Fig. 6: RhoB promotes bacterial clearance in vivo.
a, b Protein level of RhoB, Beclin 1 and LC3 in CFT073-infected wild-type mice. Female C57BL/6J mice were transurethrally inoculated with 108 CFU of CFT073 cultured overnight. a Immunohistofluorescence analysis. Levels of indicated proteins were quantified by measurement of fluorescent intensity using Image Pro Plus software. Expression of each indicated protein in control mice was set to 100% (n = 6 images assessed from 3 mice per group) (left). Representative confocal images were shown (right). Indicated proteins were stained with appropriate primary antibodies plus Alexa Fluor-488-conjugated secondary antibody (green). Nuclei were counterstained with DAPI (blue). Scale bar, 25 μm. RhoB P = 0.0087, Beclin 1 P = 0.0022, LC3 P = 0.0022. b Western blotting analysis of indicated proteins in CFT073-infected mice. n = 3 mice. c Protein level of RhoB, Beclin 1 and LC3 in RhoB+/− and RhoB−/− mice infected by CFT073 through immunohistofluorescence analysis. Quantification of indicated proteins was shown, and expression of each indicated protein in WT mice was set to 100% (n = 6 images assessed from 3 mice per group) (left). Representative confocal images were shown (right). Scale bar, 25 μm. RhoB: WT vs. RhoB+/− P < 1.0e−15, WT vs. RhoB−/− P < 1.0e−15; Beclin 1: WT vs. RhoB+/− P < 1.0e−15, WT vs. RhoB−/− P < 1.0e−15; LC3: WT vs. RhoB+/− P < 1.0e−15, WT vs. RhoB−/− P < 1.0e−15. d Western blotting analysis of indicated proteins in CFT073-infected RhoB−/− mice. n = 3 mice. e Bacterial titers in bladders of WT, RhoB+/− and RhoB−/− mice infected by CFT073 (n = 5 mice per group). WT vs. RhoB+/− P = 0.0492, WT vs. RhoB−/− P < 1.0e−15. f Schematic diagram illustrating RhoB-induced UPEC clearance. During UPEC infections, RhoB is rapidly induced upon LPS stimulation through TLR4-mediated signaling, possibly through the activated NF-κB pathway. RhoB forms a complex with Beclin 1 and Hsp90, which leads to increased stability of Beclin 1. As a result, more autophagosomes are formed, resulting in enhanced clearance of intracellular UPEC. Data are the mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed unpaired Mann−Whitney test (a), two-way ANOVA (c), or one-way ANOVA (e).
