Cystic fibrosis (CF) is the most common fatal genetic disorder that afflicts young Canadians. The major cause of morbidity and mortality in patients with CF is chronic pulmonary infection with the opportunistic Gram-negative pathogen Pseudomonas aeruginosa. Once established, P. aeruginosa lung infections cannot be cleared despite sustained and aggressive antimicrobial therapy. Treatment failure of P. aeruginosa lung infections is caused by a combination of antibiotic resistance and tolerance mechanisms. Antibiotic resistance is mainly mediated by multidrug efflux pumps such as MexAB-OprM. Antibiotic tolerance has been attributed to biofilms and to nutrient starvation. In this thesis, I present an analysis of three transcriptional regulators (PA3225, RpoS, and RpoN) and their contributions to resistance and tolerance in P. aeruginosa. PA3225 is a transcriptional regulator that I initially identified as a candidate regulator of a type VI secretion system (T6SS) that had been previously implicated in biofilm tolerance. While a ΔPA3225 deletion mutant did not, unfortunately, have dysregulated expression of the T6SS, I fortuitously discovered that the mutant displayed increased resistance to various antibiotics from different functional classes. I linked the increased antibiotic resistance of ΔPA3225 to upregulation of MexAB-OprM and provided evidence that PA3225 may be a direct repressor of mexAB-oprM. Next, I sought to identify a transcriptional regulator of ndvB, which is another gene that plays a role in biofilm tolerance. I found that the stationary phase sigma factor, RpoS, was essential for expression of ndvB in stationary phase and biofilm cells. Moreover, RpoS was important for tolerance of stationary phase cells to tobramycin (TOB), an aminoglycoside antibiotic that is used to treat CF patients. In recent years, several groups have sought to identify novel treatments to combat antibiotic tolerance in P. aeruginosa. A popular strategy is metabolic potentiation, which involves co-administration of an antibiotic with a metabolite to reverse tolerance due to nutrient starvation. For example, one group found that fumarate (FUM) combined with TOB (TOB+FUM) was highly effective at killing tolerant P. aeruginosa. FUM uptake depends on C4-dicarboyxlate transporters, which are transcriptionally regulated by the alternative sigma factor, RpoN. Importantly, rpoN loss-of-function mutations are a recognised mechanism of pathoadaptation in CF clinical isolates. I demonstrated that TOB+FUM was unable to kill ΔrpoN stationary phase and biofilm cells due to loss of FUM uptake and that rpoN alleles from CF clinical isolates were unable to complement the ΔrpoN mutant. These findings could have important implications for TOB+FUM as a treatment modality in CF patients with a high burden of rpoN mutants. Overall, my work has provided interesting and, in the case of RpoN, clinically relevant insights into the regulatory networks that determine antibiotic susceptibility in P. aeruginosa.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/39483 |
| Date | 31 July 2019 |
| Creators | Hall, Clayton Wallace |
| Contributors | Mah, Thien-Fah |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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