lipid-a and Tularemia

Francisella tularensis causes the zoonotic disease tularemia. Arthropod vectors are important transmission routes for the disease, although it is not known how Francisella survives the efficient arthropod immune response. Here, we used Drosophila melanogaster as a model host for Francisella infections and investigated whether the bacteria are resistant to insect humoral immune responses, in particular to the antimicrobial peptides (AMPs) secreted into the insect hemolymph. Moreover, we asked to what extent such resistance might depend on lipopolysaccharide (LPS) structure and surface characteristics of the bacteria. We analyzed Francisella novicida mutant strains in genes, directly or indirectly involved in specific steps of LPS biosynthesis, for virulence in wild-type and Relish(E20) immune-deficient flies, and tested selected mutants for sensitivity to AMPs in vitro. We demonstrate that Francisella is sensitive to specific fly AMPs, i.e. Attacin, Cecropin, Drosocin and Drosomycin. Furthermore, six bacterial genes, kpsF, manB, lpxF, slt, tolA and pal, were found to be required for resistance to Relish-dependent immune responses, illustrating the importance of structural details of Francisella lipid A and Kdo core for interactions with AMPs. Interestingly, a more negative surface charge and lack of O-antigen did not render mutant bacteria more sensitive to cationic AMPs and did not attenuate virulence in flies.

Topics: Animals; Antimicrobial Cationic Peptides; Arthropod Vectors; Disease Models, Animal; Drosophila melanogaster; Drosophila Proteins; Francisella tularensis; Genes, Bacterial; Immunity; Insect Proteins; Lipid A; Lipopolysaccharides; Mutation; Organisms, Genetically Modified; Sugar Acids; Transcription Factors; Tularemia

Francisella tularensis (Ft) is a highly infectious gram-negative bacterium and the causative agent of the human disease tularemia. Ft is designated a class A select agent by the Centers for Disease Control and Prevention. Human clinical isolates of Ft produce lipid A of similar structure to Ft subspecies novicida (Fn), a pathogen of mice. We identified three enzymes required for Fn lipid A carbohydrate modifications, specifically the presence of mannose (flmF1), galactosamine (flmF2), or both carbohydrates (flmK). Mutants lacking either galactosamine (flmF2) or galactosamine/mannose (flmK) addition to their lipid A were attenuated in mice by both pulmonary and subcutaneous routes of infection. In addition, aerosolization of the mutants (flmF2 and flmK) provided protection against challenge with wild-type (WT) Fn, whereas subcutaneous administration of only the flmK mutant provided protection from challenge with WT Fn. Furthermore, infection of an alveolar macrophage cell line by the flmK mutant induced higher levels of tumor necrosis factor-alpha (TNF-alpha) and macrophage inhibitory protein-2 (MIP-2) when compared to infection with WT Fn. Bone marrow-derived macrophages (BMMø) from Toll-like receptor 4 (TLR4) and TLR2/4 knockout mice infected with the flmK mutant also produced significantly higher amounts of interleukin-6 (IL-6) and MIP-2 than BMMø infected with WT Fn. However, production of IL-6 and MIP-2 was undetectable in BMMø from MyD88(-/-) mice infected with either strain. MyD88(-/-) mice were also susceptible to flmK mutant infection. We hypothesize that the ability of the flmK mutant to activate pro-inflammatory cytokine/chemokine production and innate immune responses mediated by the MyD88 signaling pathway may be responsible for its attenuation, leading to the induction of protective immunity by this mutant.

Topics: Animals; Biomarkers; Bone Marrow Cells; Cell Line; Disease Models, Animal; Female; Francisella tularensis; Gene Silencing; Genes, Bacterial; Immunity, Innate; Lipid A; Macrophages, Alveolar; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Mutation; Myeloid Differentiation Factor 88; Signal Transduction; Specific Pathogen-Free Organisms; Tularemia

The present study examines the relationship between the structure and important biological effects of the lipopolysaccharide (LPS) of the intracellular bacterial pathogen, Francisella tularensis LVS. It shows that treating mice with sub-immunogenic amounts of intact F. tularensis LPS rapidly induces an enhanced resistance to intradermal or aerogenic challenge with strains of the pathogen of varying virulence. However, neither the free Lipid A nor core-O-chain produced by mild acid hydrolysis of LPS appeared able to elicit this host defense mechanism.

Topics: Aerosols; Animals; Antigens, Bacterial; Bacterial Vaccines; Female; Francisella tularensis; Immunity, Innate; Injections, Intradermal; Lipid A; Mice; Mice, Inbred BALB C; O Antigens; Specific Pathogen-Free Organisms; Tularemia; Vaccination; Virulence