Figure 2. The Two-Component System RprXY Is a BFT Suppressor and Protects Susceptible Mice from Lethal ETBF Colonization.

(A) EMSA was performed with labeled bft promoter and increasing concentrations of rRprY with (rRprY~P) or without acetyl-phosphate treatment. Results are representative of three independent experiments.

(B) RprX or RprY were overexpressed downstream of the GAPDH promoter on a plasmid in ETBF ATCC 43858 (ETBF pRprX and ETBF RprY, respectively) and compared with ETBF encoding empty vector (ETBF EV). RNA was extracted from these ETBF clones and bft transcript quantified using qRT-PCR, normalized to 16S RNA. Results are pooled from four biological replicates of independent trials. Data are presented as mean ± SD; p value was calculated using one-way ANOVA with Dunnett’s multiple comparisons test, **p < 0.01.

(C) WT mice were orally gavaged with ETBF ATCC 43858 WT (n = 12) or ETBF ΔrprX (n = 12). Two days after colonization, fecal RNA was extracted and quantified for bft mRNA using qRT-PCR, normalized to B. fragilis 16S rRNA. The results are pooled from three independent experiments. All data points are shown. p Value was calculated using the Mann-Whitney U test, *p < 0.05.

(D) WT mice were orally gavaged with ETBF EV (n = 9), ETBF pRprX (n = 9), or ETBF pRprY (n = 9). Two days after colonization, fecal RNA was extracted and quantified for bft mRNA using qRT-PCR, normalized to B. fragilis 16S rRNA. Results are pooled from two independent experiments. Data are presented as the mean. p Value was calculated using one-way ANOVA with Dunnett’s multiple comparisons test: *p < 0.05; n.s., not significant.

(E) Survival of Muc2^−/− mice after oral gavage with ETBF EV (n = 14), ETBF pRprX (n = 13) or ETBF pRprY (n = 14) clones at 120 hr post colonization. Results depict pooled data of three independent experiments. p Values were calculated by log-rank Mantel-Cox test comparing ETBF EV and ETBF RprX or ETBF EV and ETBF RprY groups: *p < 0.05, ***p < 0.001.