1887

Microbiology Society logo

Volume 155, Issue 12

RelA regulates virulence and intracellular survival of Free

Abstract

Analysis of the genome of has revealed few regulatory systems, and how the organism adapts to conditions in different niches is poorly understood. The stringent response is a global stress response mediated by (p)ppGpp. The enzyme RelA has been shown to be involved in generation of this signal molecule in a range of bacterial species. We investigated the effect of inactivation of the gene in by generating a mutant in . Under amino acid starvation conditions, the mutant was defective for (p)ppGpp production. Characterization showed the mutant to grow similarly to the wild-type, except that it entered stationary phase later than wild-type cultures, resulting in higher cell yields. The mutant showed increased biofilm formation, which may be linked to the delay in entering stationary phase, which in turn would result in higher cell numbers present in the biofilm and reduced resistance to stress. The mutant was attenuated in the J774A macrophage cell line and was shown to be attenuated in the mouse model of tularaemia, but was able to induce a protective immune response. Therefore, (p)ppGpp appears to be an important intracellular signal, integral to the pathogenesis of .

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): CV, crystal violet , FPI, Francisella pathogenicity island , MLD, median lethal dose and SPI, Salmonella pathogenicity island
Crown copyright Dstl
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.031021-0
2009-12-01
2024-04-20
Loading full text...

Full text loading...

/deliver/fulltext/micro/155/12/4104.html?itemId=/content/journal/micro/10.1099/mic.0.031021-0&mimeType=html&fmt=ahah

