Date of Award

2006

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

Elizabeth Bryda

Third Advisor

Todd Green

Fourth Advisor

Susan Jackman

Fifth Advisor

Donald Primerano

Abstract

Cystic fibrosis (CF) is the most common, autosomal recessive lethal genetic disease in the Caucasian population, resulting from a malfunctioned cystic fibrosis transmembrane conductance regulator (CFTR) and leading to bacterial lung infections. P. aeruginosa, an opportunistic pathogen, establishes a chronic infection in CF with a phenotype of overproduction of an exopolysaccharide (alginate) due to host-directed mutagenesis. While free-floating planktonic bacteria can be properly cleared from the CF lung, P. aeruginosa, along with alginate production, establishes an infection in the form of a biofilm which supports its survival in nature and in vivo. As a result, genomic structure, gene and protein expression and pathogenicity vary from strain to strain, potentially leading to differential roles in the CF lung. Unrelated phenotypic differences in biofilm formation and lung colonization as well as variations in genome size, all within different sources of P. aeruginosa were discovered and could lead to variations in morbidity and mortality related to P. aeruginosa infections in CF. A key factor in alginate regulation is AlgU/AlgT, a sigma factor that also impacts the expression of many other involved proteins. One such protein, AlgB, is a response regulator of a two- component system together with KinB, responsible for regulating the expression of algD, involved in alginate biosynthesis, through algU. AlgB, when nonphosphorylated, interacts through algU to synthesize alginate. Additional alginate loci were located within the P. aeruginosa PAO1 genome utilizing isogenic mucoid mutants coupled with PAO1-derived cosmid complementation. Another factor, MucD, was further classified in its role in alginate suppression and temperature sensitivity. P. aeruginosa small colony variants (SCVs) which prefer the biofilm mode of growth, thrive in harsh environmental conditions and are found in the chronically infected CF lung, were generated in the laboratory for further study. Addtionally, alginate alone was enough to modify the ability to colonize a susceptible mouse lung, resulting in varied mortality and virulence. Finally, novel genetic tools were fashioned for genomic mutagenesis and rapid gene knockouts. Taken together, the phenotypic and genotypic analysis of the regulation of alginate may further lead to treatments to prevent biofilm formation and subsequent CF host colonization with P. aeruginosa.

Subject(s)

Cystic fibrosis.

Lungs - Diseases.

Biofilms.

Pseudomonas aeruginosa.

Download

Share

COinS