Identification of a Novel Lipoprotein Regulator of Spore Germination

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Clostridium difficile is a spore-forming bacterium capable of causing severe diarrhea. The dormant spore-form of C. difficile is necessary to cause infection, since vegetative cells of this organism cannot survive in the presence of oxygen. Spores are difficult to eradicate because they can withstand extreme environmental conditions and chemical insults including antibiotics. However, since spores cannot grow, they must transform back into actively replicating cells once the appropriate environmental conditions are sensed through a process called germination. A key step during germination is the break-down of a specialized cell wall layer in the spore known as cortex by the SleC hydrolase. In this paper, we identify GerS as a novel lipid-modified protein that is important for C. difficile germination to occur. GerS is made at high levels during spore formation and gets packaged into mature spores. We show that GerS is required for the cortex hydrolase SleC to degrade the protective cortex layer, since a strain lacking GerS does not lose its cortex layer. Loss of GerS prevents C. difficile from causing infection in a hamster model of infection, suggesting that GerS is a novel target for drug development.

Vyšlo v časopise: Identification of a Novel Lipoprotein Regulator of Spore Germination. PLoS Pathog 11(10): e32767. doi:10.1371/journal.ppat.1005239
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1005239

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1. Carroll K, Bartlett J (2011) Biology of Clostridium difficile: implications for epidemiology and diagnosis. Annual review of microbiology 65 : 501–521. doi: 10.1146/annurev-micro-090110-102824 21682645

2. Rupnik M, Wilcox M, Gerding D (2009) Clostridium difficile infection: new developments in epidemiology and pathogenesis. Nature reviews Microbiology 7 : 526–536. doi: 10.1038/nrmicro2164 19528959

3. Warny M, Pepin J, Fang A, Killgore G, Thompson A, et al. (2005) Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe. Lancet 366 : 1079–1084. 16182895

4. Badger VO, Ledeboer NA, Graham MB, Edmiston CE Jr. (2012) Clostridium difficile: epidemiology, pathogenesis, management, and prevention of a recalcitrant healthcare-associated pathogen. JPEN J Parenter Enteral Nutr 36 : 645–662. doi: 10.1177/0148607112446703 22577120

5. Freeman J, Bauer M, Baines S, Corver J, Fawley W, et al. (2010) The changing epidemiology of Clostridium difficile infections. Clinical microbiology reviews 23 : 529–549. doi: 10.1128/CMR.00082-09 20610822

6. Gupta A, Khanna S (2014) Community-acquired Clostridium difficile infection: an increasing public health threat. Infect Drug Resist 7 : 63–72. doi: 10.2147/IDR.S46780 24669194

7. (CDC) CfDC (2013) Antibiotic Resistance Threats in the United States. http://wwwcdcgov/drugresistance/threat-report-2013:.

8. Dubberke E (2012) Clostridium difficile infection: the scope of the problem. J Hosp Med 7 Suppl 3: S1–4.

9. Lessa FC, Mu Y, Bamberg WM, Beldavs ZG, Dumyati GK, et al. (2015) Burden of Clostridium difficile infection in the United States. N Engl J Med 372 : 825–834. doi: 10.1056/NEJMoa1408913 25714160

10. Louie TJ, Miller MA, Mullane KM, Weiss K, Lentnek A, et al. (2011) Fidaxomicin versus vancomycin for Clostridium difficile infection. N Engl J Med 364 : 422–431. doi: 10.1056/NEJMoa0910812 21288078

11. Deakin LJ, Clare S, Fagan RP, Dawson LF, Pickard DJ, et al. (2012) The Clostridium difficile spo0A gene is a persistence and transmission factor. Infect Immun 80 : 2704–2711. doi: 10.1128/IAI.00147-12 22615253

12. Paredes-Sabja D, Shen A, Sorg JA (2014) Clostridium difficile spore biology: sporulation, germination, and spore structural proteins. Trends Microbiol 22 : 406–416. doi: 10.1016/j.tim.2014.04.003 24814671

13. Setlow P (2014) Germination of spores of Bacillus species: what we know and do not know. J Bacteriol 196 : 1297–1305. doi: 10.1128/JB.01455-13 24488313

14. Kelly C, LaMont J (2008) Clostridium difficile—more difficult than ever. The New England journal of medicine 359 : 1932–1940. doi: 10.1056/NEJMra0707500 18971494

