Characterization of Protein Glycosylation in Francisella tularensis subsp. holarctica; Identification of a Novel Glycosylated Lipoprotein Required for Virulence

Authors

Balonova, L., Mann, B.F., Cerveny, L., Alley, W.R., Chovancova, E., Forslund, A.-L., Salomonsson, E.N., Forsberg, Å., Damborsky, J., Novotny, M.V., Hernychova, L., Stulik, J.

Source

MOLECULAR & CELLULAR PROTEOMICS 11: M111.015016 (2012)

Abstract

FTH_0069 is a previously uncharacterized strongly immunoreactive protein that has been proposed to be a novel virulence factor in Francisella tularensis. Here, the glycan structure modifying two C-terminal peptides of FTH_0069 was identified utilizing a high resolution, high mass accuracy mass spectrometry, combined with an in-source CID tandem MS experiments. The glycan observed at an m/z value of 1156 was determined to be a hexasaccharide, consisting of two hexoses, three N-acetylhexosamines, and an unknown monosaccharide containing a phosphate group. The monosaccharide sequence of the glycan is tentatively proposed as X-P-HexNAc-HexNAc-Hex-Hex-HexNAc, where X denotes the unknown monosaccharide. The glycan is identical with that of DsbA glycoprotein, as well as with one of the multiple glycan structures modifying the type IV pilin PilA, suggesting a common biosynthetic pathway for the protein modification. Here, we demonstrate that the glycosylation of FTH_0069, DsbA, and PilA was affected in an isogenic mutant with a disrupted wbtDEF gene cluster encoding O-antigen synthesis and in a mutant with a deleted pglA gene encoding pilin oligosaccharyltransferase PglA. Based on our findings we propose that PglA is involved in both pilin and general F. tularensis protein glycosylation and we further suggest an interrelationship between the O-antigen and the glycan synthesis in early steps in their biosynthetic pathways.

Full text

Citation

Balonova, L., Mann, B.F., Cerveny, L., Alley, W.R., Chovancova, E., Forslund, A.-L., Salomonsson, E.N., Forsberg, Å., Damborsky, J., Novotny, M.V., Hernychova, L., Stulik, J., 2012: Characterization of Protein Glycosylation in Francisella tularensis subsp. holarctica; Identification of a Novel Glycosylated Lipoprotein Required for Virulence. Molecular & Cellular Proteomics 11: M111.015016.

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