A Genome-Wide Approach to Discovery of Small RNAs Involved in Regulation of Virulence in

English version České info

Small RNAs (sRNAs) are becoming increasingly recognized as important regulators in bacteria. To investigate the contribution of sRNA mediated regulation to virulence in Vibrio cholerae, we performed high throughput sequencing of cDNA generated from sRNA transcripts isolated from a strain ectopically expressing ToxT, the major transcriptional regulator within the virulence gene regulon. We compared this data set with ToxT binding sites determined by pulldown and deep sequencing to identify sRNA promoters directly controlled by ToxT. Analysis of the resulting transcripts with ToxT binding sites in cis revealed two sRNAs within the Vibrio Pathogenicity Island. When deletions of these sRNAs were made and the resulting strains were competed against the parental strain in the infant mouse model of V. cholerae colonization, one, TarB, displayed a variable colonization phenotype dependent on its physiological state at the time of inoculation. We identified a target of TarB as the mRNA for the secreted colonization factor, TcpF. We verified negative regulation of TcpF expression by TarB and, using point mutations that disrupted interaction between TarB and tpcF mRNA, showed that loss of this negative regulation was primarily responsible for the colonization phenotype observed in the TarB deletion mutant.

Vyšlo v časopise: A Genome-Wide Approach to Discovery of Small RNAs Involved in Regulation of Virulence in. PLoS Pathog 7(7): e32767. doi:10.1371/journal.ppat.1002126
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1002126

Souhrn

Zdroje

1. KochR 1883 An address on cholera and its bacillus. BMJ 2 403 407

2. KaperJBMorrisJGJrLevineMM 1995 Cholera. Clin Microbiol Rev 8 48 86

3. ChildersBMKloseKE 2007 Regulation of virulence in Vibrio cholerae: the ToxR regulon. Future Microbiol 2 335 344

4. HigginsDEDiRitaVJ 1994 Transcriptional control of toxT, a regulatory gene in the ToxR regulon of Vibrio cholerae. Mol Microbiol 14 17 29

5. LowdenMJSkorupskiKPellegriniMChiorazzoMGTaylorRK 2009 Structure of Vibrio cholerae ToxT reveals a mechanism for fatty acid regulation of virulence genes. Proc Natl Acad Sci U S A 107 2860 2865

6. HigginsDENazarenoEDiRitaVJ 1992 The virulence gene activator ToxT from Vibrio cholerae is a member of the AraC family of transcriptional activators. J Bacteriol 174 6974 6980

7. WitheyJHDiRitaVJ 2006 The toxbox: specific DNA sequence requirements for activation of Vibrio cholerae virulence genes by ToxT. Mol Microbiol 59 1779 1789

8. WitheyJHDiRitaVJ 2005 Activation of both acfA and acfD transcription by Vibrio cholerae ToxT requires binding to two centrally located DNA sites in an inverted repeat conformation. Mol Microbiol 56 1062 1077

9. HsiaoAXuXKanBKulkarniRVZhuJ 2009 Direct regulation by the Vibrio cholerae regulator ToxT to modulate colonization and anticolonization pilus expression. Infect Immun 77 1383 1388

10. NielsenATDolganovNARasmussenTOttoGMillerMC 2010 A bistable switch and anatomical site control Vibrio cholerae virulence gene expression in the intestine. PLoS Pathog 6 e1001102

11. LeeSHHavaDLWaldorMKCamilliA 1999 Regulation and temporal expression patterns of Vibrio cholerae virulence genes during infection. Cell 99 625 634

12. SchildSTamayoRNelsonEJQadriFCalderwoodSB 2007 Genes induced late in infection increase fitness of Vibrio cholerae after release into the environment. Cell Host Microbe 2 264 277

13. VogelJ 2009 A rough guide to the non-coding RNA world of Salmonella. Mol Microbiol 71 1 11

14. VogelJPapenfortK 2006 Small non-coding RNAs and the bacterial outer membrane. Curr Opin Microbiol 9 605 611

15. VogelJArgamanLWagnerEGAltuviaS 2004 The small RNA IstR inhibits synthesis of an SOS-induced toxic peptide. Curr Biol 14 2271 2276

