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.
Highlights
Francisella tularensis is an extremely infectious gram-negative bacterium which is readily aerosolized
Host-adapted F. tularensis display a phenotype distinct from that of bacteria cultivated in vitro using standard media such as in Mueller Hinton broth (MHB) or Chamberlain’s defined media (CDM) [31,32]
We reported that growth of F. tularensis in BHI induces a phenotype that is indistinguishable from that of bacteria which have emerged from infected MW [12]
Summary
Francisella tularensis is an extremely infectious gram-negative bacterium which is readily aerosolized. Inhalation of this Category A select agent can lead to pulmonary tularemia which has a mortality rate of ,35% in the absence of treatment. Antibiotic-resistant strains of this bacterium were developed by at least one nation’s biological weapons program [1]. The specter of such an agent being maliciously employed, in concert with the current lack of a licensed tularemia vaccine, has evoked a ground swell of interest in F. tularensis. The 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. 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
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