1887

Abstract

type A can cause both food poisoning (FP) and non-food-borne (NFB) gastrointestinal diseases. Our previous study reported that a mixture of -asparagine and KCl (AK)-germinated spores of FP and NFB isolates well, but KCl and, to a lesser extent, -asparagine induced spore germination only in FP isolates. We now report that the germination response of FP and NFB spores differsignificantly in several defined germinants and rich media. Spores of NFB strain F4969 , or mutants lacking specific germinant receptor proteins germinated more slowly than wild-type spores with rich media, did not germinate with AK and germinated poorly compared to wild-type spores with -cysteine. The germination defects in the spores were largely due to loss of GerKC as (i) spores germinated significantly with all tested germinants, while spores exhibited poor or no germination; and (ii) germination defects in spores were largely restored by expressing the wild-type operon . We also found that , and spores, but not spores, released dipicolinic acid at a slower rate than wild-type spores with AK. The colony-forming efficiency of F4969 spores was also ~35-fold lower than that of wild-type spores, while and wild-type spores had similar viability. Collectively, these results suggest that the GerAA and GerKC proteins play roles in normal germination of NFB isolates and that GerKC, but not GerAA, is important in these spores' apparent viability.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000378
2016-11-23
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/162/11/1972.html?itemId=/content/journal/micro/10.1099/mic.0.000378&mimeType=html&fmt=ahah

