1887

Abstract

type A is the causative agent of a variety of histotoxic and enteric diseases. The ability of spores to germinate might be due to the presence of nutrient germinants in the host tissue and blood. In the current study, we investigated the ability of spores of wild-type and mutation strains to germinate in blood. Results indicate that spores of all three surveyed wild-type isolates germinated better in blood than in brain heart infusion (BHI) broth. However, as expected, spores lacking germinant receptor (GR) protein GerAA or GerKB germinated like wild-type spores in BHI broth and blood. Strikingly, while spores lacking GR proteins GerKA and GerKC showed significantly decreased germination in BHI broth, these spores germinated well in blood, suggesting that blood factor(s) can trigger spore germination through a GR-independent pathway. Using spores lacking cortex lytic enzymes (Δ or Δ Δ), we were able to identify a host serum germination factor with peptidoglycan hydrolysing activity that (i) restored the colony-forming efficiencies of Δ and Δ Δ spores up to ~5–20 % of that of total colony-forming spores; (ii) increased the number of c.f.u. of decoated Δ and Δ Δ spores to ~99 % of that of colony-forming spores; (iii) and finally lost enzymic activity after heat inactivation, consistent with serum germination factor being an enzyme. Further characterization demonstrated that serum germination factor is very likely lysozyme, which can form a stable high molecular mass complex of ~120 kDa in serum. In conclusion, the current study indicates that a host serum germination factor with peptidoglycan hydrolysing activity is capable of triggering germination of spores by directly degrading the spore peptidoglycan cortex. Collectively, this study contributes to our understanding of the mechanism of germination of spores of .

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2011-12-01
2019-10-21
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References

  1. Amimoto K. , Noro T. , Oishi E. , Shimizu M. . ( 2007; ). A novel toxin homologous to large clostridial cytotoxins found in culture supernatant of Clostridium perfringens type C. . Microbiology 153:, 1198–1206. [CrossRef] [PubMed]
    [Google Scholar]
  2. Awad M. M. , Ellemor D. M. , Boyd R. L. , Emmins J. J. , Rood J. I. . ( 2001; ). Synergistic effects of alpha-toxin and perfringolysin O in Clostridium perfringens-mediated gas gangrene. . Infect Immun 69:, 7904–7910. [CrossRef] [PubMed]
    [Google Scholar]
  3. Cassier M. , Ryter A. . ( 1971; ). [A Clostridium perfringens mutant producing coatless spores by lysozyme-dependent germination]. . Ann Inst Pasteur (Paris) 121:, 717–732 (in French).[PubMed]
    [Google Scholar]
  4. Cassier M. , Sebald M. . ( 1969; ). [Lysozyme-dependent germination of spores of Clostridium perfringens ATCC 3624 after heat treatment]. . Ann Inst Pasteur (Paris) 117:, 312–324 (in French).[PubMed]
    [Google Scholar]
  5. Cole A. M. , Ganz T. . ( 2005; ). Defensins and other antimicrobial peptides: innate defense of mucosal surfaces. . In Colonization of Mucosal Surfaces, pp. 17–34. Edited by Nataro J. P. , Cohen P. S. , Mobley H. L. T. , Weiser J. N. . . Washington, DC:: American Society for Microbiology;.
    [Google Scholar]
  6. Duncan C. L. , Strong D. H. . ( 1968; ). Improved medium for sporulation of Clostridium perfringens . . Appl Microbiol 16:, 82–89.[PubMed]
    [Google Scholar]
  7. Giebel J. D. , Carr K. A. , Anderson E. C. , Hanna P. C. . ( 2009; ). The germination-specific lytic enzymes SleB, CwlJ1, and CwlJ2 each contribute to Bacillus anthracis spore germination and virulence. . J Bacteriol 191:, 5569–5576. [CrossRef] [PubMed]
    [Google Scholar]
  8. Hankiewicz J. , Swierczek E. . ( 1974; ). Lysozyme in human body fluids. . Clin Chim Acta 57:, 205–209. [CrossRef] [PubMed]
    [Google Scholar]
  9. Huang I. H. , Waters M. , Grau R. R. , Sarker M. R. . ( 2004; ). Disruption of the gene (spo0A) encoding sporulation transcription factor blocks endospore formation and enterotoxin production in enterotoxigenic Clostridium perfringens type A. . FEMS Microbiol Lett 233:, 233–240. [CrossRef] [PubMed]
    [Google Scholar]
  10. Inoue K. , Tanigawa Y. , Takubo M. , Satani M. , Amano T. . ( 1959; ). Quantitative studies on immune bacteriolysis. II. The role of lysozyme in immune bacteriolysin. . Biken's J 2:, 1–20.
