1887

Abstract

causes the disease tularaemia. Type IV pili (Tfp) genes are present in the genomes of all subspecies. We show that the wild-type subsp. expresses pilus fibres on its surface, and mutations in the Tfp genes and disrupt pilus biogenesis. Mutations in other Tfp genes ( and ) do not eliminate pilus expression. A mutation in eliminates pilus expression, whereas mutations in the other pilin subunits and do not, suggesting that is the major pilus structural subunit. The virulence regulator MglA is required for pilus expression, and it regulates the transcription of a putative Tfp glycosylation gene (FTN0431). However, MglA does not regulate transcription of , or , and a strain lacking FTN0431 still expresses pili; thus, it is unclear how MglA regulates pilus expression. Only was also required for protein secretion, while and were not, indicating that there is very little overlap of the protein secretion/Tfp functions of the genes. The protein secretion component was more important for intramacrophage growth and mouse virulence than the Tfp component . Our results provide the first genetic characterization of the novel Tfp system of .

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2008/018077-0
2008-07-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/154/7/2139.html?itemId=/content/journal/micro/10.1099/mic.0.2008/018077-0&mimeType=html&fmt=ahah

References

  1. Abd H., Johansson T., Golovliov I., Sandstrom G., Forsman M. 2003; Survival and growth of Francisella tularensis in Acanthamoeba castellanii. Appl Environ Microbiol 69:600–606
    [Google Scholar]
  2. Anthony L. S., Kongshavn P. A. 1987; Experimental murine tularemia caused by Francisella tularensis, live vaccine strain: a model of acquired cellular resistance. Microb Pathog 2:3–14
    [Google Scholar]
  3. Anthony L. D., Burke R. D., Nano F. E. 1991a; Growth of Francisella spp. in rodent macrophages. Infect Immun 59:3291–3296
    [Google Scholar]
  4. Anthony L. S., Gu M. Z., Cowley S. C., Leung W. W., Nano F. E. 1991b; Transformation and allelic replacement in Francisella spp. J Gen Microbiol 137:2697–2703
    [Google Scholar]
  5. Assalkhou R., Balasingham S., Collins R. F., Frye S. A., Davidsen T., Benam A. V., Bjoras M., Derrick J. P., Tonjum T. 2007; The outer membrane secretin PilQ from Neisseria meningitidis binds DNA. Microbiology 153:1593–1603
    [Google Scholar]
  6. Averhoff B., Friedrich A. 2003; Type IV pili-related natural transformation systems: DNA transport in mesophilic and thermophilic bacteria. Arch Microbiol 180:385–393
    [Google Scholar]
  7. Balasingham S. V., Collins R. F., Assalkhou R., Homberset H., Frye S. A., Derrick J. P., Tonjum T. 2007; Interactions between the lipoprotein PilP and the secretin PilQ in Neisseria meningitidis. J Bacteriol 189:5716–5727
    [Google Scholar]
  8. Baron G. S., Nano F. E. 1998; MglA and MglB are required for the intramacrophage growth of Francisella novicida. Mol Microbiol 29:247–259
    [Google Scholar]
  9. Brotcke A., Weiss D. S., Kim C. C., Chain P., Malfatti S., Garcia E., Monack D. M. 2006; Identification of MglA-regulated genes reveals novel virulence factors in Francisella tularensis. Infect Immun 74:6642–6655
    [Google Scholar]
  10. Burrows L. L. 2005; Weapons of mass retraction. Mol Microbiol 57:878–888
    [Google Scholar]
  11. Carbonnelle E., Helaine S., Nassif X., Pelicic V. 2006; A systematic genetic analysis in Neisseria meningitidis defines the Pil proteins required for assembly, functionality, stabilization and export of type IV pili. Mol Microbiol 61:1510–1522
    [Google Scholar]
  12. Chakraborty S., Monfett M., Maier T. M., Benach J. L., Frank D. W., Thanassi D. G. 2008; Type IV pili in Francisella tularensis: roles of pilF and pilT in fiber assembly, host cell adherence and virulence. Infect Immun in press
    [Google Scholar]
