WciG O-Acetyltransferase Functionality Differentiates Pneumococcal Serotypes 35C and 42

Abstract

Streptococcus pneumoniae expresses capsular polysaccharides (CPSs) to protect itself from opsonophagocytic killing. The genes responsible for capsules synthesized by the Wzy-dependent mechanism, which accounts for 96 of the 98 known pneumococcal capsule types, are in a chromosomal region known as the cps locus. The nucleotide sequence in this region has been determined for all serotypes. In contrast, not all CPS structures have been defined. The structure of the serotype 35C polysaccharide was recently reported, but the presence of O-acetyltransferase genes in the serotype 35C cps locus suggested that it could be incomplete, as the reported structure contains no O-acetylation. In addition, the genetic distinction of serotype 35C from the closely related serotype 42 was unclear, as their reported cps loci are nearly identical. To clarify these discrepancies, we obtained serotype 35C and 42 clinical and reference isolates and studied their serological and genetic properties, as well as the structures of CPSs purified from reference isolates. We demonstrated that the O-acetyltransferase WciG was functional in serotype 35C but nonfunctional in serotype 42 due to a deletion in wciG Serotype 35C was O-acetylated at the 5- and 6-positions of 3-β-galactofuranose, as well as the 2-position of 6-β-galactofuranose. However, serotype 42 has only O-acetylation at 3-β-galactofuranose, an observation consistent with its loss of WciG functionality, which is associated with O-acetylation at the 2-position and subsequent reaction with typing antiserum 35a. These findings provide a comprehensive view of the genetic, biochemical structural, and serological bases of serotypes 35C and 42.

Keywords: O-acetyltransferase; Streptococcus pneumoniae; biochemical structure; capsular polysaccharide; genetic basis; serological profile; serotyping.

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).