Evolution of the capsular gene locus of Streptococcus pneumoniae serogroup 6.

Streptococcus pneumoniae expressing serogroup 6 capsules frequently causes pneumococcal infections and the evolutionary origins of the serogroup 6 strains have been extensively studied. However, these studies were performed when serogroup 6 had only two known members (serotypes 6A and 6B) and before the two new members (serotypes 6C and 6D) expressing wciN(ß) were found. We have therefore reinvestigated the evolutionary origins of serogroup 6 by examining the profiles of the capsule gene loci and the multilocus sequence types (MLSTs) of many serogroup 6 isolates from several continents. We confirmed that there are two classes of cps locus sequences for serogroup 6 isolates. In our study, class 2 cps sequences were limited to a few serotype 6B isolates. Neighbour-joining analysis of cps sequence profiles showed a distinct clade for 6C and moderately distinct clades for class 1 6A and 6B sequences. The serotype 6D cps profile was found within the class 1 6B clade, suggesting that it was created by recombination between 6C and 6B cps loci. Interestingly, all 6C isolates also had a unique wzy allele with a 6 bp deletion. This suggests that serotype switching to 6C involves the transfer of a large (>4 kb) gene segment that includes both the wciN(ß) allele and the 'short' wzy allele. The MLST studies of serotype 6C isolates suggest that the 6C cps locus is incorporated into many different pneumococcal genomic backgrounds but that, interestingly, 6C cps may have preferentially entered strains of the same genomic backgrounds as those of serotype 6A.

Fusion proteins containing family 1 and family 2 PspA fragments elicit protection against Streptococcus pneumoniae that correlates with antibody-mediated enhancement of complement deposition.

PspA is an important pneumococcal vaccine candidate that is capable of inducing protection in different animal models. Because of its structural diversity, a PspA-based vaccine should contain at least one fragment from each of the two major families (1 and 2) in order to elicit broader protection. In the present work, we have tested the potential of PspA hybrids containing fused portions of family 1 and 2 (PspA1ABC-4B and PspA1ABC-3AB) PspA fragments to induce protection against pneumococci bearing distinct PspA fragments. Sera from mice immunized with these hybrid PspA fragments were able to increase C3 deposition on pneumococci bearing PspA fragments from both families, in contrast with sera made against the PspA family 1 (PspA1ABC) and PspA family 2 (PspA3ABC) fragments, which were effective only within the same family. Although PspA hybrids were able to extend protection against pneumococcal infection with strains bearing diverse PspA fragments, the immunity elicited by family 2 was clade dependent, suggesting that PspA fragments from family 2 clades 3 and 4 should both be included in a comprehensive PspA vaccine. These results indicate that PspA fusion proteins constitute an efficient immunization strategy for future PspA-based antipneumococcal vaccines since they are able to extend protection provided by a protein derived from a single transcript.

Intranasal immunization with the cholera toxin B subunit-pneumococcal surface antigen A fusion protein induces protection against colonization with Streptococcus pneumoniae and has negligible impact on the nasopharyngeal and oral microbiota of mice.

One of the candidate proteins for a mucosal vaccine antigen against Streptococcus pneumoniae is PsaA (pneumococcal surface antigen A). Vaccines targeting mucosal immunity may raise concerns as to possible alterations in the normal microbiota, especially in the case of PsaA, which was shown to have homologs with elevated sequence identity in other viridans group streptococci. In this work, we demonstrate that intranasal immunization with a cholera toxin B subunit-PsaA fusion protein is able to protect mice against colonization with S. pneumoniae but does not significantly alter the natural oral or nasopharyngeal microbiota of mice.

Intranasal immunization with the cholera toxin B subunit-pneumococcal surface antigen A fusion protein induces protection against colonization with Streptococcus pneumoniae and has negligible impact on the nasopharyngeal and oral microbiota of mice

One of the candidate proteins for a mucosal vaccine antigen against Streptococcus pneumoniae is PsaA (pneumococcal surface antigen A). Vaccines targeting mucosal immunity may raise concerns as to possible alterations in the normal microbiota, especially in the case of PsaA, which was shown to have homologs with elevated sequence identify in other viridans group streptococci. In this work, we demonstrate that intranasal immunization with a cholera toxin B subunit-PsaA fusion protein is able to protect mice against colonization with S. pneumoniae but does not significantly alter the natural oral or nasopharyngeal microbiota of mice.

