Fig. 4: The effect of CbpD on PA pathogenesis in vivo.

a CD1 mice were inoculated intravenously (IV) with 2 × 10⁷ CFU (left panel, 10 mice/group) and intratracheally (IT) with 5 × 10⁶ CFU (right panel, 10 mice/group) PA WT or ΔCbpD per mouse. Survival is represented by Kaplan–Meier survival curves and analyzed by log-rank (Mantel–Cox) test (left panel: P = 1.118E-5, right panel: P = 0.0250). b Bacterial loads in the kidney, liver, spleen and blood (CFU/g for organs and CFU/ml for blood) of 8-week-old CD1 mice were enumerated 4 h post systemic infection (IV) with PA WT or ΔCbpD. The data are plotted as the mean ± SEM, representing one experiment performed with eight mice/group and were analyzed by two-tailed t test (kidney P = 0.0129, blood P = 0.0002, liver P = 5.827E-5, spleen: P = 1.910E-5. Mock-infected mice (n = 4) were included as a control. c The categorical heatmap shows the concentration of cytokines, chemokines or growth factors in the serum of CD1 mice 4 h post infection (as described in b). The data are depicted as the geometric mean of the cytokine values, representing one experiment performed with eight mice/group. Mock-infected mice (n = 3) were included as a control. Individual values are plotted in Supplementary Fig. 12a, in which the mean ± SEM of each cytokine is depicted. The data were analyzed by two-way ANOVA (Dunnett’s multiple comparisons test) and the significant difference between WT and ΔCbpD is indicated by asterisks (*). IL-6: P = 0.0497, IL-12 (p40): P = 0.0018, G-CSF P < 0.0001, KC: P = 0.0063, MCP-1: P < 0.0001. d Representative hematoxylin and eosin-stained sections of spleen tissues from infected (WT or ΔCbpD, n = 8 mice/group) and uninfected mice (control, n = 4 mice) collected 4 h post infection (as described in b), analyzed by light microscopy. White pulp (WP) and red pulp (RP) regions are marked. Polymorphonuclear (PMN) leukocytes are indicated with arrows. The scale bar represents 50 and 20 µm in the upper and lower panels, respectively. The histopathologic scoring analysis of PMN infiltration of the spleen tissue is shown as a histogram. e Hierarchical clustering of spleen proteome based on Spearman rank correlation coefficients. The infection status (as described in b) is indicated by color and R stands for replicate, (indicating number of mice/group). f Principal component analysis (PCA) of the identified proteins, showing segregation of the spleen proteomes into two different infected (WT or ΔCbpD) and one uninfected group. The quantified proteins are plotted in two-dimensional principal component space by PC1 = 46.9724% and PC2 = 36.0129%. g Volcano plot showing differentially abundant proteins in the spleens of WT- or ΔCbpD-infected mice relative to mock-infected mice. The red dotted line(s) through the y axis and x axis, indicate significance cutoff values at q = 0.05 (log₁₀ = 1.3) and (+/−) 1.5-fold change (log₂ = 0.58) in protein abundance, respectively. Significance was determined using the two-tailed paired t test. h Heatmap of log₂ fold change values of the 32 significantly regulated proteins in infected (WT or ΔCbpD) vs. non-infected spleen. The proteins belong to the shared category. i Heatmap of enrichment score (−log₁₀ P value, cutoff = 0.01) showing pathways/cellular processes that were enriched in the spleen proteome (Supplementary Data 10 and 11, unique category) of ΔCbpD-infected mice (down- or upregulated; arrows pointing down/up). Enrichment analysis was performed by Metascape and the P value was calculated based on the cumulative hypergeometric distribution. j Depiction of the Ingenuity Pathway Analysis (IPA) of the unique proteome showing canonical pathway webs that are significantly altered in ΔCbpD-infected mice. The P value was calculated by right-tailed fisher’s exact test and is shown in the figure. Pathways without interaction with the web (singletons) are omitted from the visualization. The average fold change value of up- or downregulated proteins associated with some of the top-scored pathways are presented as heatmap and is visualized using a blue–white–red gradient. k Heatmap of enrichment score (−log P value, cutoff = 0.01) showing pathways/cellular processes that were enriched in the unique splenic proteomes (Supplementary Data 10 and 11) of WT-infected vs. uninfected mice Enrichment analysis was performed by Metascape and the P value was calculated based on the cumulative hypergeometric distribution. l Depiction of the IPA of the unique proteome showing canonical pathway webs that are significantly altered in WT-infected mice. The P value was calculated by right-tailed fisher’s exact test and is shown in the figure. Pathways without interaction with the web (singletons) are omitted from the visualization. When applicable, the significance is indicated by asterisks (*): *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. Source data are provided as a Source Data file (a–c).