Award Date
December 2023
Degree Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Life Sciences
First Committee Member
Boo Shan Tseng
Second Committee Member
Eduardo Robleto
Third Committee Member
Helen Wing
Fourth Committee Member
Joe Harrison
Fifth Committee Member
Ronald Gary
Number of Pages
215
Abstract
The opportunistic pathogen Pseudomonas aeruginosa forms chronic lung infections in people with cystic fibrosis (CF). In the CF lung, P. aeruginosa undergoes selection, where mucA mutations often arise. MucA inhibits the sigma factor AlgU. Mutations in mucA lead to AlgU dysregulation, resulting in a mucoid phenotype that is associated with poor CF disease outcomes. While mucA is commonly mutated, in the first part of my dissertation, I show that, paradoxically, a portion of mucA is essential in P. aeruginosa. We demonstrate that mucA is no longer required in a strain lacking algU, that the interaction between MucA and AlgU is essential, and that mucA is no longer essential in mutant strains containing AlgU variants with reduced sigma factor activity. Together, these results suggest that in the absence of MucA, the inability to regulate AlgU activity results in the loss of bacterial viability.
In searching for suppressors of mucA essentiality, I isolated a strain containing a single base pair deletion in dnaJ. DnaJ is known for its role in the heat shock response. In the second part of my dissertation, I discuss preliminary findings of this non-algU suppressor of mucA essentiality. The mutation in dnaJ resulted in a protein that contained the first 108 residues of the native protein. This strain is unable to respond to envelope stress. This data suggests that the mutation in dnaJ results in a functional change in the protein such that the ability of the cell to respond to envelope stress has been eliminated. DnaJ is highly conserved across all domains of life and may inform on crosstalk between stress responses.
It is known that mucoid cells revert to a non-mucoid state in both laboratory and CF lung environments. It is commonly thought that this reversion is driven by alginate production. In the third part of my dissertation, I show that mucoid reversion occurs even in the absence of alginate production. Additionally, upon sequencing of a few nonmucoid revertants, we found that the isolates contained secondary site mutations in algU, supporting the hypothesis that AlgU activity is the driving factor for reversion. Current work is focused on determining how alginate production affects nonmucoid reversion, as well as how common CF therapies affect this rate.
Keywords
AlgU; Cystic fibrosis; Envelope stress; Toxicity
Disciplines
Microbiology
File Format
File Size
12680 KB
Degree Grantor
University of Nevada, Las Vegas
Language
English
Repository Citation
Schofield Londono, Melissa C., "Regulation of Toxicity in the Pseudomonas aeruginosa Envelope Stress Response" (2023). UNLV Theses, Dissertations, Professional Papers, and Capstones. 4912.
http://dx.doi.org/10.34917/37200538
Rights
IN COPYRIGHT. For more information about this rights statement, please visit http://rightsstatements.org/vocab/InC/1.0/