VCOM Louisiana Research Day Program

Biomedical Research

Sabra Hanna, MLS(ASCP), OMS-II 1 ; Rebekah Morrow, PhD 1 ; K. Adam Morrow, PhD 1 ; Brant Wagener, MD, PhD 2 ; Jean-Francois Pittet, MD 2 ; Chung-Sik Choi, PhD 3 ; Mike Lin, PhD 3 ; Troy Stevens, PhD 3 ; and Sarah Voth, PhD 1 1 Edward Via College of Osteopathic Medicine-Louisiana, Monroe, Louisiana; 2 University of Alabama – Birmingham, Heersink School of Medicine, Birmingham, Alabama; 3 University of South Alabama – Whiddon College of Medicine, Mobile, Alabama 04 PATHOGENESIS OF KLEBSIELLA PNEUMONIAE INDUCED PROTEINOPATHY IS AMYLOID STRAIN DEPENDENT

Background: Gram-negative opportunist Klebsiella pneumoniae has been designated a ‘Critical Priority’ pathogen by the World Health Organization due to its increasing prevalence in pneumonia and sepsis, as well as its evolving multidrug resistance. Infection with K. pneumoniae is reported to induce microtubule collapse and elicit the release of lung endothelial-derived amyloid-beta (A β ) and tau. K. pneumoniae -induced lung endothelial amyloids are heat-stable and transmissible in a prion-like manner. The mechanism(s) of these phenomena are poorly understood, and whether the propagation of the infection-generated proteinopathy is dependent upon tau and/or A β has not been investigated. Hypothesis: Here, we tested the hypothesis that virulent K. pneumoniae infection induces tau dysregulation, microtubule breakdown, increased barrier permeability, and the production and release of cytotoxic amyloids. Methods: CRISPR/Cas9 was utilized to generate tau-/- and APP-/- [APP: amyloid precursor protein (A β )] pulmonary microvascular endothelial cells. Gene deletion was confirmed and monitored via sequencing, RT-qPCR, and immunoblotting. A hypervirulent K. pneumoniae strain was isolated from the

bronchoalveolar lavage of a patient diagnosed with monomicrobial nosocomial pneumonia, deidentified, and characterized (Kp 1-008). Control, tau-/-, and APP-/- cells were infected at a multiplicity of infection of 40:1. Transwell assays were used to assess permeability and bacterial dissemination over time. Amyloids were monitored via immunoblotting with validated antibodies against recombinant controls, thioflavin T fluorescence, and Congo Red with birefringence. Infection supernatants were collected, filter-sterilized, boiled and cold shocked to denature non-amyloid proteins, and transferred to naïve wild-type cells to assess independent protein propagation potential. Cytoskeletal dynamics were monitored over time via immunocytochemistry (Anti-tau and anti tubulin antibodies for microtubules; phalloidin to label F-actin). Results: Kp 1-008 induced cytoskeletal disruption, increased permeability, and bacterial dissemination by 3-4 h post-infection. Sterile infection supernatants transferred to naïve cells induced marked barrier disruption, permeability, and cell death within 12 h post-treatment. Interestingly, supernatants from tau-/- and APP- /- cells induced different pathologies, suggesting that either tau or A β alone is sufficient to propagate injury in a strain-dependent manner.

However, maximal transmissible injury required both tau and A β together. Conclusion: K. pneumoniae induces cytoskeletal disruption to promote endothelial barrier permeability and dissemination while generating tau and A β cytotoxins. Moreover, either tau or A β alone are capable of propagating injury from cell-to-cell. However, K. pneumoniae infection-generated tau and A β synergize to facilitate the maximum virulence and potency of the consequent lung endothelial derived proteinopathy.

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