The Chaperone and Redox Properties of CnoX Chaperedoxins Are Tailored to the Proteostatic Needs of Bacterial Species.

Hypochlorous acid (bleach), an oxidizing compound produced by neutrophils, turns the Escherichia coli chaperedoxin CnoX into a powerful holdase protecting its substrates from bleach-induced aggregation. CnoX is well conserved in bacteria, even in non-infectious species unlikely to encounter this oxidant, muddying the role of CnoX in these organisms. Here, we found that CnoX in the non-pathogenic aquatic bacterium Caulobacter crescentus functions as a holdase that efficiently protects 50 proteins from heat-induced aggregation. Remarkably, the chaperone activity of Caulobacter CnoX is constitutive. Like E. coli CnoX, Caulobacter CnoX transfers its substrates to DnaK/J/GrpE and GroEL/ES for refolding, indicating conservation of cooperation with GroEL/ES. Interestingly, Caulobacter CnoX exhibits thioredoxin oxidoreductase activity, by which it controls the redox state of 90 proteins. This function, which E. coli CnoX lacks, is likely welcome in a bacterium poorly equipped with antioxidant defenses. Thus, the redox and chaperone properties of CnoX chaperedoxins were fine-tuned during evolution to adapt these proteins to the specific needs of each species.IMPORTANCE How proteins are protected from stress-induced aggregation is a crucial question in biology and a long-standing mystery. While a long series of landmark studies have provided important contributions to our current understanding of the proteostasis network, key fundamental questions remain unsolved. In this study, we show that the intrinsic features of the chaperedoxin CnoX, a folding factor that combines chaperone and redox protective function, have been tailored during evolution to fit to the specific needs of their host. Whereas Escherichia coli CnoX needs to be activated by bleach, a powerful oxidant produced by our immune system, its counterpart in Caulobacter crescentus, a bacterium living in bleach-free environments, is a constitutive chaperone. In addition, the redox properties of E. coli and C. crescentus CnoX also differ to best contribute to their respective cellular redox homeostasis. This work demonstrates how proteins from the same family have evolved to meet the needs of their hosts.

Comprehensively Characterizing the Thioredoxin Interactome In Vivo Highlights the Central Role Played by This Ubiquitous Oxidoreductase in Redox Control.

Thioredoxin (Trx) is a ubiquitous oxidoreductase maintaining protein-bound cysteine residues in the reduced thiol state. Here, we combined a well-established method to trap Trx substrates with the power of bacterial genetics to comprehensively characterize the in vivo Trx redox interactome in the model bacterium Escherichia coli Using strains engineered to optimize trapping, we report the identification of a total 268 Trx substrates, including 201 that had never been reported to depend on Trx for reduction. The newly identified Trx substrates are involved in a variety of cellular processes, ranging from energy metabolism to amino acid synthesis and transcription. The interaction between Trx and two of its newly identified substrates, a protein required for the import of most carbohydrates, PtsI, and the bacterial actin homolog MreB was studied in detail. We provide direct evidence that PtsI and MreB contain cysteine residues that are susceptible to oxidation and that participate in the formation of an intermolecular disulfide with Trx. By considerably expanding the number of Trx targets, our work highlights the role played by this major oxidoreductase in a variety of cellular processes. Moreover, as the dependence on Trx for reduction is often conserved across species, it also provides insightful information on the interactome of Trx in organisms other than E. coli.

Reducing systems protecting the bacterial cell envelope from oxidative damage.

Exposure of cells to elevated levels of reactive oxygen species (ROS) damages DNA, membrane lipids and proteins, which can potentially lead to cell death. In proteins, the sulfur-containing residues cysteine and methionine are particularly sensitive to oxidation, forming sulfenic acids and methionine sulfoxides, respectively. The presence of protection mechanisms to scavenge ROS and repair damaged cellular components is therefore essential for cell survival. The bacterial cell envelope, which constitutes the first protection barrier from the extracellular environment, is particularly exposed to the oxidizing molecules generated by the host cells to kill invading microorganisms. Therefore, the presence of oxidative stress defense mechanisms in that compartment is crucial for cell survival. Here, we review recent findings that led to the identification of several reducing pathways protecting the cell envelope from oxidative damage. We focus in particular on the mechanisms that repair envelope proteins with oxidized cysteine and methionine residues and we discuss the major questions that remain to be solved.

