title: Identification, characterization and immunogenicity of an O-antigen capsular polysaccharide of Francisella tularensis.
abstract: Capsular polysaccharides are important factors in bacterial pathogenesis and have been the target of a number of successful vaccines. Francisella tularensis has been considered to express a capsular antigen but none has been isolated or characterized. We have developed a monoclonal antibody, 11B7, which recognizes the capsular polysaccharide of F. tularensis migrating on Western blot as a diffuse band between 100 kDa and 250 kDa. The capsule stains poorly on SDS-PAGE with silver stain but can be visualized using ProQ Emerald glycoprotein stain. The capsule appears to be highly conserved among strains of F. tularensis as antibody 11B7 bound to the capsule of 14 of 14 F. tularensis type A and B strains on Western blot. The capsular material can be isolated essentially free of LPS, is phenol and proteinase K resistant, ethanol precipitable and does not dissociate in sodium dodecyl sulfate. Immunoelectron microscopy with colloidal gold demonstrates 11B7 circumferentially staining the surface of F. tularensis which is typical of a polysaccharide capsule. Mass spectrometry, compositional analysis and NMR indicate that the capsule is composed of a polymer of the tetrasaccharide repeat, 4)-alpha-D-GalNAcAN-(1->4)-alpha-D-GalNAcAN-(1->3)-beta-D-QuiNAc-(1->2)-beta-D-Qui4NFm-(1-, which is identical to the previously described F. tularensis O-antigen subunit. This indicates that the F. tularensis capsule can be classified as an O-antigen capsular polysaccharide. Our studies indicate that F. tularensis O-antigen glycosyltransferase mutants do not make a capsule. An F. tularensis acyltransferase and an O-antigen polymerase mutant had no evidence of an O-antigen but expressed a capsular antigen. Passive immunization of BALB/c mice with 75 microg of 11B7 protected against a 150 fold lethal challenge of F. tularensis LVS. Active immunization of BALB/c mice with 10 microg of capsule showed a similar level of protection. These studies demonstrate that F. tularensis produces an O-antigen capsule that may be the basis of a future vaccine.
authors.affiliation: Department of Microbiology, University of Iowa, Iowa City, Iowa, United States of America. michael-apicella@uiowa.edu
authors.forename: Michael A
authors.initials: MA
authors.lastname: Apicella
authors.forename: Deborah M B
authors.initials: DM
authors.lastname: Post
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Fowler
authors.forename: Bradley D
authors.initials: BD
authors.lastname: Jones
authors.forename: Jed A
authors.initials: JA
authors.lastname: Rasmussen
authors.forename: Jason R
authors.initials: JR
authors.lastname: Hunt
authors.forename: Sayaka
authors.initials: S
authors.lastname: Imagawa
authors.forename: Biswa
authors.initials: B
authors.lastname: Choudhury
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Inzana
authors.forename: Tamara M
authors.initials: TM
authors.lastname: Maier
authors.forename: Dara W
authors.initials: DW
authors.lastname: Frank
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Zahrt
authors.forename: Kathryn
authors.initials: K
authors.lastname: Chaloner
authors.forename: Michael P
authors.initials: MP
authors.lastname: Jennings
authors.forename: Molly K
authors.initials: MK
authors.lastname: McLendon
authors.forename: Bradford W
authors.initials: BW
authors.lastname: Gibson
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D016667:Bacterial Capsules / Q000276:immunology* / Q000378:metabolism* / Q000648:ultrastructure; D015153:Blotting, Western; D020285:Cryoelectron Microscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005604:Francisella tularensis / Q000276:immunology* / Q000378:metabolism* / Q000648:ultrastructure; D008401:Gas Chromatography-Mass Spectrometry; D006602:Hexosyltransferases / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D051379:Mice; D016253:Microscopy, Immunoelectron; D019081:O Antigens / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000911:Antibodies, Monoclonal; D019081:O Antigens; D006602:Hexosyltransferases; C085652:O-antigen polymerase