An RNA thermometer dictates production of a secreted bacterial toxin

English version

Autoři: Christian Twittenhoff ^aff001; Ann Kathrin Heroven ^aff002; Sabrina Mühlen ^aff002; Petra Dersch ^aff002; Franz Narberhaus ^aff001
Působiště autorů: Microbial Biology, Ruhr University Bochum, Bochum, Germany ^aff001; Department of Molecular Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany ^aff002; Institute of Infectiology, Center for Molecular Biology of Inflammation, University of Münster, Münster, Germany ^aff003
Vyšlo v časopise: An RNA thermometer dictates production of a secreted bacterial toxin. PLoS Pathog 16(1): e32767. doi:10.1371/journal.ppat.1008184
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.ppat.1008184

Souhrn

Frequent transitions of bacterial pathogens between their warm-blooded host and external reservoirs are accompanied by abrupt temperature shifts. A temperature of 37°C serves as reliable signal for ingestion by a mammalian host, which induces a major reprogramming of bacterial gene expression and metabolism. Enteric Yersiniae are Gram-negative pathogens accountable for self-limiting gastrointestinal infections. Among the temperature-regulated virulence genes of Yersinia pseudotuberculosis is cnfY coding for the cytotoxic necrotizing factor (CNF_Y), a multifunctional secreted toxin that modulates the host’s innate immune system and contributes to the decision between acute infection and persistence. We report that the major determinant of temperature-regulated cnfY expression is a thermo-labile RNA structure in the 5’-untranslated region (5’-UTR). Various translational gene fusions demonstrated that this region faithfully regulates translation initiation regardless of the transcription start site, promoter or reporter strain. RNA structure probing revealed a labile stem-loop structure, in which the ribosome binding site is partially occluded at 25°C but liberated at 37°C. Consistent with translational control in bacteria, toeprinting (primer extension inhibition) experiments in vitro showed increased ribosome binding at elevated temperature. Point mutations locking the 5’-UTR in its 25°C structure impaired opening of the stem loop, ribosome access and translation initiation at 37°C. To assess the in vivo relevance of temperature control, we used a mouse infection model. Y. pseudotuberculosis strains carrying stabilized RNA thermometer variants upstream of cnfY were avirulent and attenuated in their ability to disseminate into mesenteric lymph nodes and spleen. We conclude with a model, in which the RNA thermometer acts as translational roadblock in a two-layered regulatory cascade that tightly controls provision of the CNF_Y toxin during acute infection. Similar RNA structures upstream of various cnfY homologs suggest that RNA thermosensors dictate the production of secreted toxins in a wide range of pathogens.

Klíčová slova:

Bacterial pathogens – Body temperature – Point mutation – Ribonucleases – RNA structure – Thermometers – Toxins – Yersinia pseudotuberculosis

Zdroje

1. Steinmann R, Dersch P. Thermosensing to adjust bacterial virulence in a fluctuating environment. Front Microbiol. 2013; 8(1):85–105. doi: 10.2217/fmb.12.129 23252495

2. Lam O, Wheeler J, Tang CM. Thermal control of virulence factors in bacteria: A hot topic. Virulence. 2014; 5(8):852–62. doi: 10.4161/21505594.2014.970949 25494856

3. Klinkert B, Narberhaus F. Microbial thermosensors. Cell Mol Life Sci. 2009; 66(16):2661–76. doi: 10.1007/s00018-009-0041-3 19554260

4. Saita EA, De Mendoza D. Thermosensing via transmembrane protein-lipid interactions. Biochim Biophys Acta. 2015; 1848(9):1757–64. doi: 10.1016/j.bbamem.2015.04.005 25906947

5. Kortmann J, Narberhaus F. Bacterial RNA thermometers: molecular zippers and switches. Nat Rev Microbiol. 2012; 10(4):255–65. doi: 10.1038/nrmicro2730 22421878

6. Krajewski SS, Narberhaus F. Temperature-driven differential gene expression by RNA thermosensors. Biochim Biophys Acta. 2014; 1839(10):978–88. doi: 10.1016/j.bbagrm.2014.03.006 24657524

