Serum opsonic deficiency produced by Streptococcus pneumoniae and by capsular polysaccharide antigens.

The opsonic requirements for phagocytosis of S. pneumoniae types 6, 7, 18, and 23 were determined in normal and C2 deficient serum, and in normal serum chelated with magnesium ethyleneglycoltetraacetic acid. All four strains were effectively opsonized via the alternative complement pathway, a finding suggesting that the capsular polysaccharides of these strains activated complement via the alternative pathway. Since bacteremic pneumococcal disease is often associated with circulating capsular polysaccharide, it was considered that this cellular component may activate complement in vivo and impair host defenses by producing an opsonic defect for pneumococci. To examine this hypothesis, serum was incubated with suspensions of whole S. pneumoniae types 6, 7, 18, or 23 or with purified capsular polysaccharide from each of these types, and residual complement activity and opsonic capacity were measured. Hemolytic C 3--9 complement activity and opsonic capacity for 3H-thymidine labeled Salmonella typhimurium, a species effectively opsonized via the alternative pathway, were reduced in serum following incubation. Polysaccharide concentrations as low as 1 microgram/ml inhibited serum opsonic capacity for salmonella. Whole pneumococci and pneumococcal capsular polysaccharide also inhibited the opsonic activity of human C2 deficient serum for salmonella, further evidence for activation of complement via the alternative pathway. Pneumococcal capsular polysaccharide markedly inhibited the opsonic capacity of normal serum for the homologous pneumoccal type. Thus, amounts of pneumococcal capsular polysaccharide, similar to those found in the serum of patients with pneumococcal disease, bring about decomplementation of serum via activation of the alternative pathway and inhibit pneumococcal opsonization.     

Novel vaccine strategies with protein antigens of Streptococcus pneumoniae

Infections caused by Streptococcus pneumoniae (pneumococcus) are a major cause of mortality throughout the world. This organism is primarily a commensal in the upper respiratory tract of humans, but can cause pneumonia in high-risk persons and disseminate from the lungs by invasion of the bloodstream. Currently, prevention of pneumococcal infections is by immunization with vaccines which contain capsular polysaccharides from the most common serotypes causing invasive disease. However, there are more than 90 antigenically distinct serotypes and there is concern that serotypes not included in the vaccines may become more prevalent in the face of continued use of polysaccharide vaccines. Also, certain high-risk groups have poor immunological responses to some of the polysaccharides in the vaccine formulations. Protein antigens that are conserved across all capsular serotypes would induce more effective and durable humoral immune responses and could potentially protect against all clinically relevant pneumococcal capsular types. This review provides a summary of work on pneumococcal proteins that are being investigated as components for future generations of improved pneumococcal vaccines.     

Capsular polysaccharides of nongroupable streptococci that cross-react with pneumococcal group 19

The cross-reactivity and chemical characterization of the nongroupable streptococcal and pneumococcal group 19 polysaccharides (PS) have been studied. Extensive cross-reactions were observed between capsular PSs of streptococcal strains 14636/74, 4907, 4731 and pneumococcal type 19F and 19A antisera. Streptococcal 14636/74 PS had an identical composition to that of pneumococcal 19F PS. Type 19F and 14636/74 PS were composed of equimolar amounts of rhamnose, glucose, N-acetyl mannosamine, and phosphorus. The capsular PS of strains 4731 and 4907 contained rhamnose, glucose, ribose, N-acetyl mannosamine, and N-acetyl glucosamine in different molar ratios. Extensive immunologic reactivity was observed between the 19F and 14636/74 PS, as determined by light scattering rate nephelometry, passive immune hemolysis, and precipitin reaction. There was an identity reaction by immunodiffusion between type 19F and 14636/74 PS when reacted with rabbit antiserum against either organism. Biochemical studies showed that strain 14636/74 was not a pneumococcus, because it was optochin resistant, was bile insoluble, did not possess the C-carbohydrate antigen common to all pneumococci, and produced neither pneumolysin nor IgA protease. Furthermore, it grew in comparatively simple media in contrast to the complex nutritional requirements of pneumococci. The 13C-NMR spectra of the 19F and 14636/74 PS were identical. These two capsular PS can, therefore, be considered identical.     

Population-Based Analysis of Invasive Nontypeable Pneumococci Reveals That Most Have Defective Capsule Synthesis Genes

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.     

