Production of a unique pneumococcal capsule serotype belonging to serogroup 6

Abstract

Serogroup 6 of Streptococcus pneumoniae contains three serotypes, named 6A, 6B and 6C, with highly homologous capsule gene loci. The 6A and 6B capsule gene loci consistently differ from each other by only one nucleotide in the wciP gene. The 6A capsule gene locus has a galactosyltransferase, which has been replaced with a glucosyltransferase in the 6C capsule gene locus. We considered that a new serotype named '6X1' would be possible if the galactosyltransferase of the 6B capsule gene locus is replaced with the glucosyltransferase of 6C. We demonstrate that this gene transfer yields a viable pneumococcal strain and that the capsular polysaccharide (PS) from this strain has the predicted chemical structure and serological similarity to the capsular PS of the 6B serotype. The new strain (i.e. serotype 6X1) is typed as 6B by the quellung reaction, but it can be distinguished from 6B strains with mAbs to 6B PS. Reexamination of 264 pneumococcal isolates that had been previously typed as 6B with classical typing methods revealed no isolates expressing serotype 6X1. Nevertheless, this study shows that this capsular PS is biochemically possible and could exist/emerge in nature.

Figure 1

wciN region exchange experiment diagram. * at wciP gene indicates the critical codon (serine vs asparagine) associated with 6A/6B serotype difference (Mavroidi et al., 2004). kanA^R indicates kanamycin resistance gene and rpsL⁺ indicates streptomycin sensitivity gene.

Figure 2

GLC/MS chromatograms showing carbohydrate composition of capsular PS from pneumococcal strains expressing 6B and 6X1 serotypes and before and after periodate treatment. The monosaccharides are identified in the top chromatogram. Each panel is labeled with the nature of samples.

Figure 3

Panel A shows the proposed structure of the hydrated form of the repeating unit of 6X1 capsular PS. The calculated molecular weight is 701 AMU. Panel B shows Mass spectrum of the repeating units. The peaks at 683.3 m/z and 701.3 m/z respectively correspond to the anhydrous and hydrated forms of the repeating units. Panel C shows the daughter ions of the ion with 683.3 AMU shown in Panel B. Daughter ions are identified at the bottom of Panel C. The peaks with 270.825, 574.758, and 632.756 AMUs and their satellite peaks (separated by 2 AMUs due to chloride isotopes) represent sodium chloride salt clusters (Hao et al., 2001). The peaks at 270.825 represents (NaCl)₄Cl⁻. Peaks at 574.758 AMU probably represent another salt cluster, (NaCl)₉Cl⁻, with a water molecule, like salt clusters with organic solvent molecules (Shaolian Zhou, 1996). The peaks at 632.7 have one more NaCl (i.e., 58 AMUs) than the peaks at 574.758 AMU.

Figure 4

Ability of various capsular PS (2 mg/ml) to inhibit binding of mAb to ELISA plates (Y axis) after the PS was hydrolyzed for various time periods (X-axis). “Titers” indicate the dilution of a sample necessary to inhibit the binding by 50%. For 6A and 6C PSs, ELISA plates are coated with 6A PS and mAb Hyp6AG1 is used. For 6B and 6X1 PSs, ELISA plates are coated with 6B PS and mAb Hyp6BM8 is used.

Figure 5

Ability of lysates of various pneumococcal strains to inhibit the binding of mAb Hyp6BM7 (Panel A) or Hyp6BM8 (Panel B) to 6B PS coated ELISA plates. Amount of mAb bound (Y axis) is shown in the presence of varying amounts of inhibitors (X-axis). Lysates of pneumococcal strains are used as inhibitors. Pneumococcal strains are TIGR6A (open diamond), TIGR6B (solid square), TIGR6C (open triangle), TIGR6X1 (solid circle), and TIGR6BX (x symbol). TIGR6BX does not produce capsular PS.