Identification and Characterization of Glycoproteins on the Spore Surface of Clostridium difficile

Abstract

In this study, we identify a major spore surface protein, BclA, and provide evidence that this protein is glycosylated. Following extraction of the spore surface, solubilized proteins were separated by one-dimensional PAGE and stained with glycostain to reveal a reactive high-molecular-mass region of approximately 600 kDa. Tandem mass spectrometry analysis of in-gel digests showed this band to contain peptides corresponding to a putative exosporangial glycoprotein (BclA3) and identified a number of glycopeptides modified with multiple N-acetyl hexosamine moieties and, in some cases, capped with novel glycans. In addition, we demonstrate that the glycosyltransferase gene sgtA (gene CD3350 in strain 630 and CDR3194 in strain R20291), which is located immediately upstream of the bclA3 homolog, is involved in the glycosylation of the spore surface, and is cotranscribed with bclA3. The presence of anti-␤-O-GlcNAc-reactive material was demonstrated on the surface of spores by immunofluorescence and in surface extracts by Western blotting, although each strain produced a distinct pattern of reactivity. Reactivity of the spore surface with the anti-␤-O-GlcNAc antibody was abolished in the 630 and R20291 glycosyltransferase mutant strains, while complementation with a wild-type copy of the gene restored the ␤-O-GlcNAc reactivity. Phenotypic testing of R20291 glycosyltransferase mutant spores revealed no significant change in sensitivity to ethanol or lysozyme. However, a change in the resistance to heat of R20291 glycosyltransferase mutant spores compared to R20291 spores was observed, as was the ability to adhere to and be internalized by macrophages.

The Exosporium Layer of Bacterial Spores: a Connection to the Environment and the Infected Host

“…ClosTron-generated mutants of the bclA1 and bclA2 determinants produced spores with altered surfaces, including release of "sheets of coat-like material" from the spores (188). BclA3 was identified as a glycosylated protein in extracts of C. difficile spores, and the glycosyltransferase gene sgtA, carried upstream of bclA3, is at least partly responsible for glycosylation of the spore surface (189).…”

Nasal immunization with the C-terminal domain of BclA3 induced specific IgG production and attenuated disease symptoms in mice infected withClostridioides difficilespores

“…In the hyper-virulent strain R20291 of C. difficile the BclA proteins showed 56% similarity with the BclA protein of Bacillus anthracis [10], known to be highly immunogenic and to act as spore surface ligand for α2β1 integrin present in IECs driving spore entry into the epithelial barrier [11]. Further investigation needs to be done in order to fully understand the role of BclA proteins in C. difficile spores however, strong evidence suggest that these proteins are involved in the formation of the hair-like projections in most C. difficile strains such as the hyper-virulent strain R20291 [12,13] and their morphology and localization proposes them as potential antigens [14].…”

“…BclA3 is composed by an N-terminal domain, possibly oriented to the inside, a collagen-like domain formed by GXX repeats, which is highly glycosylated [10], and a C-terminal domain that is presumably faced outwards of the exosporium [17]. Mutant C. difficile spores lacking BclA3 have shown a reduced colonization efficiency in a mice infection model [16] and a recent study has shown that several glycosylated peptides from BclA3 were able to induce humoral immune response in mice [19].…”

Vegetative Cell and Spore Proteomes ofClostridioides difficileshow finite differences and reveal potential biomarkers.

“…Shortly after, we used a gel-free proteomic method and a one-pot sample processing method that focused on the spore surface layers of B. cereus and C. difficile (22). Moreover, previous studies have described exosporium removal methods for C. difficile spores, and examined the exosporium protein components (23) as well as spore surface glycoproteins (24). Yet, none of these studies focusses on a comparative analysis that functionally links the spore and vegetative cell proteome.…”