Cellular and Molecular Life Sciences

, Volume 68, Issue 6, pp 1053–1064 | Cite as

Identification of a bacterial inhibitor against g-type lysozyme

  • L. Vanderkelen
  • J. M. Van Herreweghe
  • K. G. A. Vanoirbeek
  • G. Baggerman
  • B. Myrnes
  • P. J. Declerck
  • I. W. Nilsen
  • C. W. Michiels
  • L. Callewaert
Research Article


Lysozymes are antibacterial effectors of the innate immune system in animals that hydrolyze peptidoglycan. Bacteria have evolved protective mechanisms that contribute to lysozyme tolerance such as the production of lysozyme inhibitors, but only inhibitors of chicken (c-) and invertebrate (i-) type lysozyme have been identified. We here report the discovery of a novel Escherichia coli inhibitor specific for goose (g-) type lysozymes, which we designate PliG (periplasmic lysozyme inhibitor of g-type lysozyme). Although it does not inhibit c- or i-type lysozymes, PliG shares a structural sequence motif with the previously described PliI and MliC/PliC lysozyme inhibitor families, suggesting a common ancestry and mode of action. Deletion of pliG increased the sensitivity of E. coli to g-type lysozyme. The existence of inhibitors against all major types of animal lysozyme and their contribution to lysozyme tolerance suggest that lysozyme inhibitors may play a role in bacterial interactions with animal hosts.


Goose-type lysozyme Lysozyme inhibitor Escherichia coli Lysozyme tolerance Peptidoglycan 



L.V. holds a doctoral fellowship from the Flemish Institute for the Promotion of Scientific Technological Research (IWT). J.M.V.H. was supported a doctoral and L.C. a postdoctoral fellowship of the Research Foundation-Flanders (F.W.O.-Vlaanderen). We thank Griet Compernolle for conducting SPR analysis. We also acknowledge P. Orndorff (Department Microbiology, Pathology and Parasitology, North Carolina State University, College of Veterinary Medicine, Raleigh, NC, USA) and M.A. Valvano (Departments of Microbiology and Immunology, University of Western Ontario, London, ON N6A5C1, Canada) for providing the bacterial strains Bordetella avium 197N and E. coli GL113 ΔtolA::Km, respectively. A construct for production of recombinant T7 lysozyme was kindly donated by Prof. Dr. Smita Patel (Departement of Biochemistry, Robert Wood Johnson Medical School University of Medicine and Dentisitry, NJ, USA).

Supplementary material

18_2010_507_MOESM1_ESM.doc (94 kb)
Supplementary material 1 (DOC 93 kb)


