Distinct baseline immune characteristics associated with responses to conjugated and unconjugated pneumococcal polysaccharide vaccines in older adults.

Pneumococcal infections cause serious illness and death among older adults. The capsular polysaccharide vaccine PPSV23 and conjugated alternative PCV13 can prevent these infections; yet, underlying immunological responses and baseline predictors remain unknown. We vaccinated 39 older adults (>60 years) with PPSV23 or PCV13 and observed comparable antibody responses (day 28) and plasmablast transcriptional responses (day 10); however, the baseline predictors were distinct. Analyses of baseline flow cytometry and bulk and single-cell RNA-sequencing data revealed a baseline phenotype specifically associated with weaker PCV13 responses, which was characterized by increased expression of cytotoxicity-associated genes, increased frequencies of CD16+ natural killer cells and interleukin-17-producing helper T cells and a decreased frequency of type 1 helper T cells. Men displayed this phenotype more robustly and mounted weaker PCV13 responses than women. Baseline expression levels of a distinct gene set predicted PPSV23 responses. This pneumococcal precision vaccinology study in older adults uncovered distinct baseline predictors that might transform vaccination strategies and initiate novel interventions.

Novel pneumococcal capsule type 33E results from the inactivation of glycosyltransferase WciE in vaccine type 33F.

The polysaccharide (PS) capsule is essential for immune evasion and virulence of Streptococcus pneumoniae. Existing pneumococcal vaccines are designed to elicit anticapsule antibodies; however, the effectiveness of these vaccines is being challenged by the emergence of new capsule types or variants. Herein, we characterize a newly discovered capsule type, 33E, that appears to have repeatedly emerged from vaccine type 33F via an inactivation mutation in the capsule glycosyltransferase gene, wciE. Structural analysis demonstrated that 33E and 33F share an identical repeat unit backbone [→5)-ß-D-Galf2Ac-(1→3)-ß-D-Galp-(1→3)-α-D-Galp-(1→3)-ß-D-Galf-(1→3)-ß-D-Glcp-(1→], except that a galactose (α-D-Galp) branch is present in 33F but not in 33E. Though the two capsule types were indistinguishable using conventional typing methods, the monoclonal antibody Hyp33FM1 selectively bound 33F but not 33E pneumococci. Further, we confirmed that wciE encodes a glycosyltransferase that catalyzes the addition of the branching α-D-Galp and that its inactivation in 33F strains results in the expression of the 33E capsule type. Though 33F and 33E share a structural and antigenic similarity, our pilot study suggested that immunization with a 23-valent pneumococcal PS vaccine containing 33F PS did not significantly elicit cross-opsonic antibodies to 33E. New conjugate vaccines that target capsule type 33F may not necessarily protect against 33E. Therefore, studies of new conjugate vaccines require knowledge of the newly identified capsule type 33E and reliable pneumococcal typing methods capable of distinguishing it from 33F.

Reduced quantity and function of pneumococcal antibodies are associated with exacerbations of COPD in SPIROMICS.

While hypogammaglobulinemia is associated with COPD exacerbations, it is unknown whether frequent exacerbators have specific defects in antibody production/function. We hypothesized that reduced quantity/function of serum pneumococcal antibodies correlate with exacerbation risk in the SPIROMICS cohort. We measured total pneumococcal IgG in n = 764 previously vaccinated participants with COPD. In a propensity-matched subset of n = 200 with vaccination within five years (n = 50 without exacerbations in the previous year; n = 75 with one, n = 75 with ≥2), we measured pneumococcal IgG for 23 individual serotypes, and pneumococcal antibody function for 4 serotypes. Higher total pneumococcal IgG, serotype-specific IgG (17/23 serotypes), and antibody function (3/4 serotypes) were independently associated with fewer prior exacerbations. Higher pneumococcal IgG (5/23 serotypes) predicted lower exacerbation risk in the following year. Pneumococcal antibodies are inversely associated with exacerbations, supporting the presence of immune defects in frequent exacerbators. With further study, pneumococcal antibodies may be useful biomarkers for immune dysfunction in COPD.

Discovery and Characterization of Pneumococcal Serogroup 36 Capsule Subtypes, Serotypes 36A and 36B.

