Share / Export Citation / Email / Print / Text size:

Postępy Mikrobiologii - Advancements of Microbiology

Polish Society of Microbiologists

Subject: Microbiology


ISSN: 0079-4252
eISSN: 2545-3149





Volume / Issue / page

Related articles

VOLUME 56 , ISSUE 3 (April 2017) > List of articles


Elżbieta Katarzyna Jagusztyn-Krynicka * / Anna Marta Banaś / Magdalena Joanna Grzeszczuk

Keywords : Dsb proteins, EcDsbA, disulfide bonds, protein structure, biochemical attributes

Citation Information : Postępy Mikrobiologii - Advancements of Microbiology. Volume 56, Issue 3, Pages 326-334, DOI:

License : (CC BY-NC-ND 4.0)

Published Online: 22-May-2019



Bacterial Dsb (disulfide bond) enzymes are involved in the oxidative folding of many proteins, through the formation of disulfide bonds between thiol groups of cysteine residues. This process is critical for the correct folding and structural stability of many secreted and membrane proteins. The rapidly expanding number of sequenced bacterial genomes has revealed the enormous diversity among bacterial Dsb systems. While the Escherichia coli oxidative protein folding has been studied in great details, the mechanism of the Dsb systems functioning in other bacteria are rather poorly understood. Herein, we present the current methodology, both in vivo and in vitro experimental techniques, which allow us to understand the functioning of the Dsb proteins and has broaden our knowledge in the field of biochemistry and microbiology of this posttranslational protein modification. Many bacterial virulence factors are extracytoplasmic Dsb-dependent proteins. Thus, this system plays an important role in bacterial pathogenesis and the proteins of the Dsb network represent possible targets for new drugs.

Content not available PDF Share



1. Agudo D., Mendoza M.T., Castanares C., Nombela C., Rotger R.: A proteomic approach to study Salmonella typhi periplasmic proteins altered by a lack of the DsbA thiol: disulfide isomerase. Proteomics, 4, 355–363 (2004)

2. Arts I.S., Ball G., Leverrier P., Garvis S., Nicolaes V., Vertommen D., Ize B., Tamu Dufe V., Messens J., Voulhoux R., Collet J.F.: Dissecting the machinery that introduces disulfide bonds in Pseudomonas aeruginosa. mBio, 4, e00912–00913 (2013)

3. Berkmen M.: Production of disulfide-bonded proteins in Escherichia coli. Protein Expr. Purif. 82, 240–251 (2012)

4. Bocian-Ostrzycka K.M., Grzeszczuk M.J., Banaś A.M., Jastrząb K., Pisarczyk K., Kolarzyk A., Łasica A.M., Collet J.-F., Jagusztyn-Krynicka E.K.: Engineering of Helicobacter pylori Dimeric Oxidoreductase DsbK (HP0231). Frontiers in Microbiology, 7, 1158 (2016)

5. Chim N., Harmston C.A., Guzman D.J., Goulding C.W.: Structural and biochemical characterization of the essential DsbA-like disulfide bond forming protein from Mycobacterium tuberculosis. BMC Struct. Biol. 13, 23 (2013)

6. Cho S.H., Collet J.F.: Many roles of the bacterial envelope reducing pathways. Antioxid. Redox Signal. 18, 1690–1698

7. Cho S.H., Parsonage D., Thurston C., Dutton R.J., Poole L.B., Collet J.F., Beckwith J.: A new family of membrane electron transporters and its substrates, including a new cell envelope peroxiredoxin, reveal a broadened reductive capacity of the oxidative bacterial cell envelope. mBio, 3, e00291–11. (2012)

8. Cho S.H., Szewczyk J., Pesavento C., Zietek M., Banzhaf M., Roszczenko P., Asmar A., Laloux G., Hov A.K., Leverrier P., Van der Henst C., Vertommen D., Typas A., Collet J.F.: Detecting envelope stress by monitoring beta-barrel assembly. Cell, 159, 1652–1664 (2014)

