SUMMARY Streptococcus pneumoniae (the pneumococcus) is an important human pathogen. Its virulence is largely due to its polysaccharide capsule, which shields it from the host immune system, and because of this, the capsule has been extensively studied. Studies of the capsule led to the identification of DNA as the genetic material, identification of many different capsular serotypes, and identification of the serotype-specific nature of protection by adaptive immunity. Recent studies have led to the determination of capsular polysaccharide structures for many serotypes using advanced analytical technologies, complete elucidation of genetic basis for the capsular types, and the development of highly effective pneumococcal conjugate vaccines. Conjugate vaccine use has altered the serotype distribution by either serotype replacement or switching, and this has increased the need to serotype pneumococci. Due to great advances in molecular technologies and our understanding of the pneumococcal genome, molecular approaches have become powerful tools to predict pneumococcal serotypes. In addition, more-precise and -efficient serotyping methods that directly detect polysaccharide structures are emerging. These improvements in our capabilities will greatly enhance future investigations of pneumococcal epidemiology and diseases and the biology of colonization and innate immunity to pneumococcal capsules.

Ficolin-2 mediates serum protection by recognizing specific O-acetylated epitopes of pneumococcal capsule polysaccharides, exemplifying a novel host-microbe interaction that innately offers serotype-specific immunity to IPD.

Pneumococcus (Streptococcus pneumoniae) remains a significant cause of morbidity and mortality, especially among those at the extremes of age. Its capsular polysaccharide is essential for systemic virulence. Over 90 serologically distinct pneumococcal capsular polysaccharides (serotypes) are recognized, but they are unequal in prevalence. Because antibodies against the capsule are protective, polysaccharide conjugate vaccines, which are constructed against the most prevalent serotypes, have caused great reductions in pneumococcal disease caused by these serotypes. In response, however, the relative prevalences of serotypes have shifted. Certain previously rare serotypes, such as serotype 35B, are increasing in prevalence. Serotype 35B is thus a likely future vaccine candidate, but due to their previous rarity, serotype 35B strains have not been scrutinized for underlying heterogeneity. We studied putative serotype 35B clinical isolates to assess the uniformity of their serological reactions. While most isolates exhibited the accepted serology of serotype 35B, one isolate failed to bind to critical serotyping reagents. We determined that the genetic basis for this aberrant serology was the presence of inactivating mutations in the O-acetyltransferase gene wciG. Complementation studies in a wciG deletion strain verified that the mutant WciG was nonfunctional, and the serology of the mutant could be restored through complementation with a construct encoding a functional WciG. Nuclear magnetic resonance studies confirmed that the capsule of the WciG-deficient isolate lacked O-acetylation but was otherwise identical to serotype 35B. As this isolate expresses a unique serology with unique biochemistry and a stable genetic basis, we named its novel capsule serotype 35D.KEYWORDS O-acetylation, Streptococcus pneumoniae, capsular polysaccharide, serogroup 35, serotype 35B, serotype 35D S treptococcus pneumoniae (pneumococcus) is an important human pathogen that can express many different polysaccharide capsules that protect the organism from opsonophagocytic killing (1). As antibodies to the capsule are protective, currently available vaccines against pneumococcus are designed to elicit antibodies to the capsule and have been highly successful in reducing the incidence of invasive pneumococcal disease (IPD). A pneumococcal polysaccharide-protein conjugate vaccine (PCV) with polysaccharide from the seven most commonly observed serotypes (PCV7) was introduced in the United States in 2000 and reduced the incidence of IPD in children younger than 5 by over 75% (2); the updated 13-valent PCV (PCV13) has reduced the incidence of IPD by an additional 64% in this age group in the United States (3). However, the protection by these vaccines is serotype specific, and their widespread implementation has brought shifts in the serotypes found in both nasopharyngeal carriage and IPD (see, e.g., reference 4).

Since nasopharyngeal carriage of pneumococcus precedes invasive pneumococcal disease, characteristics of carriage isolates could be incorrectly assumed to reflect those of invasive isolates. While most pneumococci express a capsular polysaccharide, nontypeable pneumococci are sometimes isolated. Carriage nontypeables tend to encode novel surface proteins in place of a capsular polysaccharide synthetic locus, the cps locus. In contrast, capsular polysaccharide is believed to be indispensable for invasive pneumococcal disease, and nontypeables from population-based invasive pneumococcal disease surveillance have not been extensively characterized. We received 14,328 invasive pneumococcal isolates through the Active Bacterial Core surveillance program during 2006–2009. Isolates that were nontypeable by Quellung serotyping were characterized by PCR serotyping, sequence analyses of the cps locus, and multilocus sequence typing. Eighty-eight isolates were Quellung-nontypeable (0.61%). Of these, 79 (89.8%) contained cps loci. Twenty-two nontypeables exhibited serotype 8 cps loci with defects, primarily within wchA. Six of the remaining nine isolates contained previously-described aliB homologs in place of cps loci. Multilocus sequence typing revealed that most nontypeables that lacked capsular biosynthetic genes were related to established non-encapsulated lineages. Thus, invasive pneumococcal disease caused by nontypeable pneumococcus remains rare in the United States, and while carriage nontypeables lacking cps loci are frequently isolated, such nontypeable are extremely rare in invasive pneumococcal disease. Most invasive nontypeable pneumococci possess defective cps locus genes, with an over-representation of defective serotype 8 cps variants.