Antibodies to the pneumococcal surface protein A, PspA, can be produced in splenectomized and can protect splenectomized mice from infection with Streptococcus pneumoniae.

Asplenic individuals have increased susceptibility to septicemia caused by encapsulated bacteria. Streptococcus pneumoniae, a pathogen carried in the nasal passages of many humans without complication, is responsible for a large proportion of infections seen in asplenic individuals. Our studies have evaluated the efficacy of antibodies to pneumococcal surface protein A (PspA) in protection of asplenic mice. In passive immunity studies, pneumococci were more completely cleared from the blood of splenectomized mice receiving passive antiserum to PspA than those receiving normal rabbit serum. From active mucosal (intranasal) and systemic (subcutaneous) immunizations with rPspA, we determined that the levels of PspA antibodies produced in splenectomized mice were not significantly different from levels seen in mock-splenectomized animals. This active immunity to PspA was able to protect splenectomized mice against death following infection with live pneumococci. Our results suggest that PspA immunization may also protect asplenic humans from pneumococcal infections.

PspA family typing and PCR-based DNA fingerprinting with BOX A1R primer of pneumococci from the blood of patients in the USA with and without sickle cell disease.

Disease and mortality rates for Streptococcus pneumoniae infections are much higher in patients with sickle cell disease (SCD) than in age-matched patients without SCD. Pneumococcal surface protein A (PspA) has been proposed as a component in human vaccines against S. pneumoniae to provide greater breadth of coverage than can be obtained with the 7-valent conjugate vaccine. The cross-reactivity of PspA is associated with the 'PspA family' structure. In this study we examined strains of S. pneumoniae from patients with and without SCD to determine whether the strains infecting the hypersusceptible population of SCD patients were limited to the same two PspA families already known to comprise over 95% of strains infecting non-SCD patients. Each strain was also evaluated according to the presence or absence of specific PCR fragments based on repetitive BOX elements to screen for possible SCD-associated clonal structure. Strains from SCD and non-SCD patients were similarly dispersed among the most common BOX PCR groups and strains from both groups expressed a similar distribution of PspA variants. Thus, a PspA vaccine designed for the population at large should also be appropriate for patients with SCD.

Natural materno-fetal transfer of antibodies to PspA and to PsaA.

UNLABELLED: PspA and PsaA are Streptococcus pneumoniae surface proteins and potential pneumococcal vaccine antigens. The aim of this study was to characterize the transplacental transfer of antibodies to PspA and to PsaA. Paired mother and cord blood sera were obtained at delivery from 28 women. Concentrations of antibodies against PspA, PsaA, tetanus toxoid (vaccine-induced antibodies) and P6-outer membrane protein (OMP) of nontypeable Haemophilus influenzae were determined by ELISA. Antibodies to PspA of the IgG, IgG1 and IgG2 antibodies were also determined. The geometric mean percentage (GM%) of the paired infant:mother antibody were calculated. RESULTS: The GM% of the infant:mother antibody concentrations against PspA, PsaA and P6-OMP antibodies were 64.7% (3.3 micro g/ml in infants vs. 5.1 micro g/ml in mothers), 50.4% (6.8 micro g/ml vs. 13.5 micro g/ml) and 66.7% (5.6 micro g/ml vs. 8.4 micro g/ml), respectively; the GM% of antibodies against tetanus toxoid was 104.5% (4.6 micro g/ml vs. 4.4 micro g/ml). Transplacental transfer of IgG1 was more efficient than that of IgG2 (approximately 120%vs. 65%). A transplacental transfer of antibodies to PspA and to PsaA exist. Moreover, these data suggest an active placental transfer of IgG1 antibodies to PspA since the concentration of these antibodies were consistently higher in cord sera than in the mother's sera.

Pneumococcal surface protein A of invasive Streptococcus pneumoniae isolates from Colombian children.

Pneumococcal surface protein A (PspA) elicits protection in mice against fatal bacteremia and sepsis caused by genetically diverse pneumococci and protects against carriage and lung infection. We determined the PspA families of invasive isolates of Streptococcus pneumoniae recovered from Colombian children <5 years of age. That 97.5% of Colombian isolates belong to PspA families 1 and 2 supports the hypothesis that a human PspA vaccine covering a few PspA families could be broadly effective.

Tandem repeat deletion in the alpha C protein of group B streptococcus is recA independent.

