Date of Award

2019

Degree Name

Biomedical Sciences

College

Joan C. Edwards School of Medicine

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Dr. Hongwei Yu, Committee Chairperson

Second Advisor

Dr. Jim Denvir

Third Advisor

Dr. Richard Egleton

Fourth Advisor

Dr. Philippe Georgel

Fifth Advisor

Dr. Timothy Long

Abstract

Cystic Fibrosis (CF) is a genetic disorder caused by mutations in the gene encoding Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). This disorder results in thick lung secretions which compromise the patient’s immune system. Chronic lung infections with Pseudomonas aeruginosa biofilms is a major factor that contributes to poor outcome for clinical treatment. The overall hypothesis of this dissertation is that small colony variant and pyrimidine biosynthesis are linked with biofilm formation, alginate production is dependent on MucA proteolysis, rifaximin can be repurposed to treat biofilm infection and multiplex PCR can be used to rapidly measure the bacterial cell density in patients’ respiratory secretions. Using transposon mutants in the de novo pyrimidine biosynthesis, it was established that two sigma factors AlgU (σ22) and RpoN (σ54) compete for alginate biosynthesis promoter binding. The balance of intracellular sigma factors created a competition that drove the bacteria to switch between small colony variance (SCV), under pyrimidine starvation, and alginate overproduction, under normal cellular conditions. We showed that the addition of uracil to the growth media is able to recover the normal mucoid phenotype in the pyrimidine mutants. Furthermore, it was shown that proteolysis of MucA22, a truncated MucA mutant, is necessary for the initiation of the signal transduction to release AlgU resulting in alginate production. Rifaximin was shown to be a potential anti-biofilm adjuvant, to the current treatment for CF lung infections, tobramycin. Finally, we developed a multiplex PCR that may be used for rapid diagnosis of lower respiratory tract infections such as CF lung infection and/or ventilator associated pneumonia. In conclusion, this dissertation studied the transcriptional and post-translational regulation of alginate production in P. aeruginosa as well as establishing a previously unknown link between alginate production and pyrimidine de novo biosynthesis. This knowledge would aid in identifying potential targets for future therapeutics. Also, we optimized current treatments of CF lung infections by repurposing a current antibiotic as an adjuvant, as well as developed a possible rapid diagnostic tool. Our work aims to help reduce mortality of CF patients from chronic infections by mucoid P. aeruginosa and improve the patients’ quality of life.

Subject(s)

Cystic fibrosis -- Diagnosis -- Research.

Cystic fibrosis -- Gene therapy -- Research.

Available for download on Wednesday, December 01, 2021

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