15. Errington J (2003) Regulation of endospore formation in Bacillus subtilis. Nature reviews Microbiology 1 : 117–126. 15035041

16. Peter S (2003) Spore germination. Current Opinion in Microbiology 6.

17. Setlow P (2006) Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals. Journal of applied microbiology 101 : 514–525. 16907802

18. Koenigsknecht MJ, Theriot CM, Bergin IL, Schumacher CA, Schloss PD, et al. (2015) Dynamics and establishment of Clostridium difficile infection in the murine gastrointestinal tract. Infect Immun 83 : 934–941. doi: 10.1128/IAI.02768-14 25534943

19. Lawley T, Clare S, Walker A, Goulding D, Stabler R, et al. (2009) Antibiotic treatment of Clostridium difficile carrier mice triggers a supershedder state, spore-mediated transmission, and severe disease in immunocompromised hosts. Infection and immunity 77 : 3661–3669. doi: 10.1128/IAI.00558-09 19564382

20. Theriot CM, Koenigsknecht MJ, Carlson PE Jr., Hatton GE, Nelson AM, et al. (2014) Antibiotic-induced shifts in the mouse gut microbiome and metabolome increase susceptibility to Clostridium difficile infection. Nat Commun 5 : 3114. doi: 10.1038/ncomms4114 24445449

21. Francis MB, Allen CA, Shrestha R, Sorg JA (2013) Bile acid recognition by the Clostridium difficile germinant receptor, CspC, is important for establishing infection. PLoS Pathog 9: e1003356. doi: 10.1371/journal.ppat.1003356 23675301

22. Paredes-Sabja D, Setlow P, Sarker M (2011) Germination of spores of Bacillales and Clostridiales species: mechanisms and proteins involved. Trends Microbiol 19 : 85–94. doi: 10.1016/j.tim.2010.10.004 21112786

23. Adams CM, Eckenroth BE, Putnam EE, Doublie S, Shen A (2013) Structural and functional analysis of the CspB protease required for Clostridium spore germination. PLoS Pathog 9: e1003165. doi: 10.1371/journal.ppat.1003165 23408892

24. Paredes-Sabja D, Setlow P, Sarker MR (2009) SleC is essential for cortex peptidoglycan hydrolysis during germination of spores of the pathogenic bacterium Clostridium perfringens. J Bacteriol 191 : 2711–2720. doi: 10.1128/JB.01832-08 19218389

25. Paredes-Sabja D, Setlow P, Sarker MR (2009) 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. doi: 10.1099/mic.0.030965-0 19628563

26. Shimamoto S, Moriyama R, Sugimoto K, Miyata S, Makino S (2001) Partial characterization of an enzyme fraction with protease activity which converts the spore peptidoglycan hydrolase (SleC) precursor to an active enzyme during germination of Clostridium perfringens S40 spores and analysis of a gene cluster involved in the activity. Journal of bacteriology 183 : 3742–3751. 11371539

27. Yutin N, Galperin MY (2013) A genomic update on clostridial phylogeny: Gram-negative spore formers and other misplaced clostridia. Environ Microbiol 15 : 2631–2641. doi: 10.1111/1462-2920.12173 23834245

28. Buffie CG, Bucci V, Stein RR, McKenney PT, Ling L, et al. (2015) Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile. Nature 517 : 205–208. doi: 10.1038/nature13828 25337874

29. Giel J, Sorg J, Sonenshein A, Zhu J (2010) Metabolism of bile salts in mice influences spore germination in Clostridium difficile. PLoS One 5.

30. Sorg JA, Sonenshein AL (2008) Bile salts and glycine as cogerminants for Clostridium difficile spores. J Bacteriol 190 : 2505–2512. doi: 10.1128/JB.01765-07 18245298

31. Banawas S, Korza G, Paredes-Sabja D, Li Y, Hao B, et al. (2015) Location and stoichiometry of the protease CspB and the cortex-lytic enzyme SleC in Clostridium perfringens spores. Food Microbiol 50 : 83–87. doi: 10.1016/j.fm.2015.04.001 25998819

32. Burns DA, Heap JT, Minton NP (2010) SleC is essential for germination of Clostridium difficile spores in nutrient-rich medium supplemented with the bile salt taurocholate. J Bacteriol 192 : 657–664. doi: 10.1128/JB.01209-09 19933358

33. Gutelius D, Hokeness K, Logan SM, Reid CW (2013) Functional Analysis of SleC from Clostridium difficile: an essential lytic transglycosylase involved in spore germination. Microbiology.

