1887

Abstract

, the causative agent of whooping cough, is highly adapted to cause human infection. The production of virulence factors, such as adhesins and toxins, is just part of an array of mechanisms by which causes infection. The stringent response is a global bacterial response to nutritional limitation that is mediated by the accumulation of cellular ppGpp and pppGpp [termed together as (p)ppGpp]. Here, we demonstrate that production of (p)ppGpp was controlled by RelA and SpoT proteins in , and that mutation-induced loss of both proteins together caused deficiencies in (p)ppGpp production. The (p)ppGpp-deficient mutants also exhibited defects in growth regulation, decreases in viability under nutritionally limited conditions, increases in susceptibility to oxidative stress and defects in biofilm formation. Analysis of the secreted proteins and the respective transcripts showed that lack of (p)ppGpp led to decreased expression of and , which encode a fimbrial subunit and the self-polymerizing type III secretion system tip protein, respectively. Moreover, electron microscopic analysis also indicated that (p)ppGpp regulated the formation of filamentous structures. Most virulence genes – including and were expressed in the Bvg phase during which the BvgAS two-component system was activated. Although and were downregulated in a (p)ppGpp-deficient mutant, normal expression of and persisted. Lack of coherence between virulence gene expression and (p)ppGpp production indicated that (p)ppGpp did not modulate the Bvg phase. Taken together, our data indicate that (p)ppGpp may govern an as-yet-unrecognized system that influences pathogenicity.

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2013-07-01
2019-10-18
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References

  1. Aberg A. , Shingler V. , Balsalobre C. . ( 2006; ). (p)ppGpp regulates type 1 fimbriation of Escherichia coli by modulating the expression of the site-specific recombinase FimB. . Mol Microbiol 60:, 1520–1533. [CrossRef] [PubMed]
    [Google Scholar]
  2. Ashworth L. A. , Irons L. I. , Dowsett A. B. . ( 1982; ). Antigenic relationship between serotype-specific agglutinogen and fimbriae of Bordetella pertussis. . Infect Immun 37:, 1278–1281.[PubMed]
    [Google Scholar]
  3. Birkebaek N. H. , Kristiansen M. , Seefeldt T. , Degn J. , Moller A. , Heron I. , Andersen P. L. , Moller J. K. , Ostergård L. . ( 1999; ). Bordetella pertussis and chronic cough in adults. . Clin Infect Dis 29:, 1239–1242. [CrossRef] [PubMed]
    [Google Scholar]
  4. Boes N. , Schreiber K. , Schobert M. . ( 2008; ). SpoT-triggered stringent response controls usp gene expression in Pseudomonas aeruginosa. . J Bacteriol 190:, 7189–7199. [CrossRef] [PubMed]
    [Google Scholar]
  5. Bordet J. , Gengou O. . ( 1906; ). Le microbe de la coqueluche. . Ann Inst Pasteur (Paris) 20:, 731–741.
    [Google Scholar]
  6. Brickman T. J. , Armstrong S. K. . ( 1996; ). The ornithine decarboxylase gene odc is required for alcaligin siderophore biosynthesis in Bordetella spp.: putrescine is a precursor of alcaligin. . J Bacteriol 178:, 54–60.[PubMed]
    [Google Scholar]
  7. Carbonetti N. H. . ( 2007; ). Immunomodulation in the pathogenesis of Bordetella pertussis infection and disease. . Curr Opin Pharmacol 7:, 272–278. [CrossRef] [PubMed]
    [Google Scholar]
  8. Cashel M. . ( 1994; ). Detection of (p)ppGpp accumulation patterns in Escherichia coli mutants. . Methods Mol Genet 3:, 341–356.