16. LiuJMLivnyJLawrenceMSKimballMDWaldorMK 2009 Experimental discovery of sRNAs in Vibrio cholerae by direct cloning, 5S/tRNA depletion and parallel sequencing. Nucleic Acids Res 37 e46

17. HammerBKBasslerBL 2007 Regulatory small RNAs circumvent the conventional quorum sensing pathway in pandemic Vibrio cholerae. Proc Natl Acad Sci U S A 104 11145 11149

18. LenzDHMillerMBZhuJKulkarniRVBasslerBL 2005 CsrA and three redundant small RNAs regulate quorum sensing in Vibrio cholerae. Mol Microbiol 58 1186 1202

19. RasmussenAAJohansenJNielsenJSOvergaardMKallipolitisB 2009 A conserved small RNA promotes silencing of the outer membrane protein YbfM. Mol Microbiol 72 566 77

20. SongTMikaFLindmarkBLiuZSchildS 2008 A new Vibrio choleraesRNA modulates colonization and affects release of outer membrane vesicles. Mol Microbiol 70 100 111

21. DornenburgJEDevitaAMPalumboMJWadeJT 2010 Widespread Antisense Transcription in Escherichia coli. MBio 1 e00024 10

22. SharmaCMHoffmannSDarfeuilleFReignierJFindeissS 2010 The primary transcriptome of the major human pathogen Helicobacter pylori. Nature 464 250 255

23. SittkaALucchiniSPapenfortKSharmaCMRolleK 2008 Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-transcriptional regulator, Hfq. PLoS Genet 4 e1000163

24. DingYDavisBMWaldorMK 2004 Hfq is essential for Vibrio cholerae virulence and downregulates sigma expression. Mol Microbiol 53 345 354

25. RichardALWitheyJHBeyhanSYildizFDiritaVJ 2010 The Vibrio cholerae virulence regulatory cascade controls glucose uptake through activation of TarA, a small regulatory RNA. Mol Microbiol 78 1171 1181

26. MajerczykCDDunmanPMLuongTTLeeCYSadykovMR 2010 Direct targets of CodY in Staphylococcus aureus. J Bacteriol 192 2861 2877

27. WitheyJHDiritaVJ 2006 The toxbox: specific DNA sequence requirements for activation of Vibrio cholerae virulence genes by ToxT. Mol Microbiol 59 1779 1789

28. IwanagaMYamamotoKHigaNIchinoseYNakasoneN 1986 Culture conditions for stimulating cholera toxin production by Vibrio cholerae O1 El Tor. Microbiol Immunol 30 1075 1083

29. KrukonisESYuRRDiritaVJ 2000 The Vibrio cholerae ToxR/TcpP/ToxT virulence cascade: distinct roles for two membrane-localized transcriptional activators on a single promoter. Mol Microbiol 38 67 84

30. AibaH 2007 Mechanism of RNA silencing by Hfq-binding small RNAs. Curr Opin Microbiol 10 134 139

31. VecerekBMollIAfonyushkinTKaberdinVBlasiU 2003 Interaction of the RNA chaperone Hfq with mRNAs: direct and indirect roles of Hfq in iron metabolism of Escherichia coli. Mol Microbiol 50 897 909

32. AndersenJBSternbergCPoulsenLKBjornSPGivskovM 1998 New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl Environ Microbiol 64 2240 2246

33. KovacikovaGLinWSkorupskiK 2010 The LysR-type virulence activator AphB regulates the expression of genes in Vibrio cholerae in response to low pH and anaerobiosis. J Bacteriol 192 4181 4191

34. KovacikovaGSkorupskiK 1999 A Vibrio cholerae LysR homolog, AphB, cooperates with AphA at the tcpPH promoter to activate expression of the ToxR virulence cascade. J Bacteriol 181 4250 4256