References

  1. Abranches J., Martinez A. R., Kajfasz J. K., Chavez V., Garsin D. A., Lemos J. A. 2009; The molecular alarmone (p)ppGpp mediates stress responses, vancomycin tolerance and virulence in Enterococcus faecalis . J Bacteriol 191:2248–2256
     [Google Scholar]
  2. Balzer G. J., Mclean R. J. C. 2002; The stringent response genes relA and spoT are important for Escherichia coli biofilms under slow-growth conditions. Can J Microbiol 48:675–680
     [Google Scholar]
  3. Baron G. S., Nano F. E. 1998; MgIA and MgIB are required for the intramacrophage growth of Francisella novicida . Mol Microbiol 29:247–259
     [Google Scholar]
  4. Battesti A., Bouveret E. 2009; Bacteria possessing two RelA/SpoT-like proteins have evolved a specific stringent response involving the acyl carrier protein–SpoT interaction. J Bacteriol 191:616–624
     [Google Scholar]
  5. Braeken K., Moris M., Daniels R., Vanderleyden J., Michiels J. 2006; New horizons for (p)ppGpp in bacterial and plant physiology. Trends Microbiol 14:45–54
     [Google Scholar]
  6. Bugrysheva J., Dobrikova E. Y., Sartakova M. L., Caimano M. J., Daniels T. J., Radolf J. D., Godfrey H. P., Cabello F. C. 2003a; Characterization of the stringent response and relBbu expression in Borrelia burgdorferi . J Bacteriol 185:957–965
     [Google Scholar]
  7. Bugrysheva J., Dobrikova E. Y., Sartakova M. L., Caimano M. J., Daniels T. J., Radolf J. D., Godfrey H. P., Cabello F. C. 2003b; Characterization of the stringent response and relBbu expression in Borrelia burgdorferi . J Bacteriol 185:957–965
     [Google Scholar]
  8. Cashel M. 1994; Detection of (p)ppGpp accumulation patterns in Escherichia coli mutants. In Methods in Molecular Genetics,vol. 3, Molecular Microbiology. Techniques, part A pp 341–356 Edited by Adolph. New York: Academic Press;
     [Google Scholar]
  9. Chamberlain R. E. 1965; Evaluation of live tularemia vaccine prepared in a chemically defined medium. Appl Microbiol 13:232–235
     [Google Scholar]
  10. Charity J. C., Costante-Hamm M. M., Balon E. L., Boyd D. H., Rubin E. J., Dove S. L. 2007; Twin RNA polymerase-associated proteins control virulence gene expression in Francisella tularensis . PLoS Pathog 3:e84
     [Google Scholar]
  11. Dalebroux Z. D., Edwards R. L., Swanson M. S. 2009; SpoT governs Legionella pneumophila differentiation in host macrophages. Mol Microbiol 71:640–658
     [Google Scholar]
  12. Davis B. D., Dulbecco R., Eisen H. N., Ginsberg H. S. 1980 Microbiology Hagerstown, MD: Harper and Row;
     [Google Scholar]
  13. Ellis J., Oyston P. C. F., Green M., Titball R. W. 2002; Tularemia. Clin Microbiol Rev 15:631–646
     [Google Scholar]
  14. Erickson D. L., Lines J. L., Pesci E. C., Venturi V., Storey D. G. 2004; Pseudomonas aeruginosa relA contributes to virulence in Drosophila melanogaster . Infect Immun 72:5638–5645
     [Google Scholar]
  15. Fisher S. D., Reger A. D., Baum A., Hill S. A. 2005; RelA alone appears essential for (p)ppGpp production when Neisseria gonorrhoeae encounters nutritional stress. FEMS Microbiol Lett 248:1–8
     [Google Scholar]
  16. Gaynor E. C., Wells D. H., MacKichan J. K., Falkow S. 2005; The Campylobacter jejuni stringent response controls specific stress survival and virulence-associated phenotypes. Mol Microbiol 56:8–27
     [Google Scholar]
  17. Gentry D. R., Hernandez V. J., Nguyen L. H., Jensen D. B., Cashel M. 1993; Synthesis of the stationary-phase sigma-factor σ s is positively regulated by ppGpp. J Bacteriol 175:7982–7989
     [Google Scholar]
  18. Golovliov I., Sjostedt A., Mokrievich A., Pavlov V. 2003; A method for allelic replacement in Francisella tularensis . FEMS Microbiol Lett 222:273–280
     [Google Scholar]
  19. Gray C. G., Cowley S. C., Cheung K. K. M., Nano F. E. 2002; The identification of five genetic loci of Francisella novicida associated with intracellular growth. FEMS Microbiol Lett 215:53–56
     [Google Scholar]
  20. Guina T., Radulovic D., Bahrami A. J., Bolton D. L., Rohmer L., Jones-Isaac K. A., Chen J., Gallagher L. A., Gallis B. other authors 2007; MglA regulates Francisella tularensis subsp novicida ( Francisella novicida) response to starvation and oxidative stress. J Bacteriol 189:6580–6586
     [Google Scholar]
  21. Hammer B. K., Swanson M. S. 1999; Co-ordination of Legionella pneumophila virulence with entry into stationary phase by ppGpp. Mol Microbiol 33:721–731
     [Google Scholar]
  22. Haralalka S., Nandi S., Bhadra R. K. 2003; Mutation in the relA gene of Vibrio cholerae affects in vitro and in vivo expression of virulence factors. J Bacteriol 185:4672–4682
     [Google Scholar]
  23. Ingraham J. L., Maaloe O., Neidhardt F. C. 1983 Growth of the Bacterial Cell Sunderland, MA: Sinauer Associates;
     [Google Scholar]
  24. Jain V., Kumar M., Chatterji D. 2006; ppGpp: stringent response and survival. J Microbiol 44:1–10
     [Google Scholar]
