VCOM Louisiana Research Day Program Book 2024

Biomedical Research: Section 1

Sabra Hanna, MLS (ASCP), OMS-III 1 ; Brant Wagener, MD, PhD 2 ; Jean-Francois Pittet, MD 2 , Stephen A. Moser, MD, PhD 3 ; Chung-Sik Choi, PhD 4 ; Mike Lin, PhD 4,5 ; Troy Stevens, PhD 4,5,6 ; Rebekah Morrow, PhD 7 ; K. Adam Morrow, PhD 1 ; Sarah Voth, PhD 1 1 Cell Biology & Physiology Dept, VCOM 2 Dept of Anesthesiology & Perioperative Medicine, UAB School of Medicine; 3 Dept of Pathology, UAB School of Medicine; 4 Dept of Physiology & Cell Biology, Univ of South Alabama COM; 5 Center for Lung Biology, Univ of South Alabama COM; 6 Dept of Internal Medicine, Univ of South Alabama COM; 7 Microbiology & Immunology Dept, VCOM 11 KLEBSIELLA PNEUMONIAE ISOLATED FROM CRITICALLY ILL PATIENT WITH NOSOCOMIAL PNEUMONIA ELICITS A PROTEINOPATHY

Background: Klebsiella pneumoniae is a Gram negative enteric bacterium and a common agent of nosocomial pneumonia. Clinical strains are often highly virulent and infiltrate the pulmonary microvasculature to promote acute respiratory distress syndrome, bacteremia, and dissemination and/or damage to downstream organs. The World Health Organization has deemed K. pneumoniae a ‘Critical Priority’ pathogen due to its alarming levels of multidrug resistance, ever increasing incidence in the healthcare setting, and the severity of associated morbidity. Survivors of nosocomial pneumonia are often plagued with long-term impairments, including neurocognitive dysfunction, following discharge. Virulent P. aeruginosa infection elicits lung endothelial tau that is cytotoxic, self replicating, and transmissible, all characteristics of a tauopathy. However, whether K. pneumoniae infection of lung endothelial cells generates a tauopathy remains unknown. Objective: We hypothesize that lung endothelial cell infection with K. pneumoniae promotes the release of pathogenic tau that contributes to an infection induced proteinopathy. Methods: ATCC strain 43816 and a clinical isolate of K. pneumoniae were used for our studies. The clinical isolate was isolated from the bronchoalveolar lavage of a patient with a monomicrobial nosocomial pneumonia, de

identified, and characterized as Kp 1-008. Rat pulmonary microvascular endothelial cells (PMVECs) were used for infection and barrier studies. Tau-/- and control PMVECs were generated with CRISPR/Cas9. Control cells underwent the gene editing process but did not result in gene deletion. Encoding and expression of the tau gene was confirmed and monitored via sequencing, RT-qPCR, and immunoblotting. PMVECs were infected with Kp 1-008 at a multiplicity of infection (MOI) of 20:1, 40:1, and 60:1 then imaged at 0-, 3-, and 6-hours post-infection. Supernatants were collected at ~ 6 h post-infection, filter sterilized, boiled, and cold-shocked to denature non-amyloid proteins and transferred to naive wild-type cells and imaged over time to determine transmissibility. Infection experiments in transwell assays (PC; 0.4 µm) were used to ascertain barrier permeability over time. We immunoblotted for tau expressing a phosphorylation associated domain (PAD; TNT1 antibody) to measure tau burden in the supernatant fraction. Congo Red staining and polarized microscopy were used to visualize amyloid proteins. Results: Primary infection of wildtype, control, and tau-/- PMVECs with clinical isolate Kp 1-008 induced interendothelial gap formation by 6 hours (n = 6; 20:1, 40:1, 60:1). Surprisingly, infection with ATCC strain 43816 did not induce gap formation even when carried out to 24 hours (n = 9; MOI 20:1, 40:1, 60:1). Plating confirmed active bacterial

doubling during infection. Although strain 43816 is highly cited as readily breaching mouse endothelia and disseminating infection systemically in lung infection studies, it was unable to breach a barrier comprised of rat derived PMVECs. Kp 1-008 infection in transwell studies revealed increasing permeability in a dose and time dependent manner, with significant increases by 4 h post-infection (p = 0.003, n = 3, two-way ANOVA with Tukey’s post hoc). PAD harboring tau and oligomeric amyloid burden increased in the supernatant fraction over time and with increasing MOI during infection with control PMVECs. However, tau was not observed in supernatant collected from either infection suspension alone (i.e., no cells were present in the well) or tau-/- cells at any MOI or timepoint. Moreover, sterile infection supernatants collected from wildtype and control PMVECs, but not from ‘no cell’ or tau-/- PMVECs, were markedly cytotoxic to naïve cells. Conclusions: The outcome of our studies brings into question the suitability of mice and/or ATCC strain 43816 as a viable model for lung infection studies focused on an acute respiratory distress syndrome model. These data also suggest that Kp 1-008 is a virulent and translationally relevant strain of K. pneumoniae capable of inducing the cytotoxic endothelial tau release characteristic of lung infection derived proteinopathy.

23 2024 Via Research Recognition Day