困難梭狀芽孢桿菌 (Clostridium difficile) 是一隻會產孢子且造成抗生素相關腹瀉的主要致病菌，大多數有致病性的菌株會釋放毒素A、毒素B，此兩毒素為造成困難梭狀芽孢桿菌感染疾病的主要毒力因子，已有研究指出毒素A和毒素B基因的啟動子之表達會受到位於細菌致病性基因座 (PaLoc) 的調控子TcdR和TcdC調控，白胺酸反應調控蛋白(Lrp)是一廣泛性調控子 (global regulator) ，已知在大腸桿菌 (E. coli) 及枯草芽孢桿菌 (B. subtilis) 中，白胺酸反應調控蛋白可正向或負向地影響許多代謝相關的操縱子，由於白胺酸反應調控蛋白是一個廣泛性調控子，我們假設它可去影響毒力相關的因子，像是對毒素A及毒素B基因的調控，有趣的是，我們發現當沒有白胺酸反應調控蛋白的情況下，毒素A及B和調控子TcdR及TcdC的基因表達上升，此結果顯示，白胺酸反應調控蛋白可能在負調控整段致病性基因座中扮演重要的角色，再者，我們的研究結果顯示白胺酸反應調控蛋白參與CcpA及CodY此兩個已知的毒力調控子對毒力的調控，我們還發現，擾亂白胺酸反應調控蛋白的表達會造成孢子形成下降，最後，我們探討白胺酸反應調控蛋白在小鼠模型試驗中是否扮演調控毒力的角色，結果顯示白胺酸反應調控蛋白的突變菌株有較高的毒性，整合來說，白胺酸反應調控蛋白首次在困難梭狀芽孢桿菌中被探討，並且參與細菌的毒力調控，包含毒素的生產及產生芽孢。

Clostridium difficile is a spore-forming pathogen and leading cause of antibiotic-associated diarrhea. Most pathogenic strains secret toxin A (TcdA) and toxin B (TcdB), which are the primary virulence factors responsible for disease symptoms of C. difficile infections (CDI). Published studies have suggested the expression of tcdA and tcdB promoter is controlled by TcdR and TcdC, two transcriptional regulators located on C. difficile pathogenicity locus (PaLoc). Leucine-responsive regulatory protein (Lrp) is a global regulator which has been indicated to positively or negatively affect many metabolic associated operons in E. coli and B. subtilis. Since Lrp is considered to be a global regulator, we hypothesized that it also affects virulent genes including tcdA and tcdB. Interestingly, our results showed that there were more tcdA, tcdB, tcdR and tcdC expressed in the absence of Lrp when compared to the wild type. These results suggest that Lrp may play an important role in downregulating the expression of genes on the whole PaLoc. Furthermore, our results showed that Lrp might affect toxin production via controlling known regulators of toxin production, CcpA and CodY. In addition, we also found that disruption of lrp reduced spore formation in C. difficile. Finally, we investigated whether Lrp is involved in virulence in vivo and the results showed that lrp mutant was more virulent than wild type in murine model of infection. In summary, Lrp was identified for the first time in C. difficile and shown to participate in its virulent factors regulation, including toxin production and sporulation.

1. Rupnik M, Wilcox MH, Gerding DN. 2009. Clostridium difficile infection: new developments in epidemiology and pathogenesis. Nat Rev Microbiol 7:526-536.
2. Lo Vecchio A, Zacur GM. 2012. Clostridium difficile infection: an update on epidemiology, risk factors, and therapeutic options. Curr Opin Gastroenterol 28:1-9.
3. Kelly CP, LaMont JT. 2008. Clostridium difficile--more difficult than ever. N Engl J Med 359:1932-1940.
4. Hensgens MP, Goorhuis A, Dekkers OM, Kuijper EJ. 2012. Time interval of increased risk for Clostridium difficile infection after exposure to antibiotics. J Antimicrob Chemother 67:742-748.
5. Khanna S, Baddour LM, Huskins WC, Kammer PP, Faubion WA, Zinsmeister AR, Harmsen WS, Pardi DS. 2013. The epidemiology of Clostridium difficile infection in children: a population-based study. Clin Infect Dis 56:1401-1406.
6. Rupnik M. 2008. Heterogeneity of large clostridial toxins: importance of Clostridium difficile toxinotypes. FEMS Microbiol Rev 32:541-555.
7. Goorhuis A, Debast SB, van Leengoed LA, Harmanus C, Notermans DW, Bergwerff AA, Kuijper EJ. 2008. Clostridium difficile PCR ribotype 078: an emerging strain in humans and in pigs? J Clin Microbiol 46:1157; author reply 1158.
