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Inflammasomes Expressed in Corneal Epithelial Cells are Targets of Pseudomonas aeruginosa Exotoxin S
Abby R Kroken1,2, Keith A Klein1, Rachel F Mazurek1, Patrick S Mitchell3, David J Evans4,5, Suzanne MJ Fleiszig5
1Department of Microbiology and Immunology, Loyola University, Chicago, Illinois; 2Department of Ophthalmology, Loyola University, Chicago, Illinois; 3Department of Microbiology, University of Washington. 4College of Pharmacy, Touro University, Vallejo, California; 5School of Optometry, University of California, Berkeley, California
Purpose: Pseudomonas aeruginosa is a leading cause of infectious keratitis. It can invade corneal epithelial cells and replicate within them. This may contribute to its ability to cause disease even when the corneal epithelium is intact, such as during contact lens use. Prior studies identified that the effector toxin ExoS is required for maintenance of these intracellular niches. When ExoS is deleted in P. aeruginosa, lytic host cell death occurs shortly after bacterial invasion. The type of regulated cell death occurring in corneal surface cells, and how ExoS subverts it, are currently unknown.
Methods: We explored which inflammasome pathways are active in telomerase-immortalized corneal epithelial cells (hTCEpi line) and used CRISPR-Cas9 genome editing to eliminate key components of inflammasome pathways. Time-lapse imaging of host cell invasion by fluorescent P. aeruginosa (strain PAO1) was used to measure host cell survival time. Analysis was aided by a computational analysis method to automate detection of invaded cells, such that >800 cells were routinely analyzed per experiment. IL-1β was monitored by ELISA and Western.
Results: Our findings show that rapid cell lysis upon bacterial invasion is dependent on the non-canonical inflammasome, caspase-4. Bacteria lacking exotoxins could proliferate in corneal epithelial cells where either caspase-4 or gasdermin D were deleted. In addition, we show that ExoS also reduces secretion of IL-1β, which is produced independently of the NLRP3 inflammasome. This suggests ExoS also interferes with an additional canonical inflammasome pathway.
Conclusions: Our data supports a model in which corneal epithelial cells can limit ocular colonization of invasive microbes through caspase-4-mediated pyroptosis, which would release intracellular bacteria into tear fluid. However, P. aeruginosa is equipped to block the lytic endpoint of host cell pyroptosis using ExoS and persists within intact corneal surface cells while suppressing cytokine secretion.
Disclosure: N
Support: Loyola University Chicago (Kroken), R01EY011221 (Fleiszig)
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