Loss of LasR function reshapes inter-species interactions in a polymicrobial cystic fibrosis airway model

The airways of people with cystic fibrosis (CF) exhibit a particular predilection for infection by certain opportunistic pathogens. One such interloper is Pseudomonas aeruginosa, a WHO high-priority pathogen, infamous for its ability to evade antibiotic clearance. However, P. aeruginosa often has to share the airway environment with a variety of other microbes: these are polymicrobial infections. We now know that the interactions between co-habiting Gram-negative, Gram-positive, and fungal species can significantly impact the outcomes of these airway infections. Factors like intra-species genetic variation and (potentially existential) environmental challenges such as antibiotic treatment further complicate the situation. However, our understanding of the biology underpinning the microbial responses in such complex environments remains limited. In this study, I investigate the impact of loss-of-function of a gene (lasR) that is frequently mutated in CF isolates of P. aeruginosa, and also the impact of anti-pseudomonal drugs on a polymicrobial ecosystem populated by a representative selection of CF-associated microbes. To do this, I utilise an in vitro continuous-flow system that faithfully captures many of the microbial dynamics seen in CF patients. Using artificial sputum medium, this robust experimental platform enables the stable co-culture of P. aeruginosa alongside two other CF airway pathogens: Staphylococcus aureus and Candida albicans. I demonstrate that P. aeruginosa has the highest abundance in the studied polymicrobial community. Unexpectedly, the transcriptome of ‘wild-type’ P. aeruginosa was only minorly altered in the presence of other species, whereas the transcriptome of a lasR mutant showed substantial changes in the polymicrobial scenario. Furthermore, I found that the population dynamics of the lasR mutant, in co-culture with the ‘wild-type’, were very similar to those seen in people with CF (with the lasR mutant never exceeding ca. 10% of the overall P. aeruginosa titres). One key LasR-regulated gene cluster that was affected in the lasR mutant was the tseT operon, which encodes a component of the Type VI Secretion System. However, I found that this secretion system apparently plays little obvious role in constraining lasR mutant titres. I also investigated the susceptibility of P. aeruginosa to clinically-deployed antibiotics. I found that P. aeruginosa exhibits reduced susceptibility to ciprofloxacin in the presence of other co-habiting species and that treatment with ciprofloxacin and colistin combined leads to a long-term increase in titres of the fungus, C. albicans. This “fungal blooming” was linked with expression of the tseT operon. In summary, my work shows the importance of considering not only inter-species variation in the response to antibiotic challenge, but also intra-species variation. This is important because although CF-associated infections are often associated with single clones of P. aeruginosa, these clonally-derived populations often display significant genetic diversity.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Bényei, Éva

Advisor dc:contributor.advisor

• Welch, Martin

• Cystic fibrosis
• Polymicrobial
• Pseudomonas aeruginosa
• Staphylococcus aureus
• Candida albicans
• In vitro

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/d254bb5b-69b3-4306-8145-c83cfe03837d/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.115544

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/379451