Evidence for Rare Capsular Switching in Streptococcus agalactiae

The polysaccharide capsule is a major antigenic factor in Streptococcus agalactiae (Lancefield group B streptococcus [GBS]). Previous observations suggest that exchange of capsular loci is likely to occur rather frequently in GBS, even though GBS is not known to be naturally transformable. We sought to identify and characterize putative capsular switching events, by means of a combination of phenotypic and genotypic methods, including pulsed-field gel electrophoretic profiling, multilocus sequence typing, and surface protein and pilus gene profiling. We show that capsular switching by horizontal gene transfer is not as frequent as previously suggested. Serotyping errors may be the main reason behind the overestimation of capsule switching, since phenotypic techniques are prone to errors of interpretation. The identified putative capsular transformants involved the acquisition of the entire capsular locus and were not restricted to the serotype-specific central genes, the previously suggested main mechanism underlying capsular switching. Our data, while questioning the frequency of capsular switching, provide clear evidence for in vivo capsular transformation in S. agalactiae, which may be of critical importance in planning future vaccination strategies against this pathogen.Streptococcus agalactiae (group B streptococcus [GBS]) is primarily a colonizing agent of the genitourinary and gastrointestinal tracts, but it is also a leading cause of bacterial sepsis and meningitis in neonates and is increasingly associated with invasive infections in adults (39). The capsular polysaccharide is a major GBS virulence factor and also the main target of antibody-mediated killing (11). In the last decade, conjugated multivalent vaccines have been developed and proved to be highly immunogenic, raising the possibility of the prevention of perinatal GBS disease through maternal immunization (38).Nine capsular types are recognized: Ia, Ib and II to VIII, along with a new provisional serotype IX, recently proposed (19). Comparison of the capsular locus genes suggested that the structural diversity of the capsular polysaccharide is associated with the genetic diversity of the capsular locus, possibly driven by horizontal gene transfer (9, 24). Capsular serotyping has been the classical method used in epidemiological studies to differentiate GBS isolates, although further characterization of GBS diversity includes the use of a broad range of DNA-based typing methods, such as restriction fragment length polymorphisms (RFLP), pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). Both PFGE and MLST have provided new clues about the population structure of S. agalactiae, particularly the recognition of diverse lineages among serotype III that were shown to differ in virulence potential and tropism (16, 25, 26, 31, 41). Although the distinction of lineages within a particular serotype has proved useful, a complete correlation between capsular type and the lineages defined by MLST was not found (4, 21, 22). Moreover, whole-genome comparative analysis of isolates expressing different serotypes showed that they sometimes share more genes than strains of the same serotype, suggesting a serotype-independent clustering of strains (43). These observations support the hypothesis that closely and divergently related clones may share the genes coding for a particular capsular type, suggesting that exchange of capsular genes in vivo may have occurred (16, 21, 22). We refer to these phenomena here as capsular switching in vivo, recognizable by the expression of different serotypes and the presence of different capsular loci in otherwise indistinguishable isolates when sampling a set of 11 loci distributed in the genome.The changes at the capsular locus were proposed to be driven by the equilibrium between the selective pressure imposed by host immunity and conservation of a particular capsular polysaccharide, as an adaptive advantage of virulent clones (4, 9, 21). Capsular switching by homologous recombination would be facilitated by the organization of the locus encoding the capsular polysaccharide synthesis genes (cps), where the highly variable serotype determining region (cpsG-cpsK) is flanked by conserved genes (9, 24). This led to the suggestion that genetic exchange of the central part of the cps operon could be driving capsular switching (9, 22). According to Luan et al., who specifically addressed this issue, horizontal transfer of capsular genes occurs at a high level within a population without restriction to genetic background. The authors of that study also suggest that since only advantageous combinations of genotype-serotype persist, these altered serotypes, due to capsular switching, are recognized at a lower frequency among stable clones (21).Capsular switching is well established in other streptococcal species such as Streptococcus pneumoniae, where spontaneous in vivo capsular transformation events were observed and characterized (28, 34). In contrast to GBS, S. pneumoniae is naturally transformable, and this is widely believed to be responsible for the ease with which this species exchanges DNA. Capsular switching may have serious impact in pneumococcal vaccination programs since it may provide the selective pressure for virulent genotypes to switch capsules and escape vaccine coverage (6), and a similar response could be seen with a future introduction of GBS vaccination (38).The aim of the present study was to evaluate the concordance between serotype and the clusters defined by PFGE and to further characterize any putative transformants to establish unequivocally that capsular switching occurs in GBS. We combined PFGE with the analysis of multiple genes spread across the GBS genome in order to identify capsular transformants and concluded that capsular switching events occur less frequently than previously thought.