dStreptococcus pneumoniae clinical isolates were recently described that produced capsular polysaccharide with properties of both serotypes 6A and 6B. Their hybrid serological property correlated with mutations affecting the glycosyltransferase WciP, which links rhamnose to ribitol by an ␣(1-3) linkage for serotypes 6A and 6C and an ␣(1-4) linkage for serotypes 6B and 6D. The isolates had mutations in the triad residues of WciP that have been correlated with enzyme specificity. The canonical triad residues of WciP are Ala192-Ser195-Arg254 for serotypes 6A and 6C and Ser192-Asn195-Gly254 for serotypes 6B and 6D. To prove that the mutations in the triad residues are responsible for the hybrid serotype, we introduced the previously described Ala192-Cys195-Arg254 triad into a 6A strain and found that the change made WciP bispecific, resulting in 6A and 6B repeat unit expression, although 6B repeat unit production was favored over production of 6A repeat units. Likewise, this triad permitted a 6C strain to express 6C and 6D repeat units. With reported bispecificity in WciN, which adds either glucose or galactose as the second sugar in the serogroup 6 repeat unit, the possibility exists for a strain to simultaneously produce all four serogroup 6 repeat units; however, when genes encoding both bispecific enzymes were introduced into a 6A strain, only 6A, 6B, and 6D repeat units were detected serologically. Nonetheless, this may be the first example of a bacterial polysaccharide with three different repeat units. This strategy of expressing multiple repeat units in a single polymer is a novel approach to broadening vaccine coverage by eliminating the need for multiple polysaccharide sources to cover multiple serogroup members. Streptococcus pneumoniae (pneumococcus) is a major human pathogen due to its being a leading cause of pneumonia, meningitis, otitis media, and sepsis. The capsular polysaccharide (PS) is an important virulence factor that protects pneumococci from the host innate immune response and greatly enhances their pathogenicity (1, 2). Over 90 capsular serotypes have been defined based on serological properties (3-8). In addition to unique serological properties, each serotype produces a capsular PS with a distinct biochemical structure and has a unique capsule biosynthesis locus (cps) that encodes the enzymes required for capsule synthesis (3). In most serotypes, these enzymes assemble the repeat units on the cytoplasmic leaflet of the membrane through stepwise addition of each sugar, export the completed repeat units to the outer leaflet, and polymerize the repeat units (9).Serogroup 6 contains four serotypes with unique repeat units-6A, 6B, 6C, and 6D-each with distinct serological, chemical, and genetic features (Fig. 1). The genetic basis for the four serotypes is the allelism of wciN and wciP. Serotypes 6A and 6B encode WciN␣, which adds galactose to the repeat unit, whereas serotypes 6C and 6D encode WciN␤, which adds a second glucose (Glc=) (5, 10, 11). wciP allelism is the basis for differentiatin...