Type-specific enzyme immunoassay for detection of pneumococcal capsular polysaccharide antigens in nasopharyngeal specimens

We developed a new competitive EIA method for the demonstration of pneumococcal capsular polysaccharides from respiratory samples. The pediatric types 4, 6B, 9V, 14, 18C, 19F and 23F were selected for this study, because these capsular polysaccharides were included in the first heptavalent pneumococcal conjugate vaccines, which were used in the Finnish Otitis Media Vaccine Trial. Sensitivity of the EIA tests for purified polysaccharide antigens varied between 5 and 100 ng/ml, depending on the type. The assays performed well in 100 nasopharyngeal samples (NPS) samples processed through an enrichment culture, with an almost 100% sensitivity compared with routine culture. The method appeared type-specific, except that EIA for 6B capsule also detected 6A. The method is applicable for type-specific identification of pneumococcus in carriage studies.     

Pneumococcal surface protein A and new approaches for pneumococcal vaccine development

The problem of pneumococcal infections is pressing for the whole world. Existing vaccines based only on pneumococci polysaccharide antigens or polysaccharide antigens and diphtherial anatoxin are not capable of protecting from all serotypes of the microorganism. Reasonability of creation of pneumococcal vaccine based on surface proteins of Streptococcus pneumoniae is discussed in the literature. One of such key pneumococcal proteins is pneumococcal surface protein A (PSPA), because it is detected in all the S. pneumoniae strains, has cross activity and switches B-cell immune response to T-cell. Currently the development of conjugated vaccine based on surface proteins and capsule polysaccharides of pneumococcus seems promising.     

Sedimentation and viscosity studies on the capsular and somatic polysaccharides of pneumococcus Type III

Sedimentation constants at infinite dilution have been found to be 1.89 and 4.06 for the somatic and capsular polysaccharides, respectively, from pneumococcus Type III. Intrinsic viscosities have been determined for the somatic and capsular polysaccharides of pneumococcus Type III using the Ostwald viscometer. Molecular weights and dimensions have been calculated for the somatic and capsular polysaccharides of pneumococcus Type III assuming the molecules to be prolate ellipsoids of revolution. Values for the somatic polysaccharide are: molecular weight, 26,400; diameter, 0.97 mmicro; and length, 36.18 mmicro. Values for the capsular polysaccharide are: molecular weight, 171,800; diameter, 1.04 mmicro; and length, 177.87 mmicro. The molecular weights were calculated for the somatic and capsular polysaccharides of pneumococcus Type III assuming the molecules to be flexible chains. The value of the molecular weight of the somatic polysaccharide is 31,500 and the value for the molecular weight of the capsular polysaccharide is 267,500. The molecules of both the somatic and capsular polysaccharides exhibit high degrees of asymmetry.     

Purification and characterization of macrolide 2''-phosphotransferase type II from a strain of Escherichia coli highly resistant to macrolide antibiotics

Hyperimmune and high-titered polyclonal pneumococcal antisera, specific for cross-reactive types within groups, were produced in adult rabbits. Purified capsular polysaccharide was injected intravenously into adult rabbits. One week later, these rabbits were given multiple intravenous injections of formalin-inactivated pneumococci of the cross-reactive type by an established method. Each of the resultant antisera were specific for the cross-reactive type indicating that the previous injection of the polysaccharide had induced epitope-specific tolerance. This method was successful for production of antisera against pneumococcal types 6A, 6B, 9N, 9V, 19F and 19A. Polyclonal rabbit pneumococcal antisera have some advantages over murine monoclonal antibodies for serologic studies and this method should be applicable for producing type-specific antibodies to cross-reactive polysaccharides of clinical interest. Further, this method is simpler and generally produces higher titered monovalent (factor) reagents than absorbed antisera.     

Genetic relatedness of the Streptococcus pneumoniae capsular biosynthetic loci

Streptococcus pneumoniae (the pneumococcus) produces 1 of 91 capsular polysaccharides (CPS) that define the serotype. The cps loci of 88 pneumococcal serotypes whose CPS is synthesized by the Wzy-dependent pathway were compared with each other and with additional streptococcal polysaccharide biosynthetic loci and were clustered according to the proportion of shared homology groups (HGs), weighted for the sequence similarities between the genes encoding the shared HGs. The cps loci of the 88 pneumococcal serotypes were distributed into eight major clusters and 21 subclusters. All serotypes within the same serogroup fell into the same major cluster, but in six cases, serotypes within the same serogroup were in different subclusters and, conversely, nine subclusters included completely different serotypes. The closely related cps loci within a subcluster were compared to the known CPS structures to relate gene content to structure. The Streptococcus oralis and Streptococcus mitis polysaccharide biosynthetic loci clustered within the pneumococcal cps loci and were in a subcluster that also included the cps locus of pneumococcal serotype 21, whereas the Streptococcus agalactiae cps loci formed a single cluster that was not closely related to any of the pneumococcal cps clusters.     

