PiuA and PiaA, iron uptake lipoproteins of Streptococcus pneumoniae, elicit serotype independent antibody responses following human pneumococcal septicaemia

Streptococcus pneumoniae causes considerable morbidity and mortality worldwide. The need for a cheap and effective pneumococcal vaccine has necessitated the evaluation of common virulence-associated proteins as potential vaccine antigens. PiuA and PiaA are the lipoprotein components of two pneumococcal iron ABC transporters. Here, we show that patients with culture confirmed pneumococcal septicaemia have elevated levels of antibody to PiuA and PiaA in convalescent-phase, compared with acute-phase serum. Additionally, sera from septicaemic patients infected with 13 pneumococcal strains covering eight different serotypes, cross-reacted with recombinant PiuA-His(6) and PiaA-His(6) from a single pneumococcal strain, indicating that this immune response is serotype independent. Anti-PiuA and anti-PiaA antibodies were also found in healthy seven-month-old infants, indicating that they are immunogenic at a very early age.     

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.     

Extensive variation in the ddl gene of penicillin-resistant Streptococcus pneumoniae results from a hitchhiking effect driven by the penicillin-binding protein 2b gene

An internal fragment of the ddl gene, encoding the cytoplasmic enzyme D-alanyl-D-alanine ligase, was sequenced from 566 isolates of Streptococcus pneumoniae and single isolates of Streptococcus mitis and Streptococcus oralis. The 52 alleles found among the S. pneumoniae isolates fell into two groups. Group A alleles were very uniform in sequence and were present in both penicillin-susceptible and penicillin-resistant pneumococci. Group B alleles were much more diverse and were found only in penicillin-resistant isolates. The Streptococcus oralis and Streptococcus mitis alleles were less diverged from group A alleles than some of the group B pneumococcal alleles, suggesting that the latter alleles contain interspecies recombinational replacements. The ddl gene was located 783 bp downstream of the penicillin-binding protein 2b gene (pbp2b). Sequencing of the pbp2b-recR-ddl-murF region of three penicillin-resistant pneumococci that had diverged ddl alleles showed that the whole region from pbp2b to ddl (or beyond) was highly diverged (about 8%) compared with the sequences from three penicillin-susceptible isolates. The high levels of diversity in the group B ddl alleles from penicillin-resistant isolates were ascribed to a hitchhiking effect whereby interspecies recombinational exchanges at pbp2b, selected by penicillin usage, often extend into, or through, the ddl gene. The data allow the average size of the interspecies recombinational replacements to be estimated at about 6 kb.     

Versatility of choline metabolism and choline-binding proteins in Streptococcus pneumoniae and commensal streptococci

The pneumococcal choline-containing teichoic acids are targeted by choline-binding proteins (CBPs), major surface components implicated in the interaction with host cells and bacterial cell physiology. CBPs also occur in closely related commensal species, Streptococcus oralis and Streptococcus mitis , and many strains of these species contain choline in their cell wall. Physiologically relevant CBPs including cell wall lytic enzymes are highly conserved between Streptococcus pneumoniae and S. mitis . In contrast, the virulence-associated CBPs, CbpA, PspA and PcpA, are S. pneumoniae specific and are thus relevant for the characteristic properties of this species.     

Phenotypic and genotypic discrepancy of Streptococcus pneumoniae strains isolated from Asian countries

Non-typeable isolates of Streptococcus pneumoniae collected from Asian countries were characterized by optochin susceptibility test, bile solubility test, multilocus sequence typing of housekeeping genes, amplification of virulence-related genes, 16S rDNA-RsaI digestion, and 16S rDNA sequencing. Six of 54 non-typeable pneumococcal isolates showed divergence of gene sequences of recP and xpt from typical pneumococcal strains. Of these six atypical pneumococcal strains, two showed different results in optochin susceptibility or bile solubility test from typical pneumococcal strains. All six isolates showed high sequence dissimilarities of multilocus sequence typing, 16S rDNA sequences, and lytA sequences from typical S. pneumoniae strains. Data from this study suggest that classic tests such as optochin susceptibility and bile solubility tests may lead to incorrect identification of S. pneumoniae. These atypical strains may belong to different bacterial species from S. pneumoniae.     

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.     

