A Francisella Mutant in Lipid A Carbohydrate Modification Elicits Protective Immunity

Abstract

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-α (TNF-α) 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. Bacterial pathogens modify outer membrane components, such as lipid A or endotoxin, the lipid anchor of lipopolysaccharide, to enhance the ability to colonize, spread to different tissues, and/or avoid the host's immune defenses. Lipopolysaccharide also plays an essential role in maintaining membrane integrity and is a key factor in host innate immune recognition of Gram-negative bacterial infections. Francisella tularensis is the causative agent of the human disease tularemia and is classified as a category A select agent. Francisella novicida (Fn) is the murine counterpart of F. tularensis. The structure of Francisella spp. lipid A is unique in that it is modified by various carbohydrates that play a role in virulence and altered endotoxicity. In our study, we identified and defined the role of three genes involved in the carbohydrate modification of the base Fn lipid A structure. We showed that the lack of specific modification(s) of the Fn lipid A molecule lead to bacterial attenuation and activation of a protective immune response against a lethal wild-type infection. Therefore, alteration of Francisella lipid A structure may represent a pathogenesis strategy common to the Francisella species, and specific lipid A mutant strains may be candidates for inclusion in future vaccine studies.