Group B streptococci (GBS) contain a family of protective surface proteins characterized by variable numbers of repeating units within the proteins. The prototype alpha C protein of GBS from the type Ia/C strain A909 contains a series of nine identical 246-bp tandem repeat units. We have previously shown that deletions in the tandem repeat region of the alpha C protein affect both the immunogenicity and protective efficacy of the protein in animal models, and these deletions may serve as a virulence mechanism in GBS. The molecular mechanism of tandem repeat deletion is unknown. To determine whether RecA-mediated homologous recombination is involved in this process, we identified, cloned, and sequenced the recA gene homologue from GBS. A strain of GBS with recA deleted, A909DeltarecA, was constructed by insertional inactivation in the recA locus. A909DeltarecA demonstrated significant sensitivity to UV light, and the 50% lethal dose of the mutant strain in a mouse intraperitoneal model of sepsis was 20-fold higher than that of the parent strain. The spontaneous rate of tandem repeat deletion in the alpha C protein in vitro, as well as in our mouse model of immune infection, was studied using A909DeltarecA. We report that tandem repeat deletion in the alpha C protein does occur in the absence of a functional recA gene both in vitro and in vivo, indicating that tandem repeat deletion in GBS occurs by a recA-independent recombinatorial pathway.

Complete genome sequence of a virulent isolate of Streptococcus pneumoniae.

The 2,160,837-base pair genome sequence of an isolate of Streptococcus pneumoniae, a Gram-positive pathogen that causes pneumonia, bacteremia, meningitis, and otitis media, contains 2236 predicted coding regions; of these, 1440 (64%) were assigned a biological role. Approximately 5% of the genome is composed of insertion sequences that may contribute to genome rearrangements through uptake of foreign DNA. Extracellular enzyme systems for the metabolism of polysaccharides and hexosamines provide a substantial source of carbon and nitrogen for S. pneumoniae and also damage host tissues and facilitate colonization. A motif identified within the signal peptide of proteins is potentially involved in targeting these proteins to the cell surface of low-guanine/cytosine (GC) Gram-positive species. Several surface-exposed proteins that may serve as potential vaccine candidates were identified. Comparative genome hybridization with DNA arrays revealed strain differences in S. pneumoniae that could contribute to differences in virulence and antigenicity.

The autolytic enzyme LytA of Streptococcus pneumoniae is not responsible for releasing pneumolysin.

It was previously proposed that autolysin's primary role in the virulence of pneumococci was to release pneumolysin to an extracellular location. This interpretation came into question when pneumolysin was observed to be released in significant amounts from some pneumococci during log-phase growth, because autolysis was not believed to occur at this time. We have reexamined this phenomenon in detail for one such strain, WU2. This study found that the extracellular release of pneumolysin from WU2 was not dependent on autolysin action. A mutant lacking autolysin showed the same pattern of pneumolysin release as the wild-type strain. Addition of mitomycin C to a growing WU2 culture did not induce lysis, indicating the absence of resident bacteriophages that could potentially harbor lytA-like genes. Furthermore, release of pneumolysin was unaltered by growth in 2% choline, a condition which is reported to inactivate autolysin, as well as most known pneumococcal phage lysins. Profiles of total proteins in the cytoplasm and in the supernatant media supported the hypothesis that release of pneumolysin is independent of pneumococcal lysis. Finally, under some infection conditions, mutations in pneumolysin and autolysin had different effects on virulence.

Clones of Streptococcus pneumoniae isolated from nasopharyngeal carriage and invasive disease in young children in central Tennessee.

To determine whether nasopharyngeal carriage isolates of Streptococcus pneumoniae are of the same genetic background as isolates that caused invasive disease in one community, IS1167 and boxA genotypes were obtained for 182 pneumococcal isolates from children living in central Tennessee. The isolates represented 70 combined IS1167-boxA genotypes. The genotypic diversity of the invasive isolates was significantly less than that of the total population (P=.003). Most of the carriage isolates belonged to genotypes unique to carriage, whereas most of the invasive isolates belonged to genotypes common to carriage and disease (P=.02). Monte Carlo simulations showed a greater number of genotypes unique to carriage than can be explained by chance (P<.05 in all cases). Two genotypes were identified by multilocus sequence typing as members of globally disseminated clones, and one such genotype that was strictly carriage in this sample caused disease in other studies. Thus, clones can have different propensities for carriage and invasion.

Streptococcus pneumoniae: new tools for an old pathogen.