A solute binding protein of Streptococcus pneumoniae iron transport

Streptococcus pneumoniae causes considerable morbidity and mortality throughout the world. Iron acquisition is an important virulence factor for bacterial pathogens. Two loci, piu and pia, were identified as responsible for the hemoglobin utilization of S. pneumoniae. The binding activity and surface accessibility of the solute binding protein of PiuA were studied. PiuA is a lipoprotein, binds hemin and hemoglobin, resides on the cytoplasmic membrane, and is not exposed on the surface of S. pneumoniae. The localization of PiuA has implications in its role in hemoglobin utilization and possible use as a pneumococcal vaccine.     

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.     

Allelic variation of polymorphic locus lytB, encoding a choline-binding protein, from streptococci of the mitis group

The choline-binding protein LytB, an N-acetylglucosaminidase of Streptococcus pneumoniae, is the key enzyme for daughter cell separation and is believed to play a critical pathogenic role, facilitating bacterial spreading during infection. Because of these peculiarities LytB is a putative vaccine target. To determine the extent of LytB polymorphism, the lytB alleles from seven typical, clinical pneumococcal isolates of various serotypes and from 13 additional streptococci of the mitis group (12 atypical pneumococci and the Streptococcus mitis type strain) were sequenced. Sequence alignment showed that the main differences among alleles were differences in the number of repeats (range, 12 to 18) characteristic of choline-binding proteins. These differences were located in the region corresponding to repeats 11 to 17. Typical pneumococcal strains contained either 14, 16, or 18 repeats, whereas all of the atypical isolates except strains 1283 and 782 (which had 14 and 16 repeats, respectively) and the S. mitis type strain had only 12 repeats; atypical isolate 10546 turned out to be a DeltalytB mutant. We also found that there are two major types of alternating repeats in lytB, which encode 21 and 23 amino acids. Choline-binding proteins are linked to the choline-containing cell wall substrate through choline residues at the interface of two consecutive choline-binding repeats that create a choline-binding site. The observation that all strains contained an even number of repeats suggests that the duplication events that gave rise to the choline-binding repeats of LytB involved two repeats simultaneously, an observation that is in keeping with previous crystallographic data. Typical pneumococcal isolates usually grew as diplococci, indicating that an active LytB enzyme was present. In contrast, most atypical isolates formed long chains of cells that did not disperse after addition of purified LytB, suggesting that in these strains chains were produced through mechanisms unrelated to LytB.     

Characterization of LytA-like N-acetylmuramoyl-L-alanine amidases from two new Streptococcus mitis bacteriophages provides insights into the properties of the major pneumococcal autolysin

Two new temperate bacteriophages exhibiting a Myoviridae (phiB6) and a Siphoviridae (phiHER) morphology have been isolated from Streptococcus mitis strains B6 and HER 1055, respectively, and partially characterized. The lytic phage genes were overexpressed in Escherichia coli, and their encoded proteins were purified. The lytAHER and lytAB6 genes are very similar (87% identity) and appeared to belong to the group of the so-called typical LytA amidases (atypical LytA displays a characteristic two-amino-acid deletion signature). although they exhibited several differential biochemical properties with respect to the pneumococcal LytA, e.g., they were inhibited in vitro by sodium deoxycholate and showed a more acidic pH for optimal activity. However, and in sharp contrast with the pneumococcal LytA, a short dialysis of LytAHER or LytAB6 resulted in reversible deconversion to the low-activity state (E-form) of the fully active phage amidases (C-form). Comparison of the amino acid sequences of LytAHER and LytAB6 with that of the pneumococcal amidase suggested that Val317 might be responsible for at least some of the peculiar properties of S. mitis phage enzymes. Site-directed mutagenesis that changed Val317 in the pneumococcal LytA amidase to a Thr residue (characteristic of LytAB6 and LytAHER) produced a fully active pneumococcal enzyme that differs from the parental one only in that the mutant amidase can reversibly recover the low-activity E-form upon dialysis. This is the first report showing that a single amino acid residue is involved in the conversion process of the major S. pneumoniae autolysin. Our results also showed that some lysogenic S. mitis strains possess a lytA-like gene, something that was previously thought to be exclusive to Streptococcus pneumoniae. Moreover, the newly discovered phage lysins constitute a missing link between the typical and atypical pneumococcal amidases known previously.     

