1887

Abstract

Enterohaemorrhagic (EHEC) is a life-threatening human pathogen worldwide. The locus of enterocyte effacement (LEE) in EHEC encodes a type three secretion system and effector proteins, all of which are essential for bacterial adherence to host cells. When LEE expression is activated, flagellar gene expression is down-regulated because bacterial flagella induce the immune responses of host cells at the infection stage. Therefore, this inverse regulation is also important for EHEC infection. We report here that a small regulatory RNA (sRNA), Esr41, mediates LEE repression and flagellar gene activation. Multiple copies of abolished LEE expression by down-regulating the expression of and , which encode positive regulators of LEE. This regulation led to reduced EHEC adhesion to host cells. Translational gene-reporter fusion experiments revealed that Esr41 regulates expression at a post-transcriptional level, and transcription, probably via an unknown target of Esr41. Esr41-mediated and repression was not observed in cells lacking , which encodes an RNA-binding protein essential for most sRNA functions, indicating that Esr41 acts in an Hfq-dependent manner. We previously reported an increase in cell motility induced by Esr41. This motility enhancement was also observed in EHEC lacking , showing that Esr41-mediated enhancement of cell motility is in a independent manner. In addition, Esr41 activated the expression of flagellar Class 3 genes by indirectly inducing the transcription of , which encodes the sigma factor for flagellar synthesis. These results suggest that Esr41 plays important roles in the inverse regulation of LEE and flagellar gene expression.

Keyword(s): EHEC , flagellar , LEE and small RNA
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000652
2018-05-01
2019-12-05
Loading full text...

Full text loading...

/deliver/fulltext/micro/164/5/821.html?itemId=/content/journal/micro/10.1099/mic.0.000652&mimeType=html&fmt=ahah

References

  1. Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev 1998;11:142–201[PubMed]
    [Google Scholar]
  2. Frankel G, Phillips AD, Rosenshine I, Dougan G, Kaper JB et al. Enteropathogenic and enterohaemorrhagic Escherichia coli: more subversive elements. Mol Microbiol 1998;30:911–921 [CrossRef][PubMed]
    [Google Scholar]
  3. Hansen AM, Kaper JB. Hfq affects the expression of the LEE pathogenicity island in enterohaemorrhagic Escherichia coli. Mol Microbiol 2009;73:446–465 [CrossRef][PubMed]
    [Google Scholar]
  4. Lodato PB, Hsieh PK, Belasco JG, Kaper JB. The ribosome binding site of a mini-ORF protects a T3SS mRNA from degradation by RNase E. Mol Microbiol 2012;86:1167–1182 [CrossRef][PubMed]
    [Google Scholar]
  5. Shakhnovich EA, Davis BM, Waldor MK. Hfq negatively regulates type III secretion in EHEC and several other pathogens. Mol Microbiol 2009;74:347–363 [CrossRef][PubMed]
    [Google Scholar]
  6. Mellies JL, Elliott SJ, Sperandio V, Donnenberg MS, Kaper JB. The Per regulon of enteropathogenic Escherichia coli : identification of a regulatory cascade and a novel transcriptional activator, the locus of enterocyte effacement (LEE)-encoded regulator (Ler). Mol Microbiol 1999;33:296–306 [CrossRef][PubMed]
    [Google Scholar]