9. Daniels R., Mellroth P., Bernsel A., Neiers F., Normark S., von Heijne G., Henriques-Normark B.: Disulfide bond formation and cysteine exclusion in gram-positive bacteria. J. Biol. Chem. 285, 3300–3309 (2010)

10. Denoncin K., Collet J.F.: Disulfide bond formation in the bacterial periplasm: major achievements and challenges ahead. Antioxid. Redox Signal. 19, 63–71 (2013)

11. Denoncin K., Vertommen D., Paek E., Collet J.F.: The protein-disulfide isomerase DsbC cooperates with SurA and DsbA in the assembly of the essential beta-barrel protein LptD. J. Biol. Chem. 285, 29425–29433 (2010)

12. Denoncin K., Nicolaes V., Cho S.H., Leverrier P., Collet J.F.: Protein disulfide bond formation in the periplasm: determination of the in vivo redox state of cysteine residues. Methods Mol. Biol. 966, 325–336 (2013)

13. Depuydt M., Leonard S.E., Vertommen D., Denoncin K., Morsomme P., Wahni K., Messens J., Carroll K.S., Collet J.F.: A periplasmic reducing system protects single cysteine residues from oxidation. Science, 326, 1109–1111 (2009)

14. Duprez W., Bachu P., Stoermer M.J., Tay S., McMahon R.M., Fairlie D.P., Martin J.L.: Virtual Screening of Peptide and Peptidomimetic Fragments Targeted to Inhibit Bacterial Dithiol Oxidase DsbA. PLoS ONE, 10, e0133805 (2015)

15. Duprez W., Premkumar L., Halili M.A., Lindahl F., Reid R.C., Fairlie D.P., Martin J.L.: Peptide Inhibitors of the Escherichia coli DsbA Oxidative Machinery Essential for Bacterial Virulence. J. Med. Chem. 58, 577–587 (2015)

16. Dutton R.J., Boyd D., Berkmen M., Beckwith J.: Bacterial species exhibit diversity in their mechanisms and capacity for protein disulfide bond formation. P. Natl. Acad. Sci. USA, 105, 11933–11938 (2008)

17. Grabowska A.D., Wywial E., Dunin-Horkawicz S., Lasica A.M., Wosten M.M., Nagy-Staron A., Godlewska R., Bocian-Ostrzycka K., Pienkowska K., Laniewski P., Bujnicki J.M., van Putten J.P., Jagusztyn-Krynicka E.K.: Functional and bioinformatics analysis of two Campylobacter jejuni homologs of the thiol-disulfide oxidoreductase, DsbA. PLoS ONE, 9, e106247 (2014)

18. Greiner-Stoeffele T., Grunow M., Hahn U.: A general ribonuclease assay using methylene blue. Anal. Biochem. 240, 24–28 (1996)

19. Grimshaw J.P., Stirnimann C.U., Brozzo M.S., Malojcic G., Grutter M.G., Capitani G., Glockshuber R.: DsbL and DsbI form a specific dithiol oxidase system for periplasmic arylsulfate sulfotransferase in uropathogenic Escherichia coli. J. Mol. Biol. 380, 667–680 (2008)

20. Hatahet F., Boyd D., Beckwith J.: Disulfide bond formation in prokaryotes: history, diversity and design. Biochim. Biophys. Acta, 1844, 1402–1414 (2014)

21. Heras B., Edeling M.A., Schirra H.J., Raina S., Martin J.L.: Crystal structures of the DsbG disulfide isomerase reveal an unstable disulfide. P. Natl. Acad. Sci. USA, 101, 8876–8881 (2004)

22. Heras B., Shouldice S.R., Totsika M., Scanlon M.J., Schembri M.A., Martin J.L.: DSB proteins and bacterial pathogenicity. Nat. Rev. Microbiol. 7, 215–225 (2009)

23. Heras B., Kurz M., Jarrott R., Shouldice S.R., Frei P., Robin G., Cemazar M., Thony-Meyer L., Glockshuber R., Martin J.L.: Staphylococcus aureus DsbA does not have a destabilizing disulfide. A new paradigm for bacterial oxidative folding. J. Biol. Chem. 283, 4261–4271 (2008)