  25. Jishage M., Kvint K., Shingler V., Nystrom T. 2002; Regulation of σ factor competition by the alarmone ppGpp. Genes Dev 16:1260–1270
     [Google Scholar]
  26. Kazmierczak M. J., Wiedmann M., Boor K. J. 2005; Alternative sigma factors and their roles in bacterial virulence. Microbiol Mol Biol Rev 69:527
     [Google Scholar]
  27. Kim S., Watanabe K., Suzuki H., Watarai M. 2005; Roles of Brucella abortus SpoT in morphological differentiation and intramacrophagic replication. Microbiology 151:1607–1617
     [Google Scholar]
  28. Larsson P., Oyston P. C. F., Chain P., Chu M., Duffield M. L., Gabbert N. M., Fuxelius H. H., Garcia E., Halltorp G. other authors 2005; The complete genome sequence of Francisella tularensis, the causative agent of tularemia. Nat Genet 37:153–159
     [Google Scholar]
  29. Lauriano C. M., Barker J. R., Yoon S. S., Nano F. E., Arulanandam B. P., Hassettt D. J., Klose K. E. 2004; MglA regulates transcription of virulence factors necessary for Francisella tularensis intraamoebae and intramacrophage survival. Proc Natl Acad Sci U S A 101:4246–4249
     [Google Scholar]
  30. Lemos J. A. C., Brown T. A., Burne R. A. 2004; Effects of RelA on key virulence properties of planktonic and biofilm populations of Streptococcus mutans . Infect Immun 72:1431–1440
     [Google Scholar]
  31. Li W., Liu L., Chen H., Zhou R. 2009; Identification of Streptococcus suis genes preferentially expressed under iron starvation by selective capture of transcribed sequences. FEMS Microbiol Lett 292:123–133
     [Google Scholar]
  32. Lindgren H., Golovliov I., Baranov V., Ernst R. K., Telepnev M., Sjostedt A. 2004; Factors affecting the escape of Francisella tularensis from the phagolysosome. J Med Microbiol 53:953–958
     [Google Scholar]
  33. Magnusson L. U., Farewell A., Nystrom T. 2005; ppGpp: a global regulator in Escherichia coli . Trends Microbiol 13:236–242
     [Google Scholar]
  34. Manganelli R. 2007; Polyphosphate and stress response in mycobacteria. Mol Microbiol 65:258–260
     [Google Scholar]
  35. Meibom K. L., Dubail I., Dupuis M., Barel M., Lenco J., Stulik J., Golovliov I., Sjostedt A., Charbit A. 2008; The heat-shock protein ClpB of Francisella tularensis is involved in stress tolerance and is required for multiplication in target organs of infected mice. Mol Microbiol 67:1384–1401
     [Google Scholar]
  36. Milne T. S., Michell S. L., Diaper H., Wikstrom P., Svensson K., Oyston P. C. F., Titball R. W. 2007; A 55 kDa hypothetical membrane protein is an iron-regulated virulence factor of Francisella tularensissubsp. novicida U112. J Med Microbiol 56:1268–1276
     [Google Scholar]
  37. Milton D. L., O'Toole R., Horstedt P., Wolf-Watz H. 1996; Flagellin A is essential for the virulence of Vibrio anguillarum . J Bacteriol 178:1310–1319
     [Google Scholar]
  38. Mittenhuber G. 2001; Comparative genomics and evolution of genes encoding bacterial (p)ppGpp synthetases/hydrolases (the Rel, RelA and SpoT proteins. J Mol Microbiol Biotechnol 3:585–600
     [Google Scholar]
  39. Mohapatra N. P., Soni S., Bell B. L., Warren R., Ernst R. K., Muszynski A., Carlson R. W., Gunn J. S. 2007; Identification of an orphan response regulator required for the virulence of Francisella spp. and transcription of pathogenicity island genes. Infect Immun 75:3305–3314
     [Google Scholar]
  40. Mouery K., Rader B. A., Gaynor E. C., Guillemin K. 2006; The stringent response is required for Helicobacter pylori survival of stationary phase, exposure to acid, and aerobic shock. J Bacteriol 188:5494–5500
     [Google Scholar]
  41. Nano F. E., Schmerk C. 2007; The Francisella pathogenicity island. Ann N Y Acad Sci 1105122–137
     [Google Scholar]
  42. Nano F. E., Zhang N., Cowley S. C., Klose K. E., Cheung K. K. M., Roberts M. J., Ludu J. S., Letendre G. W., Meierovics A. I. other authors 2004; A Francisella tularensis pathogenicity island required for intramacrophage growth. J Bacteriol 186:6430–6436
     [Google Scholar]
  43. Nylund A., Ottem K. F., Watanabe K., Karlsbakk E., Krossoy B. 2006; Francisella sp (Family Francisellaceae) causing mortality in Norwegian cod ( Gadus morhua) farming. Arch Microbiol 185:383–392
     [Google Scholar]
  44. Ojha A. K., Mukherjee T. K., Chatterji D. 2000; High intracellular level of guanosine tetraphosphate in Mycobacterium smegmatis changes the morphology of the bacterium. Infect Immun 68:4084–4091
     [Google Scholar]
  45. Okada Y., Makino S., Tobe T., Okada N., Yamazaki S. 2002; Cloning of rel from Listeria monocytogenes as an osmotolerance involvement gene. Appl Environ Microbiol 68:1541–1547
     [Google Scholar]
  46. Ostland V. E., Stannard J. A., Creek J. J., Hedrick R. P., Ferguson H. W., Carlberg J. M., Westerman M. E. 2006; Aquatic Francisella-like bacterium associated with mortality of intensively cultured hybrid striped bass Morone chrysops × M. saxatilis . Dis Aquat Organ 72:135–145
     [Google Scholar]
  47. Oyston P. C. F. 2008; Francisella tularensis: unravelling the secrets of an intracellular pathogen. J Med Microbiol 57:921–930