  7. Bustamante VH, Santana FJ, Calva E, Puente JL. Transcriptional regulation of type III secretion genes in enteropathogenic Escherichia coli: Ler antagonizes H-NS-dependent repression. Mol Microbiol 2001;39:664–678 [CrossRef][PubMed]
    [Google Scholar]
  8. Umanski T, Rosenshine I, Friedberg D. Thermoregulated expression of virulence genes in enteropathogenic Escherichia coli. Microbiology 2002;148:2735–2744 [CrossRef][PubMed]
    [Google Scholar]
  9. Haack KR, Robinson CL, Miller KJ, Fowlkes JW, Mellies JL. Interaction of Ler at the LEE5 (tir) operon of enteropathogenic Escherichia coli. Infect Immun 2003;71:384–392 [CrossRef][PubMed]
    [Google Scholar]
  10. Stoebel DM, Free A, Dorman CJ. Anti-silencing: overcoming H-NS-mediated repression of transcription in Gram-negative enteric bacteria. Microbiology 2008;154:2533–2545 [CrossRef][PubMed]
    [Google Scholar]
  11. Tree JJ, Wolfson EB, Wang D, Roe AJ, Gally DL. Controlling injection: regulation of type III secretion in enterohaemorrhagic Escherichia coli. Trends Microbiol 2009;17:361–370 [CrossRef][PubMed]
    [Google Scholar]
  12. Islam MS, Bingle LE, Pallen MJ, Busby SJ. Organization of the LEE1 operon regulatory region of enterohaemorrhagic Escherichia coli O157:H7 and activation by GrlA. Mol Microbiol 2011;79:468–483 [CrossRef][PubMed]
    [Google Scholar]
  13. Mellies JL, Barron AM, Carmona AM. Enteropathogenic and enterohemorrhagic Escherichia coli virulence gene regulation. Infect Immun 2007;75:4199–4210 [CrossRef][PubMed]
    [Google Scholar]
  14. Iyoda S, Watanabe H. ClpXP protease controls expression of the type III protein secretion system through regulation of RpoS and GrlR levels in enterohemorrhagic Escherichia coli. J Bacteriol 2005;187:4086–4094 [CrossRef][PubMed]
    [Google Scholar]
  15. Honda N, Iyoda S, Yamamoto S, Terajima J, Watanabe H. LrhA positively controls the expression of the locus of enterocyte effacement genes in enterohemorrhagic Escherichia coli by differential regulation of their master regulators PchA and PchB. Mol Microbiol 2009;74:1393–1341 [CrossRef][PubMed]
    [Google Scholar]
  16. Iyoda S, Watanabe H. Positive effects of multiple pch genes on expression of the locus of enterocyte effacement genes and adherence of enterohaemorrhagic Escherichia coli O157:H7 to HEp-2 cells. Microbiology 2004;150:2357–2571 [CrossRef][PubMed]
    [Google Scholar]
  17. Kutsukake K, Ohya Y, Iino T. Transcriptional analysis of the flagellar regulon of Salmonella typhimurium. J Bacteriol 1990;172:741–747 [CrossRef][PubMed]
    [Google Scholar]
  18. Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, Ec Y et al. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 2001;410:1099–1103 [CrossRef][PubMed]
    [Google Scholar]
  19. Zhou X, Girón JA, Torres AG, Crawford JA, Negrete E et al. Flagellin of enteropathogenic Escherichia coli stimulates interleukin-8 production in T84 cells. Infect Immun 2003;71:2120–2129 [CrossRef][PubMed]
    [Google Scholar]
  20. Iyoda S, Koizumi N, Satou H, Lu Y, Saitoh T et al. The GrlR-GrlA regulatory system coordinately controls the expression of flagellar and LEE-encoded type III protein secretion systems in enterohemorrhagic Escherichia coli. J Bacteriol 2006;188:5682–5692 [CrossRef][PubMed]
    [Google Scholar]
