A new pneumococcal serotype, 11E, has a variably inactivated wcjE gene

Abstract

Recently, 2 serologically and biochemically distinct subtypes-11Aalpha and 11Abeta-were discovered among serotype 11A isolates of Streptococcus pneumoniae. Sequence comparison of the capsular polysaccharide synthesis (cps) loci of the 2 subtypes identified disruption of the wcjE gene, a putative O-acetyltransferase, as the genetic hallmark of the 11Abeta phenotype. Directed disruption of wcjE in vitro in an 11Aalpha strain switched the strain to the 11Abeta phenotype, confirming the role played by the gene in the divergence between the subtypes. Furthermore, sequences from 7 11Abeta clinical strains each contained unrelated disruptive mutations in the wcjE gene, displaying an unprecedented degree of genetic heterogeneity in a pneumococcal serotype. We propose to name the 11Aalpha subtype as serotype 11A and the 11Abeta subtype as 11E, a new serotype. Our findings also suggest that the diversity of pneumococcal capsules is much greater than was previously recognized.

Figure 1. Comparison of cps PCR products identify discrepancy in 3′ region of 11A subtypes

A) The 11A cps locus (GenBank accession # CR931653) [8]. The nine regions amplified by PCR and their assigned numbers are shown. Putative O-acetyltransferase genes are marked with a white asterisk. B) PCR products of regions 1-8 obtained from MNZ272 (α) and MNZ264 (β) were run on 1% agarose gel for size comparison.

Figure 2. O-acetylated 1-P-glycerol is the major distinction between the biochemical capsule structures of 11Aα (A) and 11Aβ (B) subtypes

pGlc, glucose pyranose; pGal, galactose pyranose; Gro, glycerol; Ac, O-acetyl group [13].

Figure 3. Sequencing of the cps loci of 11A subtypes reveals disruption of wcjE in 11Aβ

Sequence alignment of a portion of MNZ272 (11Aα) and MNZ264 (11Aβ) cps locus revealing a 332 bp deletion in MNZ264, which includes the start codon (black box) of the wcjE gene (highlighted in gray). The single nucleotide polymorphism [-302] C∷A is highligthed in a box. Nucleotide numbers are assigned according to distance from the wcjE start codon, with the initial thymine being nucleotide ‘0’ (arrow). Nucleotide ‘0’-T corresponds to nucleotide 16325-T of the published 11A cps locus, GenBank accession #CR931653 [8].

Figure 4. wcjE disruption was achieved using a Janus (JS) cassette construct

A) This diagram depicts JS cassette construction using the 5′ and 3′ regions flanking wcjE, and a JS core containing the genes kanA^R (kanamycin resistance) and rpsL⁺ (streptomycin sensitivity). The cassette was transformed into MNZ270 (11Aα), resulting in MNZ786 (11Aβ). Smaller arrows indicate corresponding binding sites of primers used in this assay (Table 2). B) Primers 5310 and 3309 PCR products from MNZ270 and MNZ786.

Figure 5. 11Aβ strains display high heterogeneity in disruption of wcjE

A) PCR products of region 9 from seven 11Aβ strains (β, bold) and in four 11Aα strains (α) were run on 1% agarose gel for size comparison. B) Depiction of the unique mutations affecting wcjE, including transposable element insertions, multiple-nucleotide deletions, a single nucleotide polymorphism (SNP) and nucleotide duplication (Dup). The 11Aα strain MNZ272 is depicted for comparison. Asterisks mark primer binding sites for region 9 PCR amplification. Values correspond to nucleotide distance from the wcjE start codon.