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

Abstract

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.

Francisella tularensis D-Ala D-Ala Carboxypeptidase DacD Is Involved in Intracellular Replication and It Is Necessary for Bacterial Cell Wall Integrity

“…Therefore, we performed the serum bactericidal assay in which the wild-type, FSC200/ in dacD mutant, both complemented mutant strains, and the FSC200/ wbtDEF::Cm strain lacking LPS, were exposed to human nonimmune serum. The significant sensitivity of FSC200/ wbtDEF::Cm strain to human serum on the one hand and the resistance of FSC200 on the other hand were consistent with previously published data (Dankova et al, 2016 ). The FSC200/ in dacD mutant strain also exhibited some degree of sensitivity to complement-mediated lysis.…”

Understanding Protein Glycosylation Pathways in Bacteria

“…Interestingly, the mutation of wbtC gene from LPS biosynthetic locus resulted in not only altered LPS but also reduced protein glycosylation [66]. Similarly the mutation of the putative glycan flippase gene FTS_1402 in the FCS200 strain lead to alteration in protein glycosylation and also a decrease in LPS production [65], suggesting a cross-talk between LPS biosynthesis and protein glycosylation in F.…”

“…The OST involved in the en bloc glycosylation was identified as PglA (encoded by FTL_0425 in FCS200 strain, and FTT_0905 in SchuS4 strain) [36]. The 12.5 kB gene cluster FTT0789-FTT0800 in the SchuS4 strain was shown to be involved in the assembly and translocation of the hexasaccharide [64,65]. Interestingly, the mutation of wbtC gene from LPS biosynthetic locus resulted in not only altered LPS but also reduced protein glycosylation [66].…”

Role of Glycosylation/Deglycolysation Processes in Francisella tularensis Pathogenesis

“…OSTs substrates are lipid-linked oligosaccharides while the GTs substrates are usually nucleotide-activated sugars. It was only very recently (Dankova et al, 2016) that the glycosylation machinery of Francisella was found to involve a variety of sugar biogenesis enzymes, glycosyltransferases, a flippase, and a protein-targeting oligosaccharyltransferase. As both type A and type B strains of F. tularensis subspecies expressed an O -linked protein glycosylation system, which utilizes core biosynthetic and assembly pathways, O -linked protein glycosylation may be a feature common to members of the Francisella genus (Egge-Jacobsen et al, 2011).…”

“…This protein is part of a gene cluster, which is thought to encode a polysaccharide additional to the lipopolysaccharide O antigen. Another protein, encoded by FTS_1402 , was found to be involved in glycoprotein synthesis and to also contribute in part to LPS/capsule and/or Capsule Like Complex (CLC) production (Dankova et al, 2016). The resulting FTS_1402 mutant presented more sensitivity to serum complement.…”