Graduation Year
2024
Document Type
Dissertation
Degree
Ph.D.
Degree Name
Doctor of Philosophy (Ph.D.)
Degree Granting Department
Medical Sciences
Major Professor
Sophie E. Darch, Ph.D.
Committee Member
Larry Dishaw, Ph.D.
Committee Member
Kami Kim, Ph.D.
Committee Member
Tara Randis, Ph.D.
Keywords
Biofilm, ESCAPE, hypothetical protein, spatial structure
Abstract
Bacterial aggregates are observed in both natural and artificial environments. In the context of disease, aggregates have been isolated from both chronic and acute infections. Pseudomonas aeruginosa (Pa) aggregates contribute significantly to chronic infections, particularly in the lungs of people with cystic fibrosis (CF). Unlike the large biofilm structures observed in vitro, Pa in CF sputum forms smaller aggregates (~10-1000 cells), and the mechanisms behind their formation remain underexplored. This study aims to identify genes essential and unique to Pa aggregate formation in a synthetic CF sputum media (SCFM2). We cultured Pa strain PAO1 in SCFM2 and LB, both with and without mucin, and used RNA sequencing (RNA-seq) to identify differentially expressed genes. The presence of mucin revealed hundreds of significantly differentially expressed (DE) genes, predominantly downregulated, with 40% encoding hypothetical proteins unique to aggregates. Using high-resolution microscopy, we assessed the ability of mutants to form aggregates and identified 13 that were unable to form WT aggregates. Notably, no mutant exhibited a completely planktonic phenotype. Instead, we identified multiple spatial phenotypes described as 'normal,' 'entropic,' or 'impaired.' Entropic mutants displayed tightly packed, raft-like structures, while impaired mutants had loosely packed cells. Predictive modeling linked the prioritized genes to metabolic shifts, iron acquisition, surface modification, and quorum sensing. Co-culture experiments with wild-type PAO1 revealed further spatial heterogeneity and the ability to 'rescue' some mutant phenotypes, suggesting cooperative interactions during growth. This study enhances our understanding of Pa aggregate biology, specifically the genes and pathways unique to aggregation in CF-like environments. Importantly, it provides insights for developing therapeutic strategies targeting aggregate-specific pathways.
Scholar Commons Citation
Gannon, Alexa D., "Pseudomonas aeruginosa Aggregate Formation in the Cystic Fibrosis Airway" (2024). USF Tampa Graduate Theses and Dissertations.
https://digitalcommons.usf.edu/etd/11179
