Discovery of Novel Pneumococcal Serotype 35D, a Natural WciG-Deficient Variant of Serotype 35B

Abstract

Pneumococcus (Streptococcus pneumoniae) remains a significant cause of morbidity and mortality, especially among those at the extremes of age. Its capsular polysaccharide is essential for systemic virulence. Over 90 serologically distinct pneumococcal capsular polysaccharides (serotypes) are recognized, but they are unequal in prevalence. Because antibodies against the capsule are protective, polysaccharide conjugate vaccines, which are constructed against the most prevalent serotypes, have caused great reductions in pneumococcal disease caused by these serotypes. In response, however, the relative prevalences of serotypes have shifted. Certain previously rare serotypes, such as serotype 35B, are increasing in prevalence. Serotype 35B is thus a likely future vaccine candidate, but due to their previous rarity, serotype 35B strains have not been scrutinized for underlying heterogeneity. We studied putative serotype 35B clinical isolates to assess the uniformity of their serological reactions. While most isolates exhibited the accepted serology of serotype 35B, one isolate failed to bind to critical serotyping reagents. We determined that the genetic basis for this aberrant serology was the presence of inactivating mutations in the O-acetyltransferase gene wciG Complementation studies in a wciG deletion strain verified that the mutant WciG was nonfunctional, and the serology of the mutant could be restored through complementation with a construct encoding a functional WciG. Nuclear magnetic resonance studies confirmed that the capsule of the WciG-deficient isolate lacked O-acetylation but was otherwise identical to serotype 35B. As this isolate expresses a unique serology with unique biochemistry and a stable genetic basis, we named its novel capsule serotype 35D.

Keywords: O-acetylation; Streptococcus pneumoniae; capsular polysaccharide; serogroup 35; serotype 35B; serotype 35D.

FIG 1

Isolate 3431-06 fails to bind to group 35 antisera and factor serum 35a by flow cytometric serotyping assay. Histograms are presented for the staining of indicated strains (serotype in parentheses) by the indicated serological reagent. Gray shading indicates staining of an irrelevant serotype control (serotype 4 strain TIGR4 [34]) to control for noncapsular antibodies in the typing antisera. Staining of strain 35B/2 (a serotype 35B reference strain) is representative of results obtained with strains listed in Table 1. fs, factor serum.

FIG 2

The unique serology of serotype 35D is due to loss of WciG functionality in a serotype 35B background. The flow cytometric staining of bacteria (strain description in parentheses) by the indicated serological reagents is presented as histograms. The serotype 4 isolate TIGR4 (34), with gray shading, was used to control for binding by noncapsular antibodies. A strain staining at or below the irrelevant serotype control was considered negative for binding. fs, factor serum.

FIG 3

(A) Overlay of ¹H NMR spectra of serotypes 35B (top) and 35D (bottom) polysaccharides at 25°C. The serotype 35D spectrum has lost the O-acetyl peak at 2.12 ppm (arrowhead), and shifts in the anomeric region (especially ≥5 ppm [¹H]) are observed in consequence (see Table 3). (B) The ¹H-¹³C HMQC spectra of 35B (left) and 35D (right) show the loss of the O-Ac peak at 2.12 ppm (¹H).

FIG 4

Predicted epitope correlations among serogroup 35 capsules. Capsular structures of serotypes 35F, 35A, 35B, and 35C were previously described (13). Epitopes were deduced by comparisons within serogroup 35 and with the other known structures of pneumococcal capsules (13). *, we have determined that the serotype 35C capsule is acetylated at the indicated sites (K. A. Geno, C. A. Bush, M. H. Nahm, and J. Yang, unpublished data). Gal, galactose; Ac, acetyl; Rib-ol, ribitol; P, phosphate; f, furanose; Man-ol, mannitol; Glc, glucose; GalNAc, N-acetyl galactosamine.