Spectrum of pneumococcal serotype 11A variants results from incomplete loss of capsule O-acetylation

Abstract

Streptococcus pneumoniae is a significant bacterial pathogen that expresses >90 capsule serotypes. Conventional serotyping methods assume that each serotype is a genetically and antigenically distinct entity; however, recent investigations have revealed pneumococcal isolates that cannot be unambiguously serotyped because they share the properties of more than one serotype. Here, we employed a novel serotyping method and NMR spectroscopy to examine clinical isolates sharing properties of serotypes 11A and 11E. These ambiguous clinical isolates were provisionally named 11A variant (11Av) isolates. Serotype 11A pneumococci characteristically express capsule β-galactose-6-O-acetylation (βGal6OAc) mediated by the capsule synthesis gene wcjE, while 11E strains contain loss-of-function mutations in wcjE and completely lack the expression of βGal6OAc. Although 11Av isolates also contained mutated wcjE alleles, 11Av clinical isolates were composed of antigenically homogeneous bacteria expressing reduced amounts of 11A-specific capsule antigen. NMR data confirmed reduced but detectable amounts of βGal6OAc on 11Av capsule polysaccharide. Furthermore, the transformation of strains with wcjE alleles from 11Av strains was sufficient to restore partial βGal6OAc in an 11E background. We conclude that, instead of being distinct entities, serotypes 11A and 11E represent two extremes of an antigenic spectrum resulting from variable capsule O-acetylation secondary to heterologous wcjE mutations. These findings challenge whether all clinically relevant pneumococci can be definitively categorized into distinct serotypes.

FIG 1

Serotype 11A polysaccharide repeat unit structure and cps locus. (A) Serotype 11A polysaccharide repeat unit structure. Dotted box, the O-acetyl substitution present in serotype 11A PS and absent in serotype 11E PS. Glc, glucose; Gal, galactose; P-Gro, 1-phosphoglycerol. (B) Serotype 11A cps locus. Horizontal line, the region of the serotype 11A cps locus containing serotype-specific genes examined in this study. cpsA to cpsD are genes conserved across the cps loci of almost all pneumococcal serotypes and are implicated in modulating capsule synthesis. All 11E cps loci contain mutations affecting wcjE (*). tnp, transposable element.

FIG 2

Reduced expression of 11A-specific epitopes on 11Av pneumococci. FCSA histograms depict Hyp11AM1 and Hyp11AG2 MAb binding to strains JC03 (11A), JC04 (11E), JC12 (11Av), JC13 (11E), AMB01 (11A), AMB02 (11Av), and ABM03 (isogenic, nonencapsulated control). Black curves, bacteria incubated with the corresponding MAb and secondary antibody; gray curves, negative-control preparations incubated with secondary antibody only.

FIG 3

Alignment of protein sequences encoded by various wcjE alleles. The complete protein sequence encoded by the wcjE allele of the 11A-1 cps locus is bold, and asterisks indicate every tenth amino acid. Amino acid substitutions encoded by the wcjE alleles of strain 3102-06 and the 11A-2 cps locus are shown.

FIG 4

¹H NMR spectra of de-O-acetylated serotype 11A (MNZ2293-A), 11Av (MNZ2293-C), and 11E (MNZ264) capsule PSs, which are indistinguishable. #, NMR signals arising from residual water; *, NMR signals arising from residual Tris-HCl from buffer; PC, NMR signals arising from phosphocholine residues on contaminating teichoic acid.

FIG 5

NMR data of 11Av PS, revealing biochemical signatures of both 11A and 11E PSs. (A and B) The anomeric (A) and methyl (B) regions of the ¹H NMR spectra of native capsule PSs purified from strains 3102-06 (11Av), MNZ2293-C (11Av), MNZ264 (11E), and MNZ2293-A (11A). O-Acetylation-associated signals discussed in the text are labeled with their respective chemical shift values (in ppm). *, hydrolysis-resistant signals arising from contaminating teichoic acid. (C) Select regions of the 2D ¹H-¹³C HMQC NMR spectra of native capsule PSs purified from strains MNZ2293-A, 3102-06, and MNZ264. Signals are labeled with their corresponding shift values (¹H/¹³C, in ppm). In A to C, all spectra are normalized according to the signal intensity arising from the H2 proton of βGlc at 3.36 ppm (see Fig. 4). #, signals present in 11E and 11Av spectra that are grossly absent in 11A spectra.

FIG 6

¹H NMR spectra of native PSs purified from serogroup 11 recombinant strains. The anomeric (left) and methyl (right) regions of the ¹H NMR spectra of native capsule polysaccharide expressed by strains JC03 (11A), JC04 (11E), JC12 (11Av), and JC13 (11E) are shown. *, serotype 11A-specific peaks that are present in the JC03 and JC12 spectra but absent in the JC04 and JC13 spectra. Spectra are normalized according to the signal intensity arising from the H2 proton of βGlc at 3.36 ppm (see Fig. 4).