Oxidation of Thiols and Modification of Redox-Sensitive Signaling in Human Lung Epithelial Cells Exposed to Pseudomonas pyocyanin

Abstract

The aim of this study was to examine the effects of pyocyanin exposure on mitochondrial GSH, other cellular thiols (thioredoxin-1, Trx-1), and oxidant-sensitive signaling pathways hypoxia inducible factor (HIF-1α) and heme oxygenase (HO-1) in A549 and HBE cell lines. A549 human type II alveolar epithelial cells and human bronchial epithelial (HBE) cells were treated with varying concentrations of pyocyanin extracted from Pseudomonas aeruginosa bacteria. Cytoplasmic and mitochondrial thiols and oxidant sensitive signal transduction proteins (HIF-1α and HO-1) were measured. Exposure to pyocyanin generated reactive oxygen species (ROS) in cellular mitochondria and altered total cellular glutathione (GSH). Pyocyanin, at concentrations present in conditions in vivo, increased oxidized Trx-1 in A549 human type II alveolar epithelial cells and HBE cells by 184 and 74%, respectively. Oxidized mitochondrial glutathione (GSSG) was elevated more than twofold in both cell types. Pyocyanin also increased the cellular oxidant-sensitive proteins HIF-1α and HO-1. Data indicate that pyocyanin-induced alterations in mitochondrial and cytosolic thiols, as well as oxidant-sensitive proteins, may contribute to P. aeruginosa-mediated lung injury.

Acknowledgments

This work was supported in part by grants from the NIH (AI34954 and AI60770) and VA Research service to BEB. The authors thank Yunxia O'Malley for technical assistance in the development and performance of the HO-1 and transcription factor protocols.