Nonnative Disulfide Bond Formation Activates the σ ³² -Dependent Heat Shock Response in Escherichia coli

Abstract

c Formation of nonnative disulfide bonds in the cytoplasm, so-called disulfide stress, is an integral component of oxidative stress. Quantification of the extent of disulfide bond formation in the cytoplasm of Escherichia coli revealed that disulfide stress is associated with oxidative stress caused by hydrogen peroxide, paraquat, and cadmium. To separate the impact of disulfide bond formation from unrelated effects of these oxidative stressors in subsequent experiments, we worked with two complementary approaches. We triggered disulfide stress either chemically by diamide treatment of cells or genetically in a mutant strain lacking the major disulfide-reducing systems TrxB and Gor. Studying the proteomic response of E. coli exposed to disulfide stress, we found that intracellular disulfide bond formation is a particularly strong inducer of the heat shock response. Real-time quantitative PCR experiments showed that disulfide stress induces the heat shock response in E. coli 32 dependently. However, unlike heat shock treatment, which induces these genes transiently, transcripts of 32 -dependent genes accumulated over time in disulfide stress-treated cells. Analyzing the stability of 32 , we found that this constant induction can be attributed to an increase of the half-life of 32 upon disulfide stress. This is concomitant with aggregation of E. coli proteins treated with diamide. We conclude that oxidative stress triggers the heat shock response in E. coli 32 dependently. The component of oxidative stress responsible for the induction of heat shock genes is disulfide stress. Nonnative disulfide bond formation in the cytoplasm causes protein unfolding. This stabilizes 32 by preventing its DnaK-and FtsH-dependent degradation.O xidative stress is defined as an imbalance between the generation of reactive oxygen species (ROS), such as superoxide anion, hydrogen peroxide, and hydroxyl radicals, and their detoxification by cellular antioxidant systems. ROS have the potential to attack virtually any cellular macromolecule, including DNA, lipids, and proteins. To counteract the damaging effects, microorganisms respond to oxidative stress in various ways. The classical oxidative stress response is regulated by transcription factors, such as OxyR from Escherichia coli, OhrR from Bacillus subtilis, and Yap1 from Saccharomyces cerevisiae, which all act as reversible redox switches (1-3). Activated by the oxidation of highly conserved cysteines, these regulators induce the expression of antioxidant enzymes, such as peroxiredoxins, glutaredoxins, and thioredoxins.A separate class of transcription factors which specifically sense disulfide stress has been reported in a number of organisms. These regulators are activated when the thiol-disulfide balance is perturbed, resulting in the formation of inter-and intramolecular disulfides within proteins or thiolation of proteinogenic amino acids with small-molecule thiols such as glutathione. This condition may be caused by an accumulation of naturally occurring electrophiles, such as...

Peptidoglycan Recognition Proteins Kill Bacteria by Inducing Oxidative, Thiol, and Metal Stress

“…We then tested the hypothesis that PGRPs cause thiol (disulfide) stress by inducing depletion of intracellular thiols, because the pattern of gene induction by PGRP was similar to the previously reported pattern of gene induction by diamide (a thiol-depleting electrophile), including activation of genes for the same metal detoxification systems, chaperones, protein quality control, and thiol stress responses [19], [20]. PGRP, similar to diamide, depleted over 90% of intracellular thiols in E. coli and B. subtilis within 30 min of exposure (Figure 4A), and these low levels of thiols were maintained for at least 2 hrs both in PGRP- and diamide-treated bacteria (data not shown).…”

“…Oxidative and thiol stress not only directly damage cells, but also release Fe from proteins, increase intracellular concentration of Zn and Cu, and increase toxicity of most metals [19], [21], [35]–[37]. Thiols bind free metal ions and protect cells from metal toxicity [38], and for this reason thiol stress induces the same genes for metal detoxification and protein refolding and repair [19], [20], [31] as the genes induced by PGRP (Tables 1, S1, and S2). …”

Bactericidal peptidoglycan recognition protein induces oxidative stress in Escherichia coli through a block in respiratory chain and increase in central carbon catabolism

“…However, the mechanism of PGRP‐induced depletion of thiols is unknown. Because PGRP does not enter the cytoplasm (Kashyap et al ., ) and also because PGRP by itself is not a thiol‐oxidizing electrophile, the simplest hypothesis to explain PGRP‐induced thiol depletion would be through induction of intracellular reactive oxygen species (ROS), as thiol stress is often considered a consequence and a component of oxidative stress (Leichert et al ., ; Müller et al ., ). Here we tested this hypothesis for PGRP‐induced depletion of thiols, using deletion mutants for respiratory chain, TCA cycle, their regulators and other flavoproteins that had severely diminished PGRP‐induced H 2 O 2 production (Fig.…”

“…Depletion of intracellular thiols, known as thiol (or disulfide) stress, can be induced by oxidative stress through ROS‐mediated oxidation of thiols, or by electrophiles, such as quinones, aldehydes, and azo‐ or amido‐compounds. These electrophiles can also induce ROS or can directly modify thiols by oxidation, alkylation, or arylation (Leichert et al ., ; Liebeke et al ., ; Pöther et al ., ; Faulkner and Helmann, ; Müller et al ., ).…”

Protein Quality Control under Oxidative Stress Conditions

“…This result was independently confirmed by the fact that HOCl-treatment of bacteria triggers the heat shock response, a highly conserved transcriptional response which is known to be induced by the accumulation of protein folding intermediates (64, 65). While H 2 O 2 treatment in E. coli yields in little to no protein aggregation and no significant heat shock response, exposure to disulfide stress (either caused by diamide or genetic depletion of the Trx/GSH systems) revealed a considerable overlap between heat shock and oxidative stress response pathways in both Gram-positive and Gram-negative bacteria (66–68). Eukaryotes appear to experience oxidative protein unfolding during peroxide treatment (69).…”