    [Google Scholar]
  9. Cashel M. , Gallant J. . ( 1969; ). Two compounds implicated in the function of the RC gene of Escherichia coli. . Nature 221:, 838–841. [CrossRef] [PubMed]
    [Google Scholar]
  10. Chatterji D. , Fujita N. , Ishihama A. . ( 1998; ). The mediator for stringent control, ppGpp, binds to the beta-subunit of Escherichia coli RNA polymerase. . Genes Cells 3:, 279–287. [CrossRef] [PubMed]
    [Google Scholar]
  11. Chen Q. , Decker K. B. , Boucher P. E. , Hinton D. , Stibitz S. . ( 2010; ). Novel architectural features of Bordetella pertussis fimbrial subunit promoters and their activation by the global virulence regulator BvgA. . Mol Microbiol 77:, 1326–1340. [CrossRef] [PubMed]
    [Google Scholar]
  12. Cherry J. D. . ( 1999; ). Epidemiological, clinical, and laboratory aspects of pertussis in adults. . Clin Infect Dis 28: (Suppl 2), S112–S117. [CrossRef] [PubMed]
    [Google Scholar]
  13. Deora R. , Bootsma H. J. , Miller J. F. , Cotter P. A. . ( 2001; ). Diversity in the Bordetella virulence regulon: transcriptional control of a Bvg-intermediate phase gene. . Mol Microbiol 40:, 669–683. [CrossRef] [PubMed]
    [Google Scholar]
  14. Field, L. H. & Parker, C. D. (1978). Differences observed between fresh isolates of Bordetella pertussis and their laboratory passaged derivatives. In International Symposium on Pertussis U S Department of Health, Education, and Welfare, Washington, DC, pp. 124–132. Edited by C. R. Manclark and J. C. Hill.
  15. Figurski D. H. , Helinski D. R. . ( 1979; ). Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. . Proc Natl Acad Sci U S A 76:, 1648–1652. [CrossRef] [PubMed]
    [Google Scholar]
  16. Halperin S. A. . ( 2007; ). The control of pertussis–2007 and beyond. . N Engl J Med 356:, 110–113. [CrossRef] [PubMed]
    [Google Scholar]
  17. He Q. , Mertsola J. . ( 2008; ). Factors contributing to pertussis resurgence. . Future Microbiol 3:, 329–339. [CrossRef] [PubMed]
    [Google Scholar]
  18. He H. , Cooper J. N. , Mishra A. , Raskin D. M. . ( 2012; ). Stringent response regulation of biofilm formation in Vibrio cholerae. . J Bacteriol 194:, 2962–2972. [CrossRef] [PubMed]
    [Google Scholar]
  19. Ho S. N. , Hunt H. D. , Horton R. M. , Pullen J. K. , Pease L. R. . ( 1989; ). Site-directed mutagenesis by overlap extension using the polymerase chain reaction. . Gene 77:, 51–59. [CrossRef] [PubMed]
    [Google Scholar]
  20. Irie Y. , Mattoo S. , Yuk M. H. . ( 2004; ). The Bvg virulence control system regulates biofilm formation in Bordetella bronchiseptica. . J Bacteriol 186:, 5692–5698. [CrossRef] [PubMed]
    [Google Scholar]
  21. Jennings M. E. , Quick L. N. , Ubol N. , Shrom S. , Dollahon N. , Wilson J. W. . ( 2012; ). Characterization of Salmonella type III secretion hyper-activity which results in biofilm-like cell aggregation. . PLoS ONE 7:, e33080. [CrossRef] [PubMed]
    [Google Scholar]
  22. Justo P. , Maria M. , Yust, Hassane L. , Julio G. , Javier V. , Manuel A. , Francisco M. . ( 2004; ). Production and characterization of casein hydrolysates with a high amino acid fischer's ration using immobilized proteases. . International Daily Journal 14(6):, 527–533. [CrossRef] [PubMed]
    [Google Scholar]
  23. Keen N. T. , Tamaki S. , Kobayashi D. , Trollinger D. . ( 1988; ). Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. . Gene 70:, 191–197. [CrossRef] [PubMed]
    [Google Scholar]
  24. Kuwae A. , Matsuzawa T. , Ishikawa N. , Abe H. , Nonaka T. , Fukuda H. , Imajoh-Ohmi S. , Abe A. . ( 2006; ). BopC is a novel type III effector secreted by Bordetella bronchiseptica and has a critical role in type III-dependent necrotic cell death. . J Biol Chem 281:, 6589–6600. [CrossRef] [PubMed]