  13. Chamberlain R. E. 1965; Evaluation of live tularemia vaccine prepared in a chemically defined medium. Appl Microbiol 13:232–235
    [Google Scholar]
  14. 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]
  15. Collins R. F., Davidsen L., Derrick J. P., Ford R. C., Tonjum T. 2001; Analysis of the PilQ secretin from Neisseria meningitidis by transmission electron microscopy reveals a dodecameric quaternary structure. J Bacteriol 183:3825–3832
    [Google Scholar]
  16. Collins R. F., Frye S. A., Balasingham S., Ford R. C., Tonjum T., Derrick J. P. 2005; Interaction with type IV pili induces structural changes in the bacterial outer membrane secretin PilQ. J Biol Chem 280:18923–18930
    [Google Scholar]
  17. Corpet F. 1988; Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 16:10881–10890
    [Google Scholar]
  18. Cowley S. C., Gray C. J., Nano F. E. 2000; Isolation and characterization of Francisella novicida mutants defective in lipopolysaccharide biosynthesis. FEMS Microbiol Lett 182:63–67
    [Google Scholar]
  19. Dennis D. T., Inglesby T. V., Henderson D. A., Bartlett J. G., Ascher M. S., Eitzen E., Fine A. D., Friedlander A. M., Hauer J. other authors 2001; Tularemia as a biological weapon: medical and public health management. JAMA 285:2763–2773
    [Google Scholar]
  20. Eigelsbach H. T., Braun W., Herring R. D. 1951; Studies on the variation of Bacterium tularense. J Bacteriol 61:557–569
    [Google Scholar]
  21. Ellis J., Oyston P. C., Green M., Titball R. W. 2002; Tularemia. Clin Microbiol Rev 15:631–646
    [Google Scholar]
  22. Forsberg A., Guina T. 2007; Type II secretion and type IV pili of Francisella. Ann N Y Acad Sci 1105187–201
    [Google Scholar]
  23. Forslund A. L., Kuoppa K., Svensson K., Salomonsson E., Johansson A., Bystrom M., Oyston P. C., Michell S. L., Titball R. W. other authors 2006; Direct repeat-mediated deletion of a type IV pilin gene results in major virulence attenuation of Francisella tularensis. Mol Microbiol 59:1818–1830
    [Google Scholar]
  24. Fullner K. J., Mekalanos J. J. 1999; Genetic characterization of a new type IV-A pilus gene cluster found in both classical and El Tor biotypes of Vibrio cholerae. Infect Immun 67:1393–1404
    [Google Scholar]
  25. Gallagher L. A., Ramage E., Jacobs M. A., Kaul R., Brittnacher M., Manoil C. 2007; A comprehensive transposon mutant library of Francisella novicida, a bioweapon surrogate. Proc Natl Acad Sci U S A 104:1009–1014
    [Google Scholar]
  26. Gil H., Benach J. L., Thanassi D. G. 2004; Presence of pili on the surface of Francisella tularensis. Infect Immun 72:3042–3047
    [Google Scholar]
  27. Golovliov I., Sjostedt A., Mokrievich A., Pavlov V. 2003a; A method for allelic replacement in Francisella tularensis. FEMS Microbiol Lett 222:273–280
    [Google Scholar]
  28. Golovliov I., Baranov V., Krocova Z., Kovarova H., Sjostedt A. 2003b; An attenuated strain of the facultative intracellular bacterium Francisella tularensis can escape the phagosome of monocytic cells. Infect Immun 71:5940–5950
    [Google Scholar]
  29. Gray C. G., Cowley S. C., Cheung K. K., 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]
  30. Hager A. J., Bolton D. L., Pelletier M. R., Brittnacher M. J., Gallagher L. A., Kaul R., Skerrett S. J., Miller S. I., Guina T. 2006; Type IV pili-mediated secretion modulates Francisella virulence. Mol Microbiol 62:227–237
    [Google Scholar]
  31. Hanahan D. 1983; Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580
    [Google Scholar]
  32. Hansen J. K., Forest K. T. 2006; Type IV pilin structures: insights on shared architecture, fiber assembly, receptor binding and type II secretion. J Mol Microbiol Biotechnol 11:192–207
    [Google Scholar]
  33. Kieffer T. L., Cowley S., Nano F. E., Elkins K. L. 2003; Francisella novicida LPS has greater immunobiological activity in mice than F. tularensis LPS, and contributes to F. novicida murine pathogenesis. Microbes Infect 5:397–403
