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.Methods and FindingsA 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.ConclusionsOur 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.
Highlights
Francisella tularensis is a Gram-negative coccobacillus, and the etiologic agent of tularemia in a wide variety of animals and humans
Our results indicated that the capsule-like complex (CLC) may be a glycoprotein, FTL_1422 and -FTL_1423 were involved in CLC biosynthesis, the CLC contributed to the virulence of F. tularensis live vaccine strain (LVS), and a CLC-deficient mutant of LVS can protect mice against challenge with the parent strain
Cherwonogrodzky [27] reported that daily passage of F. tularensis LVS in Chamberlain’s defined medium broth (CDMB), followed by growth on Chamberlain’s defined medium agar (CDMA), resulted in an increase of the electron dense material surrounding the bacteria
Summary
Francisella tularensis is a Gram-negative coccobacillus, and the etiologic agent of tularemia in a wide variety of animals and humans. The most pathogenic isolates of F. tularensis are type A1 strains (subspecies tularensis), which may cause human infection with as few as 10 organisms [2,3], and are associated with 30% mortality in the absence of antibiotics following pneumonic tularemia [1,4]. Type B strains (subspecies holarctica) are highly virulent, but are not associated with the same level of mortality as subspecies tularensis [2]. Due to their ease of culture and dispersal, persistence in the environment, and high virulence, F. tularensis is classified as a Category-A select agent by the CDC [2]. 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
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