experiments. ExoY generated oligomeric amyloid constituents in the supernatant fraction were likewise abrogated with SIRT1 agonist SRT2104. In transmissibility experiments, neither the vehicle- nor ‘cell free’ derived supernatants induced injury. However, heat-stable transmissible cytotoxins generated from ExoY competent induced significant injury, toxicity, and cell death. Damage from boiled/ shocked supernatants from SRT2104 treated ExoY+ infections of PMVECs were commensurate with those from cyclase-null control strain ExoYK81M infection. Conclusions: ExoY intoxication of PMVECs suppresses endothelial SIRT1 and disrupts the turnover of pathogenic amyloid species to promote PAD-harboring tau production and release. Moreover, SIRT1 activation during ExoY-competent infection mitigates the burden of PAD in PMVECs and protects downstream endothelium from secondary amyloid/ tau mediated injury. Thus, the agonist SRT2104 may provide a clinically relevant means of preventing the acute secondary injury and long-term sequelae common to survivors of nosocomial pneumonia/ARDS.

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