  1. 1.
    Weaver L, Grütter M, Matthews B (1995) The refined structures of goose lysozyme and its complex with a bound trisaccharide show that the “goose-type” lysozymes lack a catalytic aspartate residue. J Mol Biol 245(1):54–68CrossRefPubMedGoogle Scholar
  2. 2.
    Goto T, Abe Y, Kakuta Y, Takeshita K, Imoto T, Ueda T (2007) Crystal structure of Tapes japonica lysozyme with substrate analogue: structural basis of the catalytic mechanism and manifestation of its chitinase activity accompanied by quaternary structural change. J Biol Chem 282(37):27459–27467CrossRefPubMedGoogle Scholar
  3. 3.
    Savan R, Aman A, Sakai M (2003) Molecular cloning of G type lysozyme cDNA in common carp (Cyprinus carpio L.). Fish Shellfish Immunol 15(3):263–268CrossRefPubMedGoogle Scholar
  4. 4.
    Grütter M, Weaver L, Matthews B (1983) Goose lysozyme structure: an evolutionary link between hen and bacteriophage lysozymes? Nature 303(5920):828–831CrossRefPubMedGoogle Scholar
  5. 5.
    Bachali S, Jager M, Hassanin A, Schoentgen F, Jollès P, Fiala-Medioni A, Deutsch J (2002) Phylogenetic analysis of invertebrate lysozymes and the evolution of lysozyme function. J Mol Evol 54(5):652–664CrossRefPubMedGoogle Scholar
  6. 6.
    Callewaert L, Michiels CW (2010) Lysozymes in the animal kingdom. J Biosci 35(1):127–160CrossRefPubMedGoogle Scholar
  7. 7.
    Zhao J, Song L, Li C, Zou H, Ni D, Wang W, Xu W (2007) Molecular cloning of an invertebrate goose-type lysozyme gene from Chlamys farreri, and lytic activity of the recombinant protein. Mol Immunol 44(6):1198–1208CrossRefPubMedGoogle Scholar
  8. 8.
    Hikima S, Hikima J, Rojtinnakorn J, Hirono I, Aoki T (2003) Characterization and function of kuruma shrimp lysozyme possessing lytic activity against Vibrio species. Gene 2003(316):187–195CrossRefGoogle Scholar
  9. 9.
    Canfield R, McMurry S (1967) Purification and characterization of a lysozyme from goose egg white. Biochem Biophys Res Commun 26(1):38–42CrossRefPubMedGoogle Scholar
  10. 10.
    Nile C, Townes C, Michailidis G, Hirst B, Hall J (2004) Identification of chicken lysozyme g2 and its expression in the intestine. Cell Mol Life Sci 61(21):2760–2766CrossRefPubMedGoogle Scholar
  11. 11.
    Vollmer W (2008) Structural variation in the glycan strands of bacterial peptidoglycan. FEMS Microbiol Rev 32(2):287–306CrossRefPubMedGoogle Scholar
  12. 12.
    Monchois V, Abergel C, Sturgis J, Jeudy S, Claverie J (2001) Escherichia coli ykfE ORFan gene encodes a potent inhibitor of C-type lysozyme. J Biol Chem 276(21):18437–18441CrossRefPubMedGoogle Scholar
  13. 13.
    Deckers D, Vanlint D, Callewaert L, Aertsen A, Michiels CW (2008) Role of the lysozyme inhibitor Ivy in growth or survival of Escherichia coli and Pseudomonas aeruginosa bacteria in hen egg white and in human saliva and breast milk. Appl Environ Microbiol 74(14):4434–4439CrossRefPubMedGoogle Scholar
  14. 14.
    Deckers D, Masschalck B, Aertsen A, Callewaert L, Van Tiggelen CGM, Atanassova M, Michiels CW (2004) Periplasmic lysozyme inhibitor contributes to lysozyme resistance in Escherichia coli. Cell Mol Life Sci 61(10):1229–1237CrossRefPubMedGoogle Scholar
  15. 15.
    Callewaert L, Aertsen A, Deckers D, Vanoirbeek KGA, Vanderkelen L, Van Herreweghe JM, Masschalck B, Nakimbugwe D, Robben J, Michiels CW (2008) A new family of lysozyme inhibitors contributing to lysozyme tolerance in gram-negative bacteria. PLoS Pathogens 4(3):e1000019. doi: 10.1371/journal.ppat.1000019 CrossRefPubMedGoogle Scholar
  16. 16.
    Finn R, Tate J, Mistry J, Coggill P, Sammut S, Hotz H, Ceric G, Forslund K, Eddy S, Sonnhammer E, Bateman A (2008) The Pfam protein families database. Nucleic Acids Res 36(Database issue):D281–D288PubMedGoogle Scholar
