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Secretome analysis of Clostridium difficile strains

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Abstract

Clostridium difficile causes infections ranging from mild C. difficile-associated diarrhea to severe pseudomembranous colitis. Since 2003 new hypervirulent C. difficile strains (PCR ribotype 027) emerged characterized by a dramatically increased mortality. The secretomes of the three C. difficile strains CDR20291, CD196, and CD630 were analyzed and compared. Proteins were separated and analyzed by means of SDS--PAGE and LC–MS. MS data were analyzed using Mascot and proteins were checked for export signals with SecretomeP and SignalP. LC–MS analysis revealed 158 different proteins in the supernatant of C. difficile. Most of the identified proteins originate from the cytoplasm. Thirty-two proteins in CDR20291, 36 in CD196 and 26 in CD630 were identified to be secreted by C. difficile strains. Those were mainly S-layer proteins, substrate-binding proteins of ABC-transporters, cell wall hydrolases, pilin and unknown hypothetical proteins. Toxin A and toxin B were identified after growth in brain heart infusion medium using immunological techniques. The ADP-ribosyltransferase-binding component protein, which is a part of the binary toxin CDT, was only identified in the hypervirulent ribotype 027 strains. Further proteins that are secreted specifically by hypervirulent strains were identified.

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References

  • Akerlund T, Persson I, Unemo M, Norén T, Svenungsson B, Wullt M, Burman LG (2008) Increased sporulation rate of epidemic Clostridium difficile Type 027/NAP1. J Clin Microbiol 46:1530–1533

    Article  PubMed  Google Scholar 

  • Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783–795

    Article  PubMed  Google Scholar 

  • Bendtsen JD, Kiemer L, Fausbøll A, Brunak S (2005) Non-classical protein secretion in bacteria. BMC Microbiol 5:58–64

    Article  PubMed  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248

    Article  PubMed  CAS  Google Scholar 

  • Cartman ST, Heap JT, Kuehne SA, Cockayne A, Minton NP (2010) The emergence of ‘hypervirulence’ in Clostridium difficile. Int J Med Microbiol 300:387–395

    Article  PubMed  Google Scholar 

  • Frei R, Nagy E, Bouza E, Marin M, Akerlund T, Virolainen-Julkunen A, Lyytikäinen O, Kotila S, Ingebretsen A, Smyth B et al (2008) Update of Clostridium difficile infection due to PCR ribotype 027 in Europe. Euro Surveill 13:18942–18951

    PubMed  Google Scholar 

  • Gerhard R, Burger S, Tatge H, Genth H, Just I, Hofmann F (2005) Comparison of wild type with recombinant Clostridium difficile toxin. Microb Pathog 38:77–83

    Article  PubMed  CAS  Google Scholar 

  • Gerhard R, Nottrott S, Schoentaube J, Tatge H, Olling A, Just I (2008) Glucosylation of Rho GTPases by Clostridium difficile toxin A triggers apoptosis in intestinal epithelial cells. J Med Microbiol 57:765–770

    Article  PubMed  CAS  Google Scholar 

  • Hall A (2005) Rho GTPases and the control of cell behaviour. Biochem Soc Trans 33:891–895

    Article  PubMed  CAS  Google Scholar 

  • Hammond GA, Johnson JL (1995) The toxigenic element of Clostridium difficile strain VPI10463. Microb Pathog 19:203–213

    Article  PubMed  CAS  Google Scholar 

  • He M, Sebaihia M, Lawley TD, Stabler RA, Dawson LF, Martin MJ, Holt KE, Seth-Smith HM, Quail MA, Rance R et al (2010) Evolutionary dynamics of Clostridium difficile over short and long time scales. Proc Natl Acad Sci USA 107:7527–7532

    Article  PubMed  CAS  Google Scholar 

  • Jain S, Graham RLJ, McMullan G, Ternan NG (2010) Proteomic analysis of the insoluble subproteome of Clostridium difficile strain 630. FEMS Microbiol Lett 312:151–159

    Article  PubMed  CAS  Google Scholar 

  • Jain S, Graham C, Graham RLJ, McMullan G, Ternan NG (2011) Quantitative proteomics analysis of the heat stress response in Clostridium difficile strain 630. J Proteom Res 10:3880–3890

    Article  CAS  Google Scholar 

  • Jeffrey CJ (2005) Mass spectrometry and the search for moonlighting proteins. Mass Spec Rev 24:772–782

    Article  Google Scholar 

  • Just I, Gerhard R (2004) Large clostridial cytotoxins. Rev Physiol Biochem Pharmacol 152:23–47

    Article  PubMed  CAS  Google Scholar 

  • Karlsson S, Burman LG, Akerlund T (1999) Suppression of toxin production in Clostridium difficile VPI 10463 by amino acids. Microbiology 145:1683–1693

    Article  PubMed  CAS  Google Scholar 

  • Kelly CP, LaMont JT (2008) Clostridium difficile-more difficult than ever. N Engl J Med 359:1932–1940

    Article  PubMed  CAS  Google Scholar 

  • King RN, Lager SL (2011) Incidence of Clostridium difficile infections in patients receiving antimicrobial and acid suppression therapy. Pharmacotherapy 31:642–648

    Article  PubMed  Google Scholar 

  • Kuehne SA, Cartman ST, Heap JT, Kelly ML, Cockayne A, Minton NP (2010) The role of toxin A and toxin B in Clostridium difficile infection. Nature 467:711–713

