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Global regulation of the Lon protease of Pseudomonas aeruginosa and its influence on ciprofloxacin resistance and virulence Breidenstein, Elena Bernadette Monika

Abstract

This thesis focuses on investigating global regulation, ciprofloxacin resistance and virulence by the ATP-dependent Lon protease of Pseudomonas aeruginosa. Screening a P. aeruginosa PA14 mutant library for non-essential genes involved in altered ciprofloxacin susceptibility identified more than 100 genes (35 involved in intrinsic and 79 in mutational resistance). The identification of known and novel genes involved in resistance through mutant library screening provided new insights into the ciprofloxacin resistome. These mutants provide insights into adaptive resistance mechanisms and the clinical phenomenon of creeping baselines. The ATP-dependent Lon protease, which showed a 4-8 fold increase in ciprofloxacin susceptibility upon mutation, was chosen for more detailed studies. This study showed that Lon protease regulated antibiotic resistance and virulence properties despite the fact that it is not a traditional regulator. The ciprofloxacin susceptible phenotype of a lon mutant could be complemented. Furthermore, the lon mutant was identified to influence cytotoxicity, adhesion, anaerobic growth and metabolism as well as impact on global regulation. Microarray analysis showed that Lon is at the top of a transcriptional hierarchy dysregulating around 200 genes. Proteomic profiling showed that Lon appeared to be involved in cleaving GroEL, Hfq and KatA amongst others. Furthermore, mechanistic studies on the involvement of Lon protease in the SOS response under sub-inhibitory concentrations of ciprofloxacin revealed that SOS response was less induced in the lon mutant which could be explained by the action of Lon on the key SOS regulator RecA. Lon protease influences virulence in vivo as shown in a lettuce leaf model, an amoeba assay as well as a rat model of chronic infection. The alterations in virulence-related processes in vitro in a lon mutant were also paralleled by defective virulence in vivo. Antibiotic resistance and motility phenotypes were also investigated for other proteases and mutations in pfpI, clpP and clpS had distinct, but overlapping phenotypes cf. the lon mutant. Overall, my results suggest that while the Lon protease is not a traditional regulator, it is still involved in a multitude of cellular processes highlighting its importance for the bacterial cell. Thus, it would be a good target for therapy.

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Attribution-NonCommercial-NoDerivatives 4.0 International

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