From the Department of Life Sciences, Imperial College London, SW7 2AZ London, United Kingdom, the Vaccine Program, Human Health Therapeutics Research Centre, National Research Council, Ottawa, Ontario K1A 0R6, Canada, Biopharmaspec, Suite 3.1, Lido Medical Centre, St. Saviours Road, JE2 7LA Jersey, United Kingdom, the London School of Hygiene and Tropical Medicine, WC1E 7HT, London, United Kingdom
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.
13C NMR, 1H NMR, gel filtration, NMR-2D, GC-MS, SDS-PAGE, sugar analysis, DNA techniques, anion-exchange chromatography, MS/MS, LC-ESI-MS, MALDI-TOF/TOF MS, biofilm assays, bioinformatics analysis, adhesion assays, sporulation assays
13C NMR data:
Linkage Residue C1 C2 C3 C4 C5 C6
3,3,3,3,4,4,3,4,4,4,3,4 bDRhap 101.9 71.2 72.7 83.7 72.1 17.9
3,3,3,3,4,4,3,4,4,4,3 aDGlcp 96.5 72.5 73.9 77.9 71.6 61.6
3,3,3,3,4,4,3,4,4,4 bDRhap 101.0 68.1 78.9 73.5 73.6 17.9
3,3,3,3,4,4,3,4,4,3 aDRibf 99.0 72.5 71.1 86.4 62.6
3,3,3,3,4,4,3,4,4 bDRhap 101.4 68.1 75.5 77.8 72.9 18.2
3,3,3,3,4,3 aDRibf 99.0 72.5 71.1 86.4 62.6
3,3,3,3,4,4,3,4,4,4,3,4,4,3 aDRibf 99.4 72.6 71.5 86.5 62.6
3,3,3,3,4,4,3,4,4,4,3,4,4,4,3 P
3,3,3,3,4,4,3,4,4,4,3,4,4,4 bDRhap 99.6 75.8 81.1 74.1 72.3 17.9
3,3,3,3,4,4,3,4,4,4,3,4,4 bDRhap 101.4 68.4 75.5 78.2 72.9 18.2
3,3,3,3,4,4,3,4 bDRhap 101.9 71.2 72.7 83.7 72.1 17.9
3,3,3,3,4,4,3 aDGlcp 96.5 72.5 73.9 77.9 71.6 61.6
3,3,3,3,4,4 bDRhap 101.0 68.1 78.9 73.5 73.6 17.9
3,3,3,3,4 bDRhap 101.4 68.1 75.5 77.8 72.9 18.2
3,3,3,3 aDRhap 103.3 71.2 70.2 83.8 68.7 17.9
3,3,3 aDRhap 103.3 71.2 79.7 72.5 70.5 17.9
3,3 aDRhap 103.3 71.2 79.4 72.5 70.4 17.9
3 bDGalp 102.0 71.3 81.3 69.6 76.3 62.1
x?Thr ? 59.5 75.3 17.0
1H NMR data:
Linkage Residue H1 H2 H3 H4 H5 H6
3,3,3,3,4,4,3,4,4,4,3,4 bDRhap 4.90 4.15 3.72 3.54 3.54 1.34
3,3,3,3,4,4,3,4,4,4,3 aDGlcp 5.10 3.59 3.93 3.68 4.01 3.80-3.89
3,3,3,3,4,4,3,4,4,4 bDRhap 4.77 4.24 3.68 3.44 3.43 1.34
3,3,3,3,4,4,3,4,4,3 aDRibf 5.31 4.14 3.99 4.35 3.70-3.76
3,3,3,3,4,4,3,4,4 bDRhap 4.76 4.34 3.94 3.74 3.62 1.38
3,3,3,3,4,3 aDRibf 5.30 4.14 3.99 4.35 3.70-3.76
3,3,3,3,4,4,3,4,4,4,3,4,4,3 aDRibf 5.26 4.11 3.97 4.46 3.74-3.74
3,3,3,3,4,4,3,4,4,4,3,4,4,4,3 P
3,3,3,3,4,4,3,4,4,4,3,4,4,4 bDRhap 4.97 4.68 4.34 3.62 3.45 1.33
