Investigating Pseudomonas aeruginosa physiology and the impact of pathoadaptive mutations, in a polymicrobial airway environment

Bioscience for an integrated understanding of health

The airways of people with cystic fibrosis (CF) are often colonised by a diverse "zoo" of microbes, especially during their early and teenage years. These polymicrobial communities have historically proven difficult to recapitulate in the laboratory environment, so, until now, most research has involved monoculture of key pathogens such as Pseudomonas aeruginosa. It is becoming increasingly clear, however, that pathogens in multi-species systems often behave radically differently than when grown in pure axenic cultures, and, as such, recent years have seen a growing interest in efforts to recapitulate the complex CF microbiota in vitro.

The Welch Lab has developed an in vitro laboratory model that accurately captures the long-term stability of the polymicrobial community present in CF airways. In brief, the setup consists of a continuous flow bioreactor in which the rate of fluid - specifically, artificial sputum media - replacement reflects that seen in the airways. The current model incorporates three major CF-associated pathogens: P. aeruginosa, Staphylococcus aureus, and Candida albicans. This system has opened new possibilities for interrogation of the biology of CF-associated polymicrobial airway infections, and has already yielded some intriguing new insights.

In this project, I will introduce other common CF-associated pathogens into the in vitro model system, alongside the current three, for example: Haemophilus influenzae, Rothia mucilaginosa, and Streptococcus milleri. I will then use this "improved" model system to examine the impacts of the presence of P. aeruginosa mutants in the polymicrobial community. The P. aeruginosa population observed in CF airway infections is not homogenous, with mutant variants frequently observed. Loss-of-function mutations in certain genes are commonly over-represented in CF, indicating they may confer increased fitness in this environment; however, we still have very little idea as to why this is. Key "pathoadaptive" genes include mutS (mutations leading to hypermutability), lasR (affecting virulence quorum sensing), mucA (mucoidy), mexT (ciprofloxacin resistance), and metF (methionine auxotrophy).

To investigate the impact of these pathoadaptive mutations on P. aeruginosa fitness, I will generate loss-of-function mutants for each target gene, then examine how introduction of these mutants (or mixtures of both wild type and mutant(s)) influences the diversity and species trajectory of the polymicrobial culture. This will further our understanding of how the arrival of P. aeruginosa into the CF airway influences the existing microbiome, and whether some mutants are more successful at colonising this niche than others. Preliminary data from the Welch Group indicate that lasR mutants do indeed show majorly altered inter-species interactions as compared to the wild type, and I hope to see similarly interesting results for other pathoadaptive mutants. I will also examine how the presence of pathoadaptive mutants in the model system impacts clinically relevant factors, such as response of the involved pathogens to antimicrobials.

This project will help us understand why some mutants arise with such high frequency in CF, the advantages these mutants potentially confer on P. aeruginosa, and their impact on therapeutic interventions: it therefore has both biological and clinical interest and importance.