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Repairing oxidized proteins in the bacterial envelope using respiratory chain electrons

Abstract

The reactive species of oxygen and chlorine damage cellular components, potentially leading to cell death. In proteins, the sulfur-containing amino acid methionine is converted to methionine sulfoxide, which can cause a loss of biological activity. To rescue proteins with methionine sulfoxide residues, living cells express methionine sulfoxide reductases (Msrs) in most subcellular compartments, including the cytosol, mitochondria and chloroplasts1,2,3. Here we report the identification of an enzymatic system, MsrPQ, repairing proteins containing methionine sulfoxide in the bacterial cell envelope, a compartment particularly exposed to the reactive species of oxygen and chlorine generated by the host defence mechanisms. MsrP, a molybdo-enzyme, and MsrQ, a haem-binding membrane protein, are widely conserved throughout Gram-negative bacteria, including major human pathogens. MsrPQ synthesis is induced by hypochlorous acid, a powerful antimicrobial released by neutrophils. Consistently, MsrPQ is essential for the maintenance of envelope integrity under bleach stress, rescuing a wide series of structurally unrelated periplasmic proteins from methionine oxidation, including the primary periplasmic chaperone SurA. For this activity, MsrPQ uses electrons from the respiratory chain, which represents a novel mechanism to import reducing equivalents into the bacterial cell envelope. A remarkable feature of MsrPQ is its capacity to reduce both rectus (R-) and sinister (S-) diastereoisomers of methionine sulfoxide, making this oxidoreductase complex functionally different from previously identified Msrs. The discovery that a large class of bacteria contain a single, non-stereospecific enzymatic complex fully protecting methionine residues from oxidation should prompt a search for similar systems in eukaryotic subcellular oxidizing compartments, including the endoplasmic reticulum.

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Figure 1: The MsrPQ system reduces free Met-O and is induced by HOCl.
Figure 2: MsrP non-stereospecifically reduces protein-bound Met-O.
Figure 3: The MsrPQ system rescues oxidized Met residues in SurA and Pal.
Figure 4: The reducing activity of the MsrPQ system is important for envelope integrity.

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Acknowledgements

We thank A. Boujtat, G. Herinckx and J.-P. Szikora for technical and computational help and the members of the Barras and Collet laboratories for discussions. We are indebted to M. Sabaty and D. Pignol for sharing unpublished information, to T. Silhavy, T. Palmer, E. Bouveret and D. Hughes for providing strains and plasmids, to T. Lowther and M. Réglier for advice and discussions and to N. Typas, T. Mignot, J. Messens, J. Bardwell and F.-A. Wollman for reading the manuscript and providing comments. A.G. and J.S. are research fellows of the Fonds pour la formation à la Recherche dans l’Industrie et dans l’Agriculture, P.L. is ‘Chargée de Recherche’ and J.-F.C. is ‘Maître de Recherche’ of the Fonds de la Recherche Scientifique-FNRS (FRS-FNRS). E.O. is supported by a grant from the Indo-French Center for the Promotion of Advanced Research CEFIPRA ‘(5105-2)’. R.A. is supported by the Fonds Maurange, Fondation Roi Baudouin. This work was supported, in part, by grants from the FRS-FNRS and from the European Research Council (FP7/2007–2013) ERC independent researcher starting grant 282335–Sulfenic to J.-F.C. and funding by the Centre National de la Recherche Scientifique (CNRS), Fondation pour la Recherche Médicale (FRM) and Aix-Marseille Université to the F.B. team.

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Authors and Affiliations

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Contributions

F.B., J.-F.C., A.G. and B.E. wrote the paper. A.G., B.E., C.H., A.V., L.E., J.-F.C. and F.B. designed and performed the experiments. A.G., B.E., C.H., J.B., P.L. and J.S. constructed the strains and cloned the constructs. F.B., J.-F.C., A.G., B.E. and C.H. analysed and interpreted the data. D.V. performed MS analyses. E.O. and O.I. prepared the diastereoisomers. R.A. performed bioinformatic analyses. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Jean-François Collet or Frédéric Barras.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Induction of MsrPQ by HOCl is dependent on the presence of a functional YedVW two-component system.

Top, immunoblot analysis shows that the induction of MsrP synthesis by HOCl (0.2 mM) is yedW-dependent. The image is representative of experiments made in biological triplicate. Bottom, an msrP::lacZ fusion was used as a read-out for msrP expression. Deletion of yedV upregulates msrP expression, while deletion of yedW prevents its induction by HOCl. Error bars, mean ± s.e.m.; n = 4. The uncropped blot is shown in Supplementary Fig. 4.

Extended Data Figure 2 Respiratory chain-powered, non-stereospecific reduction of Met-O in periplasmic proteins by the MsrPQ system maintains envelope integrity.

Upon exposure to reactive species of chlorine (RCS) and/or reactive species of oxygen (ROS), methionine residues (Met) in periplasmic proteins such as SurA and Pal get oxidized and randomly form either the R- or the S- diastereoisomer of Met-O. This results in the loss of function of some proteins important for maintaining the integrity of the envelope, such as SurA, giving rise to envelope defects. MsrP catalyses the reduction of both diastereoisomers of Met-O with the help of its molybdenum-molybdopterin (Mo-MPT) cofactor. Electrons for reduction are provided by the quinone (Q) pool of the respiratory chain through MsrQ, the inner membrane haem b-containing partner of MsrP. PG, peptidoglycan.

