Genetic Diversity of the Pneumococcal Capsule: Implications for Molecular-based Serotyping

Mary Catherine McEllistrem

Disclosures

Future Microbiol. 2009;4(7):857-865. 

In This Article

Insight into Limitations of PCR-based Serotyping Methods

Recent studies that have completely sequenced the capsular operon of 91 serotypes[11,12] provide insight into the inability for PCR-based assays to classify some of the PCV7 serotypes ( Table 2 & Figure 1). Namely, the expression of a serotype 6A or 6B capsule correlates with a single nucleotide polymorphism (SNP) within the wciP gene, which encodes rhamnosyl transferase.[29] While pyrosequencing serogroup 6 strains can correctly classify 6A and 6B strains by identifying the SNP in the wciP gene,[30] it is unlikely this technique could realistically be utilized to differentiate serotypes from all related serogroups. To compound the difficulty associated with serogroup 6, the recently discovered serotype 6C is quite similar to 6A and only differs in the WciN gene. Specifically, the 6A capsular operon encodes a galactosyltransferase, while the 6C capsular operon encodes a glucosyltransferase. The genotypic difference likely arose through a single recombination event.[12]

Figure 1.

Selected genes within the capsular operon of serotypes 3, 6B, 9V, 15C, 18C and 19F. Spaces between the boxes indicate intervening sequences between the genes.
Adapted from [31,59].

Moreover, serotypes 9L, 9N, 9V and 9A are highly genetically related. Serotypes 9L and 9N only differ by 79 bp, most of which are present in the glycosyltransferase gene, wcjA. Serotypes 9V and 9A differ only in acetylation. Specifically, the O-acetyl transferase-encoding wcjE gene is intact and functional in 9V, and disrupted by frameshift mutation in 9A (deletion of nt 726).[11,31] Likewise, a frameshift mutation in the wciX gene, which results in an O-acetylation state, differentiates 18B from 18C. Furthermore, among prominent PPS23 serotypes, serotypes 15B and 15C only differ by the presence (15B) and absence (15C) of acetylation.[32,33] The difference is due to a variable-length tandem repeat at the 5′ end of wciZ.[11,31]

Sequenced-based Serotyping Methods

Since many clinically relevant serotypes within a serogroup have only small genetic differences, sequence-based assays have been pursued in addition to pyrosequencing. For example, one study sequenced part of the wzg (cpsA), wzh (cpsB), wzy and wzx genes and attempted to correlate the genotype with the serotype. Unfortunately, the assay was unfeasible as serotyping a strain required nearly 50 different PCR reactions. Moreover, the sensitivity was only 73%, even with a small number of samples (Table 1).[34,35] As more strains are included, it is likely that clinically insignificant SNPs will be detected, resulting in classification of strains as 'unrelated' despite being the same serotype by quellung reaction.

Significant Polymorphisms in the Capsular Operon

While a variety of SNPs in the capsular operon are not meaningful, biofilm-derived pneumococcal strains have been detected with key mutations in this operon. The serotype 3 capsular operon was characterized nearly 25 years ago;[14,15] however, sequencing of these capsular genes has become essential to further our understanding of biofilm formation. Acapsular strains, also called rough variants or small colony variants, which emerge during the formation of serotype 3 biofilms, arise owing to tandem duplications,[36] deletions[37] and/or SNPs in the serotype-specific capsular gene, cps3D (Figure 1).[38] It is likely that when future clinically relevant 'nontypeable' strains are detected in natural populations, sequencing the fragment between dexB and the aliA genes will provide insight into whether this strain would have been of a known serotype if not for a mutation in a type-specific capsular gene.

Immunology-based Serotyping Methods

Given the limitations of PCR and sequencing assays, immunologic assays have been developed to serotype the pneumococci.[39] The multibead assay is a semiautomated, reliable and validated method that can accurately identify 24 different serotypes.[40,41] In essence, this multiplex assay uses flow cytometry to serotype the strains using monoclonal antibodies and pneumococcal polysaccharides covalently linked to a variety of latex beads. The capacity of pneumococcal lysates to prevent anticapsule antibodies to bind to latex beads is the basis for serotype designation for the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 20, 22F, 19A, 19F, 23F and 33F. Since serotype 15C and other emerging serotypes were not detected with this technique, a single multiplex PCR reaction for five additional serotypes (15A, 15C, 16F, 35B and 38) was also included. Together, the immunology- and molecular-based approach reliably serotyped strains from 29 serotypes. It is likely that this combined approach, which utilizes both the immunologic and genetic differences in capsule to accurately serotype a strain, will be needed to reliably serotype all 91 serotypes (Table 1). Alternatively, perhaps a comprehensive immunology-based serotyping method could be developed. The main limitations to this approach are the challenges associated with the development of all the necessary monoclonal antibodies, and the difficulty of performing this assay in a clinical laboratory.

Discovery of New Serotypes while Developing an Immunology-based Serotyping Assay

In an effort to create the novel immunology-based multibead serotyping method,[39–41] previously unknown serotypes (6C and 11A) have been discovered.[12,42] The presence of these newly recognized serotypes may partially explain unexpected seroepidemiology trends in the PCV7 era. Specifically, PCV7 provides cross-protection against serotype 6A; however, serotype '6A', as classified by the quellung reaction, is actually both 6A and 6C strains in studies published before 2007. With the use of monoclonal antibodies to differentiate serotype 6C from 6A, Park et al. demonstrated that serotype 6A disease is actually declining while serotype 6C disease is increasing in some age groups in the PCV7 era.[43] It is likely that more serotypes will be discovered as more monoclonal antibodies are developed.

Implications of Capsular Recombination for Molecular-based Serotyping Assays

The ability of the pneumococci to incorporate DNA from the environment has been known for over 75 years. It is now estimated that recombination occurs ten-times more frequently than mutation in pneumococcus.[44] Penicillin resistance via altered penicillin-binding proteins (encoded by pbp), serotype capsular transformation and pneumococcal surface protein A gene diversity can all occur by this mechanism.[45–47] Moreover, the housekeeping gene, ddl, has been shown to 'hitchhike' with one of the penicillin-binding protein genes, pbp2b, in natural populations.[48] Since two of the penicillin-binding protein genes (pbp2x and pbp1a) flank the capsular operon, located between dexB and aliA, a 39-kB recombination event can simultaneously alter both the serotype and penicillin resistance level.[49] The crossover sites implicated in capsular transformation for several strains have been determined.[45,46,49] To date, none of the crossover sites have been within the type-specific capsular genes.

However, it is possible that a strain could emerge that combines part of the capsule from two different serotypes.[50] For example, serotype 14 and serogroup 15 differ only at the 3′ end of the capsular operon. Likewise, serogroup 12 and serotypes 44 and 46 only differ in the insertion-sequence transposase genes.[4] Therefore, recombination events with crossover sites within the capsular loci of two different serotypes could result in a strain that has unique immunologic properties. Depending on the type of genotype-based serotyping method used, this strain could be misclassified. By contrast, the detection of these novel serotypes could be greatly enhanced with an immunology-based serotyping technique.

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