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.

Structural, Biosynthetic, and Serological Cross-Reactive Elucidation of Capsular Polysaccharides from Streptococcus pneumoniae Serogroup 16.

Capsular polysaccharides (CPS) are crucial virulence factors of Streptococcus pneumoniae The previously unknown CPS structures of the pneumococcal serogroup 16 (serotypes 16F and 16A) were thoroughly elucidated by nuclear magnetic resonance (NMR) spectroscopy and verified by chemical analysis. The following repeat unit structures were determined: 16F, -3)-α-l-Rhap-[4-P-1-Gro]-(1-3)-α-d-Glcp-[(6-P-1)-Gro]-(1-3)-ß-l-Rhap-[2-OAc]-(1-4)-ß-d-Glcp-(1-; 16A, -3)-ß-d-Galf-[2-OAc (70%)]-(1-3)-α-l-Rhap-(1-2)-α-l-Rhap-(1-3)-α-d-Galp-[(6-P-1)-Gro]-(1-3)-ß-d-Galp-(1-4)-ß-d-Glcp-(1- (OAc, O-acetyl substitution; P-1-Gro, glycerol-1-phosphate substitution) A further analysis of CPS biosynthesis of serotypes 16F and 16A, in conjunction with published cps gene bioinformatics analysis and structures of related serotypes, revealed presumable specific function of glycosyltransferase, acetyltransferase, phosphotransferase, and polymerase. The functions of glycosyltransferases WcxN and WcxT were proposed for the first time, and they were assigned to catalyze linkage of α-l-Rhap-(1-3)-α-d-Glcp and α-l-Rhap-(1-2)-α-l-Rhap, respectively. Furthermore, since serotype 16F was genetically close to serogroup 28, cross-reactions between serogroup 16 and serogroup 28 were studied using diagnostic antisera, which provided further understanding of antigenic properties of CPS and diagnostic antisera. Interestingly, serotype 16F cross-reacted with factor antisera 28b and 11c. Meanwhile, serotype 16A cross-reacted with factor antiserum 11c.IMPORTANCE The vaccine pressure against Streptococcus pneumoniae could result in a change of prevalence in carriage and invasive serotypes. As such, it is necessary to monitor the distribution to achieve successful vaccination of the population, and similarly, it is important to increase the knowledge of even the currently less prevalent serotypes. The CPS are vital for the virulence of the pathogen, and antigenic properties of CPS are based on the structure. Consequently, a better understanding of the structure, biosynthesis, and serology of the capsular polysaccharides can be of great importance toward developing future diagnostic tools and vaccines.

Full NMR assignment, revised structure and biosynthetic analysis for the capsular polysaccharide from Streptococcus Pneumoniae serotype 15F.

Upon investigation of Streptococcus pneumoniae serotype 15F capsular polysaccharide (CPS), we discovered that it had a different phosphorylation substituent, namely glycerol-2-phosphate like the other serogroup 15 CPS rather than the originally reported 0.2 equivalent of phosphate or phosphocholine. Furthermore, we also determined the locations of the two previously unassigned O-acetyl groups present in the repeating unit of the 15F CPS, and carried out full NMR assignments of the 15F as well as 15A CPS. Lastly, a biosynthetic analysis of serotypes 15F and 15A was performed and used to make a prediction for the structure of the recently discovered serotype 15D.

Structural, biosynthetic and serological cross-reactive elucidation of capsular polysaccharides from Streptococcus pneumoniae serogroup 28.

Capsular polysaccharides (CPS) are the key virulent factors in the pathogenesis of Streptococcus pneumoniae. The previously unknown CPS structures of the pneumococcal serotype 28F and 28A were thoroughly characterized by NMR spectroscopy, chemical analysis and AF4-MALS-dRI. The following repeat unit structures were determined: -4)[α-l-Rhap-[4-P-2-Gro]]-(1-3)-α-d-Sug-[6-P-Cho]-(1-3)-ß-l-Rhap-[2-OAc]-(1-4)-ß-d-Glcp-(1-; 28F: Sug = Glcp, Mw: 540.5 kDa; 28A: Sug = GlcpNAc, Mw: 421.9 kDa; The correlation of CPS structures with biosynthesis showed that glycosyltransferase WciU in serotypes 28F and 28A had different sugar donor specificity toward α-d-Glcp and α-d-GlcNAcp, respectively. Furthermore, latex agglutination tests of de-OAc and de-PO4 CPS were conducted to understand cross-reactions between serogroup 28 with factor antiserum 23d. Interestingly, the de-OAc 28F and 28A CPS can still weakly react with factor antiserum 23d, while de-PO4 CPS did not react with factor antiserum 23d. This indicated that OAc group could affect the affinity and P-2-Gro was crucial for cross-reacting with factor antiserum 23d.

Discovery and description of a new serogroup 7 Streptococcus pneumoniae serotype, 7D, and structural analysis of 7C and 7D.

