Streptococcus pneumoniae (pneumococcus) is a major human pathogen producing more than 100 biochemically unique capsule types, which are the targets of pneumococcal conjugate vaccines (PCVs). Structural elucidation of capsule polysaccharides (PSs) is a prerequisite to determine the appearance of novel pneumococcal capsule types and unravel both antigenic and genetic relationships that exist between different capsule types. Therefore, structural characterization of pneumococcal capsule PS is fundamental for developing better diagnostic methods and future pneumococcal vaccines. Herein, we illustrate the importance of nuclear magnetic resonance (NMR) in studying the new pneumococcal capsule types.
Previously, bacterial serotypes were often assigned to be new based only on the genetic findings. Using serotype “6E”, we showed that genetic information alone is not a good predictor of a new serotype, and structural characterization by NMR is essential to determine the uniqueness. From the Pneumococcal Molecular Epidemiology Network collection, several serotype 6B strains were found to have significant genetic differences and were labeled as serotype “6E”. We showed that SPEC6B, which is widely used as a target strain for serotype 6B opsonophagocytic assay, has the genetic features of the putative serotype “6E” but produces capsular PS identical to serotype 6B as determined by the one-dimensional (1D) and 2D NMR studies. Thus, putative serotype “6E” is a mere genetic variant of serotype 6B.
Recently, we discovered two novel pneumococcal capsule types, named 35D and 10D, by determining their unique chemical structure with NMR. Capsule type 35D was identified while assessing the serological properties of 35B clinical isolates. One isolate failed to bind to the critical serotyping reagents for 35B. 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. NMR studies established that the capsule of wciG defective isolates 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 the novel capsule type 35D.
Similarly, NMR data unambiguously confirmed that the 100th pneumococcal capsule type, named serotype 10D, produces a chemically distinct capsule. The name 10D reflects its serological cross-reaction with serotype 10A and the appearance of cross-opsonic antibodies in response to immunization with 10A PS in a 23-valent pneumococcal vaccine. Genetic analysis showed that 10D cps has two large regions that are syntenic and highly homologous to cps loci of serotypes 6C and 39. In addition, the 10D cps has ~6 kb region and a short gene fragment of wciNα at the 5´ end syntenic to oral streptococcus strain (S. mitis SK145). The presence of this nonfunctional wciNα fragment provides compelling evidence for a recent interspecies genetic transfer from oral streptococcus to pneumococcus. NMR analysis revealed that 10D capsule PS shares the structure with serotypes 6C and 39. Serotype 10D has two glucose residues at the reducing end created by two glycosyltransferases, wchA and wciNβ which are shared with 6C. The remaining structure at the non-reducing end is formed by the products of genes (wcrC, wcrD, wciF, and wcrG), which are shared between serotypes 10A and 39. The two parts are linked by a ribitol-5-phosphate catalyzed by ribitol phosphotransferase, wcrO10D. In addition, the chemical structure explains its serologic properties: its reaction with polyclonal serum (FS10d) and monoclonal antibody (Hyp10AG1), which targets βGal(1-6) created by wcrG.
Since discoveries of novel capsule types can directly impact pneumococcal vaccine usage and design, biochemical characterization of putatively novel serotypes circulating in the population by NMR is an unarguably essential task to confirm their novelty. The chemical description of the S. pneumoniae capsular PS is crucial in establishing relations between the capsule genetic locus and the repeating unit structure. Thereby providing insights into the epitopes of capsular PSs detected by the immune system.