Fig. 6

Benchmarking co-culture model with in vivo P. aeruginosa transcriptomic datasets directly isolated from the airways of CF subjects. (a) Accuracy analysis using gene-wise mean and standard deviation values from in vivo samples in Lewin et al., 2023⁵⁸ as reference. Genes were considered accurate if their expression was within 2 standard deviations of the mean in the in vivo sample. (b) PCA plots showing sample distribution by condition (Magenta: co-culture; green: bacterial culture in isolates; purple: in vivo) or by study of origin (Orange: this study; purple: Cornforth et al., 2018¹⁹; pink: Kordes et al., 2019⁵⁹; blue: Rossi et al., 2018⁴⁹). (c) Volcano plots displaying gene log₂ fold change and -log₁₀ adjusted p-value comparing transcriptomes of in vivo P. aeruginosa (left) or co-cultured PAO1 (right) to those of all pure bacterial culture samples. Green indicates differentially expressed genes (DEGs) (log₂ fold change > 1 and adjusted p value < 0.05). Indicated in the boxes the number of up- or downregulated DEGs. (d) Venn diagrams displaying the overlap between genes that are upregulated (left), downregulated (middle), or both (right) in the previous in vivo and co-culture comparison to in vitro and mono-culture samples (b). (e) Expression heat map displaying the common up-(left) and downregulated (right) genes. Samples clustered based on the expression of all genes plotted per heat map. Color-code indicates condition (Magenta: co-culture; green: pure bacteria; purple: in vivo) and study of origin (Orange: this study; purple: Cornforth et al., 2018; pink: Kordes et al., 2019; blue: Rossi et al., 2018). Heatmaps were plotted using the pheatmap function of DESeq2⁷⁹ (version 1.36.0) in Rstudio. (f) Protein-protein interaction network of common DEGs (in vivo and co-culture. Each node represents a protein encoded by a DEG. Edges represent known protein-protein association (either physical or functional) with a confidence level higher than 0.7. Node color represent clusters generated MCL method. Highlighted the pathway to which the cluster proteins belong. (g) Log₂ normalized count plots of representative genes from pathways highlighted by the network analysis. Color-code indicates Magenta: co-culture, green: pure bacteria and purple: in vivo.