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 high resolution, high mass accuracy mass spectrometry, combined with in-source CID tandem MS experiments. The glycan observed at m/z 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 to that of DsbA glycoprotein, as well as to 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 inter-relationship between the O-antigen and the glycan synthesis in the early steps in their biosynthetic pathways.

• MeSH
• faktory virulence chemie   genetika   metabolismus   MeSH
• Francisella tularensis genetika   metabolismus   patogenita   MeSH
• glykosylace MeSH
• molekulární sekvence - údaje MeSH
• multigenová rodina MeSH
• mutace MeSH
• O-antigeny chemie   genetika   metabolismus   MeSH
• proteiny fimbrií chemie   genetika   metabolismus   MeSH
• sacharidové sekvence MeSH
• sekvence aminokyselin MeSH
• tandemová hmotnostní spektrometrie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• faktory virulence MeSH
• O-antigeny MeSH
• PilA protein, Francisella tularensis MeSH Prohlížeč
• proteiny fimbrií MeSH

It appears that most glycoproteins found in pathogenic bacteria are associated with virulence. Despite the recent identification of novel virulence factors, the mechanisms of virulence in Francisella tularensis are poorly understood. In spite of its importance, questions about glycosylation of proteins in this bacterium and its potential connection with bacterial virulence have not been answered yet. In the present study, several putative Francisella tularensis glycoproteins were characterized through the combination of carbohydrate-specific detection and lectin affinity with highly sensitive mass spectrometry utilizing the bottom-up proteomic approach. The protein PilA that was recently found as being possibly glycosylated, as well as other proteins with designation as novel factors of virulence, were among the proteins identified in this study. The reported data compile the list of potential glycoproteins that may serve as a takeoff platform for a further definition of proteins modified by glycans, faciliting a better understanding of the function of protein glycosylation in pathogenicity of Francisella tularensis.

• MeSH
• 2D gelová elektroforéza MeSH
• bakteriální proteiny chemie   metabolismus   MeSH
• chromatografie afinitní MeSH
• fluorescenční barviva MeSH
• Francisella tularensis chemie   metabolismus   MeSH
• glykoproteiny chemie   metabolismus   MeSH
• glykosylace MeSH
• lektiny MeSH
• molekulární sekvence - údaje MeSH
• polysacharidy metabolismus   MeSH
• proteom chemie   metabolismus   MeSH
• proteomika metody   MeSH
• sekvence aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• fluorescenční barviva MeSH
• glykoproteiny MeSH
• lektiny MeSH
• polysacharidy MeSH
• proteom MeSH