Abstract
Pseudomonas aeruginosa is a versatile opportunistic pathogen capable of causing a variety of serve and chronic infections in healthy and immunocompromised hosts. Individuals with the genetic disorder, cystic fibrosis, are particularly susceptible to P. aeruginosa infections, leading to higher morbidity and mortality in this patient population. In the CF lung environment, this microorganism is subjected to stressful conditions including chronic antibiotic exposure, elevated sodium chloride levels, reduced oxygen availability, and variable nutrient content. Under these variable and often hostile conditions, P. aeruginosa is able to rapidly develop resistance to antibiotics and alter its cellular physiology and metabolism to successfully colonize, grow, and survive. These adaptations have been associated with specific outer membrane porins of P. aeruginosa and their ability to affect cell structure and stability, adhesion to surfaces, and transport of small molecules. However, little is known about how P. aeruginosa is able to adapt and persist within the CF airways. In part, the lack of knowledge in this area can be attributed to the majority of studies conducted on laboratory adapted and/or derived P. aeruginosa strains. The experiments described in this dissertation sought to determine how P. aeruginosa emerges resistant to antibiotics and how specific porins are regulated under different growth conditions typically found in the CF lung environment. It was hypothesized that the regulation of antibiotic resistance determinants and outer membrane porins of P. aeruginosa involves multiple regulatory mechanisms and is dependent on specific environmental cues found in the hostile CF lung environment. Multiple isogenic panels of carbapenem-resistant P. aeruginosa mutants and clinical P. aeruginosa strains in addition to the prototypic strain PAO1were used to test this hypothesis. Four main findings resulted from testing this hypothesis. 1) The novel ISPa8 element was associated with the emergence of carbapenem resistance through the inactivation of the OprD porin in P. aeruginosa, along with an increased expression of the mexAB-oprM efflux pump with a concomitant down-regulation or loss of the OprD porin. 2) Carbapenem exposure can select mutants that exhibit increased susceptibility to multiple antibiotics, which is not associated with modifications in the expression of chromosomal resistance mechanisms or loss of plasmid(s). 3) The OpdQ porin was found to be associated with but not required for P. aeruginosa to exhibit increased susceptibility to antibiotics. Transcriptional analysis demonstrated opdQ transcription was initiated from a single σ70-like promoter, and requires DNA sequences directly upstream of the -35 promoter element. Under elevated sodium chloride, nitrate-rich, and oxygen-poor conditions, transcript and protein levels varied within and among the clinical strains indicating OpdQ is regulated by multiple regulatory mechanisms at the transcriptional and post-transcriptional levels, and its regulation is dependent on specific growth environments. 4) Transcription of the OprF porin gene was initiated equally by σ70 and σX-dependent promoters, and only weakly by a distal σ22-dependent promoter. In addition, the -10 element of the σ70-dependent promoter was shown to be required for oprF transcription. The transcription and production of OprF were affected by the presence of sodium chloride in a majority of the clinical strains, whereas nitrate-rich and oxygen-poor conditions did not. Taken together, these findings suggest that P. aeruginosa perceives and responds to specific environmental cues by modulating antibiotic resistance determinants and porins through multiple levels of regulation. The mechanisms that contribute to the regulation of porins in the presence of nitrate, sodium chloride, and/or oxygen-limiting conditions will still need to be elucidated.