Surface-bound capsular polysaccharide of type Ia group B Streptococcus mediates C1 binding and activation of the classic complement pathway

The role of surface-bound type Ia group B Streptococcus (GBS) capsular polysaccharide in antibody-independent binding of C1 and activation of the classic complement pathway was investigated. In a radiolabeled bacterial-polymorphonuclear leukocyte (PMN) association assay, a measure of bacterial opsonization, preincubation of 3H-type Ia GBS with purified F(ab')2 to the organism blocked the association of the bacteria with PMN', and the inhibitory effect was dose dependent. The specificity of F(ab')2 blocking was shown after adsorption of F(ab')2 with type Ia polysaccharide-sensitized erythrocytes. Polysaccharide-adsorbed F(ab')2 had a 70% decrease in ability to block the association of bacteria with PMN. Evidence for the requirement of the capsular polysaccharide in classic complement pathway activation came from a C1 transfer assay with the use of neuraminidase-digested type Ia GBS. Neuraminidase digestion removed 80% of the terminal sialic acid residues from the native polysaccharide. These neuraminidase-digested organisms had a 72% decrease in binding and transfer of purified C1 compared with non-enzyme-treated organisms. Type Ia capsular polysaccharide bound to sheep erythrocytes promoted classic complement pathway-mediated hemolysis of the cells. The role of C1 inhibitor (INH) in modulation of C1 activation by the organisms was investigated. The possibility existed that the C1 INH could be bound by the bacteria, allowing C1 activation to occur in the fluid phase. The inhibitor was purified from human serum, and its activity was measured before and after incubation with type Ia GBS. The organisms had no effect on C1 INH activity. Thus surface-bound capsular polysaccharide of type Ia GBS mediates C1 binding and classic pathway activation, and this does not involve the C1 INH.     

Activation of the alternative pathway by pneumococcal cell walls.

The present studies were performed in order to identify the pneumococcal subcellular component responsible for activating the alternative pathway. Purified pneumococcal cell walls were able to activate the alternative pathway at a concentration as low as 5 mug/ml and were more active than crude cell walls, which in turn were more active than the whole organism. Purified pneumococcal cell membranes also were able to activiate the alternative pathway but had less than 10% of the activity of the purified walls. Thus, the cell wall appears to play a major role in pneumococcal activation of the alternative pathway. Pneumococcal cell walls containing ethanolamine were as effective as cell walls containing choline in activating the alternative pathway. Since C-reactive protein binds specifically to the phosphorylcholine residue of pneumococcal C-polysaccharide, it is unlikely that pneumococcal cell walls must combine with C-reactive protein in order to activate the alternative pathway.     

SEDIMENTATION AND VISCOSITY STUDIES ON THE CAPSULAR AND SOMATIC POLYSACCHARIDES OF PNEUMOCOCCUS TYPE III

Sedimentation constants at infinite dilution have been found to be 1.89 and 4.06 for the somatic and capsular polysaccharides, respectively, from pneumococcus Type III. Intrinsic viscosities have been determined for the somatic and capsular polysaccharides of pneumococcus Type III using the Ostwald viscometer. Molecular weights and dimensions have been calculated for the somatic and capsular polysaccharides of pneumococcus Type III assuming the molecules to be prolate ellipsoids of revolution. Values for the somatic polysaccharide are: molecular weight, 26,400; diameter, 0.97 mµ; and length, 36.18 mµ. Values for the capsular polysaccharide are: molecular weight, 171,800; diameter, 1.04 mµ; and length, 177.87 mµ. The molecular weights were calculated for the somatic and capsular polysaccharides of pneumococcus Type III assuming the molecules to be flexible chains. The value of the molecular weight of the somatic polysaccharide is 31,500 and the value for the molecular weight of the capsular polysaccharide is 267,500. The molecules of both the somatic and capsular polysaccharides exhibit high degrees of asymmetry.     

Activation by some T-independent antigens and B cell mitogens of the alternative pathway of the complement system.

