Abstract
Clostridium difficile is a bacterial pathogen that causes major health challenges worldwide. It has a well-characterized surface (S)-layer, a para-crystalline proteinaceous layer surrounding the cell wall. In many bacterial and archaeal species, the S-layer is glycosylated, but no such modifications have been demonstrated in C. difficile. Here, we show that a C. difficile strain of S-layer cassette type 11, Ox247, has a complex glycan attached via an O-linkage to Thr-38 of the S-layer low-molecular-weight subunit. Using MS and NMR, we fully characterized this glycan. We present evidence that it is composed of three domains: (i) a core peptide–linked tetrasaccharide with the sequence -4-α-Rha-3-α-Rha-3-α-Rha-3-β-Gal-peptide; (ii) a repeating pentasaccharide with the sequence -4-β-Rha-4-α-Glc-3-β-Rha-4-(α-Rib-3-)β-Rha-; and (iii) a nonreducing end–terminal 2,3 cyclophosphoryl-rhamnose attached to a ribose-branched sub-terminal rhamnose residue. The Ox247 genome contains a 24-kb locus containing genes for synthesis and protein attachment of this glycan. Mutations in genes within this locus altered or completely abrogated formation of this glycan, and their phenotypes suggested that this S-layer modification may affect sporulation, cell length, and biofilm formation of C. difficile. In summary, our findings indicate that the S-layer protein of SLCT-11 strains displays a complex glycan and suggest that this glycan is required for C. difficile sporulation and control of cell shape, a discovery with implications for the development of antimicrobials targeting the S-layer.
Highlights
Clostridium difficile is a bacterial pathogen that causes major health challenges worldwide
Our findings indicate that the S-layer protein of SLC types (SLCTs)-11 strains displays a complex glycan and suggest that this glycan is required for C. difficile sporulation and control of cell shape, a discovery with implications for the development of antimicrobials targeting the S-layer
No single function has been described for S-layers; 8 The abbreviations used are: CDI, C. difficile infection; S-layer proteins (SLPs), S-layer protein; ETD, electron transfer dissociation; LMW, low-molecular-weight; HMW, high-molecular-weight; SLC, S-layer cassette; SLCT, SLC type; dH2O, distilled H2O; DMEM, Dulbecco’s modified Eagle’s medium; HSQC, heteronuclear single quantum coherence; TOCSY, total correlation spectroscopy; ROESY, rotating frame nuclear Overhauser enhancement spectroscopy; COSY, two-dimensional correlated spectroscopy; HMBC, heteronuclear multiple bond correlation; Hex, hexose; dHex, deoxyhexose; Pent, pentose
Summary
Clostridium difficile is a bacterial pathogen that causes major health challenges worldwide It has a well-characterized surface (S)-layer, a para-crystalline proteinaceous layer surrounding the cell wall. S-layers are composed primarily of a single species of (glyco)protein covering the entire surface of the cell and present as a two-dimensional array [12, 13]. Species expressing glycosylated S-layers contain a gene locus specifying the synthesis of the glycan chain, its export through the membrane(s), and ligation to the S-layer protein [27, 29]. The genes contained within this locus resemble most closely those present in other Gram-positive species that produce glycosylated S-layers, for example G. stearothermophilus and P. alvei [27]. The roles of key genes are defined by deletion analysis, and the phenotypes associated with glycosylation are investigated
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