7. Grosso-Becera MV, Servin-González L, Soberón-Chávez G. RNA structures are involved in the thermoregulation of bacterial virulence-associated traits. Trends Microbiol. 2015; 23(8):509–18. doi: 10.1016/j.tim.2015.04.004 25999019

8. Loh E, Righetti F, Eichner H, Twittenhoff C, Narberhaus F. RNA thermometers in bacterial pathogens. Microbiol Spectr. 2018; 6(2):RWR-0012-2017. doi: 10.1128/microbiolspec.RWR-0012-2017 29623874

9. Pechous RD, Sivaraman V, Stasulli NM, Goldman WE. Pneumonic plague: the darker side of Yersinia pestis. Trends Microbiol. 2016; 24(3):190–7. doi: 10.1016/j.tim.2015.11.008 26698952

10. Fredriksson-Ahomaa M. Yersinia enterocolitica and Yersinia pseudotuberculosis. In: Foodborne Diseases. 2007. p. 79–112 doi: 10.1128/9781555815936.ch11

11. Atkinson S, Williams P. Yersinia virulence factors—a sophisticated arsenal for combating host defences. F1000Res. 2016; 5:1370. doi: 10.12688/f1000research.8466.1 27347390

12. Straley SC, Plano G V., Skrzypek E, Haddix PL, Fields KA. Regulation by Ca2+ in the Yersinia low-Ca2+ response. Mol Microbiol. 1993; 8(6):1005–10. doi: 10.1111/j.1365-2958.1993.tb01644.x 8361348

13. Straley SC. The low-Ca2+ response virulence regulon of human-pathogenic yersiniae. Microb Pathog. 1991; 10(2):87–91. doi: 10.1016/0882-4010(91)90069-m 1890954

14. Straley SC, Perry RD. Environmental modulation of gene expression and pathogenesis in Yersinia. Trends Microbiol. 1995; 3(8):310–7. doi: 10.1016/s0966-842x(00)88960-x 8528615

15. Böhme K, Steinmann R, Kortmann J, Seekircher S, Heroven AK, Berger E, Pisano F, Thiermann T, Wolf-Watz H, Narberhaus F, Dersch P. Concerted actions of a thermo-labile regulator and a unique intergenic RNA thermosensor control Yersinia virulence. PLoS Pathog. 2012; 8(2):e1002518. doi: 10.1371/journal.ppat.1002518 22359501

16. Hoe NP, Goguen JD. Temperature sensing in Yersinia pestis: translation of the LcrF activator protein is thermally regulated. J Bacteriol. 1993; 175(24):7901–9. doi: 10.1128/jb.175.24.7901-7909.1993 7504666

17. Kertesz M, Wan Y, Mazor E, Rinn JL, Nutter RC, Chang HY, Segal E. Genome-wide measurement of RNA secondary structure in yeast. Nature. 2010; 467(7311):103–7. doi: 10.1038/nature09322 20811459

18. Righetti F, Nuss AM, Twittenhoff C, Beele S, Urban K, Will S, Bernhart SH, Stadler PF, Dersch P, Narberhaus F. Temperature-responsive in vitro RNA structurome of Yersinia pseudotuberculosis. Proc Natl Acad Sci USA. 2016; 113(26):7237–42. doi: 10.1073/pnas.1523004113 27298343

19. Monnappa AK, Bari W, Seo JK, Mitchell RJ. The cytotoxic necrotizing factor of Yersinia pseudotuberculosis (CNFY) is carried on extracellular membrane vesicles to host cells. Sci Rep. 2018; 8(1):14186. doi: 10.1038/s41598-018-32530-y 30242257

20. Schweer J, Kulkarni D, Kochut A, Pezoldt J, Pisano F, Pils MC, Genth H, Huehn J, Dersch P. The Cytotoxic necrotizing factor of Yersinia pseudotuberculosis (CNFY) enhances inflammation and Yop delivery during infection by activation of Rho GTPases. PLoS Pathog. 2013; 9(11):e1003746. doi: 10.1371/journal.ppat.1003746 24244167

