Date of Award
Fall 9-17-2025
Degree Type
Thesis
Degree Name
Master of Science (MS)
Department
Microbiology and Immunology
First Advisor
Abby Kroken
Abstract
Pseudomonas aeruginosa is a leading contributor to nosocomial infections. Generally thought of as an extracellular pathogen, a growing number of studies have observed P. aeruginosa surviving and replicating within host cells. Investigations characterizing the intracellular lifestyle of P. aeruginosa found two populations: cytoplasmic bacteria replicating in a manner requiring the type-three secretion system (T3SS) and the effector ExoS, while others remain in a T3SS negative state and reside within host cell vacuoles without inducing host cell death. Here, we used experimental models to test our hypothesis that P. aeruginosa strains encoding the phospholipase ExoU (instead of ExoS) may also be internalized and are able to persist within compartments if they remain negative for T3SS effector secretion. We primarily investigated two lab strains (PA103 and PA14) and two clinical isolates from keratitis: 6206 and 6452. Using time-lapse microscopy, we demonstrate that some cytotoxic strains of P. aeruginosa are capable of persisting within intracellular compartments of host cells for at least 20 hours post-infection (PA14, 6452), while others do not (PA103, 6206). Using a fluorescent reporter plasmid to quantify T3SS transcriptional activation by flow cytometry, we found correlations between the T3SS status of clinical isolate strains and the tendency of each isolate to become intracellular. Populations of strains capable of invading (PA14 and 6452) remained with the majority T3SS-off even when exposed to an in vitro stimulus, whereas classically cytotoxic strains (PA103 and 6206) were comprised of more T3SS-on bacteria. Strains able to invade also exhibited lower basal transcriptional activity of the T3SS effectors, with non-invasive strains displaying up to a 10-fold increase in transcriptional activity of T3SS effectors even when uninduced. Interestingly, the mutation of a known regulator of the T3SS in PA103, vfr, lead to an increase in bacterial invasion and persistence; the same mutation in 6206, however, did not yield similar results, indicating that other factors may be in play and differ between strains. Taken together, our results reveal a potential intracellular lifestyle for some cytotoxic strains of P. aeruginosa, which may be related to intrinsic strain differences in bistability of T3SS activity.
Recommended Citation
Serrato, Gustavo, "Intracellular Persistence of Cytotoxic Strains of P. Aeruginosa Isolates Is Enabled by Bistable Expression of the Type Three Secretion System" (2025). Dissertations. 4271.
https://ecommons.luc.edu/luc_diss/4271
