1887

Abstract

During conditions of nutrient limitation bacteria undergo a series of global gene expression changes to survive conditions of amino acid and fatty acid starvation. Rapid reallocation of cellular resources is brought about by gene expression changes coordinated by the signalling nucleotides' guanosine tetraphosphate or pentaphosphate, collectively termed (p)ppGpp and is known as the stringent response. The stringent response has been implicated in bacterial virulence, with elevated (p)ppGpp levels being associated with increased virulence gene expression. This has been observed in the highly pathogenic sub spp. SCHU S4, the causative agent of tularaemia. Here, we aimed to artificially induce the stringent response by culturing in the presence of the amino acid analogue -serine hydroxamate. Serine hydroxamate competitively inhibits tRNA aminoacylation, causing an accumulation of uncharged tRNA. The uncharged tRNA enters the A site on the translating bacterial ribosome and causes ribosome stalling, in turn stimulating the production of (p)ppGpp and activation of the stringent response. Using the essential virulence gene , which is encoded on the pathogenicity island (FPI) as a marker of active stringent response, we optimized the culture conditions required for the investigation of virulence gene expression under conditions of nutrient limitation. We subsequently used whole genome RNA-seq to show how alters gene expression on a global scale during active stringent response. Key findings included up-regulation of genes involved in virulence, stress responses and metabolism, and down-regulation of genes involved in metabolite transport and cell division. is a highly virulent intracellular pathogen capable of causing debilitating or fatal disease at extremely low infectious doses. However, virulence mechanisms are still poorly understood. The stringent response is widely recognized as a diverse and complex bacterial stress response implicated in virulence. This work describes the global gene expression profile of SCHU S4 under active stringent response for the first time. Herein we provide evidence for an association of active stringent response with FPI virulence gene expression. Our results further the understanding of the molecular basis of virulence and regulation thereof in . These results also support research into genes involved in (p)ppGpp production and polyphosphate biosynthesis and their applicability as targets for novel antimicrobials.

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2017-11-01
2019-12-08
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References

  1. Mörner T. The ecology of tularaemia. Rev Sci Tech 1992;11:1123–1130 [CrossRef][PubMed]
    [Google Scholar]
  2. Oyston PC. Francisella tularensis: unravelling the secrets of an intracellular pathogen. J Med Microbiol 2008;57:921–930 [CrossRef][PubMed]
    [Google Scholar]
  3. Postic G, Dubail I, Frapy E, Dupuis M, Dieppedale J et al. Identification of a novel small RNA modulating Francisella tularensis pathogenicity. PLoS One 2012;7:e41999 [CrossRef][PubMed]
    [Google Scholar]
  4. Olsufiev NG, Emelyanova OS, Dunayeva TN. Comparative study of strains of B. tularense in the old and new world and their taxonomy. J Hyg Epidemiol Microbiol Immunol 1959;3:138[PubMed]
    [Google Scholar]
  5. Dennis DT, Inglesby TV, Henderson DA, Bartlett JG, Ascher MS et al. Tularemia as a biological weapon: medical and public health management. JAMA 2001;285:2763–2773[PubMed][Crossref]