Streptococcus pneumoniae can produce a wide breadth of antigenically diverse capsule types, a fact that poses a looming threat to the success of vaccines that target pneumococcal polysaccharide (PS) capsule. Yet, many pneumococcal capsule types remain undiscovered and/or uncharacterized. Prior sequence analysis of pneumococcal capsule synthesis (cps) loci suggested the existence of capsule subtypes among isolates identified as "serotype 36" according to conventional capsule typing methods. We discovered these subtypes represent two antigenically similar but distinguishable pneumococcal capsule serotypes, 36A and 36B. Biochemical analysis of their capsule PS structure reveals that both have the shared repeat unit backbone [→5)-α-d-Galf-(1→1)-d-Rib-ol-(5→P→6)-ß-d-ManpNAc-(1→4)-ß-d-Glcp-(1→] with two branching structures. Both serotypes have a ß-d-Galp branch to Ribitol. Serotypes 36A and 36B differ by the presence of a α-d-Glcp-(1→3)-ß-d-ManpNAc or α-d-Galp-(1→3)-ß-d-ManpNAc branch, respectively. Comparison of the phylogenetically distant serogroup 9 and 36 cps loci, which all encode this distinguishing glycosidic bond, revealed that the incorporation of Glcp (in types 9N and 36A) versus Galp (in types 9A, 9V, 9L, and 36B) is associated with the identity of four amino acids in the cps-encoded glycosyltransferase WcjA. Identifying functional determinants of cps-encoded enzymes and their impact on capsule PS structure is key to improving the resolution and reliability of sequencing-based capsule typing methods and discovering novel capsule variants indistinguishable by conventional serotyping methods.

A Novel Pneumococcal Surface Protein K of Nonencapsulated Streptococcus pneumoniae Promotes Transmission among Littermates in an Infant Mouse Model with Influenza A Virus Coinfection.

We established an infant mouse model for colonization and transmission by nonencapsulated Streptococcus pneumoniae (NESp) strains to gain important information about its virulence among children. Invasive pneumococcal diseases have decreased dramatically since the worldwide introduction of pneumococcal capsular polysaccharide vaccines. Increasing prevalence of nonvaccine serotypes, including NESp, has been highlighted as a challenge in treatment strategy, but the virulence of NESp is not well understood. Protective strategies against NESp colonization and transmission between children require particularly urgent evaluation. NESp lacks capsules, a major virulence factor of pneumococci, but can cause a variety of infections in children and older people. PspK, a specific surface protein of NESp, is a key factor in establishing nasal colonization. In our infant mouse model for colonization and transmission by NESp strains, NESp could establish stable nasal colonization at the same level as encapsulated serotype 6A in infant mice and could be transmitted between littermates. Transmission was promoted by NESp surface virulence factor PspK and influenza virus coinfection. However, PspK deletion mutants lost the ability to colonize and transmit to new hosts. Promotion of NESp transmission by influenza was due to increased susceptibility of the new hosts. PspK was a key factor not only in establishment of nasal colonization but also in transmission to new hosts. PspK may be targeted as a new candidate vaccine for NESp infection in children.

Anatomical Site-Specific Carbohydrate Availability Impacts Streptococcus pneumoniae Virulence and Fitness during Colonization and Disease.

Streptococcus pneumoniae colonizes the nasopharynx asymptomatically but can also cause severe life-threatening disease. Importantly, stark differences in carbohydrate availability exist between the nasopharynx and invasive disease sites, such as the bloodstream, which most likely impact S. pneumoniae's behavior. Herein, using chemically defined medium (CDM) supplemented with physiological levels of carbohydrates, we examined how anatomical site-specific carbohydrate availability impacted S. pneumoniae physiology and virulence. S. pneumoniae cells grown in CDM modeling the nasopharynx (CDM-N) had reduced metabolic activity and a lower growth rate, demonstrated mixed acid fermentation with marked H2O2 production, and were in a carbon-catabolite repression (CCR)-derepressed state versus S. pneumoniae cells grown in CDM modeling blood (CDM-B). Using transcriptome sequencing (RNA-seq), we determined the transcriptome for the S. pneumoniae wild-type (WT) strain and its isogenic CCR-deficient mutant in CDM-N and CDM-B. Genes with altered expression as a result of changes in carbohydrate availability or catabolite control protein deficiency, respectively, were primarily involved in carbohydrate metabolism, but also encoded established virulence determinants, such as polysaccharide capsule and surface adhesins. We confirmed that anatomical site-specific carbohydrate availability directly influenced established S. pneumoniae virulence traits. S. pneumoniae cells grown in CDM-B formed shorter chains, produced more capsule, were less adhesive, and were more resistant to macrophage killing in an opsonophagocytosis assay. Moreover, growth of S. pneumoniae in CDM-N or CDM-B prior to the challenge of mice impacted relative fitness in a colonization model and invasive disease model, respectively. Thus, anatomical site-specific carbohydrate availability alters S. pneumoniae physiology and virulence, in turn promoting anatomical site-specific fitness.