Genetic studies of serogroup 6 isolates of Streptococcus pneumoniae identified putative serotype 6E. Although its capsular polysaccharide structure has not been elucidated, putative serotype 6E is described in an increasing number of studies as a potentially new serotype. We show here that SPEC6B, which is widely used as a target strain for serotype 6B opsonophagocytosis assays, has the genetic features of the putative serotype 6E but produces capsular polysaccharide identical to 6B capsular polysaccharide as determined by one-dimensional (1D) and 2D nuclear magnetic resonance (NMR). Thus, putative serotype 6E is a mere genetic variant of serotype 6B. Also, SPEC6B is appropriate as a target strain for serotype 6B opsonophagocytosis assays. This example illustrates the difficulties of assigning new bacterial serotypes based on genetic findings alone. Streptococcus pneumoniae is a Gram-positive bacterial species capable of producing more than 90 distinct capsule types (1). Due to the significance of S. pneumoniae as a human pathogen, the capsule diversity of pneumococci has been extensively studied serologically, biochemically, and genetically (1). In 2006, the genetic loci for capsule synthesis (cps loci) were sequenced for all known pneumococcal capsule types (2). Subsequent studies have found that genetic information in the cps locus correlates well with serologic and biochemical data for the capsular polysaccharide (PS). Thus, several well-described PCR protocols have been developed for serotyping purposes (e.g., see reference 3 and http://www.cdc.gov/streplab/downloads/pcr-oligonucleotide-primers.pdf; reviewed in reference 1), and it has become common to use genetic information to determine capsule serotypes.Genetic studies of serotype 6B cps have revealed two subgroups of cps loci that differ by Ͼ5% in the cps locus (4). The major subgroup was called class 1; the minor subgroup was called class 2 and has an ϳ300-bp indel element between wciN and wciO as its genetic hallmark. In addition, the class 2 locus contains a 9-nucleotide (nt) in-frame deletion in wze and four open reading frames upstream of wzg (5). Because of the stark genetic difference from class 1, it was suggested that class 2 may be a new serotype, "serotype 6E" (6). Although the authors of this study concluded that "serologic and biochemical characterization" is needed to confirm it as a new serotype (6), the putative serotype 6E is being increasingly treated as a distinct serotype in publications describing its discovery in all parts of the world (5, 7-10) and its association with antibiotic resistance (7, 10). Consequently, a recent study stated that there is an urgent need to determine the structure of capsular polysaccharide from the putative serotype 6E (10).Recently, several serotype 6B reference strains from the Pneumococcal Molecular Epidemiology Network collection were found to have "serotype 6E" genetic loci (10). Furthermore, SPEC6B, which is used as the serotype 6B reference target strain for the multiplexed opsonophagocytosis assa...

Streptococcus pneumoniae produces a protective capsular polysaccharide whose production must be modulated for bacterial survival within various host niches. Capsule production is affected in part by a phosphoregulatory system comprised of CpsB, CpsC, and CpsD. Here, we found that growth of serotype 2 strain D39 under conditions of increased oxygen availability resulted in decreased capsule levels concurrent with an ϳ5-fold increase in Cps2B-mediated phosphatase activity. The change in Cps2B phosphatase activity did not result from alterations in the levels of either the cps2B transcript or the Cps2B protein. Recombinant Cps2B expressed in Escherichia coli similarly exhibited increased phosphatase activity under conditions of high-oxygen growth. S. pneumoniae D39 derivatives with defined deletion or point mutations in cps2B demonstrated reduced phosphatase activity with corresponding increases in levels of Cps2D tyrosine phosphorylation. There was, however, no correlation between these phenotypes and the level of capsule production. During growth under reduced-oxygen conditions, the Cps2B protein was essential for parental levels of capsule, but phosphatase activity alone could be eliminated without an effect on capsule. Under increased-oxygen conditions, deletion of cps2B did not affect capsule levels. These results indicate that neither Cps2B phosphatase activity nor Cps2D phosphorylation levels per se are determinants of capsule levels, whereas the Cps2B protein is important for capsule production during growth under conditions of reduced but not enhanced oxygen availability. Roles for factors outside the capsule locus, possible interactions between capsule regulatory proteins, and links to other cellular processes are also suggested by the results described in this study.

Streptococcus pneumoniae expresses capsular polysaccharides (CPSs) to protect itself from opsonophagocytic killing. The genes responsible for capsules synthesized by the Wzy-dependent mechanism, which accounts for 96 of the 98 known pneumococcal capsule types, are in a chromosomal region known as the cps locus. The nucleotide sequence in this region has been determined for all serotypes. In contrast, not all CPS structures have been defined. The structure of the serotype 35C polysaccharide was recently reported, but the presence of O-acetyltransferase genes in the serotype 35C cps locus suggested that it could be incomplete, as the reported structure contains no O-acetylation. In addition, the genetic distinction of serotype 35C from the closely related serotype 42 was unclear, as their reported cps loci are nearly identical. To clarify these discrepancies, we obtained serotype 35C and 42 clinical and reference isolates and studied their serological and genetic properties, as well as the structures of CPSs purified from reference isolates. We demonstrated that the Oacetyltransferase WciG was functional in serotype 35C but nonfunctional in serotype 42 due to a deletion in wciG. Serotype 35C was O-acetylated at the 5-and 6-positions of 3-␤-galactofuranose, as well as the 2-position of 6-␤-galactofuranose. However, serotype 42 has only O-acetylation at 3-␤-galactofuranose, an observation consistent with its loss of WciG functionality, which is associated with O-acetylation at the 2-position and subsequent reaction with typing antiserum 35a. These findings provide a comprehensive view of the genetic, biochemical structural, and serological bases of serotypes 35C and 42.