FIG 2.

T4519 is an inducible Ser/Thr kinase. (A to F) PMA-differentiated THP-1 cells were infected with strain Ty2 and were subjected to a gentamicin protection assay as for Fig. 1. Error bars, means ± standard deviations for three independent experiments. Asterisks indicate significant differences by Student's t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (A and D) Reverse transcription-qPCR results show 16S rRNA-normalized expression of t4519 mRNA at the indicated time points (A) or 12 h postinfection (D). (B and E) Immunoblots using total lysates of Ty2-infected cells. T2544 was used as a control. Results of one of three independent experiments are shown. The rightmost lane in panel B shows recombinant proteins. (C and F) Densitometry of the immunoblots. (D to F) Cells were pretreated with either the vehicle (dimethyl sulfoxide [DMSO]) (14 μM), the ROS inhibitor apocyanin (Apo) (1 mM), the RNS inhibitor ammonium pyrrolidine dithiocarbamate (APDC) (100 μM), the vacuolar H⁺-ATPase inhibitor bafilomycin A1 (Baf1) (50 nM), the protein translation inhibitor CHX (25 μg/ml), or the iron chelator desferrioxamine (DFO) (40 μM) for 1 h. (G) Reverse transcription-qPCR shows 16S rRNA-normalized expression of t4519 mRNA from S. Typhi either grown in LB medium and treated with H₂O₂ (5 mM), Na-HOCl (0.5 mM), or ferric chloride (50 mM) for 10 min or cultured in N minimal medium (low pH, low Mg²⁺).