References

  1. Asha N. J., Wilcox M. H. 2002; Laboratory diagnosis of Clostridium perfringens antibiotic-associated diarrhoea. J Med Microbiol 51:891–894 [View Article][PubMed]
    [Google Scholar]
  2. Banawas S., Paredes-Sabja D., Korza G., Li Y., Hao B., Setlow P., Sarker M. R. 2013; The Clostridium perfringens germinant receptor protein GerKC is located in the spore inner membrane and is crucial for spore germination. J Bacteriol 195:5084–5091 [View Article][PubMed]
    [Google Scholar]
  3. Bannam T. L., Rood J. I. 1993; Clostridium perfringens– Escherichia coli shuttle vectors that carry single antibiotic resistance determinants. Plasmid 29:233–235 [View Article][PubMed]
    [Google Scholar]
  4. Cabrera-Martinez R. M., Tovar-Rojo F., Vepachedu V. R., Setlow P. 2003; Effects of overexpression of nutrient receptors on germination of spores of Bacillus subtilis . J Bacteriol 185:2457–2464 [View Article][PubMed]
    [Google Scholar]
  5. Carman R. J. 1997; Clostridium perfringens in spontaneous and antibiotic-associated diarrhoea of man and other animals. Rev Med Microbiol 8:S46–S45 [View Article]
    [Google Scholar]
  6. Chen Y., McClane B. A., Fisher D. J., Rood J. I., Gupta P. 2005; Construction of an alpha toxin gene knockout mutant of Clostridium perfringens type A by use of a mobile group II intron. Appl Environ Microbiol 71:7542–7547 [View Article][PubMed]
    [Google Scholar]
  7. Cheung J. K., Keyburn A. L., Carter G. P., Lanckriet A. L., Van Immerseel F., Moore R. J., Rood J. I. 2010; The VirSR two-component signal transduction system regulates NetB toxin production in Clostridium perfringens . Infect Immun 78:3064–3072 [View Article][PubMed]
    [Google Scholar]
  8. Collie R. E., McClane B. A. 1998; Evidence that the enterotoxin gene can be episomal in Clostridium perfringens isolates associated with non-food-borne human gastrointestinal diseases. J Clin Microbiol 36:30–36[PubMed]
    [Google Scholar]
  9. Cornillot E., Saint-Joanis B., Daube G., Katayama S., Granum P. E., Canard B., Cole S. T. 1995; The enterotoxin gene (cpe) of Clostridium perfringens can be chromosomal or plasmid-borne. Mol Microbiol 15:639–647 [View Article][PubMed]
    [Google Scholar]
  10. Czeczulin J. R., Collie R. E., McClane B. A. 1996; Regulated expression of Clostridium perfringens enterotoxin in naturally cpe-negative type A, B, and C isolates of C. perfringens . Infect Immun 64:3301–3309[PubMed]
    [Google Scholar]
  11. Duncan C. L., Strong D. H. 1968; Improved medium for sporulation of Clostridium perfringens . Appl Microbiol 16:82–89[PubMed]
    [Google Scholar]
  12. Huang I. H., Sarker M. R. 2006; Complementation of a Clostridium perfringens spo0A mutant with wild-type spo0A from other Clostridium species. Appl Environ Microbiol 72:6388–6393 [View Article][PubMed]
    [Google Scholar]
  13. 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 [View Article][PubMed]
    [Google Scholar]
  14. 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]
  15. Li J., McClane B. A. 2006a; Comparative effects of osmotic, sodium nitrite-induced, and pH-induced stress on growth and survival of Clostridium perfringens type A isolates carrying chromosomal or plasmid-borne enterotoxin genes. Appl Environ Microbiol 72:7620–7625 [View Article][PubMed]
    [Google Scholar]
  16. Li J., McClane B. A. 2006b; Further comparison of temperature effects on growth and survival of Clostridium perfringens type A isolates carrying a chromosomal or plasmid-borne enterotoxin gene. Appl Environ Microbiol 72:4561–4568 [View Article][PubMed]
    [Google Scholar]
  17. Li J., McClane B. A. 2008; A novel small acid soluble protein variant is important for spore resistance of most Clostridium perfringens food poisoning isolates. PLoS Pathog 4:e1000056 [View Article][PubMed]
    [Google Scholar]
  18. Liu H., Ray W. K., Helm R. F., Popham D. L., Melville S. B. 2016; Analysis of the spore membrane proteome in Clostridium perfringens implicates cyanophycin in spore assembly. J Bacteriol 198:1773–1782 [View Article][PubMed]
    [Google Scholar]
  19. 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: ASM Press; [CrossRef]
    [Google Scholar]
  20. McClane B., Uzal F. A., Miyakawa M. F., Lyerly D., Wilkins T. 2004; The enterotoxic clostridia. In The Prokaryotes , pp. 698–752 Edited by Dworkin M., Falkow S., Rosenberg E., Schleifer K.-H., Stackebrandt E. New York: Springer;
    [Google Scholar]
  21. McDonnell J. L. 1986; Toxins of Clostridium perfringens type A, B, C, D, and E. In Pharmacology of Bacterial Toxins , pp. 477–517 Edited by Dorner F., Drews J. Oxford: Pergamon Press;
    [Google Scholar]
  22. Olguín-Araneda V., Banawas S., Sarker M. R., Paredes-Sabja D. 2015; Recent advances in germination of Clostridium spores. Res Microbiol 166:236–243 [View Article][PubMed]