The pneumococcus is one of the longest-known pathogens. It has been instrumental to our understanding of biology in many ways, such as in the discovery of the Gram strain and the identification of nucleic acid as the hereditary material. Despite major advances in our understanding of pneumococcal pathogenesis, the need for vaccines and antibiotics to combat this pathogen is still vital. Genomics is beginning to uncover new virulence factors to advance this process, and it is enabling the development of DNA chip technology, which will permit the analysis of gene expression in specific tissues and in virulence regulatory circuits.

Production, characterization, and crystallization of truncated forms of pneumococcal surface protein A from Escherichia coli.

Streptococcus pneumoniae is a major bacterial pathogen that causes diseases such as pneumonia and meningitis in humans. One of the antigens of this organism is pneumococcal surface protein A (PspA). PspA is a virulence factor of the bacteria that has been shown to protect mice against pneumococcal infection. Among several domains of the protein, the amino-terminal part of PspA has been found to be a functional module which is essential for full pneumococcal infectivity. In order to investigate the properties of this protein, several internal fragments of the pspA gene were amplified from S. pneumoniae strain Rxl using the polymerase chain reaction (PCR). The fragments were then cloned and expressed in Escherichia coli in a soluble form using the T7 RNA polymerase pET15b and pET21a vector systems. The size of these fragments ranges from 24 to 32 kDa corresponding to amino acids 67-272 (PspA-206), 1-236 (PspA-236), and 1-272 (PspA-272). The fragments were purified to homogeneity using nickel chelating affinity, size exclusion, and anion-exchange chromatographic methods. During the course of expression of some of the PspA constructs, a shorter fragment was coexpressed due to translational pausing and subsequent secondary translation initiation. Two of the constructs, PspA-206 and PspA-272, were also crystallized allowing for the initiation of a structural elucidation of PspA.

Immunization of humans with recombinant pneumococcal surface protein A (rPspA) elicits antibodies that passively protect mice from fatal infection with Streptococcus pneumoniae bearing heterologous PspA.

Pneumococcal surface protein A (PspA), a cross-reactive protein expressed by all pneumococci, is known to elicit an antibody in animals that can passively protect mice from infection with Streptococcus pneumoniae. A phase I trial with recombinant PspA showed the protein to be immunogenic in humans. Pre- and postimmune serum samples from this trial were examined, and human antibody to PspA could protect mice from pneumococcal infection. The serum samples of subjects immunized twice with 125 microg of PspA had >100 times as much antibody per milliliter as was required to consistently protect mice from fatal infection (1.3 microg/dose). At least 98% of PspAs fall into PspA sequence/serologic families 1 or 2. Human antibodies elicited by a family 1 PspA protected against infection with S. pneumoniae expressing either family 1 or 2 PspAs and with strains of all 3 capsular types tested: 3, 6A, and 6B. These studies suggest that PspA may have efficacy as a human vaccine.

Pneumococcal pspA sequence types of prevalent multiresistant pneumococcal strains in the United States and of internationally disseminated clones.

In a recent genotypic survey of beta-lactam-resistant pneumococci recovered in different areas of United States during 1997, eight clonal types that each represented 3 to 40 isolates accounted for 134 of 144 isolates (G. Gherardi, C. Whitney, R. Facklam, and B. Beall, J. Infect. Dis. 181:216-229, 2000). We determined the degree of pspA gene diversity among these 134 isolates and for 11 previously characterized internationally disseminated multiresistant strains. Thirty-four different pspA restriction profiles were determined for an amplicon encompassing the variable portion of the structural gene that encodes the surface-exposed domain of PspA and a variable-length proline-rich putative cell wall-associated domain. These restriction profiles closely correlated with those of 33 different pspA sequence types of an approximately 230-residue region corresponding to residues 182 to 410 of the strain Rx1 PspA. These residues encompass a 100-residue clade-defining region known to contain cross-protective epitopes for which 17 sequence types were found. Distinct, conserved pspA sequence types were found for the majority of strains within seven of the eight U.S. clonal types assessed, while one pulsed-field gel electrophoresis type was represented by isolates of three distinct PspA clades. Sequence typing of pspA provides an added level of specificity in the subtyping of isolates and is a necessary first step in determining the components needed in a PspA vaccine which could elicit effective cross-protective coverage.

Diversity of PspA: mosaic genes and evidence for past recombination in Streptococcus pneumoniae.

