Fig. 2: NsrP negatively regulates virulence by inhibiting purD expression.

a The regions of base pairing between NsrP and purD as predicted by the TargetRNA3 with the probability threshold of 0.25 and p-value of 0.05. Point mutations to generate the disrupted alleles. b qRT-PCR analysis of purD in the NMEC WT, ΔNsrP, and cNsrP. c Western blot analysis and quantification of PurD-FLAG in the NMEC WT, ΔNsrP, and cNsrP. DnaK was used as the loading control. d, e Bacterial counts in the blood (CFU/mL, d) and meningitis development (e) were determined 4 h after intravenous injection of 1 × 10⁶ CFU of the WT, ΔNsrP, ΔpurD, ΔNsrPΔpurD or ΔpurD complemented (cpurD) strain (n = 8 for each group in d). CSF culture positivity was defined as meningitis. f H&E staining of the brain sections was performed 4 h after intravenous injection of 1 × 10⁶ CFU of the WT or ΔpurD. The right panels are higher-magnification images of the boxed regions, showing meningeal thickening and neutrophil infiltration (arrows). Scale bar, 100 μm. The images shown are representative of three independent experiments. g Survival curves of 2- to 5-day-old rats subcutaneously injected with 1 × 10⁵ CFU of the WT or ΔpurD (n = 16 for each group). The number of living animals was recorded every 8 h. h Growth curves of the WT and ΔpurD in M9 medium with or without 40 mM IMP. i, j Replication of the WT and ΔpurD in 60% NMS with (j) or without (i) 40 mM IMP. k, l Bacterial counts in the blood (CFU/mL, k) and meningitis development (l) were determined 4 h after intravenous injection of 1 × 10⁶ CFU of the WT, ΔpurD, or cpurD with or without IMP (n = 8 for each group in k). CSF culture positivity was defined as meningitis (l). ns nonsignificant. In b, c, h–j, data were presented as the means ± SDs (n = 3 independent experiments). One-way ANOVA (b, c, i, j), two-tailed Mann–Whitney U-test (d, k), log-rank (Mantel–Cox) test (g), and two-way ANOVA (h) were applied.