3,3,3,3,4,4,3,4,4,4,3,4,4 bDRhap 4.78 4.33 3.90 3.80 3.62 1.40
3,3,3,3,4,4,3,4 bDRhap 4.90 4.15 3.72 3.54 3.54 1.34
3,3,3,3,4,4,3 aDGlcp 5.10 3.59 3.93 3.68 4.01 3.80-3.89
3,3,3,3,4,4 bDRhap 4.77 4.24 3.68 3.44 3.43 1.34
3,3,3,3,4 bDRhap 4.77 4.34 3.94 3.74 3.62 1.38
3,3,3,3 aDRhap 5.06 4.14 3.96 3.62 3.95 1.32
3,3,3 aDRhap 5.04 4.15 3.92 3.56 3.86 1.31
3,3 aDRhap 5.03 4.17 3.91 3.58 3.88 1.30
3 bDGalp 4.56 3.65 3.72 4.03 3.73 3.78-3.78
x?Thr - 4.60 4.34 1.29
1H/13C HSQC data:
Linkage Residue C1/H1 C2/H2 C3/H3 C4/H4 C5/H5 C6/H6
3,3,3,3,4,4,3,4,4,4,3,4 bDRhap 101.9/4.90 71.2/4.15 72.7/3.72 83.7/3.54 72.1/3.54 17.9/1.34
3,3,3,3,4,4,3,4,4,4,3 aDGlcp 96.5/5.10 72.5/3.59 73.9/3.93 77.9/3.68 71.6/4.01 61.6/3.80-3.89
3,3,3,3,4,4,3,4,4,4 bDRhap 101.0/4.77 68.1/4.24 78.9/3.68 73.5/3.44 73.6/3.43 17.9/1.34
3,3,3,3,4,4,3,4,4,3 aDRibf 99.0/5.31 72.5/4.14 71.1/3.99 86.4/4.35 62.6/3.70-3.76
3,3,3,3,4,4,3,4,4 bDRhap 101.4/4.76 68.1/4.34 75.5/3.94 77.8/3.74 72.9/3.62 18.2/1.38
3,3,3,3,4,3 aDRibf 99.0/5.30 72.5/4.14 71.1/3.99 86.4/4.35 62.6/3.70-3.76
3,3,3,3,4,4,3,4,4,4,3,4,4,3 aDRibf 99.4/5.26 72.6/4.11 71.5/3.97 86.5/4.46 62.6/3.74-3.74
3,3,3,3,4,4,3,4,4,4,3,4,4,4,3 P
3,3,3,3,4,4,3,4,4,4,3,4,4,4 bDRhap 99.6/4.97 75.8/4.68 81.1/4.34 74.1/3.62 72.3/3.45 17.9/1.33
3,3,3,3,4,4,3,4,4,4,3,4,4 bDRhap 101.4/4.78 68.4/4.33 75.5/3.90 78.2/3.80 72.9/3.62 18.2/1.40
3,3,3,3,4,4,3,4 bDRhap 101.9/4.90 71.2/4.15 72.7/3.72 83.7/3.54 72.1/3.54 17.9/1.34
3,3,3,3,4,4,3 aDGlcp 96.5/5.10 72.5/3.59 73.9/3.93 77.9/3.68 71.6/4.01 61.6/3.80-3.89
3,3,3,3,4,4 bDRhap 101.0/4.77 68.1/4.24 78.9/3.68 73.5/3.44 73.6/3.43 17.9/1.34
3,3,3,3,4 bDRhap 101.4/4.77 68.1/4.34 75.5/3.94 77.8/3.74 72.9/3.62 18.2/1.38
3,3,3,3 aDRhap 103.3/5.06 71.2/4.14 70.2/3.96 83.8/3.62 68.7/3.95 17.9/1.32
3,3,3 aDRhap 103.3/5.04 71.2/4.15 79.7/3.92 72.5/3.56 70.5/3.86 17.9/1.31
3,3 aDRhap 103.3/5.03 71.2/4.17 79.4/3.91 72.5/3.58 70.4/3.88 17.9/1.30
3 bDGalp 102.0/4.56 71.3/3.65 81.3/3.72 69.6/4.03 76.3/3.73 62.1/3.78-3.78
x?Thr 59.5/4.60 75.3/4.34 17.0/1.29
report error
Found 1 record.
Displayed record 1
]; infection due to Clostridium difficile [ICD11: XN0SE 
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