Isolation and Mutagenesis of a Capsule-Like Complex (CLC) from Francisella tularensis, and Contribution of the CLC to F. tularensis Virulence in Mice

dc.contributor.authorBandara, Aloka B.en
dc.contributor.authorChampion, Anna E.en
dc.contributor.authorWang, X.en
dc.contributor.authorBerg, G.en
dc.contributor.authorApicella, Michael A.en
dc.contributor.authorMcLendon, M.en
dc.contributor.authorAzadi, P.en
dc.contributor.authorSnyder, D. S.en
dc.contributor.authorInzana, Thomas J.en
dc.contributor.departmentBiomedical Sciences and Pathobiologyen
dc.contributor.departmentHuman Nutrition, Foods, and Exerciseen
dc.contributor.departmentFralin Life Sciences Instituteen
dc.description.abstractBackground: 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. Methods and Findings: 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 32uC 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 (LVSD1423/1422_P10) lacked most or all of the CLC, as determined by electron microscopy, and CLC isolation and analysis. Complementation of LVSD1423/1422 and subsequent passage in broth restored CLC expression. LVSD1423/1422_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.en
dc.description.versionPublished versionen
dc.format.extent? - ? (14) page(s)en
dc.rightsCreative Commons Attribution 4.0 Internationalen
dc.subjectlive vaccine strainen
dc.subjectlipopolysaccharide o-antigenen
dc.subjectgram-negative bacteriaen
dc.subjectinfluenzae type-ben
dc.subjectprotective immunityen
dc.subjectsubspecies tularensisen
dc.subjectenhanced sensitivityen
dc.subjectaerosol challengeen
dc.titleIsolation and Mutagenesis of a Capsule-Like Complex (CLC) from Francisella tularensis, and Contribution of the CLC to F. tularensis Virulence in Miceen
dc.title.serialPLOS ONEen
dc.typeArticle - Refereeden
pubs.organisational-group/Virginia Techen
pubs.organisational-group/Virginia Tech/Agriculture & Life Sciencesen
pubs.organisational-group/Virginia Tech/Agriculture & Life Sciences/Human Nutrition, Foods, & Exerciseen
pubs.organisational-group/Virginia Tech/All T&R Facultyen
pubs.organisational-group/Virginia Tech/Faculty of Health Sciencesen
pubs.organisational-group/Virginia Tech/University Research Institutesen
pubs.organisational-group/Virginia Tech/University Research Institutes/Fralin Life Sciencesen
pubs.organisational-group/Virginia Tech/University Research Institutes/Fralin Life Sciences/Fralin Affiliated Facultyen
pubs.organisational-group/Virginia Tech/Veterinary Medicineen
pubs.organisational-group/Virginia Tech/Veterinary Medicine/Biomedical Sciences and Pathobiologyen
pubs.organisational-group/Virginia Tech/Veterinary Medicine/CVM T&R Facultyen


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Isolation and mutagenesis of a capsule-like complex (CLC) from Francisella tularensis, and contribution of the CLC to F. tularensis virulence in mice.pdf
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