Effect of Oral Streptococci Expressing Pneumococcus-like Cross-Reactive Capsule Types on World Health Organization Recommended Pneumococcal Carriage Detection Procedure.

BACKGROUND: Carriage studies are fundamental to assessing the effects of pneumococcal vaccines. Because a large proportion of oral streptococci carry homologues of pneumococcal genes, non-culture-based detection and serotyping of upper respiratory tract (URT) samples can be problematic. In the current study, we investigated whether culture-free molecular methods could differentiate pneumococci from oral streptococci carried by adults in the URT. METHODS: Paired nasopharyngeal (NP) and oropharyngeal (OP) samples were collected from 100 older adults twice a month for 1 year. Extracts from the combined NP + OP samples (n = 2400) were subjected to lytA real-time polymerase chain reaction (PCR). Positive samples were subjected to pure culture isolation, followed by species confirmation using multiple approaches. Multibead assays and whole-genome sequencing were used for serotyping. RESULTS: In 20 of 301 combined NP + OP extracts with positive lytA PCR results, probable pneumococcus-like colonies grew, based on colony morphology and biochemical tests. Multiple approaches confirmed that 4 isolates were Streptococcus pneumoniae, 3 were Streptococcus pseudopneumoniae, 12 were Streptococcus mitis, and 1 were Streptococcus oralis. Eight nonpneumococcal strains carried pneumococcus-like cps loci (approximate size, 18-25 kb) that showed >70% nucleotide identity with their pneumococcal counterparts. While investigating the antigenic profile, we found that some S. mitis strains (P066 and P107) reacted with both serotype-specific polyclonal (type 39 and FS17b) and monoclonal (Hyp10AG1 and Hyp17FM1) antisera, whereas some strains (P063 and P074) reacted only with polyclonal antisera (type 5 and FS35a). CONCLUSION: The extensive capsular overlap suggests that pneumococcal vaccines could reduce carriage of oral streptococci expressing cross-reactive capsules. Furthermore, direct use of culture-free PCR-based methods in URT samples has limited usefulness for carriage studies.

Investigation on the virulence of non-encapsulated Streptococcus pneumoniae using liquid agar pneumonia model.

INTRODUCTION: Since the introduction of pneumococcal conjugate vaccine, there have been warnings of an increase in infections caused by non-vaccine type of Streptococcus pneumoniae strains. Among them, nonencapsulated Streptococcus pneumoniae (NESp) has been reported to cause invasive infections, especially in children and the elderly. Due to low virulence, however, basic experimental reports on invasive infections are limited. METHODS: We applied a liquid-agar method to establish a mouse model of invasive NESp infection. Mice were intratracheally administered a bacterial suspension including agar. With this technique, we investigated the pathogenicity of NESp and the effect of Pneumococcal surface protein K (PspK), a specific surface protein antigen of NESp. NESp wild-type strain (MNZ11) and NESp pspK-deleted mutant strain (MNZ1131) were used in this study. The survival rate, number of bacteria, cytokine/chemokine levels in the bronchoalveolar lavage fluid, and histology of the lung tissue were evaluated. RESULTS: Mice that were intratracheally administered MNZ11 developed lethal pneumonia with bacteremia within 48 h. Conversely, MNZ1131 showed predominantly low lethality without significant pro-inflammatory cytokine production. NESp was found to cause severe pneumonia and bacteremia upon reaching the lower respiratory tract, and PspK was a critical factor of NESp for developing invasive infections. CONCLUSIONS: The current study demonstrated the ability of NESp to develop invasive diseases, especially in connection with PspK by use of a mouse pneumonia model.

Structural, Genetic, and Serological Elucidation of Streptococcus pneumoniae Serogroup 24 Serotypes: Discovery of a New Serotype, 24C, with a Variable Capsule Structure.