24. Hiniker A., Bardwell J.C.: In vivo substrate specificity of periplasmic disulfide oxidoreductases. J. Biol. Chem. 279, 12967–12973 (2004)

25. Hiniker A., Collet J.F., Bardwell J.C.: Copper stress causes an in vivo requirement for the Escherichia coli disulfide isomerase DsbC. J. Biol. Chem. 280, 33785–33791 (2005)

26. Hiniker A., Ren G., Heras B., Zheng Y., Laurinec S., Jobson R.W., Stuckey J.A., Martin J.L., Bardwell J.C.: Laboratory evolution of one disulfide isomerase to resemble another. P. Natl. Acad. Sci. USA, 104, 11670–11675 (2007)

27. Inaba K., Ito K.: Structure and mechanisms of the DsbB-DsbA disulfide bond generation machine. Biochim. Biophys. Acta, 1783, 520–529 (2008)

28. Inaba K., Murakami S., Suzuki M., Nakagawa A., Yamashita E., Okada K., Ito K.: Crystal structure of the DsbB-DsbA complex reveals a mechanism of disulfide bond generation. Cell, 127, 789–801 (2006)

29. Jameson-Lee M., Garduno R.A., Hoffman P.S.: DsbA2 (27 kDa Com1-like protein) of Legionella pneumophila catalyses extracytoplasmic disulphide-bond formation in proteins including the Dot/Icm type IV secretion system. Mol. Microbiol. 80, 835–852 (2011)

30. Kadokura H., Katzen F., Beckwith J.: Protein disulfide bond formation in prokaryotes. Annu. Rev. Biochem. 72, 111–135 (2003)

31. Kadokura H., Tian H., Zander T., Bardwell J.C., Beckwith J.: Snapshots of DsbA in action: detection of proteins in the process of oxidative folding. Science, 303, 534–537 (2004)

32. Katzen F., Deshmukh M., Daldal F., Beckwith J.: Evolutionary domain fusion expanded the substrate specificity of the transmembrane electron transporter DsbD. EMBO J. 21, 3960–3969 (2002)

33. Koniger V., Holsten L., Harrison U., Busch B., Loell E., Zhao Q., Bonsor D.A., Roth A., Kengmo-Tchoupa A., Smith S.I., Mueller S., Sundberg E.J., Zimmermann W., Fischer W., Hauck C.R., Haas R.: Helicobacter pylori exploits human CEACAMs via HopQ for adherence and translocation of CagA. Nat. Microbiol. 2, 16188 (2016)

34. Kpadeh Z.Z., Day S.R., Mills B.W., Hoffman P.S.: Legionella pneumophila utilizes a single-player disulfide-bond oxidoreductase system to manage disulfide bond formation and isomerization. Mol. Microbiol. 95, 1054–1069 (2015)

35. Kpadeh Z.Z., Jameson-Lee M., Yeh A.J., Chertihin O., Shumilin I.A., Dey R., Day S.R., Hoffman P.S.: Disulfide bond oxidoreductase DsbA2 of Legionella pneumophila exhibits protein disulfide isomerase activity. J. Bacteriol. 195, 1825–1833 (2013)

36. Kurz M., Iturbe-Ormaetxe I., Jarrott R., Cowieson N., Robin G., Jones A., King G.J., Frei P., Glockshuber R., O’Neill S.L., Heras B., Martin J.L.: Cloning, expression, purification and characterization of a DsbA-like protein from Wolbachia pipientis. Protein Expr. Purif. 59, 266–273 (2008)

37. Lafaye C., Iwema T., Carpentier P., Jullian-Binard C., Kroll J.S., Collet J.F., Serre L.: Biochemical and structural study of the homologues of the thiol-disulfide oxidoreductase DsbA in Neisseria meningitidis. J. Mol. Biol. 392, 952–966 (2009)