    [Google Scholar]
  25. Liu S. , Bayles D. O. , Mason T. M. , Wilkinson B. J. . ( 2006; ). A cold-sensitive Listeria monocytogenes mutant has a transposon insertion in a gene encoding a putative membrane protein and shows altered (p)ppGpp levels. . Appl Environ Microbiol 72:, 3955–3959. [CrossRef] [PubMed]
    [Google Scholar]
  26. Livak K. J. , Schmittgen T. D. . ( 2001; ). Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔ C T method. . Methods 25:, 402–408. [CrossRef] [PubMed]
    [Google Scholar]
  27. Magnusson L. U. , Farewell A. , Nyström T. . ( 2005; ). ppGpp: a global regulator in Escherichia coli. . Trends Microbiol 13:, 236–242. [CrossRef] [PubMed]
    [Google Scholar]
  28. Makhov A. M. , Hannah J. H. , Brennan M. J. , Trus B. L. , Kocsis E. , Conway J. F. , Wingfield P. T. , Simon M. N. , Steven A. C. . ( 1994; ). Filamentous hemagglutinin of Bordetella pertussis. A bacterial adhesin formed as a 50-nm monomeric rigid rod based on a 19-residue repeat motif rich in beta strands and turns. . J Mol Biol 241:, 110–124. [CrossRef] [PubMed]
    [Google Scholar]
  29. Mattoo S. , Foreman-Wykert A. K. , Cotter P. A. , Miller J. F. . ( 2001; ). Mechanisms of Bordetella pathogenesis. . Front Biosci 6:, E168–E186. [CrossRef] [PubMed]
    [Google Scholar]
  30. Mattoo S. , Yuk M. H. , Huang L. L. , Miller J. F. . ( 2004; ). Regulation of type III secretion in Bordetella. . Mol Microbiol 52:, 1201–1214. [CrossRef] [PubMed]
    [Google Scholar]
  31. Medhekar B. , Shrivastava R. , Mattoo S. , Gingery M. , Miller J. F. . ( 2009; ). Bordetella Bsp22 forms a filamentous type III secretion system tip complex and is immunoprotective in vitro and in vivo. . Mol Microbiol 71:, 492–504. [CrossRef] [PubMed]
    [Google Scholar]
  32. Menozzi F. D. , Boucher P. E. , Riveau G. , Gantiez C. , Locht C. . ( 1994; ). Surface-associated filamentous hemagglutinin induces autoagglutination of Bordetella pertussis. . Infect Immun 62:, 4261–4269.[PubMed]
    [Google Scholar]
  33. 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.[PubMed]
    [Google Scholar]
  34. Murphy H. , Cashel M. . ( 2003; ). Isolation of RNA polymerase suppressors of a (p)ppGpp deficiency. . Methods Enzymol 371:, 596–601. [CrossRef] [PubMed]
    [Google Scholar]
  35. Nelson J. D. . ( 1978; ). The changing epidemiology of pertussis in young infants. The role of adults as reservoirs of infection. . Am J Dis Child 132:, 371–373.[PubMed] [CrossRef]
    [Google Scholar]
  36. Nomura Y. , Takabayashi T. , Kuroda H. , Yukawa Y. , Sattasuk K. , Akita M. , Nozawa A. , Tozawa Y. . ( 2012; ). ppGpp inhibits peptide elongation cycle of chloroplast translation system in vitro. . Plant Mol Biol 78:, 185–196. [CrossRef] [PubMed]
    [Google Scholar]
  37. Ooga T. , Ohashi Y. , Kuramitsu S. , Koyama Y. , Tomita M. , Soga T. , Masui R. . ( 2009; ). Degradation of ppGpp by nudix pyrophosphatase modulates the transition of growth phase in the bacterium Thermus thermophilus. . J Biol Chem 284:, 15549–15556. [CrossRef] [PubMed]
    [Google Scholar]
  38. Österberg S. , del Peso-Santos T. , Shingler V. . ( 2011; ). Regulation of alternative sigma factor use. . Annu Rev Microbiol 65:, 37–55. [CrossRef] [PubMed]
    [Google Scholar]
  39. Potrykus K. , Cashel M. . ( 2008; ). (p)ppGpp: still magical?. Annu Rev Microbiol 62:, 35–51. [CrossRef] [PubMed]
    [Google Scholar]