Pneumococcal capsules are important in pneumococcal pathogenesis and vaccine development. Although conjugate vaccines have brought about a significant reduction in invasive pneumococcal disease (IPD) caused by vaccine serotypes, the relative serotype prevalence has shifted with the dramatic emergence of serotype 24F in some countries. Here, we describe 14 isolates (13 IPD and 1 non-IPD) expressing a new capsule type, 24C, which resembles 24F but has a novel serological profile. We also describe the antigenic, biochemical, and genetic basis of 24F and 24C and the related serotypes 24A and 24B. Structural studies show that 24B, 24C, and 24F have identical polysaccharide backbones [ß-Ribf-(1→4)-α-Rhap-(1→3)-ß-GlcpNAc-(1→4)-ß-Rhap-(1→4)-ß-Glcp] but with different side chains, as follows: 24F has arabinitol-phosphate and 24B has ribitol-phosphate. 24C has a mixture of 24F and 24B repeating units, with the ratio of ribitol to arabinitol being strain dependent. In contrast, the 24A capsule has a backbone without ß-Ribf but with arabinitol-phosphate and phosphocholine side chains. These structures indicate that factor-sera 24d and 24e recognize arabinitol and ribitol, respectively, which explains the serology of serogroup 24, including those of 24C. The structures can be genetically described by the bispecificity of wcxG, which is capable of transferring arabinitol or ribitol when arabinitol is limiting. Arabinitol is likely not produced in 24B but is produced in reduced amounts in 24C due to various mutations in abpA or abpB genes. Our findings demonstrate how pneumococci modulate their capsule structure and immunologic properties with small genetic changes, thereby evading host immune responses. Our findings also suggest a potential for new capsule types within serogroup 24.

Carbon Source-Dependent Changes of the Structure of Streptococcus pneumoniae Capsular Polysaccharide with Serotype 6F.

The structure of the exopolysaccharide capsule of Streptococcus pneumoniae is defined by the genetic arrangement of the capsule operon allowing the unequivocal identification of the pneumococcal serotype. Here, we investigated the environment-dependent composition of the polysaccharide structure of S. pneumoniae serotype 6F. When grown in a chemically defined medium (CDM) with glucose versus galactose, the exopolysaccharide capsule of the serotype 6F strains reveals a ratio of 1/0.6 or 1/0.3 for galactose/glucose in the capsule by 1H-NMR analyses, respectively. Increased production of the capsule precursor UDP-glucose has been identified by 31P-NMR in CDM with glucose. Flow cytometric experiments using monoclonal antibodies showed decreased labelling of Hyp6AG4 (specific for serotype 6A) antibodies when 6F is grown in glucose as compared to galactose, which mirrors the 1H-NMR results. Whole-genome sequencing analyses of serotype 6F isolates suggested that the isolates evolved during two different events from serotype 6A during the time when the 13-valent pneumococcal conjugate vaccine (PCV-13) was introduced. In conclusion, this study shows differences in the capsular structure of serotype 6F strains using glucose as compared to galactose as the carbon source. Therefore, 6F strains may show slightly different polysaccharide composition while colonizing the human nasopharynx (galactose rich) as compared to invasive locations such as the blood (glucose rich).

Pneumococci Can Become Virulent by Acquiring a New Capsule From Oral Streptococci.

Pneumococcal conjugate vaccines have been successful, but their use has increased infections by nonvaccine serotypes. Oral streptococci often harbor capsular polysaccharide (PS) synthesis loci (cps). Although this has not been observed in nature, if pneumococcus can replace its cps with oral streptococcal cps, it may increase its serotype repertoire. In the current study, we showed that oral Streptococcus strain SK95 and pneumococcal strain D39 both produce structurally identical capsular PS, and their genetic backgrounds influence the amount of capsule production and shielding from nonspecific killing. SK95 is avirulent in a well-established in vivo mouse model. When acapsular pneumococcus was transformed with SK95 cps, the transformant became virulent and killed all mice. Thus, cps from oral Streptococcus strains can make acapsular pneumococcus virulent, and interspecies cps transfer should be considered a potential mechanism of serotype replacement. Our findings, along with publications from the US Centers for Disease Control and Prevention, highlight potential limitations of the 2013 World Health Organization criterion for studying pneumococcal serotypes carried without isolating bacteria. We show that an oral streptococcal strain, SK95, and a pneumococcal strain, D39, both produce chemically identical capsular PS. We also show that transferring SK95 cps into noncapsulated, avirulent pneumococcus gave it the capacity for virulence in a mouse model.

Classical and lectin complement pathways and markers of inflammation for investigation of susceptibility to infections among healthy older adults.

