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.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.

Whole-Genome Relationships among Francisella Bacteria of Diverse Origins Define New Species and Provide Specific Regions for Detection

“…The previously described seven unique LPS genes were present in all the F. tularensis genomes but only sporadically present among the F. novicida genomes. Only the F. tularensis genomes contained all three genes previously mentioned (13,71,72) to be involved in the formation of a cell surface capsule-like structure; these genes were FTT0794 (phosphocholine metabolism), FTT0795 (formylmethyltransferase), and FTT0796 (phosphocholine metabolism) (13,73). The F. novicida U112 genome had only the OppABCDF locus and the four R-M systems identified previously (70).…”

Glycosylation of a Capsule-Like Complex (CLC) by Francisella novicida Is Required for Virulence and Partial Protective Immunity in Mice

“…The strain was then extracted with 0.5% phenol as previously described (Bandara et al, 2011) and by a modified method using urea (Champion et al, 2015). Briefly, the bacteria were scraped off 10 agar plates and suspended in ~100 ml of 1 M urea.…”

“…Gels were stained with the Pierce Silver Stain Kit (Thermo Scientific), or 0.25% Stains-All (Sigma-Aldrich, St. Louis, MO; Bandara et al, 2011), or both.…”

Subversion of Host Recognition and Defense Systems by Francisella spp

“…This is important to note, since recent evidence has suggested that the O antigen and capsule of Francisella are structurally similar, with the capsule consisting of a polymer of O-antigen tetrasaccharide subunits (9) as well as a glycoprotein component termed a "capsulelike complex" (18). As such, some mutants deficient in LPS O-antigen production are also impaired in the production of capsule (9,130), including specific wbt operon mutants (wbtA, wbtC, wbtM, and wbtI).…”