Figure 3.

Salmonella exhibits enhanced recovery from cells undergoing UPR. (A) Pharmacological UPR-inducing agents and their mode of action. A23187 is a Ca²⁺ ionophore that disrupts intracellular Ca²⁺ levels. Thapsigargin is a non-competitive inhibitor of the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) pump leading to reduced Ca²⁺ endoplasmic reticulum (ER) concentrations. Tunicamycin inhibits the addition of carbohydrate (CHO) moities to newly synthesised proteins. MG132 inhibits proteasome-mediated degradation leading to accumulation of misfolded proteins within the ER. Triacin C is also shown and inhibits de novo lipid synthesis. (B) Treatment of cells with UPR-inducing drugs increases the number of intracellular bacteria 24 hours pi. Cells were treated with A23187 (0.5 mM), MG132 (0.5 µg/mL), TPG (200 nM) and TUN (0.5 µg/mL). The mean fold increases in mCherry MFI values±SEM are shown (n=3). ANOVA was performed on mCherry MFI values (p<0.0001) with Tukey’s multiple comparison post-test to determine significant differences between individual groups (*p<0.05m, ***p<0.001. (C–D) TPG-treated cells exhibit significant increases in MFI values of the infected cells when compared with dimethyl sulfoxide (DMSO) controls (C) and in the fold difference in MFI values between the DMSO-treated and TPG-treated cells (D) in a concentration-dependent manner. The mean MFI values±SEM (n=3) are shown and ANOVA was performed (p<0.0001) with Tukey’s multiple comparison post-test used to determine if there were significant differences between DMSO and all drug-treated groups at 24 hours pi (***p<0.001). (E) Recovery of viable bacteria from infected cells exhibits similar fold differences between the DMSO-treated and TPG-treated cells infected at 24 hours pi. The mean fold differences in cfu values (n=6)±SEM are shown. The Mann-Whitney test was used to compare cfu recoveries between DMSO-treated and TPG-treated cells and the exact p values were calculated (*p=0.004, **p=0.015). ANOVA, analysis of variance; cfu, colony-forming unit; ER, endoplasmic reticulum; MFI, mean fluorescence intensity; pi, postinfection; SERCA, sarco/endoplasmic reticulum Ca²⁺-ATPase; TPG, thapsigargin; TUN, tunicamycin; UPR, unfolded protein response.