Background: There is increasing recognition of the significance of chronic, low-level inflammation in older adults, or "inflammaging." Innate immune responses and host-bacterial interactions are recognized as key factors in inflammaging. Inflammatory cytokine IL-6, and complement protein C1q have been identified as biomarkers for the development of frailty and aging-related diseases. Older adults are also susceptible to infections with serotypes of Streptococcus pneumoniae that bind ficolin-2, a component of the lectin complement pathway, and low ficolin-2 levels could possibly be involved in such susceptibility. Methods: The aim of our study was to evaluate complement pathway components and biomarkers for inflammaging among older adults in order to investigate potential innate immune mechanisms that may account for susceptibility to infections in this population. We compared inflammatory markers, as well as components/activity of the classical and lectin complement pathways between healthy older and younger adults. We hypothesized that older adults would have higher levels of inflammatory markers and C1q, and lower levels of lectin pathway components. Older (≥70 years old) and younger (19-54 years old) adults without significant smoking history or chronic medical conditions were eligible for participation. Inflammatory markers (IL-6, TNF-α, CRP), classical complement pathway activity (CH50) and protein levels (C1q, C3, C4), and lectin pathway (MBL levels/activity, CL-L1, MASP-1/2/3, MAp44, MAp19, and H/M/L-ficolin) were compared between groups. Results: Older adults had significantly higher mean levels of IL-6 and TNF-α. There were no significant differences in lectin pathway components between older and younger adults. Unexpectedly, mean C1q was significantly higher in the younger group in both unadjusted and adjusted analyses. There was also a significant association between race and C1q levels, but this association did not completely account for the observed differences between age groups. Conclusions: We did not observe deficiencies in lectin pathway components to account for increased susceptibility to ficolin-binding serotypes of S. pneumoniae. Elevated levels of inflammatory cytokines in older adults are suggestive of inflammaging. However, the observed age and race-associated changes in C1q have not been previously reported in the populations included in our study. These findings are relevant to the investigation of C1q in aging-related pathology, and for its proposed role as a biomarker for frailty and disease.

Phosphorylcholine esterase is critical for Dolichos biflorus and Helix pomatia agglutinin binding to pneumococcal teichoic acid.

Streptococcus pneumoniae (the pneumococcus) has wall teichoic acid (WTA) and lipoteichoic acid (LTA) expressing the Forssman antigen (FA). Two lectins, Dolichos biflorus agglutinin (DBA) and Helix pomatia agglutinin (HPA), are known to bind FA. To determine the molecular structure targeted by these two lectins, different pneumococcal strains were studied for DBA/HPA binding with flow cytometry and fluorescence microscopy. Genetic experiments were used to further examine the lectins' molecular target. Twelve strains were positive for DBA binding, whereas three were negative. Super-resolution microscopy showed that DBA stained only the subcapsular area of pneumococci. The three DBA nonbinders showed no phosphorylcholine esterase (Pce) activity in vitro, whereas 10 DBA binders displayed Pce activity (the remaining two strains were DBA binders with no Pce activity in vitro). The pcegene sequence for 10 representative strains revealed two functional pce alleles, the previously recognized "allele A" and a newly discovered "allele B" (with 12 additional nucleotides). Isolates with allele B showed no Pce activity in vitro but did bind to DBA, indicating allele B Pce is functional in vivo. Genetic transfer experiments confirmed that either allele is sufficient (and necessary) for DBA binding. The three DBA nonbinders had various mutations that affected Pce function. Observations with HPA were identical to those with DBA. We show that DBA and HPA bind only to the WTA/LTA of pneumococcal isolates with a functional Pce enzyme. A newly discovered Pce variant (allele B) is functional in vivo but nonfunctional when assayed in vitro.

Molecular typing, antibiotic susceptibility, and biofilm production in nonencapsulated Streptococcus pneumoniae isolated from children in Japan.