Streptococcus pneumoniae: virulence factors, pathogenesis, and vaccines.

Although pneumococcal conjugate vaccines are close to being licensed, a more profound knowledge of the virulence factors responsible for the morbidity and mortality caused by Streptococcus pneumoniae is necessary. This review deals with the major structures of pneumococci involved in the pathogenesis of pneumococcal disease and their interference with the defense mechanisms of the host. It is well known that protection against S. pneumoniae is the result of phagocytosis of invading pathogens. For this process, complement and anticapsular polysaccharide antibodies are required. Besides, relatively recent experimental data suggest that protection is also mediated by the removal of disintegrating pneumococci and their degradation products (cell wall, pneumolysin). These structures seem to be major contributors to illness and death caused by pneumococci. An effective conjugate vaccine should therefore preferably include the capsular polysaccharide and at least one of these inflammatory factors.     

Development of a DNA microarray to identify the Streptococcus pneumoniae serotypes contained in the 23-valent pneumococcal polysaccharide vaccine and closely related serotypes

Streptococcus pneumoniae is a major worldwide human pathogen. This investigation has developed a reliable and accurate DNA microarray method for inter-species differentiation of S. pneumoniae and intra-species differentiation of the 23 groups of S. pneumoniae including serotypes represented in the 23-valent pneumococcal vaccine and the other 20 closely related serotypes. In addition to 16S rDNA probes, serotype- or serogroup-specific probes targeting the capsular polysaccharide synthesis (cps) genes, wzy or capA were generated. We adopted a two-step multiplex PCR to improve the sensitivity of detection to a level of 10(5) cfu/ml in pure culture or 50 ng DNA. A total of 169 isolates (from China, Australia, Canada and New Zealand) including 147 belonging to 23-valent vaccine and closely related serotypes of S. pneumoniae, 11 belonging to other serotypes and 11 of different species commonly isolated from respiratory tract were tested to verify the method. The DNA microarray method developed provides a sensitive means to rapidly identify the members of the most common S. pneumoniae serotypes in patients and to monitor their distribution in different patient groups and geographic locations. Such information is needed for disease surveillance and to monitor vaccine efficacy.     

NanA, a neuraminidase from Streptococcus pneumoniae, shows high levels of sequence diversity, at least in part through recombination with Streptococcus oralis

Streptococcus pneumoniae, an important human pathogen, contains at least two genes, nanA and nanB, that express sialidase activity. NanA is a virulence determinant of pneumococci which is important in animal models of colonization and middle ear infections. The gene encoding NanA was detected in all 106 pneumococcal strains screened that represented 59 restriction profiles. Sequencing confirmed a high level of diversity, up to 17.2% at the nucleotide level and 14.8% at the amino acid level. NanA diversity is due to a number of mechanisms including insertions, point mutations, and recombination generating mosaic genes. The level of nucleotide divergence for each recombinant block is greater than 30% and much higher than the 20% identified within mosaic pbp genes, suggesting that a high selective pressure exists for these alterations. These data indicate that at least one of the four recombinant blocks identified originated from a Streptococcus oralis isolate, demonstrating for the first time that protein virulence determinants of pneumococci have, as identified previously for genes encoding penicillin binding proteins, evolved by recombination with oral streptococci. No amino acid alterations were identified within the aspartic boxes or predicted active site, suggesting that sequence variation may be important in evading the adaptive immune response. Furthermore, this suggests that nanA is an important target of the immune system in the interaction between the pneumococcus and host.     

Mutations in the tacF gene of clinical strains and laboratory transformants of Streptococcus pneumoniae: impact on choline auxotrophy and growth rate

