Evolution of the capsular gene locus of Streptococcus pneumoniae serogroup 6

Abstract

Streptococcus pneumoniae expressing serogroup 6 capsules frequently causes pneumococcal infections and the evolutionary origins of the serogroup 6 strains have been extensively studied. However, these studies were performed when serogroup 6 had only two known members (serotypes 6A and 6B) and before the two new members (serotypes 6C and 6D) expressing wciN(β) were found. We have therefore reinvestigated the evolutionary origins of serogroup 6 by examining the profiles of the capsule gene loci and the multilocus sequence types (MLSTs) of many serogroup 6 isolates from several continents. We confirmed that there are two classes of cps locus sequences for serogroup 6 isolates. In our study, class 2 cps sequences were limited to a few serotype 6B isolates. Neighbour-joining analysis of cps sequence profiles showed a distinct clade for 6C and moderately distinct clades for class 1 6A and 6B sequences. The serotype 6D cps profile was found within the class 1 6B clade, suggesting that it was created by recombination between 6C and 6B cps loci. Interestingly, all 6C isolates also had a unique wzy allele with a 6 bp deletion. This suggests that serotype switching to 6C involves the transfer of a large (>4 kb) gene segment that includes both the wciN(β) allele and the 'short' wzy allele. The MLST studies of serotype 6C isolates suggest that the 6C cps locus is incorporated into many different pneumococcal genomic backgrounds but that, interestingly, 6C cps may have preferentially entered strains of the same genomic backgrounds as those of serotype 6A.

Fig. 1.

Capsule gene loci of 6A (GenBank accession no. CR931638), 6B (GenBank accession no. CR931639), 6C (GenBank accession no. EF538714) and 6D (GenBank accession no. HM171374) strains. All ORFs involved in capsule synthesis are shown as horizontal arrows, and their direction indicates the transcriptional orientation. The three regions of the wciP (645 bases), wzy (492 bases) and wzx (477 bases) genes used for cps profiling are shown as three black bars above the 6A cps diagram. The wciN and wciP alleles are indicated by α and β. The size of a gene fragment from the beginning of wciN_β to the end of wzx is about 4130 bases. Bent arrows indicate potential transcription sites.

Fig. 2.

Sequence diversity of the wciN_β and its flanking regions for serotype 6C and 6D strains. The DNA sequence was determined from base 5671 to base 8452. The consensus sequence (top line) and the numbering system (numbers are given vertically) are based on the 6C cps sequence (GenBank accession no. EF538714). Heavy vertical bars at the top indicate the two ends of the wciN_β ORF. The flanking regions of the ‘foreign gene’ that produced wciN_β are shaded (between bases 6209 and 6508 and bases 7545 and 7655). Most sequences were from serotype 6C isolates except for the four sequences from 6D isolates (marked by •). One of 26 6C sequences is a published sequence (CHPA388, GenBank accession no. EF538714). Two Oceania 6D sequences are from GenBank (accession nos FJ899599 and FJ899600). X, No corresponding sequences. Sequences from 33F (GenBank accession no. CR931697) and a class 2 6B strain (GenBank accession no. AF246897) are included to show the similarity in the 5′ and 3′ flanking regions, respectively. The sequence for 6A (GenBank accession no. CR931638) is shown for comparison at the bottom.

Fig. 3.

Neighbour-joining trees of unique cps profiles. (a) Tree constructed with 22 unique cps profiles from our study, which included 23 6A (▴), 18 6B (○), 25 6C (▪) and 2 6D (+) isolates. Table 2 shows the cps profiles of all the strains included in our study. Note that three class 2 6B isolates are shown on the right of the tree and all class 1 strains are shown on the left. (b) Tree constructed with cps profile data from both our current study and a published study (Mavroidi et al., 2004). Class 2 isolates are shown on the right of the tree and two cross-over strains (profiles 8-7-5 and 7-7-6) are shown in the middle. The bar represents a genetic distance of 0.5 %. (c) Polymorphic nucleotides for all new alleles of the cps genes (wciP, wzy and wzx). Allele 1 is shown at the top for comparison. Note that wzy alleles 11 and 12, which are associated with serotype 6C, have 6 base deletions (positions 152–157). –, No 6 bp deletions; ., identical bases.

Fig. 4.

Two models for the evolution of the serogroup 6 strains. (a) The model previously proposed by Mavroidi et al. (2004) modified to include the new serotypes 6C and 6D. (b) Proposed new model based on the new data. The nature of the ‘cps gene pool’ from which the capsule genes have originated is undefined at the moment. Genetic recombination events are shown with thin arrows. Mutations are shown with dashed lines.