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.

Keywords: Clostridium difficile; Gram-positive bacteria; bacteria; flagellin; glycosylation; mass spectrometry (MS); modification; nuclear magnetic resonance (NMR); sulfonated.

FIGURE 1.

Positive ion on-line nano-LC MS/MS high resolution CAD mass spectrum of m/z 998²⁺ (main spectrum) with equivalent low resolution MS/MS spectrum of m/z 991²⁺ (inset). Note the low mass signals (below m/z 400), many of which do not correlate with either known peptide or carbohydrate fragments and which therefore indicated the discovery of novel structural features in these glycopeptides. For full interpretation, see “Results” and Scheme 1.

SCHEME 1.

Summary of the interpretation and mechanistic logic used to assess the mass spectrometric fragmentation data determined in this study (see Fig. 1) showing the probable structural assignments. Conclusions were aided by the derivation of atomic compositions of key fragments shown in Table 1.

FIGURE 2.

Positive ion nanospray CAD MS/MS spectrum of m/z 152 produced via cone voltage-induced in-source fragmentation of m/z 998²⁺. Signals at m/z 70 and 108 correspond to losses of 82 and 44 mass units, respectively, and were correlated with the low mass high resolution data from Fig. 1 to deduce atomic compositions for these ions of C₄H₈N and C₂H₆NO₂S. This suggests two overlapping fragments derived from the m/z 152 ion (C₄H₁₀NO₃S in Table 1), an alkylamine and a possible sulfonic acid. For a full interpretation, see “Results” and Scheme 1.

FIGURE 3.

Negative ion nanospray CAD MS/MS spectrum of m/z 996²⁻ (m/z 100–1200 mass range). Negative ion fragmentation data complement and expand those from positive ion experiments, as here for m/z 394 (equivalent to m/z 396 in Fig. 1), m/z 266 (m/z 268), m/z 173 (m/z 175), and m/z 150 (m/z 152). Interestingly, a moderately intense low mass fragment was observed at m/z 124 (lowest mass significant fragment), and this was therefore chosen for further CAD MS/MS analysis; see Fig. 4. The highest mass signal seen at m/z 921 corresponds to the elimination of the complete sulfonated peptidylamido-glycan from the peptide backbone to leave a dehydropeptide (not observed). For a full interpretation, see “Results.”

FIGURE 4.

The negative ion nanospray CAD MS/MS spectrum of m/z 124 produced via cone voltage induced in-source fragmentation of m/z 996²⁻. The base peak (main signal) observed is present at m/z 79.96, interpreted as SO₃^˙̄, together with minor, less informative signals. This spectrum provided the first evidence of the presence of a sulfonic acid group in the new structure. A sample of synthetic taurine was then analyzed, showing an equivalent negative ion spectrum with base peak m/z 79.96.

FIGURE 5.

C. difficile R20291 flagellar glycan compounds. Structures of isolated compounds 1, 2, and 3 are presented. A, B, C, C′, D, E, and F below the structures indicate the assignment of resonances presented in Fig. 6 and Tables 2 and 3. 381, fragment identified by MS analysis in Fig. 1 (inset, m/z 382).

FIGURE 6.

Part of the ¹H-¹³C HSQC spectrum of the mixture of compounds 2 and 3. The signal marked # probably belongs to a structure 2 with an additional Me group, not yet localized. Amino acids marked with superscript numbers are from the peptide part, sequence not determined. Thr* and Ser* are glycosylated.