    [Google Scholar]
  11. Keyburn A. L. , Boyce J. D. , Vaz P. , Bannam T. L. , Ford M. E. , Parker D. , Di Rubbo A. , Rood J. I. , Moore R. J. . ( 2008; ). NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens . . PLoS Pathog 4:, e26. [CrossRef] [PubMed]
    [Google Scholar]
  12. Klobutcher L. A. , Ragkousi K. , Setlow P. . ( 2006; ). The Bacillus subtilis spore coat provides “eat resistance” during phagocytic predation by the protozoan Tetrahymena thermophila . . Proc Natl Acad Sci U S A 103:, 165–170. [CrossRef] [PubMed]
    [Google Scholar]
  13. Kokai-Kun J. F. , Songer J. G. , Czeczulin J. R. , Chen F. , McClane B. A. . ( 1994; ). Comparison of Western immunoblots and gene detection assays for identification of potentially enterotoxigenic isolates of Clostridium perfringens . . J Clin Microbiol 32:, 2533–2539.[PubMed]
    [Google Scholar]
  14. Laaberki M. H. , Dworkin J. . ( 2008; ). Role of spore coat proteins in the resistance of Bacillus subtilis spores to Caenorhabditis elegans predation. . J Bacteriol 190:, 6197–6203. [CrossRef] [PubMed]
    [Google Scholar]
  15. Lund B. M. , Peck M. W. . ( 1994; ). Heat resistance and recovery of spores of non-proteolytic Clostridium botulinum in relation to refrigerated, processed foods with an extended shelf-life. . Soc Appl Bacteriol Symp Ser 23:, 115S–128S.[PubMed]
    [Google Scholar]
  16. McClane B. A. . ( 2007; ). Clostridium perfringens . . In Food Microbiology: Fundamentals and Frontiers, pp. 423–444. Edited by Doyle M. P. , Beuchat L. R. . . Washington, DC:: American Society for Microbiology;.
    [Google Scholar]
  17. Michael J. G. , Braun W. . ( 1964; ). Analysis of sequential stages in serum bactericidal reactions. . J Bacteriol 87:, 1067–1072.[PubMed]
    [Google Scholar]
  18. Paidhungat M. , Ragkousi K. , Setlow P. . ( 2001; ). Genetic requirements for induction of germination of spores of Bacillus subtilis by Ca2+-dipicolinate. . J Bacteriol 183:, 4886–4893. [CrossRef] [PubMed]
    [Google Scholar]
  19. Paredes-Sabja D. , Sarker M. R. . ( 2009; ). Clostridium perfringens sporulation and its relevance to pathogenesis. . Future Microbiol 4:, 519–525. [CrossRef] [PubMed]
    [Google Scholar]
  20. Paredes-Sabja D. , Setlow B. , Setlow P. , Sarker M. R. . ( 2008a; ). Characterization of Clostridium perfringens spores that lack SpoVA proteins and dipicolinic acid. . J Bacteriol 190:, 4648–4659. [CrossRef] [PubMed]
    [Google Scholar]
  21. Paredes-Sabja D. , Torres J. A. , Setlow P. , Sarker M. R. . ( 2008b; ). Clostridium perfringens spore germination: characterization of germinants and their receptors. . J Bacteriol 190:, 1190–1201. [CrossRef] [PubMed]
    [Google Scholar]
  22. Paredes-Sabja D. , Setlow P. , Sarker M. R. . ( 2009a; ). Role of GerKB in germination and outgrowth of Clostridium perfringens spores. . Appl Environ Microbiol 75:, 3813–3817. [CrossRef] [PubMed]
    [Google Scholar]
  23. Paredes-Sabja D. , Setlow P. , Sarker M. R. . ( 2009b; ). The protease CspB is essential for initiation of cortex hydrolysis and dipicolinic acid (DPA) release during germination of Clostridium perfringens type A food poisoning isolates. . Microbiology 155:, 3464–3472. [CrossRef] [PubMed]
    [Google Scholar]
  24. Paredes-Sabja D. , Setlow P. , Sarker M. R. . ( 2009c; ). SleC is essential for cortex peptidoglycan hydrolysis during germination of spores of the pathogenic bacterium Clostridium perfringens . . J Bacteriol 191:, 2711–2720. [CrossRef] [PubMed]
    [Google Scholar]
  25. Paredes-Sabja D. , Setlow P. , Sarker M. R. . ( 2011; ). Germination of spores of Bacillales and Clostridiales species: mechanisms and proteins involved. . Trends Microbiol 19:, 85–94. [CrossRef] [PubMed]
    [Google Scholar]
  26. Peck M. W. , Fairbairn D. A. , Lund B. M. . ( 1992; ). Factors affecting growth from heat-treated spores of non-proteolytic Clostridium botulinum . . Lett Appl Microbiol 15:, 152–155. [CrossRef]
    [Google Scholar]
  27. Pier G. B. , Lyczak J. B. , Wetzler L. M. . ( 2004; ). Immunology, Infection, and Immunity. Washington, DC:: American Society for Microbiology;.