  40. Ruffing A. M. , Chen R. R. . ( 2012; ). Transcriptome profiling of a curdlan-producing Agrobacterium reveals conserved regulatory mechanisms of exopolysaccharide biosynthesis. . Microb Cell Fact 11:, 17. [CrossRef] [PubMed]
    [Google Scholar]
  41. Serra D. , Bosch A. , Russo D. M. , Rodríguez M. E. , Zorreguieta A. , Schmitt J. , Naumann D. , Yantorno O. . ( 2007; ). Continuous nondestructive monitoring of Bordetella pertussis biofilms by Fourier transform infrared spectroscopy and other corroborative techniques. . Anal Bioanal Chem 387:, 1759–1767. [CrossRef] [PubMed]
    [Google Scholar]
  42. Serra D. O. , Lücking G. , Weiland F. , Schulz S. , Görg A. , Yantorno O. M. , Ehling-Schulz M. . ( 2008; ). Proteome approaches combined with Fourier transform infrared spectroscopy revealed a distinctive biofilm physiology in Bordetella pertussis. . Proteomics 8:, 4995–5010. [CrossRef] [PubMed]
    [Google Scholar]
  43. Serra D. O. , Conover M. S. , Arnal L. , Sloan G. P. , Rodriguez M. E. , Yantorno O. M. , Deora R. . ( 2011; ). FHA-mediated cell-substrate and cell-cell adhesions are critical for Bordetella pertussis biofilm formation on abiotic surfaces and in the mouse nose and the trachea. . PLoS ONE 6:, e28811. [CrossRef] [PubMed]
    [Google Scholar]
  44. Srivatsan A. , Wang J. D. . ( 2008; ). Control of bacterial transcription, translation and replication by (p)ppGpp. . Curr Opin Microbiol 11:, 100–105. [CrossRef] [PubMed]
    [Google Scholar]
  45. Stainer D. W. , Scholte M. J. . ( 1970; ). A simple chemically defined medium for the production of phase I Bordetella pertussis. . J Gen Microbiol 63:, 211–220. [CrossRef] [PubMed]
    [Google Scholar]
  46. Stanley N. R. , Lazazzera B. A. . ( 2004; ). Environmental signals and regulatory pathways that influence biofilm formation. . Mol Microbiol 52:, 917–924. [CrossRef] [PubMed]
    [Google Scholar]
  47. Stibitz S. . ( 1994; ). Use of conditionally counterselectable suicide vectors for allelic exchange. . Methods Enzymol 235:, 458–465. [CrossRef] [PubMed]
    [Google Scholar]
  48. Sukumar N. , Mishra M. , Sloan G. P. , Ogi T. , Deora R. . ( 2007; ). Differential Bvg phase-dependent regulation and combinatorial role in pathogenesis of two Bordetella paralogs, BipA and BcfA. . J Bacteriol 189:, 3695–3704. [CrossRef] [PubMed]
    [Google Scholar]
  49. Uhl M. A. , Miller J. F. . ( 1996; ). Integration of multiple domains in a two-component sensor protein: the Bordetella pertussis BvgAS phosphorelay. . EMBO J 15:, 1028–1036.[PubMed]
    [Google Scholar]
  50. Willems R. , Paul A. , van der Heide H. G. , ter Avest A. R. , Mooi F. R. . ( 1990; ). Fimbrial phase variation in Bordetella pertussis: a novel mechanism for transcriptional regulation. . EMBO J 9:, 2803–2809.[PubMed]
    [Google Scholar]
  51. 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.[PubMed]
    [Google Scholar]
  52. Yuk M. H. , Harvill E. T. , Miller J. F. . ( 1998; ). The BvgAS virulence control system regulates type III secretion in Bordetella bronchiseptica. . Mol Microbiol 28:, 945–959. [CrossRef] [PubMed]
    [Google Scholar]
  53. Zhang J. M. , Cowell J. L. , Steven A. C. , Carter P. H. , McGrath P. P. , Manclark C. R. . ( 1985; ). Purification and characterization of fimbriae isolated from Bordetella pertussis. . Infect Immun 48:, 422–427.[PubMed]
    [Google Scholar]
  54. Zhou Y. N. , Jin D. J. . ( 1998; ). The rpoB mutants destabilizing initiation complexes at stringently controlled promoters behave like “stringent” RNA polymerases in Escherichia coli. . Proc Natl Acad Sci U S A 95:, 2908–2913. [CrossRef] [PubMed]
    [Google Scholar]
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