Host-Adaptation of Francisella tularensis Alters the Bacterium's Surface-Carbohydrates to Hinder Effectors of Innate and Adaptive Immunity

Abstract

BackgroundThe gram-negative bacterium Francisella tularensis survives in arthropods, fresh water amoeba, and mammals with both intracellular and extracellular phases and could reasonably be expected to express distinct phenotypes in these environments. The presence of a capsule on this bacterium has been controversial with some groups finding such a structure while other groups report that no capsule could be identified. Previously we reported in vitro culture conditions for this bacterium which, in contrast to typical methods, yielded a bacterial phenotype that mimics that of the bacterium's mammalian, extracellular phase.Methods/FindingsSDS-PAGE and carbohydrate analysis of differentially-cultivated F. tularensis LVS revealed that bacteria displaying the host-adapted phenotype produce both longer polymers of LPS O-antigen (OAg) and additional HMW carbohydrates/glycoproteins that are reduced/absent in non-host-adapted bacteria. Analysis of wildtype and OAg-mutant bacteria indicated that the induced changes in surface carbohydrates involved both OAg and non-OAg species. To assess the impact of these HMW carbohydrates on the access of outer membrane constituents to antibody we used differentially-cultivated bacteria in vitro to immunoprecipitate antibodies directed against outer membrane moieties. We observed that the surface-carbohydrates induced during host–adaptation shield many outer membrane antigens from binding by antibody. Similar assays with normal mouse serum indicate that the induced HMW carbohydrates also impede complement deposition. Using an in vitro macrophage infection assay, we find that the bacterial HMW carbohydrate impedes TLR2-dependent, pro-inflammatory cytokine production by macrophages. Lastly we show that upon host-adaptation, the human-virulent strain, F. tularensis SchuS4 also induces capsule production with the effect of reducing macrophage-activation and accelerating tularemia pathogenesis in mice.ConclusionF. tularensis undergoes host-adaptation which includes production of multiple capsular materials. These capsules impede recognition of bacterial outer membrane constituents by antibody, complement, and Toll-Like Receptor 2. These changes in the host-pathogen interface have profound implications for pathogenesis and vaccine development.

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).…”

TolC-Dependent Modulation of Host Cell Death by the Francisella tularensis Live Vaccine Strain

“…Francisella mutants that are structurally compromised exhibit increased sensitivity to reactive oxygen and nitrogen species (ROS and RNS, respectively) and are susceptible to the bactericidal action of complement (50,54,55). We observed no differences between the WT and ⌬tolC LVS in sensitivity to ROS-or RNSinducing compounds (H 2 O 2 , tert-butyl hydroperoxide, and peroxynitrite) (see Fig.…”

“…In addition, the WT and mutant bacteria exhibited similar resistance to mouse serum (see Fig. S2E), indicating no change in complement sensitivity and suggesting that the exopolysaccharide capsule of the ⌬tolC mutant is not altered (54). To test further the integrity of the bacteria, we assessed permeability to Sytox green, a DNA binding stain normally excluded by the bacterial envelope, and examined the ultrastructure of the bacteria by electron microscopy.…”

Subversion of Host Recognition and Defense Systems by Francisella spp

“…Mechanisms that the bacteria use to resist damage by antimicrobials, and therefore the release of BLPs, are also an indirect way of evading TLR2 signaling. It has been reported that high-molecular-weight (HMW) carbohydrates from "host-adapted" LVS and F. tularensis capsules impede TLR2-dependent cytokine production in murine macrophages (240), possibly by shielding the bacteria from antimicrobials. Taken together, these data indicate that Francisella is capable of subverting TLR2 signaling, while the host uses this pathway as a mechanism of innate defense.…”