    [Google Scholar]
  28. Prager E. M. , Jolles P. . ( 1996; ). Lysozyme: a model enzyme in protein chemistry. . In Lysozymes: Model Enzymes in Biochemistry and Biology, pp. 9–87. Edited by Jolles P. . . Basel:: Birkhauser-Verlag;.
    [Google Scholar]
  29. Reitamo S. , Klockars M. , Adinolfi M. , Osserman E. F. . ( 1978; ). Human lysozyme (origin and distribution in health and disease). . Ric Clin Lab 8:, 211–231.[PubMed]
    [Google Scholar]
  30. Sacks L. E. . ( 1981; ). Influence of cations on lysozyme-induced germination of coatless spores of Clostridium perfringens 8-6. . Biochim Biophys Acta 674:, 118–127.[PubMed] [CrossRef]
    [Google Scholar]
  31. Sarker M. R. , Shivers R. P. , Sparks S. G. , Juneja V. K. , McClane B. A. . ( 2000; ). Comparative experiments to examine the effects of heating on vegetative cells and spores of Clostridium perfringens isolates carrying plasmid genes versus chromosomal enterotoxin genes. . Appl Environ Microbiol 66:, 3234–3240. [CrossRef] [PubMed]
    [Google Scholar]
  32. Setlow P. . ( 2003; ). Spore germination. . Curr Opin Microbiol 6:, 550–556. [CrossRef] [PubMed]
    [Google Scholar]
  33. Shimizu T. , Ohtani K. , Hirakawa H. , Ohshima K. , Yamashita A. , Shiba T. , Ogasawara N. , Hattori M. , Kuhara S. , Hayashi H. . ( 2002; ). Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater. . Proc Natl Acad Sci U S A 99:, 996–1001. [CrossRef] [PubMed]
    [Google Scholar]
  34. Stewart G. S. , Johnstone K. , Hagelberg E. , Ellar D. J. . ( 1981; ). Commitment of bacterial spores to germinate. A measure of the trigger reaction. . Biochem J 198:, 101–106.[PubMed]
    [Google Scholar]
  35. Yi X. , Setlow P. . ( 2010; ). Studies of the commitment step in the germination of spores of Bacillus species. . J Bacteriol 192:, 3424–3433. [CrossRef] [PubMed]
    [Google Scholar]
  36. Zhang P. , Garner W. , Yi X. , Yu J. , Li Y. Q. , Setlow P. . ( 2010; ). Factors affecting variability in time between addition of nutrient germinants and rapid dipicolinic acid release during germination of spores of Bacillus species. . J Bacteriol 192:, 3608–3619. [CrossRef] [PubMed]
    [Google Scholar]
  37. Zhao Y. , Melville S. B. . ( 1998; ). Identification and characterization of sporulation-dependent promoters upstream of the enterotoxin gene (cpe) of Clostridium perfringens . . J Bacteriol 180:, 136–142.[PubMed]
    [Google Scholar]
  38. Zhou M. , Lucas D. A. , Chan K. C. , Issaq H. J. , Petricoin E. F. III , Liotta L. A. , Veenstra T. D. , Conrads T. P. . ( 2004; ). An investigation into the human serum “interactome”. . Electrophoresis 25:, 1289–1298. [CrossRef] [PubMed]
    [Google Scholar]
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