  17. 17.
    Daigle F, Graham J, Rr Curtiss (2001) Identification of Salmonella typhi genes expressed within macrophages by selective capture of transcribed sequences (SCOTS). Mol Microbiol 41(5):1211–1222CrossRefPubMedGoogle Scholar
  18. 18.
    Van Herreweghe J, Vanderkelen L, Callewaert L, Aertsen A, Compernolle G, Declerck P, Michiels C (2010) Lysozyme inhibitor conferring bacterial tolerance to invertebrate type lysozyme. Cell Mol Life Sci. doi: 10.1007/s00018-009-0241-x
  19. 19.
    Callewaert L, Masschalck B, Deckers D, Nakimbugwe D, Atanassova M, Aertsen A, Michiels CW (2005) Purification of Ivy, a lysozyme inhibitor from Escherichia coli, and characterisation of its specificity for various lysozymes. Enzyme Microb Technol 37(2):205–211CrossRefGoogle Scholar
  20. 20.
    Kyomuhendo P, Nilsen I, Brandsdal B, Smalås A (2008) Structural evidence for lack of inhibition of fish goose-type lysozymes by a bacterial inhibitor of lysozyme. J Mol Model 14(9):777–788CrossRefPubMedGoogle Scholar
  21. 21.
    Kawamura S, Ohno K, Ohkuma M, Chijiiwa Y, Torikata T (2006) Experimental verification of the crucial roles of Glu73 in the catalytic activity and structural stability of goose type lysozyme. J Biochem 140(1):75–85CrossRefPubMedGoogle Scholar
  22. 22.
    Helland R, Larsen R, Finstad S, Kyomuhendo P, Larsen A (2009) Crystal structures of g-type lysozyme from Atlantic cod shed new light on substrate binding and the catalytic mechanism. Cell Mol Life Sci 66(15):2585–2598CrossRefPubMedGoogle Scholar
  23. 23.
    Hikima J, Minagawa S, Hirono I, Aoki T (2001) Molecular cloning, expression and evolution of the Japanese flounder goose-type lysozyme gene, and the lytic activity of its recombinant protein. Biochim Biophys Acta 1520(1):35–44PubMedGoogle Scholar
  24. 24.
    Arnheim N, Inouye M, Law L, Laudin A (1973) Chemical studies on the enzymatic specificity of goose egg white lysozyme. J Biol Chem 248(1):233–236PubMedGoogle Scholar
  25. 25.
    Jollès P, Jollès J (1984) What’s new in lysozyme research? Always a model system, today as yesterday. Mol Cell Biochem 63(2):165–189CrossRefPubMedGoogle Scholar
  26. 26.
    Nakimbugwe D, Masschalck B, Deckers D, Callewaert L, Aertsen A, Michiels CW (2006) Cell wall substrate specificity of six different lysozymes and lysozyme inhibitory activity of bacterial extracts. FEMS Microbiol Lett 259(1):41–46CrossRefPubMedGoogle Scholar
  27. 27.
    Kyomuhendo P, Myrnes B, Nilsen I (2007) A cold-active salmon goose-type lysozyme with high heat tolerance. Cell Mol Life Sci 64(21):2841–2847CrossRefPubMedGoogle Scholar
  28. 28.
    Heukeshoven J, Dernick R (1988) Improved silver staining procedure for fast staining in PhastSystem Development Unit. I. Staining of sodium dodecyl sulfate gels. Electrophoresis 9(1):28–32CrossRefPubMedGoogle Scholar
  29. 29.
    Datsenko K, Wanner B (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97(12):6640–6645CrossRefPubMedGoogle Scholar
  30. 30.
    Bendtsen J, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340(4):783–795CrossRefPubMedGoogle Scholar
  31. 31.
    Rice P, Longden I, Bleasby A (2000) EMBOSS: the European molecular biology open software suite. Trends Genet 16(6):276–277CrossRefPubMedGoogle Scholar
  32. 32.
    Thunnissen A, Isaacs N, Dijkstra B (1995) The catalytic domain of a bacterial lytic transglycosylase defines a novel class of lysozymes. Proteins 22(3):245–258CrossRefPubMedGoogle Scholar
  33. 33.
    Nilsen I, Myrnes B, Edvardsen R, Chourrout D (2003) Urochordates carry multiple genes for goose-type lysozyme and no genes for chicken- or invertebrate-type lysozymes. Cell Mol Life Sci 60(10):2210–2218CrossRefPubMedGoogle Scholar
  34. 34.