     [Google Scholar]
  48. Parker R. R., Steinhaus E. A., Kohls G. M., Jellison W. L. 1951; Contamination of natural waters and mud with Pasteurella tularensis and tularemia in beavers and muskrats in the northwestern United States. Bull Natl Inst Health 193:1–161
     [Google Scholar]
  49. Parkhill J., Achtman M., James K. D., Bentley S. D., Churcher C., Klee S. R., Morelli G., Basham D., Brown D. other authors 2000; Complete DNA sequence of a serogroup A strain of Neisseria meningitidis Z2491. Nature 404:502–506
     [Google Scholar]
  50. Pizarro-Cerda J., Tedin K. 2004; The bacterial signal molecule, ppGpp, regulates Salmonella virulence gene expression. Mol Microbiol 52:1827–1844
     [Google Scholar]
  51. Potrykus K., Cashel M. 2008; (p)ppGpp: still magical?. Annu Rev Microbiol 62:35–51
     [Google Scholar]
  52. Primm T. P., Andersen S. J., Mizrahi V., Avarbock D., Rubin H., Barry C. E. 2000; The stringent response of Mycobacterium tuberculosis is required for long-term survival. J Bacteriol 182:4889–4898
     [Google Scholar]
  53. Quarry J. E., Isherwood K. E., Michell S. L., Diaper H., Titball R. W., Oyston P. C. F. 2007; A Francisella tularensis subspecies novicida purF mutant, but not a purA mutant, induces protective immunity to tularemia in mice. Vaccine 25:2011–2018
     [Google Scholar]
  54. Richards M. I., Michell S. L., Oyston P. C. F. 2008; An intracellularly inducible gene involved in virulence and polyphosphate production in Francisella . J Med Microbiol 57:1183–1192
     [Google Scholar]
  55. Rohmer L., Brittnacher M., Svensson K., Buckley D., Haugen E., Zhou Y., Chang J., Levy R., Hayden H. other authors 2006; Potential source of Francisella tularensis live vaccine strain attenuation determined by genome comparison. Infect Immun 74:6895–6906
     [Google Scholar]
  56. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  57. Simon R., Priefer U., Puhler A. 1983; A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Biotechnology (N Y ) 1:784–791
     [Google Scholar]
  58. Sjostedt A. 2006; Intracellular survival mechanisms of Francisella tularensis, a stealth pathogen. Microbes Infect 8:561–567
     [Google Scholar]
  59. Song M. Y., Kim H. J., Kim E. Y., Shin M. S., Lee H. C., Hong Y. J., Rhee J. H., Yoon H., Ryu S. other authors 2004; ppGpp-dependent stationary phase induction of genes on Salmonella pathogenicity island 1. J Biol Chem 279:34183–34190
     [Google Scholar]
  60. Stepanovic S., Vukovic D., Dakic I., Savic B., Svabic-Vlahovic M. 2000; A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods 40:175–179
     [Google Scholar]
  61. Sureka K., Dey S., Datta P., Singh A. K., Dasgupta A., Rodrigue S., Basu J., Kundu M. 2007; Polyphosphate kinase is involved in stress-induced mprAB-sigE-rel signalling in mycobacteria. Mol Microbiol 65:261–276
     [Google Scholar]
  62. Swanson M. S., Hammer B. K. 2000; Legionella pneumophila pathogenesis: a fateful journey from amoebae to macrophages. Annu Rev Microbiol 54:567–613
     [Google Scholar]
  63. Taylor C. M., Beresford M., Epton H. A. S., Sigee D. C., Shama G., Andrew P. W., Roberts I. S. 2002; Listeria monocytogenes relA and hpt mutants are impaired in surface-attached growth and virulence. J Bacteriol 184:621–628
     [Google Scholar]
  64. Tettelin H., Saunders N. J., Heidelberg J., Jeffries A. C., Nelson K. E., Eisen J. A., Ketchum K. A., Hood D. W., Peden J. F. other authors 2000; Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. Science 287:1809–1815
     [Google Scholar]
  65. Thompson A., Rolfe M. D., Lucchini S., Schwerk P., Hinton J. C. D., Tedin K. 2006; The bacterial signal molecule, ppGpp, mediates the environmental regulation of both the invasion and intracellular virulence gene programs of Salmonella . J Biol Chem 281:30112–30121
     [Google Scholar]
  66. Wells D. H., Gaynor E. C. 2006; Helicobacter pylori initiates the stringent response upon nutrient and pH downshift. J Bacteriol 188:3726–3729
     [Google Scholar]
  67. Williams M. D., Ouyang T. X., Flickinger M. C. 1994; Starvation-induced expression of SspA and SspB – the effects of a null mutation in sspA on Escherichia coli protein synthesis and survival during growth and prolonged starvation. Mol Microbiol 11:1029–1043
     [Google Scholar]
  68. Xiao H., Kalman M., Ikehara K., Zemel S., Glaser G., Cashel M. 1991; Residual guanosine 3′,5′-bispyrophosphate synthetic activity of relA null mutants can be eliminated by spoT null mutations. J Biol Chem 266:5980–5990
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.031021-0
Loading
RelA regulates virulence and intracellular survival of Francisella novicida
Microbiology 155, 4104 (2009); https://doi.org/10.1099/mic.0.031021-0
/content/journal/micro/10.1099/mic.0.031021-0
/content/journal/micro/10.1099/mic.0.031021-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

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