Fig 1.
Schematic overview of the unfolded protein response (UPR) and integrated stress response (ISR).
Fig 2.
P. aeruginosa secreted virulence factors induce ER stress in primary bronchial epithelial cells.
A. Time-dependent induction of ER stress in primary bronchial epithelial cells, as assessed by XBP1 splicing, CHOP and GADD34 mRNA after treatment with CM-PAO1 (n = 5; mean ± SEM). B. Dose-response of spliced XBP1, CHOP and GADD34 mRNA in primary bronchial epithelial cells treated with CM-PAO1 for 12 hours (n = 5; mean ± SEM). C. Time-dependent phosphorylation of eIF2α (p-EIF2α) and synthesis of GADD34 and GRP78 (visualised with anti-KDEL antibody). Relative quantifications for each protein are shown within; representative of n = 3). D. Time-dependent decrease of puromycin incorporation in nascent proteins. Total eIF2α and β-actin serve as loading controls. * p<0.05, ** p<0.01, ***p<0.001 versus control (ctrl) with two-way repeated-measurements ANOVA (Bonferroni post-hoc) or untreated (-) with a one-way repeated-measurements ANOVA (Bonferroni post-hoc).
Fig 3.
Conditioned medium of P. aeruginosa induces ER stress via TAK1-p38 MAP kinase (MAPK).
A. Time-dependent phosphorylation of p38 MAPK in 16HBE after treatment with CM-PAO1. Total p38 MAPK and β-actin serve as loading controls. Numbers display the fold increase of phosphorylated p38 MAPK to total p38 MAPK compared to phosphorylated p38 MAPK at t = 0 (representative of n = 3). B. Western blot of phosphorylated p38 MAPK from 16HBE after pre-treatment for 30 min with the TAK-1 inhibitor LL-Z1640-2 (LL) or p38 MAPK inhibitor SB203580 (SB), followed by CM-PAO1 stimulation for 6 hours. Total p38 MAPK and β-actin serve as loading controls. Numbers display the fold increase of phosphorylated p38 MAPK to total p38 MAPK compared to phosphorylated p38 MAPK at t = 0 (representative of n = 3). C. IL-8 release of 16HBE cells treated as in B (n = 3; mean ± SEM). D. Normalised mRNA levels of spliced XBP1, CHOP, GADD34 and GRP78 in 16HBE cells, treated as B (n = 3; mean ± SEM). All values are normalised to the housekeeping genes RPL13A and ATP5B. * p<0.05, ** p<0.01, ***p<0.001 versus untreated (-) with a one-way repeated-measurements ANOVA (Bonferroni post-hoc).
Fig 4.
Pyocyanin is able to cause ER stress.
A. Quantitation of pyocyanin in CM-PAO1. Iron (Fe3+) was supplemented in the culture medium of the bacteria to inhibit virulence factor secretion (n = 3; mean ± SEM). B. Normalised mRNA expression levels of spliced XBP1, CHOP, GADD34 and GRP78 after pyocyanin treatment (0-1-3-10-30 μM) (n = 3; mean ± SEM). All values are normalised to the housekeeping genes RPL13A and ATP5B. C. IL-8 release of 16HBE cells treated as in B (n = 3; mean ± SEM). D. Western blot for GRP78/GRP94 and GAPDH (loading control) from 16HBE cell lysates treated as in B. (representative of n = 3). E. Spliced XBP1, CHOP, GADD34 and GRP78 mRNA levels in 16HBE cells exposed to CM-PAO1 derived after growth in the presence or absence of iron (Fe3+) (n = 3; mean ± SEM). All values are normalised to the housekeeping genes RPL13A and ATP5B. * p<0.05, ** p<0.01, ***p<0.001 versus untreated (-) with a one-way repeated-measurements ANOVA (Bonferroni post-hoc).
Table 1.
Bacterial strains.
Fig 5.
The P. aeruginosa secreted virulence factors alkaline protease (AprA) and pyocyanin mediate ER stress.
