Fig. 2: CbpD expression and proteomics analysis.

a Expression of cbpD in wild-type (WT) PA14 was evaluated for the mid-exponential growth phase (OD₆₀₀ = 0.6) in LB medium, and M9_PA or M9_PA supplemented with NHS (10%, 30 min incubation). The data are plotted as the mean ± SEM, representing three experiments performed in duplicate and analyzed by two-way ANOVA (Tukey’s multiple comparisons test; M9_PA vs LB P = 0.0279, M9_PA vs NHS P = 0.0410). b Histogram showing the total number of the quantified proteins per condition in WT and its isogenic mutant, ΔCbpD. c Principal component analysis (PCA) performed on the entire proteome. For each individual replicate, the quantified proteins were plotted in two-dimensional principal component space by PC1 = 43.8092% and PC2 = 31.5492% and clustered according to growth condition and strain. d Volcano plots demonstrating differentially abundant proteins and q values of significance identified by comparing the ΔCbpD vs. WT proteomes. The red dotted line(s) crossing the y axis and x axis indicate significance cutoff at q = 0.05 (log₁₀ = 1.3) and (+/−) 1.5-fold change (log₂ = 0.58) in protein abundance. Cutoff values for significance were set to fold change ≥1.5 and q ≤ 0.05 in a two-tailed paired t test. e Hierarchical clustering of proteins expressed by WT and ΔCbpD in LB, RPMI, and RPMI-NHS using agglomerative hierarchical clustering analysis. For visualization, rows (genes) have been standardized, so that the mean is 0 and the standard deviation is 1. f Heatmap showing KEGG pathways that were significantly enriched in ΔCbpD vs. WT upon growth in RPMI or RPMI/NHS. g Heatmaps showing the average fold change values (log₂) for significantly regulated proteins belong to shared proteome in the ΔCbpD compared to WT strain in all growth conditions. h, i Heatmaps showing the average fold change values (log₂) for selected regulators and virulence factors in the unique proteome that are significantly regulated in ΔCbpD relative to WT during growth in RPMI (h) or RPMI-NHS (i). The selected proteins are marked in bold in Supplementary Data 3. j Functional analysis of protein-protein interaction networks revealed some highly interconnected proteins listed in (i). The heatmap (i) was mapped to the nodes using a blue–white–red gradient that indicates fold change log2 LFQ (ΔCbpD/WT). Proteins without any interaction partners (singletons) or chains with no interaction with the main network have been omitted from the visualization. Source data are provided as a Source Data file (a) or Supplementary Data.