The nutritional requirement that Streptococcus pneumoniae has for the aminoalcohol choline as a component of teichoic and lipoteichoic acids appears to be exclusive to this prokaryote. A mutation in the tacF gene, which putatively encodes an integral membrane protein (possibly, a teichoic acid repeat unit transporter), has been recently identified as responsible for generating a choline-independent phenotype of S. pneumoniae (M. Damjanovic, A. S. Kharat, A. Eberhardt, A. Tomasz, and W. Vollmer, J. Bacteriol. 189:7105-7111, 2007). We now report that Streptococcus mitis can grow in choline-free medium, as previously illustrated for Streptococcus oralis. While we confirmed the finding by Damjanovic et al. of the involvement of TacF in the choline dependence of the pneumococcus, the genetic transformation of S. pneumoniae R6 by using S. mitis SK598 DNA and several PCR-amplified tacF fragments suggested that a minimum of two mutations were required to confer improved fitness to choline-independent S. pneumoniae mutants. This conclusion is supported by sequencing results also reported here that indicate that a spontaneous mutant of S. pneumoniae (strain JY2190) able to proliferate in the absence of choline (or analogs) is also a double mutant for the tacF gene. Microscopic observations and competition experiments during the cocultivation of choline-independent strains confirmed that a minimum of two amino acid changes were required to confer improved fitness to choline-independent pneumococcal strains when growing in medium lacking any aminoalcohol. Our results suggest complex relationships among the different regions of the TacF teichoic acid repeat unit transporter.     

Prevalence of antibiotic resistance and serotypes in pneumococci in England and Wales: results of observational surveys in 1990 and 1995.

OBJECTIVE--To assess the prevalence of antibiotic resistance and serotype distribution among pneumococci in England and Wales in 1990 and 1995. DESIGN--Observational surveys in March 1990 and March 1995. During two weeks in each survey period all pneumococci isolated in public health laboratories in England and Wales were collected and assessed for sensitivity to antibiotics and the distribution of serogroups or serotypes. SETTING--The network of public health laboratories throughout England and Wales. SUBJECTS--1127 individual patient isolates of Streptococcus pneumoniae obtained during the two surveys. MAIN OUTCOME MEASURES--Sensitivity or resistance to a range of antibiotics; serogroup or serotype. RESULTS--The prevalence of intermediate or full resistance to penicillin increased from 1.5% in 1990 to 3.9% in 1995 and resistance to erythromycin increased from 2.8% to 8.6%. About 92% of isolates belonged to serogroups or serotypes included in the currently available pneumococcal vaccine. CONCLUSION--Resistance to penicillin and erythromycin has increased among pneumococci in England and Wales. Continued surveillance to assess further increases in the prevalence of pneumococcal resistance to antibiotics is essential.     

Identification of a secreted cholesterol-dependent cytolysin (mitilysin) from Streptococcus mitis

We have detected a cholesterol-dependent cytolysin, which we have named mitilysin, in a small number of Streptococcus mitis isolates. We have sequenced the mitilysin gene from seven isolates of S. mitis. Comparisons with the pneumococcal pneumolysin gene show 15 amino acid substitutions. S. mitis appear to release mitilysin extracellularly. Certain alleles of mitilysin are not recognized by a monoclonal antibody raised to the related toxin pneumolysin. Based on enzyme-linked immunosorbent assay and neutralization assay results, one isolate of S. mitis may produce a further hemolytic toxin in addition to mitilysin. As genetic exchange is known to occur between S. mitis and Streptococcus pneumoniae, this finding may have implications for the development of vaccines or therapies for pneumococcal disease that are based on pneumolysin.     

PspA Family Distribution,Antimicrobial Resistance and Serotype of Streptococcus pneumoniae Isolated from Upper Respiratory Tract Infections in Japan

Background

The protection against pneumococcal infections provided by currently available pneumococcal polysaccharide conjugate vaccines are restricted to the limited number of the serotypes included in the vaccine. In the present study, we evaluated the distribution of the pneumococcal capsular type and surface protein A (PspA) family of pneumococcal isolates from upper respiratory tract infections in Japan.

Methods

A total of 251 S. pneumoniae isolates from patients seeking treatment for upper respiratory tract infections were characterized for PspA family, antibiotic resistance and capsular type.

Results

Among the 251 pneumococci studied, the majority (49.4%) was identified as belonging to PspA family 2, while most of the remaining isolates (44.6%) belonged to family 1. There were no significant differences between the distributions of PspA1 versus PspA2 isolates based on the age or gender of the patient, source of the isolates or the isolates’ susceptibilities to penicillin G. In contrast, the frequency of the mefA gene presence and of serotypes 15B and 19F were statistically more common among PspA2 strains.

