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

Abstract

Francisella tularensis is a Gram-negative bacterium and the etiologic agent of tularemia. F. tularensis may appear encapsulated when examined by transmission electron microscopy (TEM), which is due to production of an extracellular capsule-like complex (CLC) when the bacterium is grown under specific environmental conditions. Deletion of two glycosylation genes in the live vaccine strain (LVS) results in loss of apparent CLC and attenuation of LVS in mice. In contrast, F. novicida, which is also highly virulent for mice, is reported to be non-encapsulated. However, the F. novicida genome contains a putative polysaccharide locus with homology to the CLC glycosylation locus in F. tularensis. Following daily subculture of F. novicida in Chamberlain's defined medium, an electron dense material surrounding F. novicida, similar to the F. tularensis CLC, was evident. Extraction with urea effectively removed the CLC, and compositional analysis indicated the extract contained galactose, glucose, mannose, and multiple proteins, similar to those found in the F. tularensis CLC. The same glycosylation genes deleted in LVS were targeted for deletion in F. novicida by allelic exchange using the same mutagenesis vector used for mutagenesis of LVS. In contrast, this mutation also resulted in the loss of five additional genes immediately upstream of the targeted mutation (all within the glycosylation locus), resulting in strain F. novicida Δ1212–1218. The subcultured mutant F. novicida Δ1212–1218 was CLC-deficient and the CLC contained significantly less carbohydrate than the subcultured parent strain. The mutant was severely attenuated in BALB/c mice inoculated intranasally, as determined by the lower number of F. novicida Δ1212–1218 recovered in tissues compared to the parent, and by clearance of the mutant by 10–14 days post-challenge. Mice immunized intranasally with F. novicida Δ1212–1218 were partially protected against challenge with the parent, produced significantly reduced levels of inflammatory cytokines, and their spleens contained only areas of lymphoid hyperplasia, whereas control mice challenged with the parent exhibited hypercytokinemia and splenic necrosis. Therefore, F. novicida is capable of producing a CLC similar to that of F. tularensis, and glycosylation of the CLC contributed to F. novicida virulence and immunoprotection.

Further Characterization of the Capsule-Like Complex (CLC) Produced by Francisella tularensis Subspecies tularensis: Protective Efficacy and Similarity to Outer Membrane Vesicles

“…Colonies of F. tularensis type A strains SCHU S4 and clinical isolate TI0902 were more mucoid and iridescent when subcultured in CDMB and grown at 32 or 37°C on CDMA than when these strains were not subcultured. These results were similar to those obtained when LVS (Bandara et al, 2011 ) and F. novicida (Freudenberger Catanzaro et al, 2017 ) were similarly subcultured in CDMB and CDMA to enhance CLC expression. However, initial efforts to visualize the CLC on the surface of type A strains using negative stain TEM techniques were less successful than with LVS or F. novicida .…”

“…The CLC of F. tularensis remains a novel, elusive antigen, shown to be necessary for virulence in LVS and F. novicida (Bandara et al, 2011 ; Freudenberger Catanzaro et al, 2017 ). However, isolation and analysis of the CLC has been challenging due to self-aggregation and insolubility of the CLC during purification.…”

“…However, the TxI-SS fraction of the double mutant did demonstrate a diffuse, darker staining HMS material by SDS-PAGE and silver staining, indicating the mutant CLC contained different proteins or glycose-deficient proteins than the parent CLC. We have previously reported that WbtI G191V Δ1423-22_P10 (Bandara et al, 2011 ), and an F. novicida mutant lacking genes from the same glycosylation locus (Freudenberger Catanzaro et al, 2017 ), are deficient in, but continue to produce, a lesser amount of carbohydrate-deficient CLC. The difference in protein content between the O-antigen deficient parent and the glycose-deficient CLC mutant was evident in the ratio of TxI-SS:TxI-SI fractions.…”

“…Sucrose-resistant clones were patched on MH-kanamycin agar to verify loss of the kanamycin-resistance marker, and colony PCR was performed to detect clones with allelic replacement within the correct chromosomal locus using FTT_0798 and 0799 gene specific primers, which are identical to primers used for detection of FTL_1422 and FTL_1423 from LVS (Bandara et al, 2011 ). Mutagenesis of the glycosylation locus in one clone (SCHU S4Δ0798-99) was confirmed by the loss of carbohydrate content using the anthrone assay (Scott and Melvin, 1953 ) as previously described (Freudenberger Catanzaro et al, 2017 ).…”

“…The locus responsible for glycosylation of some proteins (FTT0789-FTT0800), such as DsbA, in F. tularensis type A and B strains is also responsible for glycosylation of the CLC (Larsson et al, 2005 ; Bandara et al, 2011 ) (Balonova et al, 2010 ; Thomas et al, 2011 ). We previously demonstrated that when two glycosyl transferases (FTT0798-99) in this locus were deleted in LVS (Bandara et al, 2011 ) or F. novicida (Freudenberger Catanzaro et al, 2017 ), CLC production was significantly diminished and both species were highly attenuated. In LVS, and to a lesser extent F. novicida , the CLC-deficient strains were protective against challenge with the parent strain.…”