FIGURE 4.

Decreased cAMP contributed to the T3SS defect in the ΔnrtR mutant. (A,B) cAMP level was decreased in ΔnrtR mutant and Δvfr mutant. β-galactosidase assay was used to examine the transcriptional activity of lacP1 promoter fused to a lacZ gene in indicated strains under T3SS inducing and non-inducing conditions (A). (B) Intracellular cAMP levels were measured using an ELISA kit. Error bars represent standard deviations. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 by Student’s t-test. (C–F) Exogenous addition of cAMP recovers the expression of ExsA, ExoS and cytotoxicity of the ΔnrtR mutant, while not of or ΔnrtRΔvfr mutant. (C) Relative mRNA levels of exsA in indicated strains with or without cAMP addition at the beginning of subculture in the presence or absence of 5 mM EGTA, with rpsL as an internal control. ns, not significant, ^∗P < 0.05, ^∗∗∗P < 0.001 by Student’s t-test. (D) Indicated strains containing an exsA-Flag driven by its native promoter were grown at 37°C with or without 5 mM EGTA and 50 mM cAMP as indicated. Protein samples from equal number of bacteria were separated by SDS-PAGE and probed with an anti-Flag antibody or an anti-RNA polymerase beta subunit antibody. (E) Expression of ExoS in indicated strains were grown with or without 5 mM EGTA and 50 mM cAMP. The protein levels were detected with an antibody against ExoS or RNA polymerase beta subunit. S, supernatant; P, pellet. (F) Cytotoxicity of indicated strains in the presence or absence of 50 mM cAMP. HeLa cells were infected with indicated strains at a MOI of 50. 50 mM final concentration of cAMP was added to DMEM medium as indicated. Three hours post infection, cells attached to the 24-well plate were washed with PBS and stained with crystal violet. The cell associated crystal violet was dissolved in ethanol and quantified by measuring OD₅₉₀. HeLa cells with no bacterial infection (blank and blank+50 mM cAMP) served as a control. ^∗∗P < 0.01, by Student’s t-test.