1887

Abstract

is a highly infectious Gram-negative pathogen that replicates intracellularly within the mammalian host. One of the factors associated with virulence of is the protein FupA that mediates high-affinity transport of ferrous iron across the outer membrane. Together with its paralogue FslE, a siderophore–ferric iron transporter, FupA supports survival of the pathogen in the host by providing access to the essential nutrient iron. The FupA orthologue in the attenuated live vaccine strain (LVS) is encoded by the hybrid gene /, the product of an intergenic recombination event that significantly contributes to attenuation of the strain. We used Fe transport assays with mutant strains complemented with the different paralogues to show that the FupA/B protein of LVS retains the capacity for high-affinity transport of ferrous iron, albeit less efficiently than FupA of virulent strain Schu S4. Fe transport assays using purified siderophore and siderophore-dependent growth assays on iron-limiting agar confirmed previous findings that FupA/B also contributes to siderophore-mediated ferric iron uptake. These assays further demonstrated that the LVS FslE protein is a weaker siderophore–ferric iron transporter than the orthologue from Schu S4, and may be a result of the sequence variation between the two proteins. Our results indicate that iron-uptake mechanisms in LVS differ from those in Schu S4 and that functional differences in the outer membrane iron transporters have distinct effects on growth under iron limitation.

Funding
This study was supported by the:
  • National Institutes of Health (Award AI067823)
  • University of Virginia School of Medicine
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/content/journal/micro/10.1099/mic.0.072835-0
2014-02-01
2024-03-28
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References

