Article Figures & Data
Figures
- FIG 1
Serological analysis of putative serotype 35C and 42 reference isolates. Indicated strains were assayed by flow cytometric staining for binding to the indicated typing antisera. Gray shading represents binding to TIGR4, which does not contain the indicated antigens (see Table 1) and represents binding by antibodies in these reagents directed against epitopes other than the capsule.
- FIG 2
Genetic and structural differences between serotypes 35C and 42. (A) Arrangement of structure-determining genes in the published cps loci of S. pneumoniae serotypes 35C (accession no. CR931706 ) and 42 (accession no. KY009533 ). The red “X” in wciG of cps42 reflects our finding in this study that wciG of serotype 42 is defective. (B) Comparison of nucleotide and amino acid sequences of wciG genes. The differences between the two sequences are highlighted in red. In cps42 of SP155, deletion of a single nucleotide “a” after nucleotide 16383 results in premature termination of WciG translation. Numbering is with respect to GenBank accession no. CR931706 . (C) Complete structures of serotype 35C and 42 polysaccharides as determined by NMR spectroscopy. The uppercase letters in the structures indicate the residues whose 1H and 13C chemical shifts are given in Table 3 and in the text. The sole difference between the two polysaccharides is the O-acetylation at 2-position of residue C, which is highlighted in red.
- FIG 3
Serological panel demonstrating that WciG functionality differentiates serotypes 35C and 42. The indicated strains were assayed by flow cytometric staining for binding to the indicated typing antisera. Gray shading represents binding to TIGR4, which does not contain the indicated antigens (see Table 1) and represents binding by antibodies to noncapsular epitopes.
- FIG 4
Anomeric and central regions of multiplicity-edited HSQC spectra of serotype 35C (A) and serotype 42 (B) polysaccharides. The chemical shift of 1H in residue C at the 2-position (C2, red circle) is affected by O-acetylation.
- FIG 5
1H-13C HSQC spectra in the O-acetyl methyl regions of serotype 35C (A) and serotype 42 (B) polysaccharides. Chemical shifts of these peaks are listed in Table 4. This peak at 2.140/21.12 ppm is assigned O-acetylation at 2-position of residue C (OAc-C2), which is clearly present in serotype 35C but completely lost in serotype 42. Both isolates contain full O-acetylation at the 5- and 6-positions of residue A (di-OAc-A5 and di-OAc-A6).
Tables
- TABLE 2
Strains used in this study
Straina Putative serotype FCSAb serotype wciG status GenBank accession no. Sourcec Reference isolates SP147 35C 35C Intact KY091876 CDC SP155* 42 42 Disrupted KY009533 CDC TIGR4 4 4 NAd NC_003028 ATCC Clinical isolates 5705-06† 35C 35C Intact KX470740 CDC PATH1895 35C 35C Intact KY091869 CDC PATH1896 35C 35C Intact KY091870 CDC PATH1901 35C 35C Intact KY091871 CDC PATH2897 35C 35C Intact KY091872 CDC PATH4040 35C 35C Intact KY091873 CDC PATH4191 35C 35C Intact KY091874 CDC PATH4348 35C 35C Intact KY091875 CDC Laboratory-derived strains KAG1017†e KY124245 This study KAG1021*f This study ↵a *, The mnp2 gene in strains SP155 and KAG1021 encodes 14637A→G with respect to the reference serotype 35C cps locus (GenBank accession no. CR931706 ); †, the mnp2 genes in strains 5705-06 and KAG1017 correspond to CR931706 .
↵b FCSA, flow cytometric serotyping assay.
↵c CDC, Centers for Disease Control and Prevention, Active Bacterial Core surveillance, Atlanta, GA; ATCC, American Type Culture Collection, Manassas, VA.
↵d NA, not applicable.
↵e 5705-06ΔwciG.
↵f SP155 aliA::pKAG2008; aliA+ wciG+.
- TABLE 3
Residue-by-residue comparison of HSQC 1H and 13C chemical shift of S. pneumoniae serotype 35C and 42 polysaccharides
Polysaccharide (isolate source) Residue/structure Chemical shift (ppm)a 1-H, 1-C 2-H, 2-C 3-H, 3-C 4-H, 4-C 5-H, 5-C 6-H, 6-C 35C (SP147) A/3-β-Galf 5.360 4.457 4.109 4.540 5.540 4.322, 4.362 108.80 79.64 85.50 82.17 70.87 64.29 42 (SP155) 5.361 4.457 4.108 4.540 5.542 4.322, 4.362 108.80 79.63 85.49 82.18 70.87 64.29 35C (SP147) B/3-β-Galp 4.692 3.824 4.241 4.231 3.721 3.763 103.55 73.06 76.76 68.21 75.25 61.41 42 (SP155) 4.703 3.820 4.243 4.229 3.722 3.765 103.57 73.07 76.80 68.20 75.25 61.41 35C (SP147) C/6-β-Galf 5.192 4.990 4.250 4.072 4.071 3.769, 4.073 106.70 84.40 76.41 84.66 70.48 72.31 42 (SP155) 5.054 4.138 4.091 4.019 4.031 3.763, 4.074 108.78 81.70 77.57 83.91 70.57 72.25 35C (SP147) D/3-β-Glcp 4.556 3.443 3.663 3.406 3.489 3.724, 3.922 103.37 74.54 81.92 68.77 76.72 61.52 42 (SP155) 4.551 3.443 3.664 3.406 3.494 3.726, 3.924 103.35 74.54 81.92 68.77 76.71 61.54 35C (SP147) E/3-α-Glcp 5.542 3.504 3.829 3.335 4.087 3.721, 3.912 97.82 72.63 73.69 70.85 72.58 61.75 42 (SP155) 5.542 3.513 3.829 3.333 4.090 3.726, 3.913 97.82 72.62 73.69 70.85 72.58 61.76 35C (SP147) F/1-Manol-6 3.669, 4.020 3.873 3.873 3.876 3.876 4.102, 4.161 70.02 69.67 70.19 70.38 69.14 68.00 42 (SP155) 3.764, 4.031 3.868 3.878 3.877 3.875 4.097, 4.158 70.52 69.72 70.26 70.39 69.12 67.97 ↵a Chemical shifts of polysaccharides were recorded at 25°C. Pairs of values separated by a comma are H, H′ values. Underlined numbers indicate O-acetylated positions, and numbers in boldface indicate di-O-acetylated positions.
- TABLE 4
O-acetyl groups identified in the serotype 35C polysaccharide
Position on residuea Chemical shift (ppm) Methyl 1H Methyl 13C Carbonyl 13C Link 1H 6-di-OAc on residue A 2.096 21.03 174.70 4.322, 4.362 5-di-OAc on residue A 2.156 21.18 174.42 5.540 2-OAc on residue C 2.140 21.12 174.13 4.990 ↵a The number indicates position of the carbon atom on sugar residues. Residue A is 3-β-galactofuranose, and residue C is 6-β-galactofuranose in the serotype 35C repeat unit. OAc, O-acetylation.
Supplemental material
- Supplemental file 1 -
Tables S1 (Genetic differences between published serotype 35C and 42 cps loci) and S2 (Primers used in this study)
PDF, 308K
- Supplemental file 1 -
