Virulence Factors of Pseudomonas aeruginosa Induce Both the Unfolded Protein and Integrated Stress Responses in Airway Epithelial Cells

Abstract

Pseudomonas aeruginosa infection can be disastrous in chronic lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease. Its toxic effects are largely mediated by secreted virulence factors including pyocyanin, elastase and alkaline protease (AprA). Efficient functioning of the endoplasmic reticulum (ER) is crucial for cell survival and appropriate immune responses, while an excess of unfolded proteins within the ER leads to “ER stress” and activation of the “unfolded protein response” (UPR). Bacterial infection and Toll-like receptor activation trigger the UPR most likely due to the increased demand for protein folding of inflammatory mediators. In this study, we show that cell-free conditioned medium of the PAO1 strain of P. aeruginosa, containing secreted virulence factors, induces ER stress in primary bronchial epithelial cells as evidenced by splicing of XBP1 mRNA and induction of CHOP, GRP78 and GADD34 expression. Most aspects of the ER stress response were dependent on TAK1 and p38 MAPK, except for the induction of GADD34 mRNA. Using various mutant strains and purified virulence factors, we identified pyocyanin and AprA as inducers of ER stress. However, the induction of GADD34 was mediated by an ER stress-independent integrated stress response (ISR) which was at least partly dependent on the iron-sensing eIF2α kinase HRI. Our data strongly suggest that this increased GADD34 expression served to protect against Pseudomonas-induced, iron-sensitive cell cytotoxicity. In summary, virulence factors from P. aeruginosa induce ER stress in airway epithelial cells and also trigger the ISR to improve cell survival of the host.

Transient Proteotoxicity of Bacterial Virulence Factor Pyocyanin in Renal Tubular Epithelial Cells Induces ER-Related Vacuolation and Can Be Efficiently Modulated by Iron Chelators

“…The observed hyperactivation of the transcription factor Nrf2, a chief regulator of the oxidative/electrophilic stress response, and a rather moderate activity for the p53 at cytotoxic PCN concentrations is also in concord with an idea that PCN disrupts the intracellular redox homeostasis by targeting thiols directly. Similar conclusions about PCN proteotoxicity have been made recently based on the experiments involving airway epithelial cells (van ‘t Wout et al , 2015) and Caernorabdus elegans (Ray et al , 2015). …”

The integrated endoplasmic reticulum stress response in Leishmania amazonensis macrophage infection: the role of X‐box binding protein 1 transcription factor

“…Total RNA was extracted from RAW 264.7 cells (1 3 10 6 ) with RNeasy Plus (Qiagen, Hilden, Germany), and 1 mg total RNA was reverse transcribed into the first-strand cDNA with ImProm (Promega, Madison WI, USA) and oligo (dT) [12][13][14][15][16][17][18] primer, according to the manufacturer's instructions. Forward 59-CCGCAGCACTCAGACTATG-39 and reverse 59-GGGTCCAACTTGTCCAGAAT-39 pairs were used to amplify uXBP1 mRNA; forward 59-CTGAGTCCGCAGCAGGT-39and reverse 59-AAACATGACAGGGTCCAACTT-39 primers were used to amplify sXBP1 mRNA; forward: 59-TCCAAGAAAGGA-CGAACATTC-39 and reverse 59-TGAGGACATCTCCCACG-TCAA-39 primers were used to amplify IFN1-b; and forward 59-GTGTCCGTCGTGGATCTG-39 and reverse 59-TGCTTCAC-CACCTTCTTGA-39 primers were used to amplify GAPDH.…”

“…For example, Listeria monocytogenes induces XBP1 splicing, which reduces the number of intracellular bacterial (17). Pseudomonas aeruginosa likewise triggers the IERSR and induces sXBP1 expression, to facilitate survival of the infected host cell (18).…”

Roles of endoplasmic reticulum stress-mediated apoptosis in M1-polarized macrophages during mycobacterial infections

“…Accumulation of unfolded proteins, hypoxia, glucose deprivation, oxidative stress, and bacterial infection has all been shown to induce ER stress and activate the unfolded protein response (UPR). For example, Pseudomonas aeruginosa infection has been shown to induce ER stress in primary bronchial epithelial cells12. Brucella melitensis infection induced UPR to promote bacterial intracellular growth13, and Simkania negevensis infection inhibited UPR to promote its intracellular proliferation14.…”