  1. Bagos P. G., Liakopoulos T. D., Spyropoulos I. C., Hamodrakas S. J. ( 2004a). PRED-TMBB: a web server for predicting the topology of beta-barrel outer membrane proteins. Nucleic Acids Res 32:Web Server issueW400–4[PubMed] [CrossRef]
    [Google Scholar]
  2. Bagos P. G., Liakopoulos T. D., Spyropoulos I. C., Hamodrakas S. J. ( 2004b). A Hidden Markov Model method, capable of predicting and discriminating beta-barrel outer membrane proteins. BMC Bioinformatics 5:29 [View Article][PubMed]
    [Google Scholar]
  3. Broekhuijsen M., Larsson P., Johansson A., Byström M., Eriksson U., Larsson E., Prior R. G., Sjöstedt A., Titball R. W., Forsman M. ( 2003). Genome-wide DNA microarray analysis of Francisella tularensis strains demonstrates extensive genetic conservation within the species but identifies regions that are unique to the highly virulent F. tularensis subsp. tularensis . J Clin Microbiol 41:2924–2931 [View Article][PubMed]
    [Google Scholar]
  4. Burke D. S. ( 1977). Immunization against tularemia: analysis of the effectiveness of live Francisella tularensis vaccine in prevention of laboratory-acquired tularemia. J Infect Dis 135:55–60 [View Article][PubMed]
    [Google Scholar]
  5. Chamberlain R. E. ( 1965). Evaluation of live tularemia vaccine prepared in a chemically defined medium. Appl Microbiol 13:232–235[PubMed]
    [Google Scholar]
  6. Chatfield C. H., Mulhern B. J., Burnside D. M., Cianciotto N. P. ( 2011). Legionella pneumophila LbtU acts as a novel, TonB-independent receptor for the legiobactin siderophore. J Bacteriol 193:1563–1575 [View Article][PubMed]
    [Google Scholar]
  7. Deng K., Blick R. J., Liu W., Hansen E. J. ( 2006). Identification of Francisella tularensis genes affected by iron limitation. Infect Immun 74:4224–4236 [View Article][PubMed]
    [Google Scholar]
  8. Faraldo-Gómez J. D., Sansom M. S. ( 2003). Acquisition of siderophores in gram-negative bacteria. Nat Rev Mol Cell Biol 4:105–116 [View Article][PubMed]
    [Google Scholar]
  9. Fortier A. H., Slayter M. V., Ziemba R., Meltzer M. S., Nacy C. A. ( 1991). Live vaccine strain of Francisella tularensis: infection and immunity in mice. Infect Immun 59:2922–2928[PubMed]
    [Google Scholar]
  10. Hubálek M., Hernychová L., Brychta M., Lenco J., Zechovská J., Stulík J. ( 2004). Comparative proteome analysis of cellular proteins extracted from highly virulent Francisella tularensis ssp. tularensis and less virulent F. tularensis ssp. holarctica and F. tularensis ssp. mediaasiatica . Proteomics 4:3048–3060 [View Article][PubMed]
    [Google Scholar]
  11. Huntley J. F., Conley P. G., Hagman K. E., Norgard M. V. ( 2007). Characterization of Francisella tularensis outer membrane proteins. J Bacteriol 189:561–574 [View Article][PubMed]
    [Google Scholar]
  12. Kiss K., Liu W., Huntley J. F., Norgard M. V., Hansen E. J. ( 2008). Characterization of fig operon mutants of Francisella novicida U112. FEMS Microbiol Lett 285:270–277 [View Article][PubMed]
    [Google Scholar]
  13. Larsson P., Oyston P. C., Chain P., Chu M. C., Duffield M., Fuxelius H. H., Garcia E., Hälltorp G., Johansson D. & other authors ( 2005). The complete genome sequence of Francisella tularensis, the causative agent of tularemia. Nat Genet 37:153–159 [View Article][PubMed]
    [Google Scholar]
  14. Lindgren H., Honn M., Golovlev I., Kadzhaev K., Conlan W., Sjöstedt A. ( 2009). The 58-kilodalton major virulence factor of Francisella tularensis is required for efficient utilization of iron. Infect Immun 77:4429–4436 [View Article][PubMed]
    [Google Scholar]
  15. Lindgren H., Honn M., Salomonsson E., Kuoppa K., Forsberg A., Sjöstedt A. ( 2011). Iron content differs between Francisella tularensis subspecies tularensis and subspecies holarctica strains and correlates to their susceptibility to H2O2-induced killing. Infect Immun 79:1218–1224 [View Article][PubMed]
    [Google Scholar]
  16. Milne T. S., Michell S. L., Diaper H., Wikström P., Svensson K., Oyston P. C., Titball R. W. ( 2007). A 55 kDa hypothetical membrane protein is an iron-regulated virulence factor of Francisella tularensis subsp. novicida U112. J Med Microbiol 56:1268–1276 [View Article][PubMed]
    [Google Scholar]
  17. Noinaj N., Guillier M., Barnard T. J., Buchanan S. K. ( 2010). TonB-dependent transporters: regulation, structure, and function. Annu Rev Microbiol 64:43–60 [View Article][PubMed]
    [Google Scholar]
  18. Qin A., Scott D. W., Rabideau M. M., Moore E. A., Mann B. J. ( 2011). Requirement of the CXXC motif of novel Francisella infectivity potentiator protein B FipB, and FipA in virulence of F. tularensis subsp. tularensis . PLoS ONE 6:e24611 [View Article][PubMed]
    [Google Scholar]
  19. Ramakrishnan G., Meeker A., Dragulev B. ( 2008). fslE is necessary for siderophore-mediated iron acquisition in Francisella tularensis Schu S4. J Bacteriol 190:5353–5361 [View Article][PubMed]
    [Google Scholar]
  20. Ramakrishnan G., Sen B., Johnson R. ( 2012). Paralogous outer membrane proteins mediate uptake of different forms of iron and synergistically govern virulence in Francisella tularensis tularensis . J Biol Chem 287:25191–25202 [View Article][PubMed]
    [Google Scholar]
  21. Ratledge C., Dover L. G. ( 2000). Iron metabolism in pathogenic bacteria. Annu Rev Microbiol 54:881–941 [View Article][PubMed]
    [Google Scholar]
  22. 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 [View Article][PubMed]
    [Google Scholar]
  23. Salomonsson E., Kuoppa K., Forslund A. L., Zingmark C., Golovliov I., Sjöstedt A., Noppa L., Forsberg A. ( 2009). Reintroduction of two deleted virulence loci restores full virulence to the live vaccine strain of Francisella tularensis . Infect Immun 77:3424–3431 [View Article][PubMed]
    [Google Scholar]
  24. Sen B., Meeker A., Ramakrishnan G. ( 2010). The fslE homolog, FTL_0439 (fupA/B), mediates siderophore-dependent iron uptake in Francisella tularensis LVS. Infect Immun 78:4276–4285 [View Article][PubMed]
    [Google Scholar]
  25. Sjöstedt A. ( 2007). Tularemia: history, epidemiology, pathogen physiology, and clinical manifestations. Ann N Y Acad Sci 1105:1–29 [View Article][PubMed]
    [Google Scholar]
  26. Sullivan J. T., Jeffery E. F., Shannon J. D., Ramakrishnan G. ( 2006). Characterization of the siderophore of Francisella tularensis and role of fslA in siderophore production. J Bacteriol 188:3785–3795 [View Article][PubMed]
    [Google Scholar]
  27. Svensson K., Larsson P., Johansson D., Byström M., Forsman M., Johansson A. ( 2005). Evolution of subspecies of Francisella tularensis . J Bacteriol 187:3903–3908 [View Article][PubMed]
    [Google Scholar]
  28. Twine S., Byström M., Chen W., Forsman M., Golovliov I., Johansson A., Kelly J., Lindgren H., Svensson K. & other authors ( 2005). A mutant of Francisella tularensis strain SCHU S4 lacking the ability to express a 58-kilodalton protein is attenuated for virulence and is an effective live vaccine. Infect Immun 73:8345–8352 [View Article][PubMed]
    [Google Scholar]
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