  21. Huang HY, Chang HY, Chou CH, Tseng CP, Ho SY et al. sRNAMap: genomic maps for small non-coding RNAs, their regulators and their targets in microbial genomes. Nucleic Acids Res 2009;37:D150–D154 [CrossRef][PubMed]
    [Google Scholar]
  22. Kawano M, Reynolds AA, Miranda-Rios J, Storz G. Detection of 5'- and 3'-UTR-derived small RNAs and cis-encoded antisense RNAs in Escherichia coli. Nucleic Acids Res 2005;33:1040–1050 [CrossRef][PubMed]
    [Google Scholar]
  23. Sittka A, Pfeiffer V, Tedin K, Vogel J. The RNA chaperone Hfq is essential for the virulence of Salmonella typhimurium. Mol Microbiol 2007;63:193–217 [CrossRef][PubMed]
    [Google Scholar]
  24. Zhang A, Wassarman KM, Rosenow C, Tjaden BC, Storz G et al. Global analysis of small RNA and mRNA targets of Hfq. Mol Microbiol 2003;50:1111–1124 [CrossRef][PubMed]
    [Google Scholar]
  25. Gottesman S. The small RNA regulators of Escherichia coli: roles and mechanisms. Annu Rev Microbiol 2004;58:303–328 [CrossRef][PubMed]
    [Google Scholar]
  26. Repoila F, Gottesman S. Temperature sensing by the dsrA promoter. J Bacteriol 2003;185:6609–6614 [CrossRef][PubMed]
    [Google Scholar]
  27. Wassarman KM. Small RNAs in bacteria: diverse regulators of gene expression in response to environmental changes. Cell 2002;109:141–144[PubMed][Crossref]
    [Google Scholar]
  28. Waters LS, Storz G. Regulatory RNAs in bacteria. Cell 2009;136:615–628 [CrossRef][PubMed]
    [Google Scholar]
  29. Vogel J, Luisi BF. Hfq and its constellation of RNA. Nat Rev Microbiol 2011;9:578–589 [CrossRef][PubMed]
    [Google Scholar]
  30. Aiba H. Mechanism of RNA silencing by Hfq-binding small RNAs. Curr Opin Microbiol 2007;10:134–139 [CrossRef][PubMed]
    [Google Scholar]
  31. Tsui HC, Leung HC, Winkler ME. Characterization of broadly pleiotropic phenotypes caused by an hfq insertion mutation in Escherichia coli K-12. Mol Microbiol 1994;13:35–49 [CrossRef][PubMed]
    [Google Scholar]
  32. Bibova I, Skopova K, Masin J, Cerny O, Hot D et al. The RNA chaperone Hfq is required for virulence of Bordetella pertussis. Infect Immun 2013;81:4081–4090 [CrossRef][PubMed]
    [Google Scholar]
  33. Meng X, Meng X, Zhu C, Wang H, Wang J et al. The RNA chaperone Hfq regulates expression of fimbrial-related genes and virulence of Salmonella enterica serovar Enteritidis. FEMS Microbiol Lett 2013;346:90–96 [CrossRef][PubMed]
    [Google Scholar]
  34. Christiansen JK, Larsen MH, Ingmer H, Søgaard-Andersen L, Kallipolitis BH. The RNA-binding protein Hfq of Listeria monocytogenes: role in stress tolerance and virulence. J Bacteriol 2004;186:3355–3362 [CrossRef][PubMed]
    [Google Scholar]
  35. Kulesus RR, Diaz-Perez K, Slechta ES, Eto DS, Mulvey MA. Impact of the RNA chaperone Hfq on the fitness and virulence potential of uropathogenic Escherichia coli. Infect Immun 2008;76:3019–3026 [CrossRef][PubMed]
    [Google Scholar]
  36. Sousa SA, Ramos CG, Moreira LM, Leitão JH. The hfq gene is required for stress resistance and full virulence of Burkholderia cepacia to the nematode Caenorhabditis elegans. Microbiology 2010;156:896–908 [CrossRef][PubMed]
    [Google Scholar]
  37. Chiang MK, Lu MC, Liu LC, Lin CT, Lai YC. Impact of Hfq on global gene expression and virulence in Klebsiella pneumoniae. PLoS One 2011;6:e22248 [CrossRef][PubMed]
    [Google Scholar]