21. Wolters M, Boyle EC, Lardong K, Trülzsch K, Steffen A, Rottner K, Ruckdeschel K, Aepfelbacher M. Cytotoxic necrotizing factor-Y boosts Yersinia effector translocation by activating Rac protein. J Biol Chem. 2013; 288(32):23543–53. doi: 10.1074/jbc.M112.448662 23803609

22. Hoffmann C, Pop M, Leemhuis J, Schirmer J, Aktories K, Schmidt G. The Yersinia pseudotuberculosis cytotoxic necrotizing factor (CNFY) selectively activates RhoA. J Biol Chem. 2004; 279(16):16026–32. doi: 10.1074/jbc.M313556200 14761941

23. Lockman HA, Gillespie RA, Baker BD, Shakhnovich E. Yersinia pseudotuberculosis produces a cytotoxic necrotizing factor. Infect Immun. 2002; 70(5):2708–14. doi: 10.1128/IAI.70.5.2708-2714.2002 11953417

24. Knust Z, Schmidt G. Cytotoxic necrotizing factors (CNFs)-a growing toxin family. Toxins (Basel). 2010; 2(1):116–27. doi: 10.3390/toxins2010116 22069550

25. Derbise A, Lesic B, Dacheux D, Ghigo JM, Carniel E. A rapid and simple method for inactivating chromosomal genes in Yersinia. FEMS Immun Med Microbiol. 2003; 38(2):113–6. doi: 10.1016/S0928-8244(03)00181-0 13129645

26. Sambrook J, W Russell D. Molecular cloning: a laboratory manual. Cold Spring Harb Lab Press Cold Spring Harb NY. 2001; doi: 10.1016/0092-8674(90)90210-6 1205

27. Gaubig LC, Waldminghaus T, Narberhaus F. Multiple layers of control govern expression of the Escherichia coli ibpAB heat-shock operon. Microbiology. 2011; 157(Pt 1):66–76. doi: 10.1099/mic.0.043802-0 20864473

28. Klinkert B, Cimdins A, Gaubig LC, Roßmanith J, Aschke-Sonnenborn U, Narberhaus F. Thermogenetic tools to monitor temperature-dependent gene expression in bacteria. J Biotechnol. 2012; 160(1–2):55–63. doi: 10.1016/j.jbiotec.2012.01.007 22285954

29. Nuss AM, Heroven AK, Waldmann B, Reinkensmeier J, Jarek M, Beckstette M, Dersch P. Transcriptomic profiling of Yersinia pseudotuberculosis reveals reprogramming of the Crp regulon by temperature and uncovers Crp as a master regulator of small RNAs. PLoS Genet. 2015; 11(3):e1005087. doi: 10.1371/journal.pgen.1005087 25816203

30. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods. 2001; 25(4):402–8. doi: 10.1006/meth.2001.1262 11846609

31. Brantl S, Wagner EG. Antisense RNA-mediated transcriptional attenuation occurs faster than stable antisense/target RNA pairing: an in vitro study of plasmid pIP501. EMBO J. 1994; 13(15):3599–607. doi: 10.1046/j.1365-2958.2000.01813.x 7520390

32. Waldminghaus T, Heidrich N, Brantl S, Narberhaus F. FourU: a novel type of RNA thermometer in Salmonella. Mol Microbiol. 2007; 65(2):413–24. doi: 10.1111/j.1365-2958.2007.05794.x 17630972

33. Krajewski SS, Nagel M, Narberhaus F. Short ROSE-like RNA thermometers control IbpA synthesis in Pseudomonas species. PLoS One. 2013; 8(5):e65168. doi: 10.1371/journal.pone.0065168 23741480

34. Hartz D, McPheeters DS, Traut R, Gold L. Extension inhibition analysis of translation initiation complexes. Methods Enzym. 1988; 164:419–25. doi: 10.1016/S0076-6879(88)64058-4 2468068