38. Lasica A.M., Jagusztyn-Krynicka E.K.: The role of Dsb proteins of Gram-negative bacteria in the process of pathogenesis. FEMS Microbiol. Rev. 31, 626–636 (2007)

39. Lasica A.M., Wyszynska A., Szymanek K., Majewski P., Jagusztyn-Krynicka E.K.: Campylobacter protein oxidation influences epithelial cell invasion or intracellular survival as well as intestinal tract colonization in chickens. J. Appl. Genet. 51, 383–393 (2010)

40. Leverrier P., Declercq J.P., Denoncin K., Vertommen D., Hiniker A., Cho S.H., Collet J.F.: Crystal structure of the outer membrane protein RcsF, a new substrate for the periplasmic protein-disulfide isomerase DsbC. J. Biol. Chem. 286, 16734–16742 (2011)

41. Lin H.H., Tseng L.Y.: DBCP: a web server for disulfide bonding connectivity pattern prediction without the prior knowledge of the bonding state of cysteines. Nucleic Acids Res. 38, W503–507 (2010)

42. Marquez-Chamorro A.E., Aguilar-Ruiz J.S.: Soft Computing Methods for Disulfide Connectivity Prediction. Evol. Bioinform. Online, 11, 223–229 (2015)

43. McCarthy A.A., Haebel P.W., Torronen A., Rybin V., Baker E.N., Metcalf P.: Crystal structure of the protein disulfide bond isomerase, DsbC, from Escherichia coli. Nat. Struct. Biol. 7, 196–199 (2000)

44. McMahon R.M., Premkumar L., Martin J.L.: Four structural subclasses of the antivirulence drug target disulfide oxidoreductase DsbA provide a platform for design of subclass-specific inhibitors. Biochim. Biophys. Acta. 1844, 1391–1401 (2014)

45. Messens J., Collet J.F.: Pathways of disulfide bond formation in Escherichia coli. Int. J. Biochem. Cell Biol. 38, 1050–1062 (2006)

46. Messens J., Collet J.F., Van Belle K., Brosens E., Loris R., Wyns L.: The oxidase DsbA folds a protein with a nonconsecutive disulfide. J. Biol. Chem. 282, 31302–31307 (2007)

47. Premkumar L., Heras B., Duprez W., Walden P., Halili M., Kurth F., Fairlie D.P., Martin J.L.: Rv2969c, essential for optimal growth in Mycobacterium tuberculosis, is a DsbA-like enzyme that interacts with VKOR-derived peptides and has atypical features of DsbA-like disulfide oxidases. Acta Crystallogr. D Biol. Crystallogr. 69, 1981–1994 (2013)

48. Quan S., Schneider I., Pan J., Von Hacht A., Bardwell J.C.: The CXXC motif is more than a redox rheostat. J. Biol. Chem.. 282, 28823–28833 (2007)

49. Raines R.T.: Ribonuclease A. Chem. Rev. 98, 1045–1066 (1998)

50. Ren G., Champion M.M., Huntley J.F.: Identification of disulfide bond isomerase substrates reveals bacterial virulence factors. Mol. Microbiol.. 94, 926–944 (2014)

51. Ren G., Stephan D., Xu Z., Zheng Y., Tang D., Harrison R.S., Kurz M., Jarrott R., Shouldice S.R., Hiniker A., Martin J.L., Heras B., Bardwell J.C.: Properties of the thioredoxin fold superfamily are modulated by a single amino acid residue. J. Biol. Chem. 284, 10150–10159 (2009)

52. Roszczenko P., Radomska K.A., Wywial E., Collet J.F., Jagusztyn-Krynicka E.K.: A novel insight into the oxidoreductase activity of Helicobacter pylori HP0231 protein. PLoS ONE, 7, e46563 (2012)

53. Roszczenko P., Grzeszczuk M., Kobierecka P., Wywial E., Urbanowicz P., Wincek P., Nowak E., Jagusztyn-Krynicka E.K.: Helicobacter pylori HP0377, a member of the Dsb family, is an untypical multifunctional CcmG that cooperates with dimeric thioldisulfide oxidase HP0231. BMC Microbiol. 15, 135 (2015)

