Evolution of Metabolism in Pseudomonas aeruginosa During Adaptation to the Cystic Fibrosis Airways

Pseudomonas aeruginosa is a highly versatile pathogen with several mechanisms both for immune escape and antibiotic resistance, making it one of the most critical pathogens to develop new treatments against to mitigate the antibiotic crisis. Because of its metabolic versatility, P. aeruginosa is able to undergo rapid adaptation and specialization toward a diverse range of environmental niches. This is evident in the case of patients with the heritable disease Cystic Fibrosis, where mutations in the Cystic Fibrosis Transmembrane Conductance Regulator dysregulates epithelial fluid transport. This leads to abnormal accumulation of dehydrated mucus in the lungs, which provides a nutrient-rich environment for infecting microbes. By adjusting its metabolism and physiology to the solutions available for the various niches that exist in the CF airways, P. aeruginosa is able to achieve persistent infections, which cause chronic lung inflammation that can last for more than 30 years and is the predominant cause of morbidity and mortality.
This study focuses on the evolution of P. aeruginosa metabolism during adaptation to the CF airways to identify key aspects of the strategies by which it achieves chronicity, and by extension how within-patient evolution of bacteria more generally leads to persistent infections, in order to identify potential targets for the development of novel therapeutic and diagnostic measures.
The first chapter gives a general introduction to the clinical relevance and molecular biology of P. aeruginosa CF airway infections and elaborates on the specific aims and design of the project. The second chapter describes a study in which we develop a high-throughput method for dynamic exo-metabolomic analysis in order to screen large collections of clinical isolates for metabolic changes. The study demonstrates how dilution-resolved sampling from several time-shifted growth curves accurately captures the metabolic dynamics of traditional low-throughput methods. The third chapter describes another study, where we select 8 pairs of clinical strains from different infection scenarios of P. aeruginosa in patients with Cystic Fibrosis based on reduction in growth rates as proxy for metabolic rewiring and apply the method described in Chapter 2 to identify commonalities in adapted metabotypes. Doing so, we identify clinical mutations in the aceEF genes encoding the key metabolic regulator pyruvate dehydrogenase as central to the adapted metabotypes and insert these mutations into the clean genetic background of PAO1. We combine this with proteomic analysis of both clinical and genetically recombinant strains and demonstrate that these mutations in PAO1 leads to a partial recapitulation of the persistent phenotype. We then verify the pathoadaptive nature of these mutations by testing the mutant strains in an air-liquid-interface airway infection model system where they show reduced virulence and immunogenicity reminiscent of persistent infections. The fourth chapter gives a discussion of specific topics, where the obtained results may have interesting implications and finally, the fifth chapter gives a summary of the key findings and conclusions of the project and suggests some important directions for future research.

Altogether, this work provides evidence that evolution of metabolism is not simply a biproduct, necessary for accommodating the persistent phenotypes, but directly promotes the establishment of persistent infections without relying on traditional antibiotic resistance mechanisms. Furthermore, it suggests that dysregulation of pyruvate dehydrogenase and the consequent change in the flux of pyruvate is a common strategy by P. aeruginosa to achieve chronicity in CF airway infections.