  38. Fantappiè L, Metruccio MM, Seib KL, Oriente F, Cartocci E et al. The RNA chaperone Hfq is involved in stress response and virulence in Neisseria meningitidis and is a pleiotropic regulator of protein expression. Infect Immun 2009;77:1842–1853 [CrossRef][PubMed]
    [Google Scholar]
  39. Meibom KL, Forslund AL, Kuoppa K, Alkhuder K, Dubail I et al. Hfq, a novel pleiotropic regulator of virulence-associated genes in Francisella tularensis. Infect Immun 2009;77:1866–1880 [CrossRef][PubMed]
    [Google Scholar]
  40. Ding Y, Davis BM, Waldor MK. Hfq is essential for Vibrio cholerae virulence and downregulates sigma expression. Mol Microbiol 2004;53:345–354 [CrossRef][PubMed]
    [Google Scholar]
  41. Laaberki MH, Janabi N, Oswald E, Repoila F. Concert of regulators to switch on LEE expression in enterohemorrhagic Escherichia coli O157:H7: interplay between Ler, GrlA, HNS and RpoS. Int J Med Microbiol 2006;296:197–210 [CrossRef][PubMed]
    [Google Scholar]
  42. Gruber CC, Sperandio V. Posttranscriptional control of microbe-induced rearrangement of host cell actin. mBio 2014;5:e01025-13 [CrossRef][PubMed]
    [Google Scholar]
  43. Gruber CC, Sperandio V. Global analysis of posttranscriptional regulation by GlmY and GlmZ in enterohemorrhagic Escherichia coli O157:H7. Infect Immun 2015;83:1286–1295 [CrossRef][PubMed]
    [Google Scholar]
  44. Tobe T, Yen H, Takahashi H, Kagayama Y, Ogasawara N et al. Antisense transcription regulates the expression of the enterohemorrhagic Escherichia coli virulence regulatory gene ler in response to the intracellular iron concentration. PLoS One 2014;9:e101582 [CrossRef][PubMed]
    [Google Scholar]
  45. Sudo N, Soma A, Muto A, Iyoda S, Suh M et al. A novel small regulatory RNA enhances cell motility in enterohemorrhagic Escherichia coli. J Gen Appl Microbiol 2014;60:44–50 [CrossRef][PubMed]
    [Google Scholar]
  46. Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 2000;97:6640–6645 [CrossRef][PubMed]
    [Google Scholar]
  47. Morita T, Maki K, Aiba H. RNase E-based ribonucleoprotein complexes: mechanical basis of mRNA destabilization mediated by bacterial noncoding RNAs. Genes Dev 2005;19:2176–2186 [CrossRef][PubMed]
    [Google Scholar]
  48. Aiba H, Adhya S, de Crombrugghe B. Evidence for two functional gal promoters in intact Escherichia coli cells. J Biol Chem 1981;256:11905–11910[PubMed]
    [Google Scholar]
  49. Iyoda S, Kutsukake K. Molecular dissection of the flagellum-specific anti-sigma factor, FlgM, of Salmonella typhimurium. Mol Gen Genet 1995;249:417–424[PubMed]
    [Google Scholar]
  50. Morita T, Kawamoto H, Mizota T, Inada T, Aiba H. Enolase in the RNA degradosome plays a crucial role in the rapid decay of glucose transporter mRNA in the response to phosphosugar stress in Escherichia coli. Mol Microbiol 2004;54:1063–1075 [CrossRef][PubMed]
    [Google Scholar]
  51. Ishikawa H, Otaka H, Maki K, Morita T, Aiba H. The functional Hfq-binding module of bacterial sRNAs consists of a double or single hairpin preceded by a U-rich sequence and followed by a 3' poly(U) tail. RNA 2012;18:1062–1074 [CrossRef][PubMed]
    [Google Scholar]
  52. Morita T, Ueda M, Kubo K, Aiba H. Insights into transcription termination of Hfq-binding sRNAs of Escherichia coli and characterization of readthrough products. RNA 2015;21:1490–1501 [CrossRef][PubMed]
    [Google Scholar]