    Article  PubMed  CAS  Google Scholar 

  • Kuijper EJ, van Dissel JT, Wilcox MH (2007) Clostridium difficile: changing epidemiology and new treatment options. Curr Opin Infect Dis 20:376–383

    PubMed  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  • Lawley TD, Croucher NJ, Yu L, Clare S, Sebaihia M, Goulding D, Pickard DJ, Parkhill J, Choudhary J, Dougan G (2009) Proteomic and genomic characterization of highly infectious Clostridium difficile 630 spores. J Bacteriol 191:5377–5386

    Article  PubMed  CAS  Google Scholar 

  • Luecke N, Neumann D, Templin C, Muetzelburg MV, Just I, Pich A (2010) Secretom analysis of the hematopoietic progenitor cell line DKmix using LC-MALDI techniques. Rapid Commun Mass Spectrom 24:561–570

    Article  PubMed  CAS  Google Scholar 

  • Macy JM, Snellen JE, Hungate RE (1972) Use of syringe methods for anaerobiosis. Am J Clin Nutr 25:1318–1323

    PubMed  CAS  Google Scholar 

  • Mukherjee K, Karlsson S, Burman LG, Akerlund T (2002) Proteins released during high toxin production in Clostridium difficile. Microbiology 148:2245–2253

    PubMed  CAS  Google Scholar 

  • Murray R, Boyd D, Levett PN, Mulvey MR, Alfa MJ (2009) Truncation in the tcdC region of the Clostridium difficile PathLoc of clinical isolates does not predict increased biological activity of Toxin B or Toxin A. BMC Infect Dis 9:103–107

    Article  PubMed  Google Scholar 

  • Perelle S, Gibert M, Bourlioux P, Corthier G, Popoff MR (1997) Production of a complete binary toxin (actin-specific ADP-ribosyltransferase) by Clostridium difficile. Infect Immun 65:1402–1407

    PubMed  CAS  Google Scholar 

  • Schwan C, Stecher B, Tzivelekidis T, van Ham M, Rohde M, Hardt WD, Wehland J, Aktories K (2009) Clostridium difficile toxin CDT induces formation of microtubule-based protrusions and increases adherence of bacteria. PLoS Pathog 5(10):e1000626

    Article  PubMed  Google Scholar 

  • Smith BE, Hill JA, Gjukich MA, Andrews PC (2011) Tranche distributed repository and proteomecommons.org. Meth Mol Biol 696:123–145

    Article  CAS  Google Scholar 

  • Stabler RA, He M, Dawson L, Martin M, Valiente E, Corton C, Sebaihia M, Quail MA, Rose G, Gerding DN et al (2009) Comparative genome and phenotypic analysis of Clostridium difficile 027 strains provides insight into the evolution of a hypervirulent bacterium. Genome Biol 10:102–108

    Article  Google Scholar 

  • Viswanathan V, Mallozzi M, Vedantam G (2010) Clostridium difficile infections: an overview of the disease and its pathogenesis, epidemiology and interventions. Gut Microbes 1:234–242

    Google Scholar 

  • Warny M, Pepin J, Fang A, Killigore G, Thompson A, Brazier J, Frost E, McDonnald IC (2005) Toxin production by emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe. Lancet 366:1079–1084

    Article  PubMed  CAS  Google Scholar 

  • Wenger CD, Phanstiel DH, Lee MV, Bailey DJ, Coon JJ (2011) Compass: a suite of pre- and post-search proteomics software tools for OMSSA. Proteomics 11:1064–1074

    Article  PubMed  CAS  Google Scholar 

  • Wessel D, Flügge UI (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138:141–143

    Article  PubMed  CAS  Google Scholar 

  • Wright A, Wait R, Begum S, Crossett B, Nagy J, Brown K (2005) Proteomic analysis of cell surface proteins from Clostridium difficile. Proteomics 5:2443–2452

    Article  PubMed  CAS  Google Scholar 

  • Yamakawa K, Karasawa T, Ikoma S, Nakamura S (1996) Enhancement of Clostridium difficile toxin production in biotin-limited conditions. J Med Microbiol 44:111–114

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We thank Karin Agternkamp and Saskia Kohlscheen for excellent technical assistance. We thank Ralf Gerhard and Harald Genth for providing the antibodies against toxin A and B.

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Authors and Affiliations

  1. Hannover Medical School, Institute of Toxicology, Carl-Neuberg-Str. 1, 30625, Hannover, Germany

    Alexander Boetzkes, Katharina Wiebke Felkel, Johannes Zeiser, Nelli Jochim, Ingo Just & Andreas Pich

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  1. Alexander Boetzkes
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  2. Katharina Wiebke Felkel
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  3. Johannes Zeiser
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  4. Nelli Jochim
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  5. Ingo Just
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  6. Andreas Pich
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Corresponding author

Correspondence to Andreas Pich.

Additional information

Communicated by Wolfgang Buckel.

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Boetzkes, A., Felkel, K.W., Zeiser, J. et al. Secretome analysis of Clostridium difficile strains. Arch Microbiol 194, 675–687 (2012). https://doi.org/10.1007/s00203-012-0802-5

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  • DOI: https://doi.org/10.1007/s00203-012-0802-5

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