Francisella tularensis, an intracellular pathogen causing the disease tularemia, utilizes surface glycoconjugates such as lipopolysaccharide, capsule, and capsule-like complex for its protection against inhospitable conditions of the environment. Francisella species also possess a functional glycosylation apparatus by which specific proteins are O-glycosidically modified. We here created a mutant with a nonfunctional FTS_1402 gene encoding for a putative glycan flippase and studied the consequences of its disruption. The mutant strain expressed diminished glycosylation similarly to, but to a lesser extent than, that of the oligosaccharyltransferase-deficient ΔpglA mutant. In contrast to ΔpglA, inactivation of FTS_1402 had a pleiotropic effect, leading to alteration in glycosylation and, importantly, to decrease in lipopolysaccharide, capsule, and/or capsule-like complex production, which were reflected by distinct phenotypes in host-pathogen associated properties and virulence potential of the two mutant strains. Disruption of FTS_1402 resulted in enhanced sensitivity to complement-mediated lysis and reduced virulence in mice that was independent of diminished glycosylation. Importantly, the mutant strain induced a protective immune response against systemic challenge with homologous wild-type FSC200 strain. Targeted disruption of genes shared by multiple metabolic pathways may be considered a novel strategy for constructing effective live, attenuated vaccines.

Department of Molecular Pathology and Biology Faculty of Military Health Sciences University of Defence Hradec Kralove 500 01 Czech Republic

Department of Phototrophic Microorganisms Institute of Microbiology The Academy of Sciences of The Czech Republic Trebon 379 81 Czech Republic

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