34. Makino S, Moriyama R (2002) Hydrolysis of cortex peptidoglycan during bacterial spore germination. Med Sci Monit 8: RA119–127. 12070452

35. Popham DL, Helin J, Costello CE, Setlow P (1996) Muramic lactam in peptidoglycan of Bacillus subtilis spores is required for spore outgrowth but not for spore dehydration or heat resistance. Proc Natl Acad Sci U S A 93 : 15405–15410. 8986824

36. Francis MB, Allen CA, Sorg JA (2015) Spore cortex hydrolysis precedes DPA release during Clostridium difficile spore germination. J Bacteriol.

37. Wang S, Shen A, Setlow P, Li YQ (2015) Characterization of the dynamic germination of individual Clostridium difficile spores using Raman spectroscopy and differential interference contrast microscopy. J Bacteriol.

38. 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. 11466292

39. Setlow P (2003) Spore germination. Curr Opin Microbiol 6 : 550–556. 14662349

40. Fimlaid KA, Bond JP, Schutz KC, Putnam EE, Leung JM, et al. (2013) Global Analysis of the Sporulation Pathway of Clostridium difficile. PLoS Genet 9: e1003660. doi: 10.1371/journal.pgen.1003660 23950727

41. Pishdadian K, Fimlaid KA, Shen A (2015) SpoIIID-mediated regulation of sigma(K) function during Clostridium difficile sporulation. Mol Microbiol 95 : 189–208. doi: 10.1111/mmi.12856 25393584

42. Saujet L, Pereira FC, Serrano M, Soutourina O, Monot M, et al. (2013) Genome-wide analysis of cell type-specific gene transcription during spore formation in Clostridium difficile. PLoS Genet 9: e1003756. doi: 10.1371/journal.pgen.1003756 24098137

43. Chesnokova ON, McPherson SA, Steichen CT, Turnbough CL Jr. (2009) The spore-specific alanine racemase of Bacillus anthracis and its role in suppressing germination during spore development. J Bacteriol 191 : 1303–1310. doi: 10.1128/JB.01098-08 19074397

44. Venir E, Del Torre M, Cunsolo V, Saletti R, Musetti R, et al. (2014) Involvement of alanine racemase in germination of Bacillus cereus spores lacking an intact exosporium. Arch Microbiol 196 : 79–85. doi: 10.1007/s00203-013-0946-y 24346000

45. Yan X, Gai Y, Liang L, Liu G, Tan H (2007) A gene encoding alanine racemase is involved in spore germination in Bacillus thuringiensis. Arch Microbiol 187 : 371–378. 17165028

46. Paidhungat M, Setlow B, Driks A, Setlow P (2000) Characterization of spores of Bacillus subtilis which lack dipicolinic acid. J Bacteriol 182 : 5505–5512. 10986255

47. Leuschner RG, Lillford PJ (1999) Effects of temperature and heat activation on germination of individual spores of Bacillus subtilis. Lett Appl Microbiol 29 : 228–232. 10583749

48. Levinson HS, Hyatt MT (1970) Effects of temperature on activation, germination, and outgrowth of Bacillus megaterium spores. J Bacteriol 101 : 58–64. 4983656

49. Yi X, Setlow P (2010) Studies of the commitment step in the germination of spores of Bacillus species. J Bacteriol 192 : 3424–3433. doi: 10.1128/JB.00326-10 20435722

50. Dembek M, Stabler RA, Witney AA, Wren BW, Fairweather NF (2013) Transcriptional analysis of temporal gene expression in germinating Clostridium difficile 630 endospores. PLoS One 8: e64011. doi: 10.1371/journal.pone.0064011 23691138

51. Mongkolthanaruk W, Robinson C, Moir A (2009) Localization of the GerD spore germination protein in the Bacillus subtilis spore. Microbiology 155 : 1146–1151. doi: 10.1099/mic.0.023853-0 19332816