54. Ruiz N., Chng S.S., Hiniker A., Kahne D., Silhavy T.J.: Nonconsecutive disulfide bond formation in an essential integral outer membrane protein. P. Natl. Acad. Sci. USA, 107, 12245–12250 (2010)

55. Ruoppolo M., Torella C., Kanda F., Panico M., Pucci P., Marino G., Morris H.R.: Identification of disulphide bonds in the refolding of bovine pancreatic RNase A. Fold. Des. 1, 381–390 (1996)

56. Segatori L., Paukstelis P.J., Gilbert H.F., Georgiou G.: Engineered DsbC chimeras catalyze both protein oxidation and disulfide-bond isomerization in Escherichia coli: Reconciling two competing pathways. P. Natl. Acad. Sci. USA, 101, 10018–10023 (2004)

57. Shouldice S.R., Heras B., Walden P.M., Totsika M., Schembri M.A., Martin J.L.: Structure and function of DsbA, a key bacterial oxidative folding catalyst. Antioxid. Redox Signal. 14, 1729–1760 (2011)

58. Singh R.: A review of algorithmic techniques for disulfide-bond determination. Brief. Funct. Genomic. Proteomic. 7, 157–172 (2008)

59. Sinha S., Langford P.R., Kroll J.S.: Functional diversity of three different DsbA proteins from Neisseria meningitidis. Microbiology, 150, 2993–3000 (2004)

60. Stirnimann C.U., Grutter M.G., Glockshuber R., Capitani G.: nDsbD: a redox interaction hub in the Escherichia coli periplasm. Cell Mol. Life Sci. 63, 1642–1648 (2006)

61. Stirnimann C.U., Rozhkova A., Grauschopf U., Bockmann R.A., Glockshuber R., Capitani G., Grutter M.G.: High-resolution structures of Escherichia coli cDsbD in different redox states: A combined crystallographic, biochemical and computational study. J. Mol. Biol. 358, 829–845 (2006)

62. Tinsley C.R., Voulhoux R., Beretti J.L., Tommassen J., Nassif X.: Three homologues, including two membrane-bound proteins, of the disulfide oxidoreductase DsbA in Neisseria meningitidis: effects on bacterial growth and biogenesis of functional type IV pili. J. Biol. Chem. 279, 27078–27087 (2004)

63. Wang X., Dutton R.J., Beckwith J., Boyd D.: Membrane topology and mutational analysis of Mycobacterium tuberculosis VKOR, a protein involved in disulfide bond formation and a homologue of human vitamin K epoxide reductase. Antioxid. Redox Signal. 14, 1413–1420 (2011)

64. Watanabe M.M., Laurindo F.R., Fernandes D.C.: Methods of measuring protein disulfide isomerase activity: a critical overview. Frontiers in chemistry. 2, 73 (2014)

65. Williamson J.A., Cho S.H., Ye J., Collet J.F., Beckwith J.R., Chou J.J.: Structure and multistate function of the transmembrane electron transporter CcdA. Nat. Struct. Mol. Biol. 22, 809–814 (2015)

66. Wunderlich M., Glockshuber R.: Redox properties of protein disulfide isomerase (DsbA) from Escherichia coli. Protein Sci. 2, 717–726 (1993)

67. Yoon J.Y., Kim J., Lee S.J., Kim H.S., Im H.N., Yoon H.J., Kim K.H., Kim S.J., Han B.W., Suh S.W.: Structural and functional characterization of Helicobacter pylori DsbG. FEBS Lett. 585, 3862–3867 (2011)

68. Zhou Y., Cierpicki T., Jimenez R.H., Lukasik S.M., Ellena J.F., Cafiso D.S., Kadokura H., Beckwith J., Bushweller J.H.: NMR solution structure of the integral membrane enzyme DsbB: functional insights into DsbB-catalyzed disulfide bond formation. Mol. Cell. 31, 896–908 (2008