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.

Genetic and molecular analyses reveal an evolutionary trajectory for glycan synthesis in a bacterial protein glycosylation system

“…Some systems display robust inter-and intrastrain glycan variability, suggesting that glycoform diversification is adaptive. For example, flagellar protein glycosylation systems in Campylobacter jejuni and Clostridium species display remarkable plasticity in glycoform expression (2)(3)(4). However, precise correlations between glycosylation gene content and glycoform phenotypes have been difficult to establish.…”

O-Glycosylation of the N-terminal Region of the Serine-rich Adhesin Srr1 of Streptococcus agalactiae Explored by Mass Spectrometry

“…This mechanism has long been considered to be restricted to O-glycosylation like in Campylobacter, Helicobacter, or Pseudomonas spp. for flagellar proteins (12)(13)(14)(15). However, it was recently demonstrated that HMW1c, a GT of Haemophilus influenzae, was responsible for the cytoplasmic N-glycosylation of the adhesin HMW1 (16,17).…”

“…GmaR was the first prokaryotic enzyme of this type to be characterized (20). The type of sugar added to flagellins and the number of modified sites is different between species and is even straindependent in Clostridium difficile (13). Genomic analysis suggests that flagellin glycosylation in Clostridium spp.…”

“…The glycosylation of flagellins has been demonstrated in Clostridium spp. and in Listeria monocytogenes (13,18); the modification is required for flagella assembly in C. difficile (13) and motility in L. monocytogenes (19,20). In L. monocytogenes, the flagellin monomer is modified with N-acetylglucosamine (GlcNAc) residues at up to six different sites by GmaR, a cytoplasmic O-GlcNAc-transferase (OGT) homologous to eukaryotic OGTs (18).…”

In Vitro Activity of Neisseria meningitidis PglL O-Oligosaccharyltransferase with Diverse Synthetic Lipid Donors and a UDP-activated Sugar

“…In this case, cytosolic Leloir (nucleotide-dependent) glycosyltransferases transfer the sugars from nucleotide-activated donors to the protein acceptors. For example, bacterial species such as Clostridium difficile, Haemophilus influenzae, and Helicobacter pylori glycosylate adhesins and flagellins using specialized N-and O-glycosyltransferases in the bacterial cytoplasm (2)(3)(4). Alternatively, glycosylation may occur via an en bloc mechanism, in which the sugars are assembled onto a lipid carrier before being transferred to acceptor proteins through the activity of specialized non-Leloir glycosyltransferases collectively known as "oligosaccharyltransferases" (OTases).…”