Streptococcus pneumoniae is characterised into 92 serotypes based on antigenic reactions of commercial rabbit sera to the capsular polysaccharides. During development of a bioinformatic serotyping tool (PneumoCaT), an isolate exhibited a novel codon at residue 385 of the glycosyltransferase gene wcwK encoding a distinct amino acid, which differentiates genogroup 7. Investigation by repeat serotyping and Quellung reaction revealed a novel pattern of factor sera with the isolate reacting very strongly with 7f, but also with 7e factor sera. The structure of the capsular polysaccharide was determined by NMR spectroscopy to be an approximately 5:1 combination of the structures of 7C and 7B, respectively, and the structure of 7C was also elucidated. All data from whole genome sequencing, NMR spectroscopy, production of antisera and serotyping of the novel 7 strain shows that it is a new serotype, which will be named in the Danish nomenclature as 7D.

Evaluation of a new lateral flow test for detection of Streptococcus pneumoniae and Legionella pneumophila urinary antigen.

Pneumonia is a major cause of morbidity and mortality worldwide. Early diagnosis of the etiologic agent is important in order to choose the correct antibiotic treatment. In this study we evaluated the first commercial combined test for the agents of pneumococcal pneumonia and Legionnaires' disease based on urinary antigen detection, the ImmuView® Streptococcus pneumoniae and Legionella pneumophila Urinary Antigen Test. In this evaluation, the new test had a significantly higher sensitivity than the BinaxNOW® lateral flow tests and the Binax® EIA test. This identifies the ImmuView® S. pneumoniae and L. pneumophila Urinary Antigen Test as a fast and sensitive point of care test for identification of the infectious agent in a major group of patients with pneumonia.

Application of a lateral flow test as an additional serological tool for diagnosis of Legionella infections.

We investigate a new serological lateral flow test for detection of Legionella infection. The sensitivity of the test was compared to existing ELISA methods, using well-defined samples from patients with Legionella infection. The lateral flow test was found to be a good supplement for fast serological diagnosis of legionellosis including Legionnaires' disease.

Sequence based typing and pre-absorption test in retrospective analysis of a pseudo-outbreak of Legionella infections differentiates true cases of legionellosis.

UNLABELLED: The aim of this study was elimination of false positive results obtained by the Chlamylege kit. Two serological kits (IgM ELISA L. pneumophila sgs1-7; ImmuView(TM) L. pneumophila sg1/sg3) and pre-absorption tests (with L. pneumophila sg1 and sg3 reference strains antigens) were used. 153 sera (79 patients) were examined. The high correlations were found between the results by both tests. Positive results by ELISA (sgs1-7) were found in 19/79 patients; by ImmuView(TM) (sg1+sg3) in 16/63. In 8 patients, the dynamics of the IgM in pairs of sera was high (ratio ≥2). In 5/8 of those patients seroconversion was determined. Selected pre-absorbed sera (15 pairs) were tested simultaneously by the same tests. In 8/15 pairs of sera, the reduction of IgM levels in pre-absorbed sera was higher than 10. The reduction of IgM differed in sg1 and sg3 tests. The probability of infections due to L. pneumophila sg3 (7 patients) and L. pneumophila sg1 (5 patients) was based on the results of pre-absorption tests. The correlation between ELISA and ImmuView(TM) tests of pre-absorbed sera was statistically significant (Po=0.0389). Moreover, genotyping of L. pneumophila (SBT) directly in the sera of selected 15 patients (high IgM reduction) was carried out. Completed 7 alleles profile (ST36) was determined in one patient. However, a second patient had the same profile of 5 alleles, and similar reactions in pre-absorption tests. At least 4 sources of infections were suggested on the base of genotyping and pre-absorption results. CONCLUSIONS: Positive results obtained by molecular techniques (eg.PCR) in the diagnosis of Legionella infections should be supplemented by other tests for confirmation of legionellosis. The sequence based typing carried out directly in clinical specimens seems to be a promising method.

Development and validation of ELISA for detection of antibodies to Legionella pneumophila serogroup 1, 3 and 6 in human sera.

The aim of this study was to determine whether separate measurement of immunoglobulin (Ig) M and G antibodies to Legionella (L.) pneumophila serogroups (sg) 1, 3 and 6 as single antigens can facilitate an early diagnosis of Legionnaires' disease. The developed ELISA was evaluated and compared with an in-house indirect Legionella immunofluorescence antibody test (IFAT) measuring Total Ig. A total of 193 sera from 128 patients with confirmed L. pneumophila infections were used to assess the sensitivity of the developed ELISA. The sensitivity was assessed in different time-periods after onset of symptoms. It was found that the sensitivity of the test increased during the first month of infection, IgM being the most sensitive; ranging from 13% in the first week after onset of symptoms, 45% in the second week to 84% in the third week; in the fourth (and beyond) week a drop to 67% was observed. The IFAT detecting L. pneumophila sg 1-6 had a sensitivity of 11%, 27%, 80% and 59%, respectively, during these time-periods. The test with the lowest sensitivity was the IgG ELISA (0%, 21%, 36% and 52%), but by combining the IgG results with the IgM results, the overall sensitivity of the assay was improved (13%, 48%, 88% and 70%). This study confirms that detection of IgG and IgM antibodies by ELISA is an important diagnostic tool especially during the initial phase of the disease, when supported by other tests like the urinary antigen test, PCR or culture. Furthermore, we showed that the ELISA is suitable for the detection of significant changes in antibody levels in paired serum samples. It was found that the sensitivity was higher for the ELISA assays than for the IFAT. Both the in-house IgM ELISA and the IFAT had a low false positive rate, while a 14% false positive rate was found for the IgG ELISA among serum samples from patients with other infections.