Inactivation of <i>Francisella tularensis</i> Gene Encoding Putative ABC Transporter Has a Pleiotropic Effect upon Production of Various Glycoconjugates

<i>Francisella tularensis</i>, 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. <i>Francisella</i> species also possess a functional glycosylation apparatus by which specific proteins are O-glycosidically modified. We here created a mutant with a nonfunctional <i>FTS_1402</i> 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 <i>ΔpglA</i> mutant. In contrast to <i>ΔpglA</i>, inactivation of <i>FTS_1402</i> 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 <i>FTS_1402</i> 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

Inactivation of <i>Francisella tularensis</i> Gene Encoding Putative ABC Transporter Has a Pleiotropic Effect upon Production of Various Glycoconjugates

<i>Francisella tularensis</i>, 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. <i>Francisella</i> species also possess a functional glycosylation apparatus by which specific proteins are O-glycosidically modified. We here created a mutant with a nonfunctional <i>FTS_1402</i> 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 <i>ΔpglA</i> mutant. In contrast to <i>ΔpglA</i>, inactivation of <i>FTS_1402</i> 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 <i>FTS_1402</i> 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

Inactivation of <i>Francisella tularensis</i> Gene Encoding Putative ABC Transporter Has a Pleiotropic Effect upon Production of Various Glycoconjugates

<i>Francisella tularensis</i>, 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. <i>Francisella</i> species also possess a functional glycosylation apparatus by which specific proteins are O-glycosidically modified. We here created a mutant with a nonfunctional <i>FTS_1402</i> 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 <i>ΔpglA</i> mutant. In contrast to <i>ΔpglA</i>, inactivation of <i>FTS_1402</i> 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 <i>FTS_1402</i> 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

Quantitative Proteomics Analysis of Macrophage-Derived Lipid Rafts Reveals Induction of Autophagy Pathway at the Early Time of Francisella tularensis LVS Infection

Francisella tularensis is a highly infectious intracellular pathogen that has evolved an efficient strategy to subvert host defense response to survive inside the host. The molecular mechanisms regulating these host–pathogen interactions and especially those that are initiated at the time of the bacterial entry via its attachment to the host plasma membrane likely predetermine the intracellular fate of pathogen. Here, we provide the evidence that infection of macrophages with F. tularensis leads to changes in protein composition of macrophage-derived lipid rafts, isolated as detergent-resistant membranes (DRMs). Using SILAC-based quantitative proteomic approach, we observed the accumulation of autophagic adaptor protein p62 at the early stages of microbe–host cell interaction. We confirmed the colocalization of the p62 with ubiquitinated and LC3-decorated intracellular F. tularensis microbes with its maximum at 1 h postinfection. Furthermore, the infection of p62-knockdown host cells led to the transient increase in the intracellular number of microbes up to 4 h after in vitro infection. Together, these data suggest that the activation of the autophagy pathway in F. tularensis infected macrophages, which impacts the early phase of microbial proliferation, is subsequently circumvented by ongoing infection