Biofilms are matrix-associated communities that enable bacteria to colonise environments unsuitable for free-living bacteria. The facultative intracellular pathogen Francisella tularensis can persist in water, amoebae, and arthropods, as well as within mammalian macrophages. F . tularensis Types A and B form poor biofilms, but F . tularensis mutants lacking lipopolysaccharide O-antigen, O-antigen capsule, and capsule-like complex formed up to 15-fold more biofilm than fully glycosylated cells. The Type B live vaccine strain was also 50% less capable of initiating surface attachment than mutants deficient in O-antigen and capsule-like complex. However, the growth medium of all strains tested also influenced the formation of biofilm, which contained a novel exopolysaccharide consisting of an amylose-like glucan. In addition, the surface polysaccharide composition of the bacterium affected the protein:DNA:polysaccharide composition of the biofilm matrix. In contrast, F . novicida attached to surfaces more efficiently and made a more robust biofilm than Type A or B strains, but loss of O-antigen or capsule-like complex did not significantly affect F . novicida biofilm formation. These results indicated that suppression of surface polysaccharides may promote biofilm formation by F . tularensis Types A and B. Whether biofilm formation enhances survival of F . tularensis in aquatic or other environmental niches has yet to be determined.

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.

SUMMARY Francisella tularensis is a tier 1 select agent responsible for tularemia in humans and a wide variety of animal species. Extensive research into understanding the virulence factors of the bacterium has been ongoing to develop an efficacious vaccine. At least two such virulence factors are described as capsules of F. tularensis: the O-antigen capsule and the capsule-like complex (CLC). These two separate entities aid in avoiding host immune defenses but have not been clearly differentiated. These components are distinct and differ in composition and genetic basis. The O-antigen capsule consists of a polysaccharide nearly identical to the lipopolysaccharide (LPS) O antigen, whereas the CLC is a heterogeneous complex of glycoproteins, proteins, and possibly outer membrane vesicles and tubes (OMV/Ts). In this review, the current understanding of these two capsules is summarized, and the historical references to “capsules” of F. tularensis are clarified. A significant amount of research has been invested into the composition of each capsule and the genes involved in synthesis of the polysaccharide portion of each capsule. Areas of future research include further exploration into the molecular regulation and pathways responsible for expression of each capsule and further elucidating the role that each capsule plays in virulence.