    [Google Scholar]
  6. Kadzhaev K, Zingmark C, Golovliov I, Bolanowski M, Shen H et al. Identification of genes contributing to the virulence of Francisella tularensis SCHU S4 in a mouse intradermal infection model. PLoS One 2009;4:e5463 [CrossRef][PubMed]
    [Google Scholar]
  7. Nano FE, Zhang N, Cowley SC, Klose KE, Cheung KK et al. A Francisella tularensis pathogenicity island required for intramacrophage growth. J Bacteriol 2004;186:6430–6436 [CrossRef][PubMed]
    [Google Scholar]
  8. Nano FE, Schmerk C. The Francisella pathogenicity Island. Ann N Y Acad Sci 2007;1105:122–137 [CrossRef][PubMed]
    [Google Scholar]
  9. Guina T, Radulovic D, Bahrami AJ, Bolton DL, Rohmer L et al. MglA regulates Francisella tularensis subsp. novicida (Francisella novicida) response to starvation and oxidative stress. J Bacteriol 2007;189:6580–6586 [CrossRef][PubMed]
    [Google Scholar]
  10. Lauriano CM, Barker JR, Yoon SS, Nano FE, Arulanandam BP et al. MglA regulates transcription of virulence factors necessary for Francisella tularensis intraamoebae and intramacrophage survival. Proc Natl Acad Sci USA 2004;101:4246–4249 [CrossRef][PubMed]
    [Google Scholar]
  11. Dean RE, Ireland PM, Jordan JE, Titball RW, Oyston PC. RelA regulates virulence and intracellular survival of Francisella novicida. Microbiology 2009;155:4104–4113 [CrossRef][PubMed]
    [Google Scholar]
  12. Larsson P, Oyston PC, Chain P, Chu MC, Duffield M et al. The complete genome sequence of Francisella tularensis, the causative agent of tularemia. Nat Genet 2005;37:153–159 [CrossRef][PubMed]
    [Google Scholar]
  13. Oyston PC, Dorrell N, Williams K, Li SR, Green M et al. The response regulator PhoP is important for survival under conditions of macrophage-induced stress and virulence in Yersinia pestis. Infect Immun 2000;68:3419–3425 [CrossRef][PubMed]
    [Google Scholar]
  14. Cirillo DM, Valdivia RH, Monack DM, Falkow S. Macrophage-dependent induction of the Salmonella pathogenicity Island 2 type III secretion system and its role in intracellular survival. Mol Microbiol 1998;30:175–188 [CrossRef][PubMed]
    [Google Scholar]
  15. Ferullo DJ, Lovett ST. The stringent response and cell cycle arrest in Escherichia coli. PLoS Genet 2008;4:e1000300 [CrossRef][PubMed]
    [Google Scholar]
  16. Dalebroux ZD, Swanson MS. ppGpp: magic beyond RNA polymerase. Nat Rev Microbiol 2012;10:203–212 [CrossRef][PubMed]
    [Google Scholar]
  17. Dalebroux ZD, Svensson SL, Gaynor EC, Swanson MS. ppGpp conjures bacterial virulence. Microbiol Mol Biol Rev 2010;74:171–199 [CrossRef][PubMed]
    [Google Scholar]
  18. Chatterji D, Ojha AK, Kumar Ojha A. Revisiting the stringent response, ppGpp and starvation signaling. Curr Opin Microbiol 2001;4:160–165 [CrossRef][PubMed]
    [Google Scholar]
  19. Kanjee U, Ogata K, Houry WA. Direct binding targets of the stringent response alarmone (p)ppGpp. Mol Microbiol 2012;85:1029–1043 [CrossRef][PubMed]
    [Google Scholar]
  20. Magnusson LU, Farewell A, Nyström T. ppGpp: a global regulator in Escherichia coli. Trends Microbiol 2005;13:236–242 [CrossRef][PubMed]
    [Google Scholar]
  21. Müller CM, Conejero L, Spink N, Wand ME, Bancroft GJ et al. Role of RelA and SpoT in Burkholderia pseudomallei virulence and immunity. Infect Immun 2012;80:3247–3255 [CrossRef][PubMed]
    [Google Scholar]
  22. Candon HL, Allan BJ, Fraley CD, Gaynor EC. Polyphosphate kinase 1 is a pathogenesis determinant in Campylobacter jejuni. J Bacteriol 2007;189:8099–8108 [CrossRef][PubMed]
