Abstract
Research into chronic infection by bacterial pathogens, such as Pseudomonas aeruginosa, uses various in vitro and live host models. While these have increased our understanding of pathogen growth, virulence, and evolution, each model has certain limitations. In vitro models cannot recapitulate the complex spatial structure of host organs, while experiments on live hosts are limited in terms of sample size and infection duration for ethical reasons; live mammal models also require specialized facilities which are costly to run. To address this, we have developed an ex vivo pig lung (EVPL) model for quantifying Pseudomonas aeruginosa growth, quorum sensing (QS), virulence factor production, and tissue damage in an environment that mimics a chronically infected cystic fibrosis (CF) lung. In a first test of our model, we show that lasR mutants, which do not respond to 3-oxo-C12-homoserine lactone (HSL)-mediated QS, exhibit reduced virulence factor production in EVPL. We also show that lasR mutants grow as well as or better than a corresponding wild-type strain in EVPL. lasR mutants frequently and repeatedly arise during chronic CF lung infection, but the evolutionary forces governing their appearance and spread are not clear. Our data are not consistent with the hypothesis that lasR mutants act as social “cheats” in the lung; rather, our results support the hypothesis that lasR mutants are more adapted to the lung environment. More generally, this model will facilitate improved studies of microbial disease, especially studies of how cells of the same and different species interact in polymicrobial infections in a spatially structured environment.
Highlights
Research into chronic infection by bacterial pathogens, such as Pseudomonas aeruginosa, uses various in vitro and live host models
We focused on the wellcharacterized PAO1 wild-type (WT) strain and two lasR-null mutant (lasR) mutant strains which do not respond to the quorum sensing (QS) signal N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) [49]
To explore the effects of lasR-mediated QS on growth and virulence, cubes were inoculated with ca. 104 washed overnight-culture cells of WT PAO1, two independent lasR mutants, a mix of PAO1 and PAO1 lasR::Gm, or a phenazine bioreporter strain constructed in a WT or ⌬lasR background
Summary
Research into chronic infection by bacterial pathogens, such as Pseudomonas aeruginosa, uses various in vitro and live host models. We have developed an ex vivo pig lung (EVPL) model for quantifying Pseudomonas aeruginosa growth, quorum sensing (QS), virulence factor production, and tissue damage in an environment that mimics a chronically infected cystic fibrosis (CF) lung. People with CF experience decades of chronic infection with repeated episodes of acute pulmonary exacerbation [1] During this time, P. aeruginosa evolves and diversifies; mutants with altered production of virulence factors are commonly isolated from patients [4,5,6,7,8,9,10,11], as are mutants that are impaired in quorum sensing (QS) [9, 10, 12].
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