    [Google Scholar]
  23. Paidhungat M., Setlow P. 1999; Isolation and characterization of mutations in Bacillus subtilis that allow spore germination in the novel germinant d-alanine. J Bacteriol 181:3341–3350[PubMed]
    [Google Scholar]
  24. Paidhungat M., Setlow P. 2000; Role of Ger proteins in nutrient and nonnutrient triggering of spore germination in Bacillus subtilis . J Bacteriol 182:2513–2519 [View Article][PubMed]
    [Google Scholar]
  25. Paidhungat M., Ragkousi K., Setlow P. 2001; Genetic requirements for induction of germination of spores of Bacillus subtilis by Ca(2+)-dipicolinate. J Bacteriol 183:4886–4893 [View Article][PubMed]
    [Google Scholar]
  26. Paredes-Sabja D., Sarker M. R. 2010; Effect of the cortex-lytic enzyme SleC from non-food-borne Clostridium perfringens on the germination properties of SleC-lacking spores of a food poisoning isolate. Can J Microbiol 56:952–958 [View Article][PubMed]
    [Google Scholar]
  27. Paredes-Sabja D., Sarker M. R. 2011a; Germination response of spores of the pathogenic bacterium Clostridium perfringens and Clostridium difficile to cultured human epithelial cells. Anaerobe 17:78–84 [View Article]
    [Google Scholar]
  28. Paredes-Sabja D., Sarker M. R. 2011b; Host serum factor triggers germination of Clostridium perfringens spores lacking the cortex hydrolysis machinery. J Med Microbiol 60:1734–1741 [View Article][PubMed]
    [Google Scholar]
  29. Paredes-Sabja D., Sarker M. R. 2012; Interactions between Clostridium perfringens spores and Raw 264.7 macrophages. Anaerobe 18:148–156 [View Article][PubMed]
    [Google Scholar]
  30. 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 [View Article]
    [Google Scholar]
  31. 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 [View Article][PubMed]
    [Google Scholar]
  32. Paredes-Sabja D., Setlow P., Sarker M. R. 2009a; SleC is essential for cortex peptidoglycan hydrolysis during germination of spores of the pathogenic bacterium Clostridium perfringens . J Bacteriol 191:2711–2720 [View Article][PubMed]
    [Google Scholar]
  33. Paredes-Sabja D., Setlow P., Sarker M. R. 2009b; Role of GerKB in germination and outgrowth of Clostridium perfringens spores. Appl Environ Microbiol 75:3813–3817 [View Article][PubMed]
    [Google Scholar]
  34. Paredes-Sabja D., Setlow P., Sarker M. R. 2009c; GerO, a putative Na+/H+-K+ antiporter, is essential for normal germination of spores of the pathogenic bacterium Clostridium perfringens . J Bacteriol 191:3822–3831 [View Article][PubMed]
    [Google Scholar]
  35. Paredes-Sabja D., Setlow P., Sarker M. R. 2009d; The protease CspB is essential for initiation of cortex hydrolysis and dipicolinic acid (DPA) release during germination of spores of Clostridium perfringens type A food poisoning isolates. Microbiology 155:3464–3472 [View Article]
    [Google Scholar]
  36. Paredes-Sabja D., Udompijitkul P., Sarker M. R. 2009e; Inorganic phosphate and sodium ions are cogerminants for spores of Clostridium perfringens type A food poisoning-related isolates. Appl Environ Microbiol 75:6299–6305 [View Article][PubMed]
    [Google Scholar]
  37. 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 [View Article][PubMed]
    [Google Scholar]
  38. Sarker M. R., Carman R. J., McClane B. A. 1999; Inactivation of the gene (cpe) encoding Clostridium perfringens enterotoxin eliminates the ability of two cpe-positive C. perfringens type A human gastrointestinal disease isolates to affect rabbit ileal loops. Mol Microbiol 33:946–958 [View Article][PubMed]
    [Google Scholar]
  39. 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 [View Article][PubMed]
    [Google Scholar]
  40. Setlow P. 2013; Summer meeting 201 – when the sleepers wake: the germination of spores of Bacillus species. J Appl Microbiol 115:1251–1268 [View Article][PubMed]
    [Google Scholar]
  41. Setlow P. 2014; Germination of spores of Bacillus species: what we know and do not know. J Bacteriol 196:1297–1305 [View Article][PubMed]
    [Google Scholar]
  42. Udompijitkul P., Alnoman M., Banawas S., Paredes-Sabja D., Sarker M. R. 2014; New amino acid germinants for spores of the enterotoxigenic Clostridium perfringens type A isolates. Food Microbiol 44:24–33 [View Article][PubMed]
    [Google Scholar]
  43. Wen Q., McClane B. A. 2004; Detection of enterotoxigenic Clostridium perfringens type A isolates in American retail foods. Appl Environ Microbiol 70:2685–2691 [View Article][PubMed]
    [Google Scholar]
  44. 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]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000378
Loading
/content/journal/micro/10.1099/mic.0.000378
Loading

Data & Media loading...

Supplements

Supplementary File 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error