    [Google Scholar]
  23. Rao NN, Liu S, Kornberg A. Inorganic polyphosphate in Escherichia coli: the phosphate regulon and the stringent response. J Bacteriol 1998;180:2186–2193[PubMed]
    [Google Scholar]
  24. Fraley CD, Rashid MH, Lee SS, Gottschalk R, Harrison J et al. A polyphosphate kinase 1 (ppk1) mutant of Pseudomonas aeruginosa exhibits multiple ultrastructural and functional defects. Proc Natl Acad Sci USA 2007;104:3526–3531 [CrossRef][PubMed]
    [Google Scholar]
  25. Brown MR, Kornberg A. The long and short of it - polyphosphate, PPK and bacterial survival. Trends Biochem Sci 2008;33:284–290 [CrossRef][PubMed]
    [Google Scholar]
  26. Manganelli R. Polyphosphate and stress response in mycobacteria. Mol Microbiol 2007;65:258–260 [CrossRef][PubMed]
    [Google Scholar]
  27. Kuroda A. A polyphosphate-lon protease complex in the adaptation of Escherichia coli to amino acid starvation. Biosci Biotechnol Biochem 2006;70:325–331 [CrossRef][PubMed]
    [Google Scholar]
  28. Kuroda A, Murphy H, Cashel M, Kornberg A. Guanosine tetra- and pentaphosphate promote accumulation of inorganic polyphosphate in Escherichia coli. J Biol Chem 1997;272:21240–21243 [CrossRef][PubMed]
    [Google Scholar]
  29. Rao NN, Gómez-García MR, Kornberg A. Inorganic polyphosphate: essential for growth and survival. Annu Rev Biochem 2009;78:605–647 [CrossRef][PubMed]
    [Google Scholar]
  30. Richards MI, Michell SL, Oyston PC. An intracellularly inducible gene involved in virulence and polyphosphate production in Francisella. J Med Microbiol 2008;57:1183–1192 [CrossRef][PubMed]
    [Google Scholar]
  31. Tosa T, Pizer LI. Biochemical bases for the antimetabolite action of L-serine hydroxamate. J Bacteriol 1971;106:972–982[PubMed]
    [Google Scholar]
  32. Tosa T, Pizer LI. Effect of serine hydroxamate on the growth of Escherichia coli. J Bacteriol 1971;106:966–971[PubMed]
    [Google Scholar]
  33. Agirrezabala X, Fernández IS, Kelley AC, Cartón DG, Ramakrishnan V et al. The ribosome triggers the stringent response by RelA via a highly distorted tRNA. EMBO Rep 2013;14:811–816 [CrossRef][PubMed]
    [Google Scholar]
  34. Faron M, Fletcher JR, Rasmussen JA, Long ME, Allen LA et al. The Francisella tularensis migR, trmE, and cphA genes contribute to F. tularensis pathogenicity Island gene regulation and intracellular growth by modulation of the stress alarmone ppGpp. Infect Immun 2013;81:2800–2811 [CrossRef][PubMed]
    [Google Scholar]
  35. Charity JC, Costante-Hamm MM, Balon EL, Boyd DH, Rubin EJ et al. Twin RNA polymerase-associated proteins control virulence gene expression in Francisella tularensis. PLoS Pathog 2007;3:e84 [CrossRef][PubMed]
    [Google Scholar]
  36. Charity JC, Blalock LT, Costante-Hamm MM, Kasper DL, Dove SL. Small molecule control of virulence gene expression in Francisella tularensis. PLoS Pathog 2009;5:e1000641 [CrossRef][PubMed]
    [Google Scholar]
  37. Wehrly TD, Chong A, Virtaneva K, Sturdevant DE, Child R et al. Intracellular biology and virulence determinants of Francisella tularensis revealed by transcriptional profiling inside macrophages. Cell Microbiol 2009;11:1128–1150 [CrossRef][PubMed]
    [Google Scholar]
  38. Brown MJ, Russo BC, O'Dee DM, Schmitt DM, Nau GJ. The contribution of the glycine cleavage system to the pathogenesis of Francisella tularensis. Microbes Infect 2014;16:300–309 [CrossRef][PubMed]
    [Google Scholar]
