Streptococcus pneumoniae serotype 33G: genetic, serological, and structural analysis of a new capsule type

Abstract

Streptococcus pneumoniae (the pneumococcus) is a bacterial pathogen with the greatest burden of disease in Asia and Africa. The pneumococcal capsular polysaccharide has biological relevance as a major virulence factor as well as public health importance as it is the target for currently licensed vaccines. These vaccines have limited valency, covering up to 23 of the >100 known capsular types (serotypes) with higher valency vaccines in development. Here, we have characterized a new pneumococcal serotype, which we have named 33G. We detected serotype 33G in nasopharyngeal swabs (n = 20) from children and adults hospitalized with pneumonia, as well as healthy children in Mongolia. We show that the genetic, serological, and biochemical properties of 33G differ from existing serotypes, satisfying the criteria to be designated as a new serotype. Future studies should focus on the geographical distribution of 33G and any changes in prevalence following vaccine introduction.

Keywords: Streptococcus pneumoniae; capsule; recombination; serotypes; vaccines.

Fig 1

Schematic representation of the 33X cps locus with the 35A (A), 10B and 33B (B) cps reference sequences (30) and Streptococcus oralis subsp. dentisani strain Y5914-11 cps locus (Genbank accession no. NZ_NCUW01000028) (31). Images were generated using Easyfig version 2.2.5 (32).

Fig 2

Competition ELISA with serum from mice colonized with 33X pneumococci pre-incubated with serotypes 33X, 10B, 33B, or 14 as a competing capsular antigen source. Data presented are mean + SD and analyzed by unpaired t test.

Fig 3

Expansion of the 1D proton NMR spectra of pneumococcal serotype 33X polysaccharide, some diagnostic anomeric, and ring protons are labeled. (A) 1D ¹H NMR at 500 MHz and 25°C, (B) 1D diffusion-ordered spectroscopy (DOSY) at 500 MHz and 25°C, (C) 1D DOSY at 600 MHz and 70°C (partially de-O-acetylated), and (D) 1D DOSY at 600 MHz and 60°C (de-O-acetylated polysaccharide).

Fig 4

The expansion of the HSQC spectrum of polysaccharide 33X recorded at 600 MHz (60°C), the cross peaks from the methyl region of the spectrum are shown in the inset. All the hexasaccharide repeat unit (including ribitol-P) proton/carbon cross peaks have been labeled according to the carbon atom of the corresponding residue (βG^I f = β-Gal^I f, αG = α-Galp, βG^II f = β-Gal^II f, βGN = β-GalNAc, β-G = β-Glc, and rib = ribitol). Additional peaks are due to buffer (B) and glycerol (G).

Fig 5

The 2D ¹H-¹³C overlay of the anomeric region for polysaccharide 33X: HSQC-DEPT (red)/HMBC (black) recorded at 600 MHz (333 K). The intra- and inter-residue HMBC cross peaks from H1 are labeled according to the carbon atom of the corresponding residue (βG^I f = β-Gal^I f, αG = α-Galp, βG^II f = β-Gal^II f, βGN = β-GalNAc, β-G = β-Glc, and rib = ribitol). The HMBC experiment was optimized for a coupling constant of 6 Hz and established the following linkages: H1 of β-Gal^I f to C3 of β-Glc, H1 of α-Galp to C4 of ribitol, H1 of β-Gal^II f to C3 of β-GalNAc, H1 of β-GalNAc to C3 of α-Galp, and H1 of β-Glc to C5 of β-Gal^II f.

Fig 6

The 2D ¹H-¹³C overlay of the anomeric region for O-acetylated polysaccharide 33X: HSQC-DEPT (red)/HMBC (black) recorded at 500 MHz (298 K). The intra- and inter-residue HMBC cross peaks from diagnostic anomeric proton signals are labeled according to the carbon atom of the corresponding residue (βG^I f, = β-Gal^I f, αG = α-Galp, βG^II f 2Ac= β-Gal^II f 2Ac, βGN = β-GalNAc, β-G = β-Glc, and rib = ribitol). The HMBC experiment was optimized for a coupling constant of 6 Hz.

Fig 7

Polysaccharide repeat unit structure of 10B (45), 33B (43), and new serotype 33G (this study), as well as proposed enzymatic assignments based on the literature (15, 44).