title: Isolation and mutagenesis of a capsule-like complex (CLC) from Francisella tularensis, and contribution of the CLC to F. tularensis virulence in mice.
abstract: BACKGROUND
  Francisella tularensis is a category-A select agent and is responsible for tularemia in humans and animals. The surface components of F. tularensis that contribute to virulence are not well characterized. An electron-dense capsule has been postulated to be present around F. tularensis based primarily on electron microscopy, but this specific antigen has not been isolated or characterized.
  
  
  RESULTS
  A capsule-like complex (CLC) was effectively extracted from the cell surface of an F. tularensis live vaccine strain (LVS) lacking O-antigen with 0.5% phenol after 10 passages in defined medium broth and growth on defined medium agar for 5 days at 32°C in 7% CO₂. The large molecular size CLC was extracted by enzyme digestion, ethanol precipitation, and ultracentrifugation, and consisted of glucose, galactose, mannose, and Proteinase K-resistant protein. Quantitative reverse transcriptase PCR showed that expression of genes in a putative polysaccharide locus in the LVS genome (FTL_1432 through FTL_1421) was upregulated when CLC expression was enhanced. Open reading frames FTL_1423 and FLT_1422, which have homology to genes encoding for glycosyl transferases, were deleted by allelic exchange, and the resulting mutant after passage in broth (LVSΔ1423/1422_P10) lacked most or all of the CLC, as determined by electron microscopy, and CLC isolation and analysis. Complementation of LVSΔ1423/1422 and subsequent passage in broth restored CLC expression. LVSΔ1423/1422_P10 was attenuated in BALB/c mice inoculated intranasally (IN) and intraperitoneally with greater than 80 times and 270 times the LVS LD₅₀, respectively. Following immunization, mice challenged IN with over 700 times the LD₅₀ of LVS remained healthy and asymptomatic.
  
  
  CONCLUSIONS
  Our results indicated that the CLC may be a glycoprotein, FTL_1422 and -FTL_1423 were involved in CLC biosynthesis, the CLC contributed to the virulence of F. tularensis LVS, and a CLC-deficient mutant of LVS can protect mice against challenge with the parent strain.
authors.affiliation: Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, United States of America.
authors.forename: Aloka B
authors.initials: AB
authors.lastname: Bandara
authors.forename: Anna E
authors.initials: AE
authors.lastname: Champion
authors.forename: Xiaoshan
authors.initials: X
authors.lastname: Wang
authors.forename: Gretchen
authors.initials: G
authors.lastname: Berg
authors.forename: Michael A
authors.initials: MA
authors.lastname: Apicella
authors.forename: Molly
authors.initials: M
authors.lastname: McLendon
authors.forename: Parastoo
authors.initials: P
authors.lastname: Azadi
authors.forename: D Scott
authors.initials: DS
authors.lastname: Snyder
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Inzana
mesh_terms: D000818:Animals; D005604:Francisella tularensis / Q000737:chemistry* / Q000472:pathogenicity* / Q000648:ultrastructure; D008401:Gas Chromatography-Mass Spectrometry; D006023:Glycoproteins / Q000235:genetics* / Q000302:isolation & purification* / Q000502:physiology; D008264:Macrophages / Q000382:microbiology; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D011135:Polysaccharides, Bacterial / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014406:Tularemia / Q000382:microbiology*; D037521:Virulence Factors / Q000235:genetics* / Q000302:isolation & purification* / Q000502:physiology
chemical_list: D006023:Glycoproteins; D011135:Polysaccharides, Bacterial; D037521:Virulence Factors