A number of T-independent antigens and B cell mitogens were examined for their ability to activate C3 via the alternative pathway of the complement system. Loss of hemolytically active C3, generation of anaphylatoxin activity, and immunoelectrophoretic conversion of C3 and factor B, were checked in normal and C4-deficient guinea pig serum, and, in some cases, in normal human serum. As judged by their activity in these assays, 10 lipopolysaccharides of different origin and constitution, pneumococcus type III polysaccharide, levan, dinitrophenylated aminoethyl-dextran, dinitrophenylated (D-glutamic acid, D-lysin) copolymer, polymerized flagellin, and pokeweed mitogen were all capable of initiating the alternative pathway, but differed with respect to their potency, their relative activity in the presence or absence of C4, and their ability to inhibit C3-turnover at high concentrations. Polyvinylpyrrolidone of intermediate molecular weight (4 x 10(4) daltons) was only active if the most sensitive assay was used (anaphylatoxin generation). Other species of polyvinylpyrrolidone, depolymerized pneumococcal polysaccharide, aminoethyl-dextran, [D-glutamic acid, D-lysin] copolymer, phytohemagglutinin and concanavalin A failed to activate C3. C3-consumption by concanavalin A was due to nonspecific binding.     

Detection of pneumococcal antibodies in children with acute pneumonia and pleurisy by an immunoenzyme method

The samples of sera and pleural fluid from sick children have been analyzed by means of EIA techniques. To detect the time course of antibody production, the antigenic preparations of pneumococci (monovalent capsular polysaccharides, polyvalent polysaccharide vaccine and complex pneumococcal antigen) have been used. Antibody response observed in the forms of pneumococcal infection, studied in this investigation, has proved to be highly variable. It is expedient to determine antibodies to polysaccharide antigens not earlier than on days 10-12 from the beginning of the disease. But, besides the positive dynamics of antibodies, their unchanged level is sometimes observed in the patients at the beginning of the disease. As a rule, there is a coincidence between the dynamics of antibody formation in response to polysaccharide antigens and to complex pneumococcal antigen.     

Monoclonal antibodies with specificities for Streptococcus pneumoniae group 9 capsular polysaccharides

Streptococcus pneumoniae group 9 includes four capsular polysaccharide types: 9A, 9L, 9N and 9V. We have generated four mouse monoclonal antibodies against group 9 polysaccharide using heat-treated S. pneumoniae strains of different capsular polysaccharides types as immunogens. The specificities of the monoclonal antibodies were determined by ELISA using capsular polysaccharide directly coated to the wells as antigens and by dot blotting with heat-treated bacteria. Two groups of monoclonal antibodies were found. The first group included two monoclonal antibodies which were found to be capsular type specific. The second group was monoclonal antibodies that bound to epitopes shared by two or three pneumococcal group 9 types. The monoclonal antibody 204,A-4 (IgM) was found to be specific for S. pneumoniae type 9N. The binding of the type 9V specific monoclonal antibody 206,F-5 (IgG1) was found to be dependent upon O-acetyl groups. Monoclonal antibody 205,F-3 (IgM) reacted also with type 9V, but was found to cross-react with types 9A and 9L. The binding of this monoclonal antibody to polysaccharide 9V was not dependent upon O-acetyl moieties. The fourth monoclonal antibody (214,G-5, isotype IgM) did not show any correlation between reactivity with isolated polysaccharides and dot blotting with relevant bacteria. The monoclonal antibody reacted with polysaccharides 9A and 9L in ELISA, but not with the homologous bacteria.     

Mutation in Pneumococcus Type III Affecting Multiple Cistrons Concerned with the Synthesis of Capsular Polysaccharide

The genetic behavior in transformation reactions of 20 noncapsulated mutants of pneumococcus type III suggests that each has a single-site mutation in the locus controlling the synthesis of uridine diphosphate glucose (UDPG) dehydrogenase. Each strain is capable of yielding transformants of the binary capsular type SI-III when exposed to deoxyribonucleic acid (DNA) from type I cells. One additional mutant reacted differently and behaved as if it were a multisite mutant with the mutation affecting both the locus for UDPG dehydrogenase and that controlling the synthesis of high molecular weight type III capsular polysaccharide. When exposed to DNA from type I pneumococci, this strain yielded transformants which were genotypically binary but which expressed only the type I capsular phenotype.     