Conclusion

The vast majority of pneumococci isolated from the middle ear fluids, nasal discharges/sinus aspirates or pharyngeal secretions represented PspA families 1 and 2. Capsular serotypes were generally not exclusively associated with certain PspA families, although some capsular types showed a much higher proportion of either PspA1 or PspA2. A PspA-containing vaccine would potentially provide high coverage against pneumococcal infectious diseases because it would be cross-protective versus invasive disease with the majority of pneumococci infecting children and adults.     

Population pattern of pneumococci with lower susceptibility to penicillin and prospects of antipneumococcal vaccination to control antibiotic resistance distribution

Large-scale antipneumococcal vaccination is followed by changes in the serotype composition and level of antibiotic resistance in pneumococci. The aim of the study was to evaluate the serotype composition and population pattern of pneumococci with lower susceptibility to penicillin before large-scale antipneumococcal vaccination. Among 260 Streptococcus pneumoniae strains isolated in the Russian Federation within 2003-2007, serotypes 23F (37.2%) and 19F (13.9%) were the most frequent ones. 19.3% of the isolates belonged to serogroup 6, 3.6% of the isolates each belonged to serotype 3 and serogroup 18, 4.9% of the isolates belonged to serotype 14 and 2.2% of the isolates belonged to serotype 19A. 66.8% of the isolates belonged to serotypes of the 7-valent conjugated pneumococcal vaccine, 67.3 and 82.1% of the isolates belonged to the 10- and 13-valent conjugated pneumococcal vaccines respectively. The isolates with lower susceptibility to penicillin were characterized by significant clonality and 56.9% of them belonged to 4 global clonal complexes (CC81, CC156, CC320 and CC315). Inclusion of the conjugated antipneumococcal vaccine to the National Vaccination Time-Table of the Russian Federation could promote lower levels of antibiotic resistance in pneumococci.     

Horizontal spread of an altered penicillin-binding protein 2B gene between Streptococcus pneumoniae and Streptococcus oralis

Abstract The region encoding the transpeptidase domain of the penicillin-binding protein 2B (PBP 2B) gene of two penicillin-resistant clinical isolates of Streptococcus oralis was > 99.6% identical in nucleotide sequence to that of a penicillin-resistant serotype 6 isolate of Streptococcus pneumoniae . The downstream 849 base pairs of these genes were identical. Analysis of the data indicates that the PBP gene has probably been transferred from S. pneumoniae into S. oralis , rather than vice versa, and shows that one region of this resistance gene has been distributed horizontally both within S. pneumoniae and into two different viridans group streptococci.     

Sequence diversity within the capsular genes of Streptococcus pneumoniae serogroup 6 and 19

The main virulence factor of Streptococcus pneumoniae is the capsule. The polysaccharides comprising this capsule are encoded by approximately 15 genes and differences in these genes result in different serotypes. The aim of this study was to investigate the sequence diversity of the capsular genes of serotypes 6A, 6B, 6C, 19A and 19F and to explore a possible effect of vaccination on variation and distribution of these serotypes in the Netherlands. The complete capsular gene locus was sequenced for 25 serogroup 6 and for 20 serogroup 19 isolates. If one or more genes varied in 10 or more base pairs from the reference sequence, it was designated as a capsular subtype. Allele-specific PCRs and specific gene sequencing of highly variable capsular genes were performed on 184 serogroup 6 and 195 serogroup 19 isolates to identify capsular subtypes. This revealed the presence of 6, 3 and a single capsular subtype within serotypes 6A, 6B and 6C, respectively. The serotype 19A and 19F isolates comprised 3 and 4 capsular subtypes, respectively. For serogroup 6, the genetic background, as determined by multi locus sequence typing (MLST) and multiple-locus variable number of tandem repeat analysis (MLVA), seemed to be closely related to the capsular subtypes, but this was less pronounced for serogroup 19 isolates. The data also suggest shifts in the occurrence of capsular subtypes within serotype 6A and 19A after introduction of the 7-valent pneumococcal vaccine. The shifts within these non-vaccine serotypes might indicate that these capsular subtypes are filling the niche of the vaccine serotypes. In conclusion, there is considerable DNA sequence variation of the capsular genes within pneumococcal serogroup 6 and 19. Such changes may result in altered polysaccharides or in strains that produce more capsular polysaccharides. Consequently, these altered capsules may be less sensitive for vaccine induced immunity.