35. Roßmanith J, Weskamp M, Narberhaus F. Design of a temperature-responsive transcription terminator. ACS Synth Biol. 2018; 7(2):613–21. doi: 10.1021/acssynbio.7b00356 29191010

36. Deana A, Belasco JG. Lost in translation: The influence of ribosomes on bacterial mRNA decay. Genes Dev. 2005; 19(21):2526–33. doi: 10.1101/gad.1348805 16264189

37. Uliczka F, Pisano F, Kochut A, Opitz W, Herbst K, Stolz T, Dersch P. Monitoring of gene expression in bacteria during infections using an adaptable set of bioluminescent, fluorescent and colorigenic fusion vectors. PLoS One. 2011; 6(6):e20425. doi: 10.1371/journal.pone.0020425 21673990

38. Cimdins A, Roßmanith J, Langklotz S, Bandow JE, Narberhaus F. Differential control of Salmonella heat shock operons by structured mRNAs. Mol Microbiol. 2013; 89(4):715–31. doi: 10.1111/mmi.12308 23802546

39. Johansson J, Mandin P, Renzoni A, Chiaruttini C, Springer M, Cossart P. An RNA thermosensor controls expression of virulence genes in Listeria monocytogenes. Cell. 2002; 110(5):551–61. doi: 10.1016/s0092-8674(02)00905-4 12230973

40. Weber GG, Kortmann J, Narberhaus F, Klose KE. RNA thermometer controls temperature-dependent virulence factor expression in Vibrio cholerae. Proc Natl Acad Sci USA. 2014; 111(39):14241–6. doi: 10.1073/pnas.1411570111 25228776

41. Loh E, Kugelberg E, Tracy A, Zhang Q, Gollan B, Ewles H, Chalmers R, Pelicic V, Tang CM. Temperature triggers immune evasion by Neisseria meningitidis. Nature. 2013; 502(7470):237–40. doi: 10.1038/nature12616 24067614

42. Kouse AB, Righetti F, Kortmann J, Narberhaus F, Murphy ER. RNA-Mediated thermoregulation of iron-acquisition genes in Shigella dysenteriae and pathogenic Escherichia coli. PLoS One. 2013; 8(5) doi: 10.1371/journal.pone.0063781 23704938

43. Wei Y, Kouse AB, Murphy ER. Transcriptional and posttranscriptional regulation of Shigella shuT in response to host-associated iron availability and temperature. Microbiologyopen. 2017; 6(3):e00442. doi: 10.1002/mbo3.442 28127899

44. Schmidt G, Sehr P, Wilm M, Mann M, Aktories K. Gln 63 of Rho is deamidated by Escherichia coli cytotoxic necrotizing factor-1. Nature. 1997; 387(June):725–9. doi: 10.1038/42735 9192900

45. Fukui A, Horiguchi Y. Bordetella dermonecrotic toxin exerting toxicity through activation of the small GTPase Rho. J Biochem. 2004; 136(4):415–9. doi: 10.1093/jb/mvh155 15625308

46. Zhang L, Krachler AM, Broberg CA, Li Y, Mirzael H, Gilpin CJ, Orth K. Type III effector VopC mediates invasion for Vibrio species. Cell Rep. 2012; 1(5):453–60. doi: 10.1016/j.celrep.2012.04.004 22787576

47. Nuss AM, Beckstette M, Pimenova M, Schmühl C, Opitz W, Pisano F, Heroven AK, Dersch P. Tissue dual RNA-seq allows fast discovery of infection-specific functions and riboregulators shaping host–pathogen transcriptomes. Proc Natl Acad Sci USA. 2017; 114(5):E791–800. doi: 10.1073/pnas.1613405114 28096329

48. Kolb A, Busby S, Buc H, Garges S, S A. Transcriptional regulation by cAMP and its receptor protein. Annu Rev Biochem. 1993; 62:749–95. doi: 10.1146/annurev.bi.62.070193.003533 8394684

