Motility and flagellar glycosylation in Clostridium difficile

Abstract

In this study, intact flagellin proteins were purified from strains of Clostridium difficile and analyzed using quadrupole time of flight and linear ion trap mass spectrometers. Top-down studies showed the flagellin proteins to have a mass greater than that predicted from the corresponding gene sequence. These top-down studies revealed marker ions characteristic of glycan modifications. Additionally, diversity in the observed masses of glycan modifications was seen between strains. Electron transfer dissociation mass spectrometry was used to demonstrate that the glycan was attached to the flagellin protein backbone in O linkage via a HexNAc residue in all strains examined. Bioinformatic analysis of C. difficile genomes revealed diversity with respect to glycan biosynthesis gene content within the flagellar biosynthesis locus, likely reflected by the observed flagellar glycan diversity. In C. difficile strain 630, insertional inactivation of a glycosyltransferase gene (CD0240) present in all sequenced genomes resulted in an inability to produce flagellar filaments at the cell surface and only minor amounts of unmodified flagellin protein.

FIG. 1.

Characterization of C. difficile isolates. The upper panel shows motility stabs of clinical isolates (0.175% agar). Motility was clearly visible in agar stabs for all strains with the exception of CM-26. The lower panel depicts an SDS-PAGE gel of FliC protein extracts. Lane 1, C. difficile 630; l ane 2, C. difficile BI-1; l ane 3, C. difficile BI-7; l ane 4, C. difficile QCD32g58; l ane 5, C. difficile M23257; l ane 6, C. difficile M46846; l ane 7, C. difficile M7465; l ane 8, C. difficile M9349; l ane 9, CM- 26; l ane 10, CM- 56; l ane 11, 06CD130. Molecular masses (in kDa) are shown to the left of the gel.

FIG. 2.

Electron microscopy of uranyl acetate-stained cells. (A) C. difficile 630. (B) C. difficile QCD32g58. (C) C. difficile 630::0240 erm. (D) C. difficile 630::fliC erm.

FIG. 3.

Electrospray MS analyses of intact flagellin from strains of C. difficile. (A) The reconstructed molecular mass profile of FliC from C. difficile 630, showing two peaks at 33,559 and 33,160 Da. These are 2,804 and 2,405 Da larger than that predicted from the translated gene sequence of fliC and correspond to the attachments of six and seven glycan moieties of 398 Da. No signal corresponding to the unmodified flagellin was observed. (B) The reconstructed molecular mass profile of FliC from C. difficile BI-1, showing intense peaks at 35,106, 35,473, and 35,841 Da and less intense peaks at 36,208 and 37,186 Da, all larger than the mass of FliC, which was predicted to be 30,901 Da. (C) Tandem MS analysis of the multiply charged protein ion from C. difficile strain 630 at m/z 1,119.7³⁰⁺. Intense ions were observed at m/z 399.1, the putative glycan observed to modify FliC from this strain, and glycan-associated fragment ions were observed at m/z 284.1, 214.1, and 116.1 (indicated by an asterisk). (D) MS/MS analysis of the multiply charged protein ion from C. difficile BI-1 m/z 1,140.7³¹⁺. Numerous ions were observed in the resulting MS/MS spectrum, including glycan-associated ions at m/z 524.1, 364.1, 204.1, and 161.1. MS/MS spectra were acquired at collision energy of 15 to 35 V by using argon as the collision gas.

FIG. 4.

MS analysis of modified tryptic peptides from flagellar proteins of C. difficile strains 630 and BI-1. (A) nLC-MS/MS spectrum of the doubly protonated T^167-190 glycopeptide ion at 1,581.8²⁺ from C. difficile 630. The spectrum is dominated in the high-molecular-m/z region by the sequential loss of two carbohydrate moieties. Two sequential losses of m/z 199.1²⁺ corresponds to the loss of a doubly charged glycan of a residue mass of 398.1 Da (indicated by the arrow). The low-m/z region of the spectrum is dominated by the carbohydrate oxonium ion (m/z 399.1) and what could be a fragmentation component (m/z 214.1). (B) nLC-MS/MS spectrum of the doubly protonated T^135-144 glycopeptide ion 807.4²⁺ from C. difficile BI-1. The high-m/z region is dominated by the sequential loss of m/z 160.1, 160.1, and 203.1 from what appears to be a trisaccharide. Oxonium ions are visible at m/z 204.1 (HexNAc) and m/z 161.1. The glycan appears to be O linked through the HexNAc residue.

FIG. 5.

Analysis of the unusual carbohydrates obtained by nano-electrospray ionization-front-end collision-induced dissociation MS/MS. (A) MS/MS spectra of the oxonium ion at m/z 399.1. Intense fragment ions were observed at m/z 284.1 and 214.1, with weaker ions at m/z 204.1 and 116.1. (B) MS/MS spectrum of glycan fragment ion at m/z 284.1. Neutral loss of 98 Da from the parent ion resulted in a fragment ion at m/z 186.1. (C) MS/MS spectrum of glycan fragment ion at m/z 214.1. A neutral loss of 98 Da resulted in an observed fragment ion at m/z 116.1.

FIG. 6.

Schematic diagram showing the genetic organization of the flagellar glycosylation locus in C. difficile strains. The diagram shows the region of the chromosome flanked by the fliC and flgB structural genes, which encode flagellin (FliC) and FlgB (rod protein), respectively. Flagellar structural genes fliC and flgB are colored black; for the flagellar glycosylation locus genes, glycosyltransferase genes are dark gray and precursor glycan biosynthesis genes are light gray. In the C. difficile 630 locus, the genes annotated as a phosphatase gene (CD0241), sugar nucleotide transferase gene (CD0242), and hypothetical ORF (CD0243) are colored light grey. The available shotgun sequence data for strain QCD32g58 (orf2/GT2 and orf3/GT3) and for CIP107932 (orf1/GT1) likely contain sequencing errors, which result in truncation of the respective ORF. The schematic displays the full-length ORF for each of these genes.

FIG. 7.

Characterization of C. difficile mutants. (A) SDS-PAGE analysis of flagellin extracts of C. difficile 630 (lane 1), C. difficile 630::0240erm (lane 2), and C. difficile 630::fliCerm (lane 3). Molecular masses (in kDa) are shown to the left of the gel. Asterisk, protein of a molecular mass corresponding to unmodified flagellin. (B) Motility stabs (0.175% agar) show C. difficile 630 (lane 1), C. difficile 630::0240erm (lane 2), and C. difficile 630::fliCerm (lane 3).