  53. Said N, Rieder R, Hurwitz R, Deckert J, Urlaub H et al. In vivo expression and purification of aptamer-tagged small RNA regulators. Nucleic Acids Res 2009;37:e133 [CrossRef][PubMed]
    [Google Scholar]
  54. Tree JJ, Granneman S, McAteer SP, Tollervey D, Gally DL. Identification of bacteriophage-encoded anti-sRNAs in pathogenic Escherichia coli. Mol Cell 2014;55:199–213 [CrossRef][PubMed]
    [Google Scholar]
  55. Sauer E, Weichenrieder O. Structural basis for RNA 3'-end recognition by Hfq. Proc Natl Acad Sci USA 2011;108:13065–13070 [CrossRef][PubMed]
    [Google Scholar]
  56. Otaka H, Ishikawa H, Morita T, Aiba H. PolyU tail of rho-independent terminator of bacterial small RNAs is essential for Hfq action. Proc Natl Acad Sci USA 2011;108:13059–13064 [CrossRef][PubMed]
    [Google Scholar]
  57. Elliott SJ, Sperandio V, Girón JA, Shin S, Mellies JL et al. The locus of enterocyte effacement (LEE)-encoded regulator controls expression of both LEE- and non-LEE-encoded virulence factors in enteropathogenic and enterohemorrhagic Escherichia coli. Infect Immun 2000;68:6115–6126 [CrossRef][PubMed]
    [Google Scholar]
  58. Barba J, Bustamante VH, Flores-Valdez MA, Deng W, Finlay BB et al. A positive regulatory loop controls expression of the locus of enterocyte effacement-encoded regulators Ler and GrlA. J Bacteriol 2005;187:7918–7930 [CrossRef][PubMed]
    [Google Scholar]
  59. Soper TJ, Doxzen K, Woodson SA. Major role for mRNA binding and restructuring in sRNA recruitment by Hfq. RNA 2011;17:1544–1550 [CrossRef][PubMed]
    [Google Scholar]
  60. Metruccio MM, Fantappiè L, Serruto D, Muzzi A, Roncarati D et al. The Hfq-dependent small noncoding RNA NrrF directly mediates Fur-dependent positive regulation of succinate dehydrogenase in Neisseria meningitidis. J Bacteriol 2009;191:1330–1342 [CrossRef][PubMed]
    [Google Scholar]
  61. Soper TJ, Woodson SA. The rpoS mRNA leader recruits Hfq to facilitate annealing with DsrA sRNA. RNA 2008;14:1907–1917 [CrossRef][PubMed]
    [Google Scholar]
  62. Desnoyers G, Massé E. Noncanonical repression of translation initiation through small RNA recruitment of the RNA chaperone Hfq. Genes Dev 2012;26:726–739 [CrossRef][PubMed]
    [Google Scholar]
  63. Rasmussen AA, Eriksen M, Gilany K, Udesen C, Franch T et al. Regulation of ompA mRNA stability: the role of a small regulatory RNA in growth phase-dependent control. Mol Microbiol 2005;58:1421–1429 [CrossRef][PubMed]
    [Google Scholar]
  64. Massé E, Gottesman S. A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli. Proc Natl Acad Sci USA 2002;99:4620–4625 [CrossRef][PubMed]
    [Google Scholar]
  65. Liu X, Matsumura P. The FlhD/FlhC complex, a transcriptional activator of the Escherichia coli flagellar class II operons. J Bacteriol 1994;176:7345–7351 [CrossRef][PubMed]
    [Google Scholar]
  66. Chilcott GS, Hughes KT. Coupling of flagellar gene expression to flagellar assembly in Salmonella enterica serovar typhimurium and Escherichia coli. Microbiol Mol Biol Rev 2000;64:694–708 [CrossRef][PubMed]
    [Google Scholar]
  67. Aldridge P, Hughes KT. Regulation of flagellar assembly. Curr Opin Microbiol 2002;5:160–165 [CrossRef][PubMed]
    [Google Scholar]
  68. Massé E, Escorcia FE, Gottesman S. Coupled degradation of a small regulatory RNA and its mRNA targets in Escherichia coli. Genes Dev 2003;17:2374–2383 [CrossRef][PubMed]
