Understanding the molecular mechanism through which aspirated bile triggers chronic Pseudomonas aeruginosa infections in respiratory disease
University College Cork
The opportunistic pathogen Pseudomonas aeruginosa is the leading cause of morbidity and mortality in Cystic Fibrosis (CF) patients. Extensive genomic adaptation of this organism facilitates its emergence as a dominant organism within the lung microbial community and to its ability to chronically persist within the CF airways. The environmental and host factors contributing to the success of this species in vivo have been the subject of intensive research efforts. Gastro-oesophageal reflux (GOR) has recently emerged as a major co-morbidity in CF and a range of other respiratory conditions and is associated with the presence of bile acids in the lungs of CF patients, a consequence of micro-aspiration of refluxed gastric contents. This thesis aimed to investigate the impact that bile exerts on the global lung microbiota and the key CF associated pathogen P. aeruginosa. The detection of bile acids in paediatric CF patients using liquid chromatography mass spectrometry (LC-MS) analysis correlated with a reduction in lung microbial biodiversity and the emergence of dominant respiratory pathogens including P. aeruginosa. Bile acids may contribute to the progressive restructuring of the lung microbiota towards a pathogen dominated state associated with worse clinical outcomes. Bile and the active component bile acids were found to be capable of triggering P. aeruginosa to transition to a chronic, antibiotic tolerant lifestyle through a combination of transcriptional and phenotypic responses. Functional screens based on biofilm formation and growth on bile identified key two component systems mediating the biofilm response to bile with a connection to central metabolism becoming apparent. The latter screen identified the glyoxylate shunt as a key breakpoint in the suppression of redox potential as part of the bile response. Bile was also found to be capable of selecting for genetic variants in an in vitro system known to mimic conditions found within the CF lung environment. Pigmented derivatives emerged exclusively in the presence of bile with genome sequencing identifying single nucleotide polymorphisms (SNPs) in quorum sensing (lasR) and both the pyocyanin (phzS) and pyomelanin (hmgA) biosynthetic pathways. These mutations have been previously described in various clinical isolates of P. aeruginosa. Loss of Pseudomonas Quinolone Signal (PQS) production in the pigmented variants underpinned the loss of redox suppression in response to bile, perhaps a consequence of the anti-oxidant/pro-oxidant activities attributed to the PQS signalling molecule. Bile is therefore capable of influencing the evolutionary trajectory of this respiratory pathogen, a key finding in understanding the emergence of genotypic and phenotypic heterogeneity within the lungs of patients with respiratory disease. Collectively, this research supports the role for bile in the progression of chronic infection in CF through its impact on P. aeruginosa and other respiratory pathogens. Therefore, the early detection and profiling of bile acids utilising rapid point of care devices could lead to the identification of high risk paediatric patients and to the development of increasingly effective intervention strategies to prevent the establishment of chronic respiratory microbiota.
Pseudomonas aeruginosa , Bile , Gastrooesophageal reflux , Chronic , Adaptation , Pigmented , Biofilm , Cystic fibrosis
Flynn, S. 2019. Understanding the molecular mechanism through which aspirated bile triggers chronic Pseudomonas aeruginosa infections in respiratory disease. PhD Thesis, University College Cork.