The prevalence of nonencapsulated Streptococcus pneumoniae (NESp) has increased with the introduction of pneumococcal conjugate vaccines in children; however, the bacteriological characteristics of NESp have not been sufficiently clarified. In this study, NESp strains isolated from the nasopharyngeal carriage of children from four nursery schools in Japan were analyzed for molecular type, antibiotic susceptibility, and biofilm productivity. A total of 152 putative S. pneumoniae strains were identified by optochin-susceptibility analysis, of which 21 were not serotypeable by slide agglutination, quellung reaction, or multiplex PCR. Among these 21 strains, three were lytA-negative and, therefore, not S. pneumoniae. The remaining 18 strains were positive for lytA, ply, pspK, and bile solubility and were confirmed as NESp. Therefore, the isolation rate of NESp in the S. pneumoniae strains in this study was 12.0% (18/149). Molecular-typing analyses classified five strains as two existing sequence types (STs; ST7502 and ST7786), and 13 strains formed four novel STs. Horizontal spread was suspected, because strains with the same ST were often isolated from the same nursery school. The NESp isolates were generally susceptible to most antimicrobials, with the exception of macrolides; however, all isolates possessed more than one abnormal penicillin-binding protein gene. Furthermore, NESp strains were more effective than encapsulated counterparts at forming biofilms, which showed obvious differences in morphology. These data indicated that NESp strains should be continuously monitored as emerging respiratory pathogens.

Measuring quantity and function of pneumococcal antibodies in immunoglobulin products.

BACKGROUND: Immunoglobulin replacement therapy is a cornerstone of the treatment of primary immunodeficiencies. Preparations used for replacement therapy are processed by purifying immunoglobulins from large pools of plasma, which were obtained from healthy donors. The constituent antibodies in these products depend on the immune history of the donor pool as well as manufacturing processes that differ among manufacturers. For these reasons various methods have been proposed to examine the levels and function of antibodies to organisms such as Streptococcus pneumoniae, which frequently causes infections in patients with immunodeficiencies. Pneumococcal antibody levels or antibody function can be measured with enzyme-linked immunosorbent assay (ELISA) or multiplexed opsonophagocytosis assay (MOPA). Although these assays were developed initially to assess the immunogenicity of pneumococcal vaccines, the techniques have been adapted to evaluate immunoglobulin products as well. STUDY DESIGN AND METHODS: This article provides a concise review of the analytic techniques for measuring pneumococcal antibodies and prior studies of immunoglobulin products utilizing these methods. RESULTS: Studies utilizing these assays have demonstrated that antibody levels of immunoglobulin products can vary with time, location, and manufacturer. CONCLUSIONS: We highlight current issues and future considerations concerning measurement of pneumococcal antibodies in immunoglobulin products, and the assays used for this purpose.

Position of O-Acetylation within the Capsular Repeat Unit Impacts the Biological Properties of Pneumococcal Serotypes 33A and 33F.

Streptococcus pneumoniae (pneumococcus) produces many capsule types that differ in their abilities to evade host immune recognition. To explain these serotype-dependent protective capacities, many studies have investigated capsular thickness or the interaction of the capsule with complement proteins, but the effects of small chemical modifications of the capsule on its function have not been studied. One small chemical modification found frequently among pneumococcal capsules is O-acetylation. Pneumococcal serotype 33A has two membrane-bound O-acetyltransferase genes, wciG and wcjE A 33A wcjE-deficient variant, 33F, occurs naturally and is increasing in prevalence in the wake of widespread conjugate vaccine use, but no wciG-deficient variants have been reported. To study the biological consequence of the loss of O-acetylation, we created wciG-deficient variants in both serotypes 33A and 33F, which we named 33X1 (ΔwciG) and 33X2 (ΔwciG ΔwcjE). Serotypes 33X1 and 33X2 express novel capsule types based on serological and biochemical analyses. We found that loss of WcjE-mediated O-acetylation appears not to affect cell wall shielding, since serotypes 33A and 33F exhibit comparable nonspecific opsonophagocytic killing, biofilm production, and adhesion to nasopharyngeal cells, though serotype 33F survived short-term drying better than serotype 33A. Loss of WciG-mediated O-acetylation in serotypes 33X1 and 33X2, however, resulted in a phenotype resembling that of nonencapsulated strains: increased cell wall accessibility, increased nonspecific opsonophagocytic killing, enhanced biofilm formation, and increased adhesion to nasopharyngeal cells. We conclude that WciG-mediated, but not WcjE-mediated, O-acetylation is important for producing protective capsules in 33A and that small chemical changes to the capsule can drastically affect its biological properties.

Discovery of Novel Pneumococcal Serotype 35D, a Natural WciG-Deficient Variant of Serotype 35B.

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.

WciG O-Acetyltransferase Functionality Differentiates Pneumococcal Serotypes 35C and 42.

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 O-acetyltransferase 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-ß-galactofuranose, as well as the 2-position of 6-ß-galactofuranose. However, serotype 42 has only O-acetylation at 3-ß-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.

Pneumococcal Capsules and Their Types: Past, Present, and Future.

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.