    Altschul S, Gish W, Miller W, Myers E, Lipman D (1990) Basic local alignment search tool. J Mol Biol 215(3):403–410PubMedGoogle Scholar
  35. 35.
    Yum S, Kim M, Xu Y, Jin X, Yoo H, Park J, Gong J, Choe K, Lee B, Ha N (2009) Structural basis for the recognition of lysozyme by MliC, a periplasmic lysozyme inhibitor in Gram-negative bacteria. Biochem Biophys Res Commun 378(2):244–248CrossRefPubMedGoogle Scholar
  36. 36.
    Frith M, Saunders N, Kobe B, Bailey T (2008) Discovering sequence motifs with arbitrary insertions and deletions. PLoS Comput Biol 4(4):e1000071CrossRefPubMedGoogle Scholar
  37. 37.
    Yeats C, Bentley S, Bateman A (2003) New knowledge from old: in silico discovery of novel protein domains in Streptomyces coelicolor. BMC Microbiol 3:3CrossRefPubMedGoogle Scholar
  38. 38.
    Tripathi L, Sowdhamini R (2008) Genome-wide survey of prokaryotic serine proteases: analysis of distribution and domain architectures of five serine protease families in prokaryotes. BMC Genomics 9:549CrossRefPubMedGoogle Scholar
  39. 39.
    Irwin D, Gong Z (2003) Molecular evolution of vertebrate goose-type lysozyme genes. J Mol Evol 56(2):234–242CrossRefPubMedGoogle Scholar
  40. 40.
    Caipang C, Brinchmann M, Kiron V (2009) Profiling gene expression in the spleen of Atlantic cod, Gadus morhua upon vaccination with Vibrio anguillarum antigen. Comp Biochem Physiol B Biochem Mol Biol 153(3):261–267CrossRefPubMedGoogle Scholar
  41. 41.
    Li L, Zhao J, Wang L, Qiu L, Zhang H, Dong C, Cong M, Song L (2009) The polymorphism of lysozyme gene in Zhikong scallop (Chlamys farreri) and its association with susceptibility/resistance to Listonella anguillarum. Fish Shellfish Immunol 27(2):136–142CrossRefPubMedGoogle Scholar
  42. 42.
    Clarke C, Scheurwater E, Clarke A (2010) The vertebrate lysozyme inhibitor Ivy functions to inhibit the activity of lytic transglycosylase. J Biol Chem 285(20):14843–14847CrossRefPubMedGoogle Scholar
  43. 43.
    Scheurwater E, Reid C, Clarke A (2008) Lytic transglycosylases: bacterial space-making autolysins. Int J Biochem Cell Biol 40(4):586–591CrossRefPubMedGoogle Scholar
  44. 44.
    Heidrich C, Ursinus A, Berger J, Schwarz H, Höltje J (2002) Effects of multiple deletions of murein hydrolases on viability, septum cleavage, and sensitivity to large toxic molecules in Escherichia coli. J Bacteriol 184(22):6093–6099CrossRefPubMedGoogle Scholar
  45. 45.
    Vinés E, Marolda C, Balachandran A, Valvano M (2005) Defective O-antigen polymerization in tolA and pal mutants of Escherichia coli in response to extracytoplasmic stress. J Bacteriol 187:3359–3368CrossRefPubMedGoogle Scholar
  46. 46.
    Thompson J, Higgins D, Gibson T (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22(22):4673–4680CrossRefPubMedGoogle Scholar
  47. 47.
    Crooks G, Hon G, Chandonia J, Brenner S (2004) WebLogo: a sequence logo generator. Genome Res 14(6):1188–1190CrossRefPubMedGoogle Scholar

Copyright information

© Springer Basel AG 2010

Authors and Affiliations

  • L. Vanderkelen
    • 1
  • J. M. Van Herreweghe
    • 1
  • K. G. A. Vanoirbeek
    • 1
  • G. Baggerman
    • 4
  • B. Myrnes
    • 2
  • P. J. Declerck
    • 3
  • I. W. Nilsen
    • 2
  • C. W. Michiels
    • 1
  • L. Callewaert
    • 1
  1. 1.Laboratory of Food Microbiology and Leuven Food Science and Nutrition Research Centre (LFoRCe)Katholieke Universiteit LeuvenLeuvenBelgium
  2. 2.Fish Health and Marine Bioprospecting, Nofima MarinTromsøNorway
  3. 3.Laboratory for Pharmaceutical BiologyKatholieke Universiteit LeuvenLeuvenBelgium
  4. 4.Prometa, Interfaculty Centre for Proteomics and MetabolomicsKatholieke Universiteit LeuvenLeuvenBelgium

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