49. Deutscher J. The mechanisms of carbon catabolite repression in bacteria. Curr Opin Microbiol. 2008; 11(2):87–93. doi: 10.1016/j.mib.2008.02.007 18359269

50. Lathem WW, Schroeder JA, Bellows LE, Ritzert JT, Koo JT, Price PA, Caulfield AJ, Goldman WE. Posttranscriptional regulation of the Yersinia pestis cyclic AMP receptor protein Crp and impact on virulence. MBio. 2014; 5(1):1–12. doi: 10.1128/mbio.01038-13 24520064

51. Heroven AK, Sest M, Pisano F, Scheb-Wetzel M, Steinmann R, Böhme K, Klein J, Münch R, Schomburg D, Dersch P. Crp induces switching of the CsrB and CsrC RNAs in Yersinia pseudotuberculosis and links nutritional status to virulence. Front Cell Infect Microbiol. 2012; 2(158):1–21. doi: 10.3389/fcimb.2012.00158 23251905

52. Zhan L, Han Y, Yang L, Geng J, Li Y, Gao H, Guo Z, Fan W, Li G, Zhang L, Qin C, Zhou D, Yang R. The cyclic AMP receptor protein, CRP, is required for both virulence and expression of the minimal CRP regulon in Yersinia pestis biovar microtus. Infect Immun. 2008; 76(11):5028–37. doi: 10.1128/IAI.00370-08 18710863

53. Donovan GT, Paul Norton J, Bower JM, Mulvey MA. Adenylate cyclase and the cyclic AMP receptor protein modulate stress resistance and virulence capacity of uropathogenic Escherichia coli. Infect Immun. 2013; 81(1):249–58. doi: 10.1128/IAI.00796-12 23115037

54. Xue J, Tan B, Yang S, Luo M, Xia H, Zhang X, Zhou X, Yang X, Yang R, Li Y, Qiu J. Influence of cAMP receptor protein (CRP) on bacterial virulence and transcriptional regulation of allS by CRP in Klebsiella pneumoniae. Gene. 2016; 593(1):28–33. doi: 10.1016/j.gene.2016.08.006 27502416

55. Ou Q, Fan J, Duan D, Xu L, Wang J, Zhou D, Yang H, Li B. Involvement of cAMP receptor protein in biofilm formation, fimbria production, capsular polysaccharide biosynthesis and lethality in mouse of Klebsiella pneumoniae serotype K1 causing pyogenic liver abscess. J Med Microbiol. 2017; 66(1):1–7. doi: 10.1099/jmm.0.000391 27902401

56. Tsai YL, Chien HF, Huang KT, Lin WY, Liaw SJ. CAMP receptor protein regulates mouse colonization, motility, fimbria-mediated adhesion, and stress tolerance in uropathogenic Proteus mirabilis. Sci Rep. 2017; 7(1):1–14. doi: 10.1038/s41598-016-0028-x 28779108

57. El Mouali Y, Gaviria-Cantin T, Sánchez-Romero MA, Gibert M, Westermann AJ, Vogel J, Balsalobre C. CRP-cAMP mediates silencing of Salmonella virulence at the post-transcriptional level. PLoS Genet. 2018; 14(6):1–26. doi: 10.1371/journal.pgen.1007401 29879120

58. Kusmierek M, Hoßmann J, Witte R, Opitz W, Vollmer I, Volk M, Heroven AK, Wolf-Watz H, Dersch P. A bacterial secreted translocator hijacks riboregulators to control type III secretion in response to host cell contact. PLoS Pathog. 2019; 15(6):e1007813. doi: 10.1371/journal.ppat.1007813 31173606

59. Waldminghaus T, Fippinger A, Alfsmann J, Narberhaus F. RNA thermometers are common in α- and γ-proteobacteria. Biol Chem. 2005; 386(12):1279–86. doi: 10.1515/BC.2005.145 16336122

