Date of Award

2013

Degree Name

Biomedical Sciences

College

Joan C. Edwards School of Medicine

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Hongwei Yu

Second Advisor

Richard Niles

Third Advisor

Richard Egleton

Fourth Advisor

Todd Green

Fifth Advisor

Wei-ping Zeng

Abstract

Cystic fibrosis is a genetic disorder that results from mutations in the CF transmembrane conductance regulator gene. These mutations cause a disruption in the chloride transport in mucosal tissues causing the accumulation of dehydrated mucus, and a decrease in the mucocilliary removal of environmental pathogens within the lungs. Additionally, the accumulation of dehydrated mucus within the lungs provides a hospitable environment for various bacteria, including the Gram-negative opportunistic pathogen Pseudomonas aeruginosa. P. aeruginosa uses the overproduction of a surface polysaccharide called alginate to form a biofilm to evade the host’s immunological defenses. The overproduction of alginate, often referred to as mucoidy, is a virulence factor that is responsible for chronic P. aeruginosa infections, as well as an increased resistance to antibiotics and phagocytosis by the host defense cells. Chronic P. aeruginosa infections are the leading cause of morbidity and mortality in CF patients, and the detection of mucoid isolates is a proven predictor of a decline in the patient’s health. The transition from the non-mucoid phenotype, found in environmental isolates, to the mucoid phenotype found within the CF lung is typically due to “loss-of-function” mutations in the transmembrane anti-sigma factor MucA. However, P. aeruginosa can overproduce alginate independent of mutations in mucA, through the regulated proteolysis of MucA. A series of proteases, beginning with AlgW, can degrade MucA, and release the alternative sigma factor AlgU to drive transcription of the alginate biosynthetic operon. It is generally accepted that the regulated proteolysis of MucA is a mechanism used by early colonizing strains prior to the selection for MucA mutations. Therefore, understanding this mechanism employed by those early colonizing strains may prove beneficial in preventing the establishment of chronic P. aeruginosa respiratory infection. In this dissertation, I identify and characterize two novel regulators of alginate overproduction in P. aeruginosa strains possessing a wildtype MucA. Using the model strain PAO579, I determined that mutations that result in the truncation of the type-IV pilin precursor protein, PilA, can induce alginate overproduction through activation of the AlgW resulting in an increased rate of proteolysis of MucA. Additionally, I identify that expression of the genetic locus PA1494, referred to as mucoid inhibitor A (muiA), can suppress mucoidy in P. aeruginosa strains with a wild-type MucA. Collectively, these findings provide needed insight into the regulation of mucoidy in those early colonizing strains, as well as identifies potential therapeutic targets for the prevention of chronic P. aeruginosa infections in the CF lung.

Subject(s)

Cystic fibrosis - Diagnosis.

Cystic fibrosis gene - Research - United States.

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