35. KovacikovaGSkorupskiK 2001 Overlapping binding sites for the virulence gene regulators AphA, AphB and cAMP-CRP at the Vibrio cholerae tcpPH promoter. Mol Microbiol 41 393 407

36. CrawfordJAKaperJBDiRitaVJ 1998 Analysis of ToxR-dependent transcription activation of ompU, the gene encoding a major envelope protein in Vibrio cholerae. Mol Microbiol 29 235 246

37. SezonovGJoseleau-PetitDD'AriR 2007 Escherichia coli physiology in Luria-Bertani broth. J Bacteriol 189 8746 8749

38. YuRRDiritaVJ 2002 Regulation of gene expression in Vibrio choleraeby ToxT involves both antirepression and RNA polymerase stimulation. Mol Microbiol 43 119 134

39. TjadenB 2008 TargetRNA: a tool for predicting targets of small RNA action in bacteria. Nucleic Acids Res 36 W109 113

40. SperandioVGironJASilveiraWDKaperJB 1995 The OmpU outer membrane protein, a potential adherence factor of Vibrio cholerae. Infect Immun 63 4433 4438

41. LivnyJTeonadiHLivnyMWaldorMK 2008 High-throughput, kingdom-wide prediction and annotation of bacterial non-coding RNAs. PLoS One 3 e3197

42. ChristiansenJKNielsenJSEbersbachTValentin-HansenPSogaard-AndersenL 2006 Identification of small Hfq-binding RNAs in Listeria monocytogenes. RNA 12 1383 1396

43. SittkaALucchiniSPapenfortKSharmaCMRolleK 2008 Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-transcriptional regulator, Hfq. PLoS genetics 4 e1000163

44. SelingerDWCheungKJMeiRJohanssonEMRichmondCS 2000 RNA expression analysis using a 30 base pair resolution Escherichia coli genome array. Nat Biotechnol 18 1262 1268

45. AbuaitaBHWitheyJH 2009 Bicarbonate Induces Vibrio cholerae virulence gene expression by enhancing ToxT activity. Infect Immun 77 4111 4120

46. MedranoAIDiRitaVJCastilloGSanchezJ 1999 Transient transcriptional activation of the Vibrio cholerae El Tor virulence regulator toxT in response to culture conditions. Infect Immun 67 2178 2183

47. IwanagaMYamamotoK 1985 New medium for the production of cholera toxin by Vibrio cholerae O1 biotype El Tor. J Clin Microbiol 22 405 408

48. MarreroKSanchezARodriguez-UlloaAGonzalezLJCastellanos-SerraL 2009 Anaerobic growth promotes synthesis of colonization factors encoded at the Vibrio pathogenicity island in Vibrio cholerae El Tor. Res Microbiol 160 48 56

49. KirnTJTaylorRK 2005 TcpF is a soluble colonization factor and protective antigen secreted by El Tor and classical O1 and O139 Vibrio cholerae serogroups. Infect Immun 73 4461 4470

50. ContagCHContagPRMullinsJISpilmanSDStevensonDK 1995 Photonic detection of bacterial pathogens in living hosts. Mol Microbiol 18 593 603

51. WilesSPickardKMPengKMacDonaldTTFrankelG 2006 In vivo bioluminescence imaging of the murine pathogen Citrobacter rodentium. Infect Immun 74 5391 5396

52. GuzmanLMBelinDCarsonMJBeckwithJ 1995 Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177 4121 4130

53. DonnenbergMSKaperJB 1991 Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector. Infect Immun 59 4310 4317

54. TerpeK 2003 Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 60 523 533

55. MillerVLMekalanosJJ 1988 A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol 170 2575 2583

56. PepkeSWoldBMortazaviA 2009 Computation for ChIP-seq and RNA-seq studies. Nat Methods 6 S22 32

57. CamilliAMekalanosJJ 1995 Use of recombinase gene fusions to identify Vibrio cholerae genes induced during infection. Mol Microbiol 18 671 683