A. Western blot analysis of conditioned medium (CM) of strains PAO25, PAN8 and PAN11 for AprA (representative of n = 2; complete blot is shown in S3B Fig). Equal volumes of the conditioned medium were loaded onto the gel. AprA displays 200 nM purified AprA and serves as a positive control. B. Normalised expression levels of XBP1 splicing, CHOP, GADD34 and GRP78 mRNA in 16HBE cells after stimulation with CM-PAO25, CM-PAN8 or CM-PAN11 (n = 3; mean ± SEM). All values are normalised to the housekeeping genes RPL13A and ATP5B. C. Normalised expression values of spliced XBP1, CHOP, GADD34 and GRP78 mRNA in 16HBE cells after stimulation with 10 nM purified AprA. All values are normalised to the housekeeping genes RPL13A and ATP5B. * p<0.05, ** p<0.01, ***p<0.001 versus untreated (-) with a one-way repeated-measurements ANOVA (Bonferroni post-hoc).
Fig 6.
Splicing of XBP1 mRNA is dependent on the ER stress responsive kinase IRE1α.
Splicing of XBP1 mRNA in 16HBE cells after treatment with CM-PAO1 in the presence of 30 μM 4μ8C, a selective inhibitor of the ER stress responsive kinase IRE1α (n = 3; mean ± SEM). All values are normalised to the housekeeping genes RPL13A and ATP5B. * p<0.05, ** p<0.01, *** p<0.001 versus untreated (-) with a two-way repeated-measurements ANOVA (Bonferroni post-hoc).
Fig 7.
GADD34 mRNA is regulated via the activation of the integrated stress response by P. aeruginosa.
B-G. Gadd34 mRNA normalised expression in Perk-/-, eIF2αAA, Atf4-/-, Pkr-/-, Gcn2-/- and Hri-/- mouse embryonic fibroblasts (MEFs) exposed to CM-PAO1 for 8, 16 or 24 hours or tunicamycin (Tm) for 6 hours as a positive control (n = 3; mean ± SEM). All values are normalised to the housekeeping genes Actb and Sdha. H. GADD34 mRNA levels in HeLa cells upon exposure to CM-PAO1 after knock-down of GCN2 or HRI with siRNA (n = 3; mean ± SEM). All values are normalised to the housekeeping genes RPL13A and ATP5B. I. Normalised expression values of spliced XBP1, CHOP, GADD34 and GRP78 mRNA in 16HBE cells after stimulation with 1–100 nM deferoxamine (DFO). All values are normalised to the housekeeping genes RPL13A and ATP5B. J. Gadd34 mRNA levels in wild-type MEFs after repletion of the cell culture medium with iron (Fe3+) when treated with CM-PAO1 (n = 3; mean ± SEM). The first column (- Fe3+,—CM-PAO1) reflects medium control cells, without adding or depleting iron from the cell culture medium. All values are normalised to the housekeeping genes Actb and Sdha. * p<0.05, ** p<0.01, *** p<0.001 versus untreated (-) with a two-way repeated-measurements ANOVA (Bonferroni post-hoc).
Fig 8.
Induction of GADD34 protects against P. aeruginosa mediated cell cytotoxicity.
A. LDH release of Gadd34+/+ and Gadd34ΔC/ΔC MEFs after stimulation with CM-PAO1 for 16 and 24 hours (n = 3; mean ± SEM). B. MTT assay assessing cell viability of HeLa cells conditionally expressing GADD34 (± dox) after treatment with CM-PAO1 (n = 3; mean ± SEM). C. LDH release (left) and cell viability assessed with a MTT assay (right) of wild-type MEFs treated with CM-PAO1 after repleting the cell culture medium with iron (Fe3+) (n = 3; mean ± SEM). * p<0.05, ** p<0.01, *** p<0.001 versus untreated (-) with a two-way repeated-measurements ANOVA (Bonferroni post-hoc).
Table 2.
qPCR primers.