A new role for Escherichia coli DsbC protein in protection against oxidative stress.

We report a new function for Escherichia coli DsbC, a protein best known for disulfide bond isomerization in the periplasm. We found that DsbC regulates the redox state of the single cysteine of the L-arabinose-binding protein AraF. This cysteine, which can be oxidized to a sulfenic acid, mediates the formation of a disulfide-linked homodimer under oxidative stress conditions, preventing L-arabinose binding. DsbC, unlike the homologous protein DsbG, reduces the intermolecular disulfide, restoring AraF binding properties. Thus, our results reveal a new link between oxidative protein folding and the defense mechanisms against oxidative stress.

Dissecting the machinery that introduces disulfide bonds in Pseudomonas aeruginosa.

UNLABELLED: Disulfide bond formation is required for the folding of many bacterial virulence factors. However, whereas the Escherichia coli disulfide bond-forming system is well characterized, not much is known on the pathways that oxidatively fold proteins in pathogenic bacteria. Here, we report the detailed unraveling of the pathway that introduces disulfide bonds in the periplasm of the human pathogen Pseudomonas aeruginosa. The genome of P. aeruginosa uniquely encodes two DsbA proteins (P. aeruginosa DsbA1 [PaDsbA1] and PaDsbA2) and two DsbB proteins (PaDsbB1 and PaDsbB2). We found that PaDsbA1, the primary donor of disulfide bonds to secreted proteins, is maintained oxidized in vivo by both PaDsbB1 and PaDsbB2. In vitro reconstitution of the pathway confirms that both PaDsbB1 and PaDsbB2 shuttle electrons from PaDsbA1 to membrane-bound quinones. Accordingly, deletion of both P. aeruginosa dsbB1 (PadsbB1) and PadsbB2 is required to prevent the folding of several P. aeruginosa virulence factors and to lead to a significant decrease in pathogenicity. Using a high-throughput proteomic approach, we also analyzed the impact of PadsbA1 deletion on the global periplasmic proteome of P. aeruginosa, which allowed us to identify more than 20 new potential substrates of this major oxidoreductase. Finally, we report the biochemical and structural characterization of PaDsbA2, a highly oxidizing oxidoreductase, which seems to be expressed under specific conditions. By fully dissecting the machinery that introduces disulfide bonds in P. aeruginosa, our work opens the way to the design of novel antibacterial molecules able to disarm this pathogen by preventing the proper assembly of its arsenal of virulence factors. IMPORTANCE: The human pathogen Pseudomonas aeruginosa causes life-threatening infections in immunodepressed and cystic fibrosis patients. The emergence of P. aeruginosa strains resistant to all of the available antibacterial agents calls for the urgent development of new antibiotics active against this bacterium. The pathogenic power of P. aeruginosa is mediated by an arsenal of extracellular virulence factors, most of which are stabilized by disulfide bonds. Thus, targeting the machinery that introduces disulfide bonds appears to be a promising strategy to combat P. aeruginosa. Here, we unraveled the oxidative protein folding system of P. aeruginosa in full detail. The system uniquely consists of two membrane proteins that generate disulfide bonds de novo to deliver them to P. aeruginosa DsbA1 (PaDsbA1), a soluble oxidoreductase. PaDsbA1 in turn donates disulfide bonds to secreted proteins, including virulence factors. Disruption of the disulfide bond formation machinery dramatically decreases P. aeruginosa virulence, confirming that disulfide formation systems are valid targets for the design of antimicrobial drugs.