Fig 3. In vitro regulation of the capsule by SpxR and CpsR through the 37-CE and in vivo imaging studies.

(A) Luciferase reporter assay in Δ37-CE, and strains where only SpxR (SpxR-only), CpsR (CpsR-only), or neither SpxR nor CpsR can bind (Neither) the 37-CE sequence. Individual data points, the mean, and SEM are plotted from three biological replicates. RLU; relative light units. (B) Western blot analysis of CpsA protein. A representative blot of three independent experiments is shown with quantification below. (C) Quantification of capsule content using dextran exclusion assay. Individual cells from five biological replicates, the mean, and SEM are plotted. (D) Representative images of C showing a fluorescence and phase contrast microscopy overlay. The dark area around the cell represents the capsule. (E) (Left) In vivo imaging quantification (total luminescence over lung normalized to CFU/mg of lung tissue) of P_cps::CBRluc reporters during a pneumonia model of infection. (Right) Example images of one set of mice. Statistical differences in A and C were determined using a one-way ANOVA with Tukey’s multiple comparisons test. Statistical differences in B were determined using a one sample t test and Wilcoxon test. ns = not significant, *** p≤0.001, **** p≤0.0001.