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1.
FIGURE 1.

FIGURE 1. From: Structural Characterization of Streptococcus pneumoniae Serotype 9A Capsule Polysaccharide Reveals Role of Glycosyl 6-O-Acetyltransferase wcjE in Serotype 9V Capsule Biosynthesis and Immunogenicity.

Schematic and written representations of the capsule pentasaccharide repeat unit of serotypes 9V and 9A according to previous reports and this study. Lines represent inter-subunit glycosidic linkages. O-Acetylation sites are denoted by R followed by a superscript number. Rates of O-acetylation at each site are listed in .

Juan J. Calix, et al. J Biol Chem. 2012 April 20;287(17):13996-14003.
2.
FIGURE 6.

FIGURE 6. From: Structural Characterization of Streptococcus pneumoniae Serotype 9A Capsule Polysaccharide Reveals Role of Glycosyl 6-O-Acetyltransferase wcjE in Serotype 9V Capsule Biosynthesis and Immunogenicity.

Proposed targets of wcjE-mediated O-acetylation. The reported structures of serotypes 11A, 9V, and 35 capsule PS repeat units (, ) are shown. Inter-subunit glycosidic linkages are represented by dotted lines. Arrows point to O-acetate substitutions proposed to be mediated by wcjE in each serotype (see text). Ac, acetate.

Juan J. Calix, et al. J Biol Chem. 2012 April 20;287(17):13996-14003.
3.
FIGURE 2.

FIGURE 2. From: Structural Characterization of Streptococcus pneumoniae Serotype 9A Capsule Polysaccharide Reveals Role of Glycosyl 6-O-Acetyltransferase wcjE in Serotype 9V Capsule Biosynthesis and Immunogenicity.

1H NMR spectra of native and de-O-acetylated (dO) 9V and 9A PS. Selected views of the anomeric (A) and acetate (B) regions and a magnified portion of the anomeric region (C) of the 1H NMR spectra in supplemental Fig. S1. Asterisks denote hydrolysis-sensitive signals shared by native 9A and 9V PS, and pound signs denote hydrolysis-sensitive signals observed only in 9V PS.

Juan J. Calix, et al. J Biol Chem. 2012 April 20;287(17):13996-14003.
4.
FIGURE 5.

FIGURE 5. From: Structural Characterization of Streptococcus pneumoniae Serotype 9A Capsule Polysaccharide Reveals Role of Glycosyl 6-O-Acetyltransferase wcjE in Serotype 9V Capsule Biosynthesis and Immunogenicity.

Binding of human serum antibodies to 9A and 9V capsule PS. Plates coated with 9V, 9A, 9VdO, and 9AdO PS were incubated with 1:50 dilutions of human serum samples, and the amount of bound IgG (A), IgA (B), and IgM (C) were compared according to A405. Values are averages of duplicate runs. P32 is a pool of 100 serum samples from individuals immunized with 9V PS. s32, s43, s44, s48, and s53 are serum samples from single healthy donors immunized with 9V PS.

Juan J. Calix, et al. J Biol Chem. 2012 April 20;287(17):13996-14003.
5.
FIGURE 3.

FIGURE 3. From: Structural Characterization of Streptococcus pneumoniae Serotype 9A Capsule Polysaccharide Reveals Role of Glycosyl 6-O-Acetyltransferase wcjE in Serotype 9V Capsule Biosynthesis and Immunogenicity.

Overlay of two-dimensional 1H-13C HMQC spectra of native 9V (blue), native 9A (red), and 9VdO (black) PS. A, signals indicate GlcUA O-acetylation. B, anomeric signals are assigned according to Rutherford et al. () as follows: 1, αGlc H1; 2, αGal next to αGlcUA-3-OAc H1; 3, α-Gal H1; 4, αGlcUA-2-OAc H1; 5, αGlcUA-3-OAc H1; 6, αGlc next to αGlcUA-3-OAc H1; 7, αGlcUA H1; 8, βManNAc H1; and 9, βGlc H1. C, signals of the –CH2 group of βManNAc-6-OAc. Cross-peaks corresponding to protons with geminal O-acetylation are labeled in A and C. Asterisks in A and B denote HDO signal. NA, not assigned.

Juan J. Calix, et al. J Biol Chem. 2012 April 20;287(17):13996-14003.
6.
FIGURE 4.

FIGURE 4. From: Structural Characterization of Streptococcus pneumoniae Serotype 9A Capsule Polysaccharide Reveals Role of Glycosyl 6-O-Acetyltransferase wcjE in Serotype 9V Capsule Biosynthesis and Immunogenicity.

Serotyping antibody binding to 9A and 9V PS is O-acetate-dependent. A, binding of polyclonal antibodies in factor sera 9d and 9g (left two columns) and the monoclonal antibodies Hyp9VM7 and Hyp9VG2 (right two columns) to bacterial strains expressing serotype 9V (SSISP 9V/4, top row) and serotype 9A (MNZ869, bottom row) was examined by FCSA. Each histogram box contains the geometric mean fluorescence intensities of negative controls (top) and test samples (bottom). B, relative binding of antibodies to ELISA wells coated with 9V PS (black bars), 9VdO PS (light gray bars), 9A PS (dark gray bars), and 9AdO (white bars). Antibodies are indicated at the bottom of the graph as follows: Sg9, antiserum specific for serogroup 9 at a 1:1000 dilution; Fs9d, factor serum 9d at a 1:1000 dilution; Fs9g, factor serum 9g at a 1:1000 dilution; 9VM7 and 9VG2 are tissue culture supernatants of monoclonal antibodies Hyp9VM7 and Hyp9VG2. C, inhibition of factor serum 9d antibodies binding to 9A PS-coated plates was examined by inhibition ELISA and is expressed as a percentage of the A405 of negative control (i.e. wells that did not receive inhibitor PS). Values in B and C are averages of duplicate runs.

Juan J. Calix, et al. J Biol Chem. 2012 April 20;287(17):13996-14003.

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