    [Google Scholar]
  69. Vanderpool CK, Gottesman S. Involvement of a novel transcriptional activator and small RNA in post-transcriptional regulation of the glucose phosphoenolpyruvate phosphotransferase system. Mol Microbiol 2004;54:1076–1089 [CrossRef][PubMed]
    [Google Scholar]
  70. Kawamoto H, Morita T, Shimizu A, Inada T, Aiba H. Implication of membrane localization of target mRNA in the action of a small RNA: mechanism of post-transcriptional regulation of glucose transporter in Escherichia coli. Genes Dev 2005;19:328–338 [CrossRef][PubMed]
    [Google Scholar]
  71. Le Derout J, Boni IV, Régnier P, Hajnsdorf E. Hfq affects mRNA levels independently of degradation. BMC Mol Biol 2010;11:17 [CrossRef][PubMed]
    [Google Scholar]
  72. Yamamoto S, Kutsukake K. FliT acts as an anti-FlhD2C2 factor in the transcriptional control of the flagellar regulon in Salmonella enterica serovar typhimurium. J Bacteriol 2006;188:6703–6708 [CrossRef][PubMed]
    [Google Scholar]
  73. Wada T, Morizane T, Abo T, Tominaga A, Inoue-Tanaka K et al. EAL domain protein YdiV acts as an anti-FlhD4C2 factor responsible for nutritional control of the flagellar regulon in Salmonella enterica Serovar Typhimurium. J Bacteriol 2011;193:1600–1611 [CrossRef][PubMed]
    [Google Scholar]
  74. Waters SA, McAteer SP, Kudla G, Pang I, Deshpande NP et al. Small RNA interactome of pathogenic E. coli revealed through crosslinking of RNase E. EMBO J 2017;36: [CrossRef][PubMed]
    [Google Scholar]
  75. Rice JB, Vanderpool CK. The small RNA SgrS controls sugar-phosphate accumulation by regulating multiple PTS genes. Nucleic Acids Res 2011;39:3806–3819 [CrossRef][PubMed]
    [Google Scholar]
  76. Wassarman KM, Repoila F, Rosenow C, Storz G, Gottesman S. Identification of novel small RNAs using comparative genomics and microarrays. Genes Dev 2001;15:1637–1651 [CrossRef][PubMed]
    [Google Scholar]
  77. Akerley BJ, Cotter PA, Miller JF. Ectopic expression of the flagellar regulon alters development of the Bordetella-host interaction. Cell 1995;80:611–620 [CrossRef][PubMed]
    [Google Scholar]
  78. Singer HM, Kühne C, Deditius JA, Hughes KT, Erhardt M. The Salmonella Spi1 virulence regulatory protein HilD directly activates transcription of the flagellar master operon flhDC. J Bacteriol 2014;196:1448–1457 [CrossRef][PubMed]
    [Google Scholar]
  79. Tobe T, Nakanishi N, Sugimoto N. Activation of motility by sensing short-chain fatty acids via two steps in a flagellar gene regulatory cascade in enterohemorrhagic Escherichia coli. Infect Immun 2011;79:1016–1024 [CrossRef][PubMed]
    [Google Scholar]
  80. Wang Q, Zhao Y, McClelland M, Harshey RM. The RcsCDB signaling system and swarming motility in Salmonella enterica serovar typhimurium: dual regulation of flagellar and SPI-2 virulence genes. J Bacteriol 2007;189:8447–8457 [CrossRef][PubMed]
    [Google Scholar]
  81. Miller EW, Cao TN, Pflughoeft KJ, Sumby P. RNA-mediated regulation in Gram-positive pathogens: an overview punctuated with examples from the group a Streptococcus. Mol Microbiol 2014;94:9–20 [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000652
Loading
/content/journal/micro/10.1099/mic.0.000652
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