Pneumococcal surface protein A (PspA) is a serologically variable protein of Streptococcus pneumoniae. Twenty-four diverse alleles of the pspA gene were sequenced to investigate the genetic basis for serologic diversity and to evaluate the potential of diversity to have an impact on PspA's use in human vaccination. The 24 pspA gene sequences from unrelated strains revealed two major allelic types, termed "families," subdivided into clades. A highly mosaic gene structure was observed in which individual mosaic sequence blocks in PspAs diverged from each other by over 20% in many cases. This level of divergence exceeds that observed for blocks in the penicillin-binding proteins of S. pneumoniae or in many cross-species comparisons of gene loci. Conversely, because the mosaic pattern is so complex, each pair of pspA genes also has numerous shared blocks, but the position of conserved blocks differs from gene pair to gene pair. A central region of pspA, important for eliciting protective antibodies, was found in six clades, which each diverge from the other clades by >20%. Sequence relationships among the 24 alleles analyzed over three windows were discordant, indicating that intragenic recombination has occurred within this locus. The extensive recombination which generated the mosaic pattern seen in the pspA locus suggests that natural selection has operated in the history of this gene locus and underscores the likelihood that PspA may be important in the interaction between the pneumococcus and its human host.

Immunization of mice with combinations of pneumococcal virulence proteins elicits enhanced protection against challenge with Streptococcus pneumoniae.

The vaccine potential of a combination of three pneumococcal virulence proteins was evaluated in an active-immunization-intraperitoneal-challenge model in BALB/c mice, using very high challenge doses of Streptococcus pneumoniae. The proteins evaluated were a genetic toxoid derivative of pneumolysin (PdB), pneumococcal surface protein A (PspA), and a 37-kDa metal-binding lipoprotein referred to as PsaA. Mice immunized with individual proteins or combinations thereof were challenged with high doses of virulent type 2 or type 4 pneumococci. The median survival times for mice immunized with combinations of proteins, particularly PdB and PspA, were significantly longer than those for mice immunized with any of the antigens alone. A similar effect was seen in a passive protection model. Thus, combinations of pneumococcal proteins may provide the best non-serotype-dependent protection against S. pneumoniae.

Immunization of healthy adults with a single recombinant pneumococcal surface protein A (PspA) variant stimulates broadly cross-reactive antibodies to heterologous PspA molecules.

Pneumococcal surface protein A (PspA) is a highly variable protein found on all strains of pneumococci. To be successful, a PspA-based vaccine for S. pneumoniae must induce antibodies that are broadly cross-reactive. To address whether cross-reactive antibodies could be induced in man, we evaluated serum from adults immunized with recombinant clade 2 PspA from strain Rx1. Immunization with 5-125 microg rPspA lead to a significant increase in circulating anti-PspA antibodies, as well as antibodies reactive to heterologous rPspA molecules. Increased binding of post-immune sera to 37 pneumococcal strains expressing a variety of PspA and capsule types was observed, versus pre-immune sera. The extent of cross-clade reactivity of human anti-rPspA followed roughly the amount of sequence homology to the non-clade 2 antigens. It is hypothesized that priming of humans by natural exposure to S. pneumoniae contributes to the breadth of the cross-reactivity of antibody to PspA.

Production and characterization of the functional fragment of pneumococcal surface protein A.

Pneumococcal surface protein A (PspA) is present on the cell wall of Streptococcus pneumoniae pathogen and has an antigenetically variable N-terminal domain. This aminoterminal domain is essential for full pneumococcal virulence, and monoclonal antibodies raised against it protect mice against pneumococcal infections. We have cloned and expressed a 34-kDa N-terminal fragment of PspA in Escherichia coli in a soluble form using the T7 RNA polymerase pET-20b vector system. Nickel chelate affinity purification followed by size exclusion and anion exchange chromatography yielded large amounts of pure and homogeneous protein. Analytical ultracentrifugation sedimentation velocity band and boundary studies showed that the molecule was present in aqueous solutions in a monomeric form with an axial shape ratio of approximately 1:12, typical of fibrous proteins. Sequence analyses indicated an alpha-helical coiled-coil structure for this monomeric molecule with only few loop-type breaks in helicity. The mostly alpha-helical structure of this PspA construct was consistent with circular dichroism spectroscopy data. Based on the ultracentrifugation studies, the circular dichroism spectra, and the PspA's sequence analyses, two structural models for the amino-terminal part of the PspA molecule are proposed. The evident highly charged and polar character of the surface of the modeled structures suggests functional properties of PspA that are related to the prevention of S. pneumoniae interactions with the host complement system.