  39. Twine SM, Mykytczuk NC, Petit MD, Shen H, Sjöstedt A et al. In vivo proteomic analysis of the intracellular bacterial pathogen, Francisella tularensis, isolated from mouse spleen. Biochem Biophys Res Commun 2006;345:1621–1633 [CrossRef][PubMed]
    [Google Scholar]
  40. Mohapatra NP, Soni S, Bell BL, Warren R, Ernst RK et al. Identification of an orphan response regulator required for the virulence of Francisella spp. and transcription of pathogenicity island genes. Infect Immun 2007;75:3305–3314 [CrossRef][PubMed]
    [Google Scholar]
  41. Argaman L, Elgrably-Weiss M, Hershko T, Vogel J, Altuvia S. RelA protein stimulates the activity of RyhB small RNA by acting on RNA-binding protein Hfq. Proc Natl Acad Sci USA 2012;109:4621–4626 [CrossRef][PubMed]
    [Google Scholar]
  42. Gandhi A, Shah NP. Integrating omics to unravel the stress-response mechanisms in probiotic bacteria: Approaches, challenges, and prospects. Crit Rev Food Sci Nutr 2016;57:3464–3471 [CrossRef][PubMed]
    [Google Scholar]
  43. Feder ME, Walser JC. The biological limitations of transcriptomics in elucidating stress and stress responses. J Evol Biol 2005;18:901–910 [CrossRef][PubMed]
    [Google Scholar]
  44. Sebbane F, Lemaître N, Sturdevant DE, Rebeil R, Virtaneva K et al. Adaptive response of Yersinia pestis to extracellular effectors of innate immunity during bubonic plague. Proc Natl Acad Sci USA 2006;103:11766–11771 [CrossRef][PubMed]
    [Google Scholar]
  45. Anders S, Pyl PT, Huber W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 2015;31:166–169 [CrossRef][PubMed]
    [Google Scholar]
  46. Peng L, Luo WY, Zhao T, Wan CS, Jiang Y et al. Polyphosphate kinase 1 is required for the pathogenesis process of meningitic Escherichia coli K1 (RS218). Future Microbiol 2012;7:411–423 [CrossRef][PubMed]
    [Google Scholar]
  47. Kristensen O, Ross B, Gajhede M. Structure of the PPX/GPPA phosphatase from Aquifex aeolicus in complex with the alarmone ppGpp. J Mol Biol 2008;375:1469–1476 [CrossRef][PubMed]
    [Google Scholar]
  48. Kristensen O, Laurberg M, Liljas A, Kastrup JS, Gajhede M. Structural characterization of the stringent response related exopolyphosphatase/guanosine pentaphosphate phosphohydrolase protein family. Biochemistry 2004;43:8894–8900 [CrossRef][PubMed]
    [Google Scholar]
  49. Malde A, Gangaiah D, Chandrashekhar K, Pina-Mimbela R, Torrelles JB et al. Functional characterization of exopolyphosphatase/guanosine pentaphosphate phosphohydrolase (PPX/GPPA) of Campylobacter jejuni. Virulence 2014;5:521–533 [CrossRef][PubMed]
    [Google Scholar]
  50. Sy J, Lipmann F. Identification of the synthesis of guanosine tetraphosphate (MS I) as insertion of a pyrophosphoryl group into the 3'-position in guanosine 5'-diphosphate. Proc Natl Acad Sci USA 1973;70:306–309 [CrossRef][PubMed]
    [Google Scholar]
  51. Haseltine WA, Block R. Synthesis of guanosine tetra- and pentaphosphate requires the presence of a codon-specific, uncharged transfer ribonucleic acid in the acceptor site of ribosomes. Proc Natl Acad Sci USA 1973;70:1564–1568 [CrossRef][PubMed]
    [Google Scholar]
  52. Haseltine WA, Block R, Gilbert W, Weber K. MSI and MSII made on ribosome in idling step of protein synthesis. Nature 1972;238:381–384 [CrossRef][PubMed]
    [Google Scholar]
  53. Cashel M, Gentry DR, Hernandez VJ, Vinella D. Escherichia coli and Salmonella: cellular and molecular biology.. 1996;11458–1496
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