60. Krajewski SS, Joswig M, Nagel M, Narberhaus F. A tricistronic heat shock operon is important for stress tolerance of Pseudomonas putida and conserved in many environmental bacteria. Env Microbiol. 2014; 16(6):1835–53. doi: 10.1111/1462-2920.12432 24612349

61. Avican K, Fahlgren A, Huss M, Heroven AK, Beckstette M, Dersch P, Fällman M. Reprogramming of Yersinia from virulent to persistent mode revealed by complex in vivo RNA-seq analysis. PLoS Pathog. 2015; 11(1):1–28. doi: 10.1371/journal.ppat.1004600 25590628

62. Loh E, Lavender H, Tan F, Tracy A, Tang CM. Thermoregulation of meningococcal fHbp, an important virulence factor and vaccine antigen, is mediated by anti-ribosomal binding site sequences in the open reading frame. PLoS Pathog. 2016; 12(8):1–21. doi: 10.1371/journal.ppat.1005794 27560142

63. Grosso-Becerra M V., Croda-Garcia G, Merino E, Servin-Gonzalez L, Mojica-Espinosa R, Soberon-Chavez G. Regulation of Pseudomonas aeruginosa virulence factors by two novel RNA thermometers. Proc Natl Acad Sci USA. 2014; 111(43):15562–7. doi: 10.1073/pnas.1402536111 25313031

64. Matsunaga J, Schlax PJ, Haake DA. Role for cis-Acting RNA sequences in the temperature-dependent expression of the multiadhesive Lig proteins in Leptospira interrogans. J Bacteriol. 2013; 195(22):5092–101. doi: 10.1128/JB.00663-13 24013626

65. Li G-W, Burkhardt D, Gross C, Weissman JS. Quantifying absolute protein synthesis rates reveals principles underlying allocation of cellular resources. Cell. 2014; 157(3):624–35. doi: 10.1016/j.cell.2014.02.033 24766808

66. Heine W, Beckstette M, Heroven AK, Thiemann S, Heise U, Nuss AM, Pisano F, Strowig T, Dersch P. Loss of CNFY toxin-induced inflammation drives Yersinia pseudotuberculosis into persistency. PLoS Pathog. 2018; 14(2):1–32. doi: 10.1371/journal.ppat.1006858 29390040

67. Blumenthal B, Hoffmann C, Aktories K, Backert S, Schmidt G. The cytotoxic necrotizing factors from Yersinia pseudotuberculosis and from Escherichia coli bind to different cellular receptors but take the same route to the cytosol. Infect Immun. 2007; 75(7):3344–53. doi: 10.1128/IAI.01937-06 17438028

68. Kouokam JC, Wai SN, Fällman M, Hacker J, Uhlin BE, Fa M, Dobrindt U. Active cytotoxic necrotizing factor 1 associated with outer membrane vesicles from uropathogenic Escherichia coli. Infect Immun. 2006; 74(4):2022–30. doi: 10.1128/IAI.74.4.2022-2030.2006 16552031

69. Davis JM, Carvalho HM, Rasmussen SB, O’Brien AD. Cytotoxic necrotizing factor type 1 delivered by outer membrane vesicles of uropathogenic Escherichia coli attenuates polymorphonuclear leukocyte antimicrobial activity and chemotaxis. Infect Immun. 2006; 74(8):4401–8. doi: 10.1128/IAI.00637-06 16861625

70. Ignatov D, Johansson J. RNA-mediated signal perception in pathogenic bacteria. Wiley Interdiscip Rev RNA. 2017; 8(6):e1429. doi: 10.1002/wrna.1429 28792118

71. Rinnenthal J, Klinkert B, Narberhaus F, Schwalbe H. Modulation of the stability of the Salmonella fourU-type RNA thermometer. Nucleic Acids Res. 2011; 39(18):8258–70. doi: 10.1093/nar/gkr314 21727085

72. Barros SA, Yoon I, Chenoweth DM. Modulation of the E. coli rpoH temperature sensor with triptycene-based small molecules. Angew Chem Int Ed. 2016; 55(29):8258–61. doi: 10.1002/anie.201601626 27240201