Date of Award

5-2011

Level of Access Assigned by Author

Open-Access Thesis

Degree Name

Master of Science (MS)

Department

Microbiology

Advisor

Carol H. Kim

Second Committee Member

John T. Singer

Third Committee Member

Paul J. Millard

Abstract

Cystic fibrosis (CF) is the most common lethal hereditary disease. CF is caused by recessive mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene and is associated with multi-­-organ defects resulting from improper ion transport across epithelial membranes. Chronic lung infection by the environmentally ubiquitous opportunistic human pathogen Pseudomonas aeruginosa and the subsequent hyperinflammation that occurs as the host immune system combats the bacterium cause substantial morbidity and mortality in CF. Despite numerous studies that have sought to elucidate the role of CFTR in the innate immune response, the links between CFTR, innate immunity, and P. aeruginosa infection remain unclear. The present work highlights the zebrafish as a powerful model organism for human infectious disease. Zebrafish embryos with reduced expression of the cftr gene (Cftr morphants) exhibited reduced respiratory burst response and directed neutrophil migration, supporting a connection between cftr and the innate immune response. Cftr morphants were also found to display a iv significant iron deficiency (ID) compared to control embryos, a symptom commonly diagnosed in CF patients. Cftr morphants were infected with P. aeruginosa or other bacterial species that are commonly associated with infections in CF patients, including Burkholderia cenocepacia, Haemophilus influenzae, and Staphylococcus aureus. Intriguingly, the bacterial burden of P. aeruginosa was found to be significantly higher in zebrafish Cftr morphants than in controls, a phenomenon that was not observed with any of the other bacterial species examined. The bacterial burden in Cftr morphants infected with a P. aeruginosa LasR mutant, a quorum sensing (QS)-­-deficient strain, was comparable to that in control fish indicating that the regulation of virulence factors through QS is required for enhancement of infection in the absence of Cftr. Cftr morphants were then challenged with P. aeruginosa mutants defective in the expression of QS regulated virulence factors. A mutant defective in the production of Exotoxin A (ETA) resulted in similar bacterial clearance in both the Cftr morphant and control embryos. The reduction in directed neutrophil migration to a P. aeruginosa infection was also restored when zebrafish embryos were challenged with the ETA mutant. Taken together, these data point towards a possible explanation for the specificity between P. aeruginosa and CFTR. The zebrafish system provides a multitude of advantages for studying the unique pathophysiology resulting from defective expression of CFTR, investigating the pathogenesis of P. aeruginosa and elucidating the role that the innate system plays in the host response to acute bacterial infections commonly associated with cystic fibrosis.

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