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

Abstract

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.

Keywords: chaperones; oxidative stress; protein folding; thioredoxin.

FIG 1

CcCnoX is a constitutive holdase. (a) CcCnoX inhibits the aggregation of thermally (43°C) denatured CS when present at high ratios. This test was performed in triplicate; this panel shows representative results. Additional results appear in the supplemental material (see Fig. S2A). (b) HOCl-treated CcCnoX inhibits the aggregation of thermally (43°C) denatured CS. This test was performed in triplicate; this panel shows representative results. Additional results are presented in Fig. S2B.

FIG 2

The surface of CcCnoX is more hydrophobic than that of EcCnoX. (a) The surface of CcCnoX is more hydrophobic than that of EcCnoX. After HOCl treatment, the hydrophobicities of both proteins are similar. Nile red was incubated with CcCnoX and EcCnoX (Materials and Methods), and fluorescence was measured in arbitrary units (A.U.) at 630 nm. This experiment was performed in triplicate; the fluorescence of EcCnoX was set to 1. Error bars denote standard deviation. Differences were evaluated with Student's t test. ns, not significant (P > 0.05). ***, P < 0.001. (b) Comparison of the surfaces of CcCnoX (model built with SWISS-MODEL) and EcCnoX (structure from reference [PDB no. 3QOU]) shows that the TPR domain of CcCnoX displays larger hydrophobic patches than EcCnoX (example in the red box). Hydrophobicity was detected and colored using YBR script (47). Hydrocarbon groups without polar substitutions are yellow, negatively charged oxygens of glutamate and aspartate are red, nitrogens of positively charged functional groups of lysine and arginine are blue, and all remaining atoms (including the polar backbone) are white (47).

FIG 3

CcCnoX cooperates with DnaK/J/GrpE and with GroEL/ES in C. crescentus. CS, chemically unfolded with guanidine hydrochloride, was diluted in refolding buffer containing various combinations of CcCnoX (6:1 ratio to CS) with DnaK/J/GprE, GroEL/ES, and ATP (see Materials and Methods). The recovered activity of CS serves as a proxy to quantify CS refolding. CcCnoX transfers CS to both CcGroEL/ES and CcDnaK/J/GrpE systems for refolding. Mean values from three independent experiments are shown; error bars denote standard deviation. Differences were evaluated with Student's t test. ns, not significant (P > 0.05). **, P < 0.01; ***, P < 0.001.

FIG 4

CcCnoX belongs to the RpoH regulon. Relative fluorescence of the CccnoX::CccnoX-gfp strain was measured in triplicate (error bars denote standard deviation) under the indicated conditions. The last two bars on the right side of the graph show the fluorescence of a CccnoX::CccnoX-gfp strain harboring rpoH on a high-copy-number plasmid, in the absence (−) or the presence (+) of inducer. Differences were evaluated with Student's t test. ns, not significant (P > 0.05). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 5

CcCnoX displays reductase activity. (a) CnoX is well conserved among Gram-negative bacteria. This schematic is an unrooted Bayesian phylogenetic tree for CnoX (Materials and Methods). The logo for the Trx catalytic site for each group appears at right. The complete tree is available in the supplemental material (Fig. S1). Red shading indicates bacteria with a CnoX version that lacks the first cysteine of the catalytic site. (b) CcCnoX catalyzes the reduction of insulin by dithiothreitol (DTT). CcTrx1 was used as a positive control, and a sample without Trx (Ctrl−) was used as a negative control. Data are the mean of three independent experiments, and error bars represent the standard deviation. (c) CcCnoX is reduced in vivo. Wild-type cultures were harvested with trichloroacetic acid and resuspended in SDS-sample buffer containing the alkylating agent Mal-PEG2k (Materials and Methods). This immunoblot was probed with anti-CcCnoX antibody (Materials and Methods); the molecular weight of CcCnoX increased in the presence of Mal-PEG2k. (d) To calculate the redox potential of CcCnoX, the protein was equilibrated in redox buffers with the indicated ratios of reduced to oxidized DTT. Redox potential was calculated from the ratio of the amounts of oxidized and reduced CcCnoX at equilibrium, determined through AMS trapping experiments (Materials and Methods). The redox potential of CcCnoX is −220 mV. Data are the mean from three independent experiments; error bars denote standard deviation. (e) Reduction of CcCnoX by CcTrxR was monitored by measuring the decrease in absorbance at 340 nm, corresponding to the decrease in reduced NADPH. We measured the initial velocities (v) of CcCnoX reduction by CcTrxR to determine the kinetic parameters of the reaction. The K_m of CcTrxR for CcCnoX is 3 μM. Data are the mean from three independent experiments; error bars denote standard deviation. (f) CcCnoX reduces MsrA and DsbDα, known substrates of Trx, as measured by the decrease in absorbance at 340 nm corresponding to the oxidation of NADPH. A sample without substrate was used as a negative control. This experiment was performed in triplicate, and error bars denote standard deviation.

FIG 6

Ninety proteins depend on the reductase activity of CcCnoX in vivo. (a) Catalytic cycle of Trxs. The first cysteine of the catalytic motif of Trx performs a nucleophilic attack on an oxidized cysteine in its substrate. This leads to the formation of a mixed-disulfide complex between the Trx and its substrate. Then, the second cysteine of the catalytic site of Trx resolves the disulfide, thereby releasing a reduced substrate. (b) Diagonal gel obtained by thiol trapping experiments. The spots analyzed by mass spectrometry are numbered, and potential candidates are identified in Table S1. Here, we show one representative example of this experiment, which has been conducted in triplicate. (c) Growth curves of wild-type cells harboring an empty vector (EV [green]), ΔEccnoX cells plus EV [blue]), and ΔEccnoX cells expressing CcCnoX in trans (orange) show that CcCnoX partially complements an EccnoX deletion under HOCl stress. Cells were grown in M9 + glucose medium after addition of 2 mM HOCl. The OD₆₀₀ was measured for 10 h. This graph shows the mean from three independent experiments; error bars are standard errors of the mean (SEM).