    [Google Scholar]
  34. Kuoppa K., Forsberg A., Norqvist A. 2001; Construction of a reporter plasmid for screening in vivo promoter activity in Francisella tularensis. FEMS Microbiol Lett 205:77–81
    [Google Scholar]
  35. Kus J. V., Tullis E., Cvitkovitch D. G., Burrows L. L. 2004; Significant differences in type IV pilin allele distribution among Pseudomonas aeruginosa isolates from cystic fibrosis (CF) versus non-CF patients. Microbiology 150:1315–1326
    [Google Scholar]
  36. Larsson P., Oyston P. C., Chain P., Chu M. C., Duffield M., Fuxelius H. H., Garcia E., Halltorp G., Johansson D. other authors 2005; The complete genome sequence of Francisella tularensis, the causative agent of tularemia. Nat Genet 37:153–159
    [Google Scholar]
  37. Lauer P., Albertson N. H., Koomey M. 1993; Conservation of genes encoding components of a type IV pilus assembly/two-step protein export pathway in Neisseria gonorrhoeae. Mol Microbiol 8:357–368
    [Google Scholar]
  38. Lauriano C. M., Barker J. R., Nano F. E., Arulanandam B. P., Klose K. E. 2003; Allelic exchange in Francisella tularensis using PCR products. FEMS Microbiol Lett 229:195–202
    [Google Scholar]
  39. Lauriano C. M., Barker J. R., Yoon S. S., Nano F. E., Arulanandam B. P., Hassett 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]
  40. Liu J., Zogaj X., Barker J., Klose K. 2007; Construction of targeted insertion mutations in Francisella tularensis subsp. novicida. Biotechniques 43:487–492
    [Google Scholar]
  41. Maier T. M., Casey M. S., Becker R. H., Dorsey C. W., Glass E. M., Maltsev N., Zahrt T. C., Frank D. W. 2007; Identification of Francisella tularensis Himar1-based transposon mutants defective for replication in macrophages. Infect Immun 75:5376–5389
    [Google Scholar]
  42. Mattick J. S. 2002; Type IV pili and twitching motility. Annu Rev Microbiol 56:289–314
    [Google Scholar]
  43. McLendon M. K., Apicella M. A., Allen L. A. 2006; Francisella tularensis: taxonomy, genetics, and immunopathogenesis of a potential agent of biowarfare. Annu Rev Microbiol 60:167–185
    [Google Scholar]
  44. Nano F. E., Zhang N., Cowley S. C., Klose K. E., Cheung K. K., 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]
  45. Nunn D. N., Lory S. 1991; Product of the Pseudomonas aeruginosa gene pilD is a prepilin leader peptidase. Proc Natl Acad Sci U S A 88:3281–3285
    [Google Scholar]
  46. Nunn D., Bergman S., Lory S. 1990; Products of three accessory genes, pilB, pilC, and pilD, are required for biogenesis of Pseudomonas aeruginosa pili. J Bacteriol 172:2911–2919
    [Google Scholar]
  47. Oyston P. C., Sjostedt A., Titball R. W. 2004; Tularaemia: bioterrorism defence renews interest in Francisella tularensis. Nat Rev Microbiol 2:967–978
    [Google Scholar]
  48. Peabody C. R., Chung Y. J., Yen M. R., Vidal-Ingigliardi D., Pugsley A. P., Saier M. H. Jr 2003; Type II protein secretion and its relationship to bacterial type IV pili and archaeal flagella. Microbiology 149:3051–3072
    [Google Scholar]
  49. Petrosino J. F., Xiang Q., Karpathy S. E., Jiang H., Yerrapragada S., Liu Y., Gioia J., Hemphill L., Gonzalez A. other authors 2006; Chromosome rearrangement and diversification of Francisella tularensis revealed by the type B (OSU18) genome sequence. J Bacteriol 188:6977–6985
    [Google Scholar]
  50. Qin A., Mann B. J. 2006; Identification of transposon insertion mutants of Francisella tularensis tularensis strain Schu S4 deficient in intracellular replication in the hepatic cell line HepG2. BMC Microbiol 6:69
    [Google Scholar]
  51. 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]
  52. Roine E., Raineri D. M., Romantschuk M., Wilson M., Nunn D. N. 1998; Characterization of type IV pilus genes in Pseudomonas syringae pv. tomato DC3000. Mol Plant Microbe Interact 11:1048–1056