8. McDonald LC, Killgore GE, Thompson A, Owens RC, Jr., Kazakova SV, Sambol SP, Johnson S, Gerding DN. 2005. An epidemic, toxin gene-variant strain of Clostridium difficile. N Engl J Med 353:2433-2441.
9. Delmee M. 2001. Laboratory diagnosis of Clostridium difficile disease. Clin Microbiol Infect 7:411-416.
10. George WL, Sutter VL, Citron D, Finegold SM. 1979. Selective and differential medium for isolation of Clostridium difficile. J Clin Microbiol 9:214-219.
11. Wilson KH. 1983. Efficiency of various bile salt preparations for stimulation of Clostridium difficile spore germination. J Clin Microbiol 18:1017-1019.
12. Tyrrell KL, Citron DM, Leoncio ES, Merriam CV, Goldstein EJ. 2013. Evaluation of cycloserine-cefoxitin fructose agar (CCFA), CCFA with horse blood and taurocholate, and cycloserine-cefoxitin mannitol broth with taurocholate and lysozyme for recovery of Clostridium difficile isolates from fecal samples. J Clin Microbiol 51:3094-3096.
13. Bartlett JG, Gerding DN. 2008. Clinical recognition and diagnosis of Clostridium difficile infection. Clin Infect Dis 46 Suppl 1:S12-18.
14. Cohen SH, Gerding DN, Johnson S, Kelly CP, Loo VG, McDonald LC, Pepin J, Wilcox MH, Society for Healthcare Epidemiology of A, Infectious Diseases Society of A. 2010. Clinical practice guidelines for Clostridium difficile infection in adults: 2010 update by the society for healthcare epidemiology of America (SHEA) and the infectious diseases society of America (IDSA). Infect Control Hosp Epidemiol 31:431-455.
15. Avila MB, Avila NP, Dupont AW. 2016. Recent advances in the diagnosis and treatment of Clostridium difficile infection. F1000Res 5.
16. Jarrad AM, Karoli T, Blaskovich MA, Lyras D, Cooper MA. 2015. Clostridium difficile drug pipeline: challenges in discovery and development of new agents. J Med Chem 58:5164-5185.
17. Cornely OA, Crook DW, Esposito R, Poirier A, Somero MS, Weiss K, Sears P, Gorbach S, Group OPTCS. 2012. Fidaxomicin versus vancomycin for infection with Clostridium difficile in Europe, Canada, and the USA: a double-blind, non-inferiority, randomised controlled trial. Lancet Infect Dis 12:281-289.
18. Khoruts A, Dicksved J, Jansson JK, Sadowsky MJ. 2010. Changes in the composition of the human fecal microbiome after bacteriotherapy for recurrent Clostridium difficile-associated diarrhea. J Clin Gastroenterol 44:354-360.
19. Tauxe WM, Haydek JP, Rebolledo PA, Neish E, Newman KL, Ward A, Dhere T, Kraft CS. 2016. Fecal microbiota transplant for Clostridium difficile infection in older adults. Therap Adv Gastroenterol 9:273-281.
20. Braun V, Hundsberger T, Leukel P, Sauerborn M, von Eichel-Streiber C. 1996. Definition of the single integration site of the pathogenicity locus in Clostridium difficile. Gene 181:29-38.
21. Matamouros S, England P, Dupuy B. 2007. Clostridium difficile toxin expression is inhibited by the novel regulator TcdC. Mol Microbiol 64:1274-1288.
22. Govind R, Dupuy B. 2012. Secretion of Clostridium difficile toxins A and B requires the holin-like protein TcdE. PLoS Pathog 8:e1002727.
23. Monot M, Eckert C, Lemire A, Hamiot A, Dubois T, Tessier C, Dumoulard B, Hamel B, Petit A, Lalande V, Ma L, Bouchier C, Barbut F, Dupuy B. 2015. Clostridium difficile: New Insights into the Evolution of the Pathogenicity Locus. Sci Rep 5:15023.
24. Brouwer MS, Roberts AP, Hussain H, Williams RJ, Allan E, Mullany P. 2013. Horizontal gene transfer converts non-toxigenic Clostridium difficile strains into toxin producers. Nat Commun 4:2601.
25. Hundsberger T, Braun V, Weidmann M, Leukel P, Sauerborn M, von Eichel-Streiber C. 1997. Transcription analysis of the genes tcdA-E of the pathogenicity locus of Clostridium difficile. Eur J Biochem 244:735-742.
