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Journal of Bacteriology, January 2007, p. 561-574, Vol. 189, No. 2
0021-9193/07/$08.00+0     doi:10.1128/JB.01505-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Characterization of Francisella tularensis Outer Membrane Proteins{triangledown} ,{dagger}

Jason F. Huntley, Patrick G. Conley, Kayla E. Hagman, and Michael V. Norgard*

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390

Received 25 September 2006/ Accepted 25 October 2006

Francisella tularensis is a gram-negative coccobacillus that is capable of causing severe, fatal disease in a number of mammalian species, including humans. Little is known about the proteins that are surface exposed on the outer membrane (OM) of F. tularensis, yet identification of such proteins is potentially fundamental to understanding the initial infection process, intracellular survival, virulence, immune evasion and, ultimately, vaccine development. To facilitate the identification of putative F. tularensis outer membrane proteins (OMPs), the genomes of both the type A strain (Schu S4) and type B strain (LVS) were subjected to six bioinformatic analyses for OMP signatures. Compilation of the bioinformatic predictions highlighted 16 putative OMPs, which were cloned and expressed for the generation of polyclonal antisera. Total membranes were extracted from both Schu S4 and LVS by spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation, which separated OMs from cytoplasmic (inner) membrane and other cellular compartments. Validation of OM separation and enrichment was confirmed by probing sucrose gradient fractions with antibodies to putative OMPs and inner membrane proteins. F. tularensis OMs typically migrated in sucrose gradients between densities of 1.17 and 1.20 g/ml, which differed from densities typically observed for other gram-negative bacteria (1.21 to 1.24 g/ml). Finally, the identities of immunogenic proteins were determined by separation on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analysis. This is the first report of a direct method for F. tularensis OM isolation that, in combination with computational predictions, offers a more comprehensive approach for the characterization of F. tularensis OMPs.

* Corresponding author. Mailing address: Department of Microbiology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390. Phone: (214) 648-5900. Fax: (214) 648-5905. E-mail: michael.norgard{at}utsouthwestern.edu.

{triangledown} Published ahead of print on 17 November 2006.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, January 2007, p. 561-574, Vol. 189, No. 2
0021-9193/07/$08.00+0     doi:10.1128/JB.01505-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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