Structural characterization of Streptococcus pneumoniae serotype 9A capsule polysaccharide reveals role of glycosyl 6-O-acetyltransferase wcjE in serotype 9V capsule biosynthesis and immunogenicity

The putative capsule O-acetyltransferase gene wcjE is highly conserved across various Streptococcus pneumoniae serotypes, but the role of the gene in capsule biosynthesis and bacterial fitness remains largely unclear. Isolates expressing pneumococcal serotype 9A arise from precursors expressing wcjE-associated serotype 9V through loss-of-function mutation to wcjE. To define the biosynthetic role of 9V wcjE, we characterized the structure and serological properties of serotype 9V and 9A capsule polysaccharide (PS). NMR data revealed that both 9V and 9A PS are composed of an identical pentasaccharide repeat unit, as reported previously. However, in sharp contrast to previous studies on 9A PS being devoid of any O-acetylation, we identified O-acetylation of α-glucuronic acid and α-glucose in 9A PS. In addition, 9V PS also contained -CH(2) O-acetylation of β-N-acetylmannosamine, a modification that disappeared following in vitro recombinatorial deletion of wcjE. We also show that serotyping sera and monoclonal antibodies specific for 9V and 9A bound capsule PS in an O-acetate-dependent manner. Furthermore, IgG and to a lesser extent IgM from human donors immunized with serotype 9V PS displayed stronger binding to 9V compared with 9A PS. We conclude that serotype 9V wcjE mediates 6-O-acetylation of β-N-acetylmannosamine. This PS modification can be selectively targeted by antibodies in immunized individuals, identifying a potential selective advantage for wcjE inactivation and serotype 9A emergence.     

Biochemical, Genetic, and Serological Characterization of Two Capsule Subtypes among Streptococcus pneumoniae Serotype 20 Strains: DISCOVERY OF A NEW PNEUMOCOCCAL SEROTYPE

The bacterial pathogen Streptococcus pneumoniae expresses one of over 90 structurally distinct polysaccharide (PS) capsule serotypes. Prior PS structural analyses of the vaccine-associated serotype 20 do not agree with reports describing the genes that mediate capsule synthesis. Furthermore, using immunized human sera-based assays, serological differences were recently noted among strains typed as serotype 20. We examined the capsule structures of two serologically dissimilar serotype 20 strains, 20α and 20β, by extensive biochemical analysis. 20α PS was composed of the previously described serotype 20 hexasaccharide repeat unit, whereas the 20β PS was composed of a novel heptasaccharide repeat unit containing an extra branching α-glucose residue. Genetic analysis of the subtypes revealed that 20α may have arisen from a 20β progenitor following loss of function mutation to the glycosyltransferase gene whaF. Conventional serotyping methods using rabbit polyclonal or mouse monoclonal antibodies were unable to distinguish the subtypes. However, genetic analysis of multiple "serotype 20" clinical isolates revealed that all strains contain the 20β genotype. We propose naming bacteria that express the previously described 20α capsule structure 20A and bacteria that express the novel 20β capsule structure 20B, a new pneumococcal serotype.     

Lipoteichoic acid is an important microbe-associated molecular pattern of Lactobacillus rhamnosus GG

Background

Receptors with a single transmembrane (TM) domain are essential for the signal transduction across the cell membrane. NMR spectroscopy is a powerful tool to study structure of the single TM domain. The expression and purification of a TM domain in Escherichia coli (E.coli) is challenging due to its small molecular weight. Although ketosteroid isomerase (KSI) is a commonly used affinity tag for expression and purification of short peptides, KSI tag needs to be removed with the toxic reagent cyanogen bromide (CNBr).

Result

The purification of the TM domain of p75 neurotrophin receptor using a KSI tag with the introduction of a thrombin cleavage site is described herein. The recombinant fusion protein was refolded into micelles and was cleaved with thrombin. Studies showed that purified protein could be used for structural study using NMR spectroscopy.

Conclusions

These results provide another strategy for obtaining a single TM domain for structural studies without using toxic chemical digestion or acid to remove the fusion tag. The purified TM domain of p75 neurotrophin receptor will be useful for structural studies.