Fig. 5. Regulation of the expression and secretion of T3SS-1 effectors by c-di-GMP signaling is dependent on the binding of c-di-GMP to SicA.
a ITC analysis of c-di-GMP binding to SicA. Data shown are one representative of three independent experiments with similar results, with Kd and complex stoichiometry (n) presented as mean ± s.d. b Co-IP of InvF-His6 with SicA-HA is impaired by c-di-GMP, but not by cGMP or c-di-AMP. The immunoprecipitated proteins (HA-IP) and the total cell lysates (Input) were assessed by western blot analysis. c–e EMSAs for InvF/SicA binding to promoters of sopB (c), sopE2 (d), and the sicAsipBCDA operon (e) in the presence and absence of nucleotides. f, g c-di-GMP decreased the Co-IP of SipB-His6 (f) and SipC-His6 (g) with SicA-HA in a dose-dependent fashion. h Western blot analysis of intracellular accumulation and secretion of SipB and SipC by strains with PsicAsipBCDA replaced by PinvF. Band intensities were presented as values relative to that of the wildtype with promoter replacement (mean ± s.d.; n = 3 independent experiments). i Surface representation of the homology model of SicA in complex with c-di-GMP. c-di-GMP is shown as purple sticks. j Schematic of the predicted contacts between c-di-GMP and SicA. Potential hydrogen bonds are indicated as green dashed lines. k Binding of c-di-GMP to SicA and its mutants, as measured by ITC (Kd = mean ± s.d.; n = 3 independent experiments). l qRT-PCR analyses of the mRNA levels of T3SS-1 genes. Expression was presented as values relative to that of sicA of the wildtype (mean ± s.e.m.; n = 3 independent experiments). m Survival curves of 6-week-old female BALB/c mice infected orally with S. Typhimurium strains. Data were illustrated as a percentage of mice survival (n = 10 mice per group). b–h Gels or blots shown are one representative of three independent experiments with similar results. Statistical significance was calculated using the Student’s t-test (h, l) or Log-rank (Mantel–Cox) test (m). h, l, m Differences were considered statistically significant at p < 0.05. Source data are provided as a Source Data file.