The Type B Flagellin of Hypervirulent Clostridium difficile Is Modified with Novel Sulfonated Peptidylamido-glycans

Abstract

Glycosylation of flagellins is a well recognized property of many bacterial species. In this study, we describe the structural characterization of novel flagellar glycans from a number of hypervirulent strains of C. difficile. We used mass spectrometry (nano-LC-MS and MS/MS analysis) to identify a number of putative glycopeptides that carried a variety of glycoform substitutions, each of which was linked through an initial N-acetylhexosamine residue to Ser or Thr. Detailed analysis of a LLDGSSTEIR glycopeptide released by tryptic digestion, which carried two variant structures, revealed that the glycopeptide contained, in addition to carbohydrate moieties, a novel structural entity. A variety of electrospray-MS strategies using Q-TOF technology were used to define this entity, including positive and negative ion collisionally activated decomposition MS/MS, which produced unique fragmentation patterns, and high resolution accurate mass measurement to allow derivation of atomic compositions, leading to the suggestion of a taurine-containing peptidylamido-glycan structure. Finally, NMR analysis of flagellin glycopeptides provided complementary information. The glycan portion of the modification was assigned as α-Fuc3N-(1→3)-α-Rha-(1→2)-α-Rha3OMe-(1→3)-β-GlcNAc-(1→)Ser, and the novel capping moiety was shown to be comprised of taurine, alanine, and glycine. This is the first report of a novel O-linked sulfonated peptidylamido-glycan moiety decorating a flagellin protein.

Role of Glycosyltransferases Modifying Type B Flagellin of Emerging Hypervirulent Clostridium difficile Lineages and Their Impact on Motility and Biofilm Formation

“…Based on the mass spectrometric data of the mutants and taking into account the flagellin type B sulfonated peptidylamido-glycan structure reported by Bouché et al (25), we conclude that the glycosyltransferases GT1 and GT2 are responsible for the sequential addition of the GlcNAc and two rhamnoses sugars, respectively, and that GT3, CDR20291_0245, and CDR20291_0246 are all involved in the biosynthesis of the terminal sulfonated peptidylamido-sugar moiety. Given that our bioinformatics analysis suggests that GT2 could also function as a methyltransferase, we hypothesize that GT2 is additionally responsible for methylation of the rhamnoses (Table 1 and Fig.…”

“…Flagellin isolated from the GT2 mutant harbored glycopeptides with a single HexNAc moiety but lacking any further extension of the glycan. Flagellin isolated from the GT3 mutant was modified by a deoxyHex-deoxyHex-HexNAc (with and without methyl groups) but lacking the terminal sulfonated peptidylamido-sugar moiety observed in the wild type (25). Similarly, the CDR20291_0245 and CDR20291_0246 mutants carried the non-extended core trisaccharide (Table 1).…”

“…Moreover, flagellin is immunogenic and protective in a murine model of C. difficile infection (34). However, little is known about the role of protein PTM in C. difficile disease, which has a type A or type B modification (23, 25). It has also been reported that type A post-translational modification of flagellin in C. difficile 630 strain has a contributory effect in pathogenesis (23).…”

“…Previous genetic analysis of the hypervirulent RT027 strain revealed that the gene lying downstream of fliC in the type B modification, glycosyltransferase 1 (GT1 (CDR20291_0241)), was highly similar to CD0240 of 630 (21). Although the function of the GT1 protein is yet to be confirmed, the first sugar of the type B PTM is also an O -linked N -acetylhexosamine (HexNAc) residue, indicating that the GT1 protein may have a function similar to the CD0240 protein (22, 25). Beyond this initial sugar, however, the RT027 flagellin does not contain a phosphodiester-linked amino acid as in type A flagellin.…”

“…Beyond this initial sugar, however, the RT027 flagellin does not contain a phosphodiester-linked amino acid as in type A flagellin. Instead, it has recently been found to be extended by two rhamnoses and a novel sulfonated peptidyl amidoglycan (25). …”