26. Bouillaut L, Dubois T, Sonenshein AL, Dupuy B. 2015. Integration of metabolism and virulence in Clostridium difficile. Res Microbiol 166:375-383.
27. Mani N, Dupuy B. 2001. Regulation of toxin synthesis in Clostridium difficile by an alternative RNA polymerase sigma factor. Proc Natl Acad Sci U S A 98:5844-5849.
28. Antunes A, Camiade E, Monot M, Courtois E, Barbut F, Sernova NV, Rodionov DA, Martin-Verstraete I, Dupuy B. 2012. Global transcriptional control by glucose and carbon regulator CcpA in Clostridium difficile. Nucleic Acids Res 40:10701-10718.
29. Dineen SS, Villapakkam AC, Nordman JT, Sonenshein AL. 2007. Repression of Clostridium difficile toxin gene expression by CodY. Mol Microbiol 66:206-219.
30. Ratnayake-Lecamwasam M, Serror P, Wong KW, Sonenshein AL. 2001. Bacillus subtilis CodY represses early-stationary-phase genes by sensing GTP levels. Genes Dev 15:1093-1103.
31. Shivers RP, Sonenshein AL. 2004. Activation of the Bacillus subtilis global regulator CodY by direct interaction with branched-chain amino acids. Mol Microbiol 53:599-611.
32. Pettit LJ, Browne HP, Yu L, Smits WK, Fagan RP, Barquist L, Martin MJ, Goulding D, Duncan SH, Flint HJ, Dougan G, Choudhary JS, Lawley TD. 2014. Functional genomics reveals that Clostridium difficile Spo0A coordinates sporulation, virulence and metabolism. BMC Genomics 15:160.
33. Mackin KE, Carter GP, Howarth P, Rood JI, Lyras D. 2013. Spo0A differentially regulates toxin production in evolutionarily diverse strains of Clostridium difficile. PLoS One 8:e79666.
34. Underwood S, Guan S, Vijayasubhash V, Baines SD, Graham L, Lewis RJ, Wilcox MH, Stephenson K. 2009. Characterization of the sporulation initiation pathway of Clostridium difficile and its role in toxin production. J Bacteriol 191:7296-7305.
35. El Meouche I, Peltier J, Monot M, Soutourina O, Pestel-Caron M, Dupuy B, Pons JL. 2013. Characterization of the SigD regulon of C. difficile and its positive control of toxin production through the regulation of tcdR. PLoS One 8:e83748.
36. Aubry A, Hussack G, Chen W, KuoLee R, Twine SM, Fulton KM, Foote S, Carrillo CD, Tanha J, Logan SM. 2012. Modulation of toxin production by the flagellar regulon in Clostridium difficile. Infect Immun 80:3521-3532.
37. McKee RW, Mangalea MR, Purcell EB, Borchardt EK, Tamayo R. 2013. The second messenger cyclic Di-GMP regulates Clostridium difficile toxin production by controlling expression of sigD. J Bacteriol 195:5174-5185.
38. Ernsting BR, Atkinson MR, Ninfa AJ, Matthews RG. 1992. Characterization of the regulon controlled by the leucine-responsive regulatory protein in Escherichia coli. J Bacteriol 174:1109-1118.
39. Newman EB, Lin R. 1995. Leucine-responsive regulatory protein: a global regulator of gene expression in E. coli. Annu Rev Microbiol 49:747-775.
40. Tani TH, Khodursky A, Blumenthal RM, Brown PO, Matthews RG. 2002. Adaptation to famine: a family of stationary-phase genes revealed by microarray analysis. Proc Natl Acad Sci U S A 99:13471-13476.
41. Calvo JM, Matthews RG. 1994. The leucine-responsive regulatory protein, a global regulator of metabolism in Escherichia coli. Microbiol Rev 58:466-490.
42. D'Ari R, Lin RT, Newman EB. 1993. The leucine-responsive regulatory protein: more than a regulator? Trends in Biochemical Sciences 18:260-263.
43. de los Rios S, Perona JJ. 2007. Structure of the Escherichia coli leucine-responsive regulatory protein Lrp reveals a novel octameric assembly. J Mol Biol 366:1589-1602.
44. Beloin C, Ayora S, Exley R, Hirschbein L, Ogasawara N, Kasahara Y, Alonso JC, Hegarat FL. 1997. Characterization of an lrp-like (lrpC) gene from Bacillus subtilis. Mol Gen Genet 256:63-71.