52. Pelczar PL, Igarashi T, Setlow B, Setlow P (2007) Role of GerD in germination of Bacillus subtilis spores. J Bacteriol 189 : 1090–1098. 17122337

53. Heeg D, Burns DA, Cartman ST, Minton NP (2012) Spores of Clostridium difficile clinical isolates display a diverse germination response to bile salts. PLoS One 7: e32381. doi: 10.1371/journal.pone.0032381 22384234

54. Behravan J, Chirakkal H, Masson A, Moir A (2000) Mutations in the gerP locus of Bacillus subtilis and Bacillus cereus affect access of germinants to their targets in spores. J Bacteriol 182 : 1987–1994. 10715007

55. Butzin XY, Troiano AJ, Coleman WH, Griffiths KK, Doona CJ, et al. (2012) Analysis of the effects of a gerP mutation on the germination of spores of Bacillus subtilis. J Bacteriol 194 : 5749–5758. doi: 10.1128/JB.01276-12 22904285

56. Carr KA, Janes BK, Hanna PC (2010) Role of the gerP operon in germination and outgrowth of Bacillus anthracis spores. PLoS One 5: e9128. doi: 10.1371/journal.pone.0009128 20161744

57. Kumazawa T, Masayama A, Fukuoka S, Makino S, Yoshimura T, et al. (2007) Mode of action of a germination-specific cortex-lytic enzyme, SleC, of Clostridium perfringens S40. Biosci Biotechnol Biochem 71 : 884–892. 17420590

58. Kamiya S, Yamakawa K, Ogura H, Nakamura S (1989) Recovery of spores of Clostridium difficile altered by heat or alkali. J Med Microbiol 28 : 217–221. 2926793

59. Miyata S, Kozuka S, Yasuda Y, Chen Y, Moriyama R, et al. (1997) Localization of germination-specific spore-lytic enzymes in Clostridium perfringens S40 spores detected by immunoelectron microscopy. FEMS microbiology letters 152 : 243–247. 9231416

60. Putnam EE, Nock AM, Lawley TD, Shen A (2013) SpoIVA and SipL are Clostridium difficile spore morphogenetic proteins. J Bacteriol 195 : 1214–1225. doi: 10.1128/JB.02181-12 23292781

61. Pereira FC, Saujet L, Tome AR, Serrano M, Monot M, et al. (2013) The Spore Differentiation Pathway in the Enteric Pathogen Clostridium difficile. PLoS Genet 9: e1003782. doi: 10.1371/journal.pgen.1003782 24098139

62. Sussman MD, Setlow P (1991) Cloning, nucleotide sequence, and regulation of the Bacillus subtilis gpr gene, which codes for the protease that initiates degradation of small, acid-soluble proteins during spore germination. J Bacteriol 173 : 291–300. 1840582

63. Hutchings MI, Palmer T, Harrington DJ, Sutcliffe IC (2009) Lipoprotein biogenesis in Gram-positive bacteria: knowing when to hold 'em, knowing when to fold 'em. Trends Microbiol 17 : 13–21. doi: 10.1016/j.tim.2008.10.001 19059780

64. Zuckert WR (2014) Secretion of bacterial lipoproteins: through the cytoplasmic membrane, the periplasm and beyond. Biochim Biophys Acta 1843 : 1509–1516. doi: 10.1016/j.bbamcr.2014.04.022 24780125

65. Buddelmeijer N (2015) The molecular mechanism of bacterial lipoprotein modification—how, when and why? FEMS Microbiol Rev 39 : 246–261. doi: 10.1093/femsre/fuu006 25670733

66. Cooper GR, Moir A (2011) Amino acid residues in the GerAB protein important in the function and assembly of the alanine spore germination receptor of Bacillus subtilis 168. J Bacteriol 193 : 2261–2267. doi: 10.1128/JB.01397-10 21378181

67. Pelczar PL, Setlow P (2008) Localization of the germination protein GerD to the inner membrane in Bacillus subtilis spores. J Bacteriol 190 : 5635–5641. doi: 10.1128/JB.00670-08 18556788

68. Leskela S, Wahlstrom E, Kontinen VP, Sarvas M (1999) Lipid modification of prelipoproteins is dispensable for growth but essential for efficient protein secretion in Bacillus subtilis: characterization of the Lgt gene. Mol Microbiol 31 : 1075–1085. 10096076