Extended Data Figure 3 MsrP non-stereospecifically reduces Met-O.

a, MsrP reduces N-acetyl-Met-O (NacMet-O), a substrate mimicking protein-bound Met-O, with Km = 3.8 ± 1.2 mM, turnover number (kcat) = 30.5 ± 3.1 s–1 and Vmax = 56.3 ± 5.8 μmol min−1 per milligram protein (error bars, mean ± s.d.; n = 3). b, MsrP is a non-stereospecific Msr, being able to reduce both S-Met-O (with Km = 8.0 ± 2.7 mM, kcat = 36.0 ± 3.6 s–1 and Vmax = 67.2 ± 6.4 μmol min−1 per milligram protein) and R-Met-O (with Km = 25.7 ± 4.7 mM, kcat = 168.3 ± 15.0 s–1 and Vmax = 313.4 ± 27.6 μmol min−1 per milligram protein). Error bars, mean ± s.d.; n = 3. c, Strain JB08 (Met MsrA MsrB BisC, producing MsrC) is able to grow only on R-Met-O, whereas strain CH193 (Met MsrA MsrB MsrC, producing BisC) in only able to grow on S-Met-O. Deletion of msrP in strain BE100 (Met Msr SupMet-O+) prevents its growth on R- and S-Met-O (strain BE104 = Met Msr SupMet-O+ ΔmsrP, compare with growth of BE100 in Fig. 2e). Images are representative of experiments made in biological triplicate. d, The periplasmic chaperone SurA was treated with H2O2, giving rise to SurA ox, a sample of which was subsequently incubated with MsrP and the inorganic reducing system in vitro. The oxidation state of specific Met residues (Met 136, 231 and 298) in the various samples was determined by LC–MS/MS analysis. Error bars, mean ± s.e.m.; n = 4.

Extended Data Figure 4 Preparation of pure diastereoisomeric forms of Met-O.

a, The Oak Ridge Thermal Ellipsoid Plot (ORTEP ellipsoid) representation with 50% probability level of the crystal structure for the isolated salt of l-methionine-S-sulfoxide (right) picrate (left). The grey, blue, red, white and yellow spheres respectively represent carbon, nitrogen, oxygen, hydrogen and sulfur atoms. b, Chemdraw representation of l-methionine-R,S-sulfoxide with proton and carbon positioning (relative to NMR assignment). c, Zoom on the 1H NMR spectra of ~150 mM solutions of l-methionine sulfoxide in D2O pD 6.5, either as a mixture of R- and S- diastereoisomers (top), isolated S- (middle) or isolated R- (bottom) (containing 30 mM dioxane as an internal reference). d, Zoom on the 13C NMR spectra of ~150 mM solutions of l-methionine sulfoxide in D2O pD 6.5, either as a mixture of R- and S- diastereoisomers (top), isolated S- (middle) or isolated R- (bottom) (containing 30 mM dioxane as an internal reference).

Extended Data Figure 5 Individual phylogenies of YedY.

Shown are unrooted Bayesian phylogenetic trees for YedY (b1971, 310 sequences, 260 positions). Numbers at nodes indicate posterior probabilities computed by MrBayes49 and bootstrap values computed by PhyML48. Only posterior probabilities and bootstrap values above 0.5 and 50%, respectively, are shown. Scale bars, average number of substitutions per site. In the phylogenetic tree, YedY from E. coli is highlighted in grey.

Extended Data Figure 6 Individual phylogenies of YedZ.

Shown are unrooted Bayesian phylogenetic trees for YedZ (b1972, 369 sequences, 135 positions). Numbers at nodes indicate posterior probabilities computed by MrBayes49 and bootstrap values computed by PhyML48. Only posterior probabilities and bootstrap values above 0.5 and 50%, respectively, are shown. Scale bars, average number of substitutions per site. In the phylogenetic tree, YedZ from E. coli is highlighted in grey.

Extended Data Table 1 The MsrPQ system uses electrons from the respiratory chain to reduce free Met-O
Extended Data Table 2 List of proteins identified as potential MsrP substrates

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1-3, Supplementary Figures 1-6, legends for Supplementary Data 1-3 (see separate files), and Supplementary References. (PDF 885 kb)

Supplementary Data 1

This file contains an exhaustive list of the homologues of YedY and YedZ proteins found in complete genomes (see Supplementary Information file for legend). (PDF 142 kb)

Supplementary Data 2

This file contains multiple sequence alignment of YedY homologs (see Supplementary Information file for legend). (PDF 9081 kb)

Supplementary Data 3

This file contains multiple sequence alignment of YedZ homologs (see Supplementary Information file for legend). (PDF 16141 kb)

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Gennaris, A., Ezraty, B., Henry, C. et al. Repairing oxidized proteins in the bacterial envelope using respiratory chain electrons. Nature 528, 409–412 (2015). https://doi.org/10.1038/nature15764

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