45. Baek CH, Wang S, Roland KL, Curtiss R, 3rd. 2009. Leucine-responsive regulatory protein (Lrp) acts as a virulence repressor in Salmonella enterica serovar Typhimurium. J Bacteriol 191:1278-1292.
46. Deng W, Wang H, Xie J. 2011. Regulatory and pathogenesis roles of Mycobacterium Lrp/AsnC family transcriptional factors. J Cell Biochem 112:2655-2662.
47. Ethapa T, Leuzzi R, Ng YK, Baban ST, Adamo R, Kuehne SA, Scarselli M, Minton NP, Serruto D, Unnikrishnan M. 2013. Multiple factors modulate biofilm formation by the anaerobic pathogen Clostridium difficile. J Bacteriol 195:545-555.
48. Heap JT, Kuehne SA, Ehsaan M, Cartman ST, Cooksley CM, Scott JC, Minton NP. 2010. The ClosTron: Mutagenesis in Clostridium refined and streamlined. J Microbiol Methods 80:49-55.
49. McBride SM, Sonenshein AL. 2011. Identification of a genetic locus responsible for antimicrobial peptide resistance in Clostridium difficile. Infect Immun 79:167-176.
50. Putnam EE, Nock AM, Lawley TD, Shen A. 2013. SpoIVA and SipL are Clostridium difficile spore morphogenetic proteins. J Bacteriol 195:1214-1225.
51. See I, Mu Y, Cohen J, Beldavs ZG, Winston LG, Dumyati G, Holzbauer S, Dunn J, Farley MM, Lyons C, Johnston H, Phipps E, Perlmutter R, Anderson L, Gerding DN, Lessa FC. 2014. NAP1 strain type predicts outcomes from Clostridium difficile infection. Clin Infect Dis 58:1394-1400.
52. Oberto J. 2013. SyntTax: a web server linking synteny to prokaryotic taxonomy. BMC Bioinformatics 14:4.
53. Beloin C, Exley R, Mahe AL, Zouine M, Cubasch S, Le Hegarat F. 2000. Characterization of LrpC DNA-binding properties and regulation of Bacillus subtilis lrpC gene expression. J Bacteriol 182:4414-4424.
54. Kuehne SA, Minton NP. 2012. ClosTron-mediated engineering of Clostridium. Bioengineered 3:247-254.
55. Slack FJ, Serror P, Joyce E, Sonenshein AL. 1995. A gene required for nutritional repression of the Bacillus subtilis dipeptide permease operon. Mol Microbiol 15:689-702.
56. Deakin LJ, Clare S, Fagan RP, Dawson LF, Pickard DJ, West MR, Wren BW, Fairweather NF, Dougan G, Lawley TD. 2012. The Clostridium difficile spo0A gene is a persistence and transmission factor. Infect Immun 80:2704-2711.
57. Paredes CJ, Alsaker KV, Papoutsakis ET. 2005. A comparative genomic view of clostridial sporulation and physiology. Nat Rev Microbiol 3:969-978.
58. Tan IS, Ramamurthi KS. 2014. Spore formation in Bacillus subtilis. Environ Microbiol Rep 6:212-225.
59. Edwards AN, McBride SM. 2014. Initiation of sporulation in Clostridium difficile: a twist on the classic model. FEMS Microbiol Lett 358:110-118.
60. Janoir C. 2016. Virulence factors of Clostridium difficile and their role during infection. Anaerobe 37:13-24.
61. Antunes A, Martin-Verstraete I, Dupuy B. 2011. CcpA-mediated repression of Clostridium difficile toxin gene expression. Mol Microbiol 79:882-899.
62. Martin-Verstraete I, Peltier J, Dupuy B. 2016. The Regulatory Networks That Control Clostridium difficile Toxin Synthesis. Toxins (Basel) 8.
63. Baban ST, Kuehne SA, Barketi-Klai A, Cartman ST, Kelly ML, Hardie KR, Kansau I, Collignon A, Minton NP. 2013. The role of flagella in Clostridium difficile pathogenesis: comparison between a non-epidemic and an epidemic strain. PLoS One 8:e73026.
64. Williams DR, Young DI, Young M. 1990. Conjugative plasmid transfer from Escherichia coli to Clostridium acetobutylicum. J Gen Microbiol 136:819-826.
65. 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.
66. Heap JT, Pennington OJ, Cartman ST, Minton NP. 2009. A modular system for Clostridium shuttle plasmids. J Microbiol Methods 78:79-85.