Discovery and characterization of sialic acid O-acetylation in group B Streptococcus

Abstract

Group B Streptococcus (GBS) is the leading cause of human neonatal sepsis and meningitis. The GBS capsular polysaccharide is a major virulence factor and the active principle of vaccines in phase II trials. All GBS capsules have a terminal alpha 2-3-linked sialic acid [N-acetylneuraminic acid (Neu5Ac)], which interferes with complement-mediated killing. We show here that some of the Neu5Ac residues of the GBS type III capsule are O-acetylated at carbon position 7, 8, or 9, a major modification evidently missed in previous studies. Data are consistent with initial O-acetylation at position 7, and subsequent migration of the O-acetyl ester at positions 8 and 9. O-acetylation was also present on several other GBS serotypes (Ia, Ib, II, V, and VI). Deletion of the CMP-Neu5Ac synthase gene neuA by precise, in-frame allelic replacement gave intracellular accumulation of O-acetylated Neu5Ac, whereas overexpression markedly decreased O-acetylation. Given the known GBS Neu5Ac biosynthesis pathway, these data indicate that O-acetylation occurs on free Neu5Ac, competing with the CMP-Neu5Ac synthase. O-acetylation often generates immunogenic epitopes on bacterial capsular polysaccharides and can modulate human alternate pathway complement activation. Thus, our discovery has important implications for GBS pathogenicity, immunogenicity, and vaccine design.

Fig. 1.

GBS Sia is endogenously O-acetylated. (A) DMB-HPLC analysis of GBS serotype III strain COH1 grown in THB (contains Sias) or RPMI medium 1640 (chemically defined, contains no Sias). Cells were pelleted, washed, and Sias were acid-hydrolyzed and DMB-derivatized. Half of each sample was treated with 0.1 M NaOH for 30 min at 37°C before derivatization to hydrolyze O-acetyl esters. R, a reagent peak of unknown identity. (B) Tandem HPLC-electrospray MS was performed on DMB-derivatized Sia peaks, each collected separately from the analysis in A. The expected m/z ratios of DMB-derivatized Neu5Ac and O-acetylated Neu5Ac are 426.0 and 468.0, respectively. The other peaks likely represent the sodium adduct (+22) or dehydrated forms (–18). (Insets) Shown are prederivatized Sia structures based on the mass/charge ratio and the HPLC retention times in A.

Fig. 2.

GBS Sia O-acetylation is not strain-specific. Sias were acid-hydrolyzed from 10 clinical GBS isolates of various serotypes (strain-serotype) and were analyzed by DMB-HPLC. The percent of total Sias that were O-acetylated is shown.

Fig. 3.

GBS capsular Sia is O-acetylated. (A) MALDI-TOF analysis of purified type III CPS repeating unit. Cell-wall preparations from COH1 were digested and the released fragments further purified as described in Materials and Methods. In negative mode, the expected m/z ratios of the GBS CPS repeating units with and without O-acetylation are 997.35 and 1039.35, respectively. (B) Revised structure of GBS type III CPS repeat unit. Note that an O-acetyl ester can migrate from carbon position 7 to position 9, but is shown here at position 9 (circled). As indicated in Figs. 1 and 2, only a portion of the capsular Sia is O-acetylated.

Fig. 4.

GBS Sia O-acetylation occurs before CMP activation. Sias were separated based on association with either the CPS or the intracellular Sia pool, and were analyzed by DMB-HPLC. This separation was carried out for the wild-type III strain COH1, an isogenic strain (COH1ΔneuA) in which neuA (the gene encoding the GBS CMP-Sia synthase) was replaced with an antibiotic resistance gene, and both of these strains were transformed with a neuA expression construct pDCneuA(+NeuA). This experiment was performed on several occasions with similar results. It should be noted that DMB derivatization is carried out under conditions that result in a combined quantitation of Neu5Ac and CMP-Neu5Ac.

Fig. 5.

A model for Sia O-acetylation in GBS. Sia biosynthesis in GBS proceeds by epimerization of UDP-GlcNAc to ManNAc, the condensation of ManNAc and phosphoenolpyruvate (PEP) to form Neu5Ac, and the CMP activation of Neu5Ac. Gene products encoded by neuC, neuB, and neuA catalyze the respective steps above. An α2–3 sialyltransferase encoded by cpsK then catalyzes the transfer of activated Neu5Ac to the assembling CPS. Sia analysis of the NeuA mutant demonstrates that O-acetylation can occur on free Sia (Neu5Ac) through the action of an unidentified O-acetyltransferase. The observed decrease in O-acetylation upon overexpression of NeuA also supports a model in which NeuA competes with the O-acetyltransferase for substrate. This model suggests that the GBS CMP-Sia synthase and the sialyltransferase can act on O-acetylated Sias.