69. Wang S, Setlow B, Conlon E, Lyon J, Imamura D, et al. (2006) The forespore line of gene expression in Bacillus subtilis. Journal of molecular biology 358 : 16–37. 16497325

70. Hudson KD, Corfe BM, Kemp EH, Feavers IM, Coote PJ, et al. (2001) Localization of GerAA and GerAC germination proteins in the Bacillus subtilis spore. J Bacteriol 183 : 4317–4322. 11418573

71. Korza G, Setlow P (2013) Topology and accessibility of germination proteins in the Bacillus subtilis spore inner membrane. J Bacteriol 195 : 1484–1491. doi: 10.1128/JB.02262-12 23335419

72. Paidhungat M, Setlow P (2001) Localization of a germinant receptor protein (GerBA) to the inner membrane of Bacillus subtilis spores. J Bacteriol 183 : 3982–3990. 11395462

73. 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. 10762253

74. Howerton A, Ramirez N, Abel-Santos E (2011) Mapping interactions between germinants and Clostridium difficile spores. J Bacteriol 193 : 274–282. doi: 10.1128/JB.00980-10 20971909

75. Yasuda Y, Kanda K, Nishioka S, Tanimoto Y, Kato C, et al. (1993) Regulation of L-alanine-initiated germination of Bacillus subtilis spores by alanine racemase. Amino Acids 4 : 89–99. doi: 10.1007/BF00805804 24190560

76. Sorg JA, Dineen SS (2009) Laboratory maintenance of Clostridium difficile. Curr Protoc Microbiol Chapter 9: Unit 9A 1.

77. Permpoonpattana P, Tolls E, Nadem R, Tan S, Brisson A, et al. (2011) Surface layers of Clostridium difficile endospores. Journal of bacteriology 193 : 6461–6470. doi: 10.1128/JB.05182-11 21949071

78. Shen A, Lupardus PJ, Morell M, Ponder EL, Sadaghiani AM, et al. (2009) Simplified, enhanced protein purification using an inducible, autoprocessing enzyme tag. PLoS One 4: e8119. doi: 10.1371/journal.pone.0008119 19956581

79. Heap JT, Pennington OJ, Cartman ST, Carter GP, Minton NP (2007) The ClosTron: a universal gene knock-out system for the genus Clostridium. J Microbiol Methods 70 : 452–464. 17658189

80. Kevorkian Y, Shirley DJ, Shen A (2015) Regulation of Clostridium difficile spore germination by the CspA pseudoprotease domain Biochimie. Epub ahead of print (S0300-9084(15)00231-X)

81. Fimlaid KA, Jensen O, Donnelly ML, Siegrist MS, Shen A (2015) A conserved channel regulates multiple stages of Clostridium difficile spore morphogenesis. PLoS Genet Accepted.

82. Sambol SP, Tang JK, Merrigan MM, Johnson S, Gerding DN (2001) Infection of hamsters with epidemiologically important strains of Clostridium difficile. J Infect Dis 183 : 1760–1766. 11372028

83. Dineen SS, Villapakkam AC, Nordman JT, Sonenshein AL (2007) Repression of Clostridium difficile toxin gene expression by CodY. Mol Microbiol 66 : 206–219. 17725558

84. Heap J, Pennington O, Cartman S, Minton N (2009) A modular system for Clostridium shuttle plasmids. Journal of microbiological methods 78 : 79–85. doi: 10.1016/j.mimet.2009.05.004 19445976

85. Underwood S, Guan S, Vijayasubhash V, Baines S, Graham L, et al. (2009) Characterization of the sporulation initiation pathway of Clostridium difficile and its role in toxin production. Journal of bacteriology 191 : 7296–7305. doi: 10.1128/JB.00882-09 19783633

86. Griffiths KK, Zhang J, Cowan AE, Yu J, Setlow P (2011) Germination proteins in the inner membrane of dormant Bacillus subtilis spores colocalize in a discrete cluster. Mol Microbiol 81 : 1061–1077. doi: 10.1111/j.1365-2958.2011.07753.x 21696470

87. Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, et al. (2011) Integrative genomics viewer. Nat Biotechnol 29 : 24–26. doi: 10.1038/nbt.1754 21221095