The virulence function of Streptococcus pneumoniae surface protein A involves inhibition of complement activation and impairment of complement receptor-mediated protection

Complement is important for elimination of invasive microbes from the host, an action achieved largely through interaction of complement-decorated pathogens with various complement receptors (CR) on phagocytes. Pneumococcal surface protein A (PspA) has been shown to interfere with complement deposition onto pneumococci, but to date the impact of PspA on CR-mediated host defense is unknown. To gauge the contribution of CRs to host defense against pneumococci and to decipher the impact of PspA on CR-dependent host defense, wild-type C57BL/6J mice and mutant mice lacking CR types 1 and 2 (CR1/2(-/-)), CR3 (CR3(-/-)), or CR4 (CR4(-/-)) were challenged with WU2, a PspA(+) capsular serotype 3 pneumococcus, and its PspA(-) mutant JY1119. Pneumococci also were used to challenge factor D-deficient (FD(-/-)), LFA-1-deficient (LFA-1(-/-)), and CD18-deficient (CD18(-/-)) mice. We found that FD(-/-), CR3(-/-), and CR4(-/-) mice had significantly decreased longevity and survival rate upon infection with WU2. In comparison, PspA(-) pneumococci were virulent only in FD(-/-) and CR1/2(-/-) mice. Normal mouse serum supported more C3 deposition on pneumococci than FD(-/-) serum, and more iC3b was deposited onto the PspA(-) than the PspA(+) strain. The combined results confirm earlier conclusions that the alternative pathway of complement activation is indispensable for innate immunity against pneumococcal infection and that PspA interferes with the protective role of the alternative pathway. Our new results suggest that complement receptors CR1/2, CR3, and CR4 all play important roles in host defense against pneumococcal infection.     

Functional organization of the gene cluster involved in the synthesis of the pneumococcal capsule.

Streptococcus pneumoniae is a major human pathogen and its capsular polysaccharide has been shown to be the main virulence factor. The molecular organization of the genes governing the formation of this capsule was not studied until the 1990s. The capsular clusters (cap) of eight of the 90 known pneumococcal types have now been studied. The cap operon, located between the dexB and aliA genes, is arranged as a central region comprising the genes coding for the specific-type polysaccharide, flanked by open reading frames that are mostly common to all of the serotypes. The biochemical functions of 24 genes required for capsular polysaccharide biosynthesis have been elucidated but the precise role of the flanking regions in capsular formation is unknown. The natural genetic transformation characteristic of pneumococci, the arrangement of the cap locus and the abundance of transposable elements at this locus favor the genetic variability of the capsule in this microorganism. These well-documented observations together with the finding that some genes located outside the cap cluster may also participate in capsule formation increase the complexity of pneumococcal infection control.     

Non-typeable pneumococci circulating in Portugal are of cps type NCC2 and have genomic features typical of encapsulated isolates

Background

Pneumococcus is a major human pathogen and the polysaccharide capsule is considered its main virulence factor. Nevertheless, strains lacking a capsule, named non-typeable pneumococcus (NT), are maintained in nature and frequently colonise the human nasopharynx. Interest in these strains, not targeted by any of the currently available pneumococcal vaccines, has been rising as they seem to play an important role in the evolution of the species. Currently, there is a paucity of data regarding this group of pneumococci. Also, questions have been raised on whether they are true pneumococci. We aimed to obtain insights in the genetic content of NT and the mechanisms leading to non-typeability and to genetic diversity.

Results

A collection of 52 NT isolates representative of the lineages circulating in Portugal between 1997 and 2007, as determined by pulsed-field gel electrophoresis and multilocus sequence typing, was analysed. The capsular region was sequenced and comparative genomic hybridisation (CGH) using a microarray covering the genome of 10 pneumococcal strains was carried out. The presence of mobile elements was investigated as source of intraclonal variation. NT circulating in Portugal were found to have similar capsular regions, of cps type NCC2, i.e., having aliB-like ORF1 and aliB-like ORF2 genes. The core genome of NT was essentially similar to that of encapsulated strains. Also, competence genes and most virulence genes were present. The few virulence genes absent in all NT were the capsular genes, type-I and type-II pili, choline-binding protein A (cbpA/pspC), and pneumococcal surface protein A (pspA). Intraclonal variation could not be entirely explained by the presence of prophages and other mobile elements.

Conclusions

NT circulating in Portugal are a homogeneous group belonging to cps type NCC2. Our observations support the theory that they are bona-fide pneumococcal isolates that do not express the capsule but are otherwise essentially similar to encapsulated pneumococci. Thus we propose that NT should be routinely identified and reported in surveillance studies.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-863) contains supplementary material, which is available to authorized users.