    [Google Scholar]
  53. Sandkvist M. 2001; Biology of type II secretion. Mol Microbiol 40:271–283
    [Google Scholar]
  54. Sandkvist M., Michel L. O., Hough L. P., Morales V. M., Bagdasarian M., Koomey M., DiRita V. J. 1997; General secretion pathway ( eps) genes required for toxin secretion and outer membrane biogenesis in Vibrio cholerae. J Bacteriol 179:6994–7003
    [Google Scholar]
  55. Sjostedt A. 2003; Virulence determinants and protective antigens of Francisella tularensis. Curr Opin Microbiol 6:66–71
    [Google Scholar]
  56. Smedley J. G. III, Jewell E., Roguskie J., Horzempa J., Syboldt A., Stolz D. B., Castric P. 2005; Influence of pilin glycosylation on Pseudomonas aeruginosa 1244 pilus function. Infect Immun 73:7922–7931
    [Google Scholar]
  57. Strom M. S., Lory S. 1993; Structure–function and biogenesis of the type IV pili. Annu Rev Microbiol 47:565–596
    [Google Scholar]
  58. Su J., Yang J., Zhao D., Kawula T. H., Banas J. A., Zhang J. R. 2007; Genome-wide identification of Francisella tularensis virulence determinants. Infect Immun 75:3089–3101
    [Google Scholar]
  59. Taylor R. K., Miller V. L., Furlong D. B., Mekalanos J. J. 1987; Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin. Proc Natl Acad Sci U S A 84:2833–2837
    [Google Scholar]
  60. Tempel R., Lai X. H., Crosa L., Kozlowicz B., Heffron F. 2006; Attenuated Francisella novicida transposon mutants protect mice against wild-type challenge. Infect Immun 74:5095–5105
    [Google Scholar]
  61. Thomas R. M., Titball R. W., Oyston P. C., Griffin K., Waters E., Hitchen P. G., Michell S. L., Grice I. D., Wilson J. C., Prior J. L. 2007; The immunologically distinct O antigens from Francisella tularensis subspecies tularensis and Francisella novicida are both virulence determinants and protective antigens. Infect Immun 75:371–378
    [Google Scholar]
  62. Tonjum T., Koomey M. 1997; The pilus colonization factor of pathogenic neisserial species: organelle biogenesis and structure/function relationships – a review. Gene 192:155–163
    [Google Scholar]
  63. Wall D., Kolenbrander P. E., Kaiser D. 1999; The Myxococcus xanthus pilQ ( sglA) gene encodes a secretin homolog required for type IV pilus biogenesis, social motility, and development. J Bacteriol 181:24–33
    [Google Scholar]
  64. Weiss D. S., Brotcke A., Henry T., Margolis J. J., Chan K., Monack D. M. 2007; In vivo negative selection screen identifies genes required for Francisella virulence. Proc Natl Acad Sci U S A 104:6037–6042
    [Google Scholar]
  65. Whitchurch C. B., Hobbs M., Livingston S. P., Krishnapillai V., Mattick J. S. 1991; Characterisation of a Pseudomonas aeruginosa twitching motility gene and evidence for a specialised protein export system widespread in eubacteria. Gene 101:33–44
    [Google Scholar]
  66. Winther-Larsen H. C., Wolfgang M., Dunham S., van Putten J. P., Dorward D., Lovold C., Aas F. E., Koomey M. 2005; A conserved set of pilin-like molecules controls type IV pilus dynamics and organelle-associated functions in Neisseria gonorrhoeae. Mol Microbiol 56:903–917
    [Google Scholar]
  67. Wolfgang M., van Putten J. P., Hayes S. F., Dorward D., Koomey M. 2000; Components and dynamics of fiber formation define a ubiquitous biogenesis pathway for bacterial pili. EMBO J 19:6408–6418
    [Google Scholar]
  68. Yoshihara S., Geng X., Okamoto S., Yura K., Murata T., Go M., Ohmori M., Ikeuchi M. 2001; Mutational analysis of genes involved in pilus structure, motility and transformation competency in the unicellular motile cyanobacterium Synechocystis sp. PCC 6803. Plant Cell Physiol 42:63–73
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2008/018077-0
Loading
/content/journal/micro/10.1099/mic.0.2008/018077-0
Loading

Data & Media loading...

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