(540) Developing a murine model for lung infection in cystic fibrosis that reflects Pseudomonas aeruginosa infection dynamics in the CF lung

Background: Developing therapies for chronic lung infection in people with Cystic Fibrosis (CF) requires understanding how infectious microbes, like Pseudomonas aeruginosa (Pa), behave in the CF lung. Pa is usually studied in laboratory models, but how well these models mimic the CF lung is not always known. To evaluate Pa behavior in CF lab models, Cornforth et al. [1] developed a quantitative framework using gene expression to compare models to a benchmark of human infection, leading them to find that synthetic CF sputum (SCFM2) captures over 85% of Pa genes expressed in the CF lung [1]. Here, we ask how the Pa strain PAO1 performs in a murine model if grown in SCFM2 before inoculation.

Methods: We grew PAO1 in SCFM2 for 6 hours, then extracted RNA (in vitro samples) or inoculated mice. Female C57BL/6 mice were infected intranasally with PAO1/SCFM2 (10⁷ CFU/mouse). We harvested lungs at 18 hours post infection for RNA or CFU counts. Libraries were prepared from total RNA of in vitro samples or lungs with the NEBNext smRNA Library Prep Set before sequencing on Illumina NovaSeq or NextSeq platforms. Reads from our samples and published in vitro samples [2] were mapped to the PAO1 genome and normalized read counts/gene were used to measure how well PAO1 gene expression from in vitro and lung samples captured Pa gene expression in sputum by calculating normalized expression per gene relative to its expression level in published sputum RNAseq data [1]. Model accuracy is the percentage of genes with normalized expression within 2 standard deviations of their expression level in sputum.

Results: CFU output from infected mouse lungs ranged from 10⁷-10⁸, and RNAseq of lungs (200 M reads) yielded 260,676 to over 4 million reads mapping to PAO1 coding sequences. This coverage was sufficient to calculate an accuracy score for our model. Both in vitro and lung samples were highly accurate, capturing 88.3% and 84.6% of Pa genes expressed in sputum, respectively. Of genes not mimicking Pa gene expression in sputum, we found some genes (323) in both in vitro and lung data sets, while some genes were unique to in vitro (320) or lung (520) data sets.

Conclusions: Deeply sequenced mouse lungs infected with PAO1 have enough reads to analyze PAO1 gene expression, and mice infected with PAO1 grown in SCFM2 reflect Pa gene expression in sputum at a comparable accuracy to published in vitro samples [1]. Numerous genes not mimicking Pa gene expression in sputum are unique to in vitro or lung data sets, suggesting that PAO1 in our mouse model may perform better in some functional categories than SCFM2, and vice versa. Further, the PAO1 transcriptome is 4% more accurate in our mouse model than in a previously published murine pneumonia model where PAO1 was grown on a standard growth medium [1, 3], raising the hypothesis that using PAO1 grown in SCFM2 as the inoculum improves accuracy of murine CF models. Work is underway to test this hypothesis.

Acknowledgements: This work was supported by a grant from the Cystic Fibrosis Foundation (WHITEL20A0) to M.W.

References: [1] Cornforth, DM, et al. 2020. Quantitative Framework for Model Evaluation in Microbiology Research Using Pseudomonas aeruginosa and Cystic Fibrosis Infection as a Test Case. mBio. 11(1): e03042-19
[2] Cornforth, DM, et al. 2018. Pseudomonas aeruginosa transcriptome during human infection. PNAS 115.22 (2018): E5125-E5134.



[3] Damron, FH, et al. 2016. Dual-seq transcriptomics reveals the battle for iron during Pseudomonas aeruginosa acute murine pneumonia. Sci. Rep. 6 :39172.