The Protective Value of Maternal Group B Streptococcus Antibodies: Quantitative and Functional Analysis of Naturally Acquired Responses to Capsular Polysaccharides and Pilus Proteins in European Maternal Sera.

BACKGROUND: Group B Streptococcus (GBS) is a major cause of neonatal sepsis and meningitis. A vaccine targeting pregnant women could protect infants through placentally transferred antibodies. The association between GBS maternal antibody concentrations and the risk of neonatal infection has been investigated in US and African populations. Here we studied naturally acquired immunoglobulin G (IgG) responses to GBS capsular polysaccharides (CPS) and pilus proteins in European pregnant women. METHODS: Maternal sera were prospectively collected in 8 EU countries from 473 GBS non-colonized and 984 colonized pregnant women who delivered healthy neonates and from 153 mothers of infants with GBS disease. GBS strains from these colonized women and infected infants were obtained in parallel and their capsular and pilus types were identified by serological and molecular methods. Maternal serum concentrations of IgG anti- Ia, -Ib, -III and -V polysaccharides and anti-BP-1, -AP1-2a and -BP-2b pilus proteins were determined by enzyme-linked immunosorbent assay. Antibody functional activity was quantified by Opsonophagocytic Killing Assay. RESULTS: Antibody levels against CPS and pilus proteins were significantly higher in GBS colonized women delivering healthy babies than in mothers of neonates with GBS disease or non-colonized women. Moreover, maternal anti-capsular IgG concentrations showed a significant correlation with functional titers measured by Opsonophagocytic Killing Assay. CONCLUSIONS: Maternal anti-capsular IgG concentrations above 1 µg/mL mediated GBS killing in vitro and were predicted to respectively reduce by 81% (95% confidence interval, 40%-100%) and 78% (45%-100%) the risk of GBS Ia and III early-onset disease in Europe.

Streptococcus agalactiae clones infecting humans were selected and fixed through the extensive use of tetracycline.

Streptococcus agalactiae (Group B Streptococcus, GBS) is a commensal of the digestive and genitourinary tracts of humans that emerged as the leading cause of bacterial neonatal infections in Europe and North America during the 1960s. Due to the lack of epidemiological and genomic data, the reasons for this emergence are unknown. Here we show by comparative genome analysis and phylogenetic reconstruction of 229 isolates that the rise of human GBS infections corresponds to the selection and worldwide dissemination of only a few clones. The parallel expansion of the clones is preceded by the insertion of integrative and conjugative elements conferring tetracycline resistance (TcR). Thus, we propose that the use of tetracycline from 1948 onwards led in humans to the complete replacement of a diverse GBS population by only few TcR clones particularly well adapted to their host, causing the observed emergence of GBS diseases in neonates.

Understanding the molecular determinants driving the immunological specificity of the protective pilus 2a backbone protein of group B streptococcus.

The pilus 2a backbone protein (BP-2a) is one of the most structurally and functionally characterized components of a potential vaccine formulation against Group B Streptococcus. It is characterized by six main immunologically distinct allelic variants, each inducing variant-specific protection. To investigate the molecular determinants driving the variant immunogenic specificity of BP-2a, in terms of single residue contributions, we generated six monoclonal antibodies against a specific protein variant based on their capability to recognize the polymerized pili structure on the bacterial surface. Three mAbs were also able to induce complement-dependent opsonophagocytosis killing of live GBS and target the same linear epitope present in the structurally defined and immunodominant domain D3 of the protein. Molecular docking between the modelled scFv antibody sequences and the BP-2a crystal structure revealed the potential role at the binding interface of some non-conserved antigen residues. Mutagenesis analysis confirmed the necessity of a perfect balance between charges, size and polarity at the binding interface to obtain specific binding of mAbs to the protein antigen for a neutralizing response.

Group B Streptococcus pilus sortase regulation: a single mutation in the lid region induces pilin protein polymerization in vitro.

Gram-positive bacteria build pili on their cell surface via a class C sortase-catalyzed transpeptidation mechanism from pilin protein substrates. Despite the availability of several crystal structures, pilus-related C sortases remain poorly characterized to date, and their mechanisms of transpeptidation and regulation need to be further investigated. The available 3-dimensional structures of these enzymes reveal a typical sortase fold, except for the presence of a unique feature represented by an N-terminal highly flexible loop known as the "lid." This region interacts with the residues composing the catalytic triad and covers the active site, thus maintaining the enzyme in an autoinhibited state and preventing the accessibility to the substrate. It is believed that enzyme activation may occur only after lid displacement from the catalytic domain. In this work, we provide the first direct evidence of the regulatory role of the lid, demonstrating that it is possible to obtain in vitro an efficient polymerization of pilin subunits using an active C sortase lid mutant carrying a single residue mutation in the lid region. Moreover, biochemical analyses of this recombinant mutant reveal that the lid confers thermodynamic and proteolytic stability to the enzyme.

Adaptive response of Group B streptococcus to high glucose conditions: new insights on the CovRS regulation network.

Although the contribution of carbohydrate catabolism to bacterial colonization and infection is well recognized, the transcriptional changes during these processes are still unknown. In this study, we have performed comparative global gene expression analysis of GBS in sugar-free versus high glucose milieu. The analysis revealed a differential expression of genes involved in metabolism, transport and host-pathogen interaction. Many of them appeared to be among the genes previously reported to be controlled by the CovRS two-component system. Indeed, the transcription profile of a ΔcovRS strain grown in high-glucose conditions was profoundly affected. In particular, of the total genes described to be regulated by glucose, ∼27% were under CovRS control with a functional role in protein synthesis, transport, energy metabolism and regulation. Among the CovRS dependent genes, we found bibA, a recently characterized adhesin involved in bacterial serum resistance and here reported to be down-regulated by glucose. ChIP analysis revealed that in the presence of glucose, CovR binds bibA promoter in vivo, suggesting that CovR may act as a negative regulator or a repressor. We also demonstrated that, as for other target promoters, chemical phosphorylation of CovR in aspartic acid increases its affinity for the bibA promoter region. The data reported in this study contribute to the understanding of the molecular mechanisms modulating the adaptation of GBS to glucose.

High-level intracellular expression of heterologous proteins in Brevibacillus choshinensis SP3 under the control of a xylose inducible promoter.

BACKGROUND: In past years research has focused on the development of alternative Gram positive bacterial expression systems to produce industrially relevant proteins. Brevibacillus choshinensis is an easy to handle non-sporulating bacterium, lacking extracellular proteases, that has been already shown to provide a high level of recombinant protein expression. One major drawback, limiting the applicability of the Brevibacillus expression system, is the absence of expression vectors based on inducible promoters. Here we used the PxylA inducible promoter, commonly employed in other Bacillae expression systems, in Brevibacillus. RESULTS: Using GFP, α-amylase and TcdA-GT as model proteins, high level of intracellular protein expression (up to 250 mg/L for the GFP) was achieved in Brevibacillus, using the pHis1522 vector carrying the B. megaterium xylose-inducible promoter (PxylA). The GFP expression yields were more than 25 fold higher than those reported for B. megaterium carrying the same vector. All the tested proteins show significant increment in their expression levels (2-10 folds) than those obtained using the available plasmids based on the P2 constitutive promoter. CONCLUSION: Combining the components of two different commercially available Gram positive expression systems, such as Brevibacillus (from Takara Bio) and B. megaterium (from Mobitec), we demonstrate that vectors based on the B. megaterium PxylA xylose inducible promoter can be successfully used to induce high level of intracellular expression of heterologous proteins in Brevibacillus.

Capsular gene typing of Streptococcus agalactiae compared to serotyping by latex agglutination.

We evaluated three different PCR-based capsular gene typing methods applied to 312 human and bovine Streptococcus agalactiae (group B Streptococcus [GBS]) isolates and compared the results to serotyping results obtained by latex agglutination. Among 281 human isolates 27% could not be typed by latex agglutination. All 312 isolates except 5 could be typed by the three PCR methods combined. Two of these methods were multiplex assays. Among the isolates that were typeable by both latex agglutination and capsular gene typing, 94% showed agreement between the two methods. However, each of the PCR methods showed limitations. One of the methods did not include all 10 recognized serotypes, one misidentified eight isolates of serotypes Ib and IV as serotype Ia, and one did not distinguish between serotypes VII and IX. For five isolates that showed aberrant patterns in the capsular gene typing, long-range PCR targeting the cps operon disclosed large insertions or deletions affecting the cps gene cluster. A sensitive flow cytometric assay based on serotype-specific antibodies applied to 76 selected isolates that were nontypeable by latex agglutination revealed that approximately one-half of these did express capsular polysaccharide. A procedure for convenient and reliable capsular gene typing to be included in epidemiological and surveillance studies of S. agalactiae is proposed.

Structural basis for group B streptococcus pilus 1 sortases C regulation and specificity.

Gram-positive bacteria assemble pili through class C sortase enzymes specialized in polymerizing pilin subunits into covalently linked, high-molecular-weight, elongated structures. Here we report the crystal structures of two class C sortases (SrtC1 and SrtC2) from Group B Streptococcus (GBS) Pilus Island 1. The structures show that both sortases are comprised of two domains: an 8-stranded ß-barrel catalytic core conserved among all sortase family members and a flexible N-terminal region made of two α-helices followed by a loop, known as the lid, which acts as a pseudo-substrate. In vitro experiments performed with recombinant SrtC enzymes lacking the N-terminal portion demonstrate that this region of the enzyme is dispensable for catalysis but may have key roles in substrate specificity and regulation. Moreover, in vitro FRET-based assays show that the LPXTG motif common to many sortase substrates is not the sole determinant of sortase C specificity during pilin protein recognition.

Streptococcus pyogenes SpyCEP influences host-pathogen interactions during infection in a murine air pouch model.

Streptococcus pyogenes is a major human pathogen worldwide, responsible for both local and systemic infections. These bacteria express the subtilisin-like protease SpyCEP which cleaves human IL-8 and related chemokines. We show that localization of SpyCEP is growth-phase and strain dependent. Significant shedding was observed only in a strain naturally overexpressing SpyCEP, and shedding was not dependent on SpyCEP autoproteolytic activity. Surface-bound SpyCEP in two different strains was capable of cleaving IL-8. To investigate SpyCEP action in vivo, we adapted the mouse air pouch model of infection for parallel quantification of bacterial growth, host immune cell recruitment and chemokine levels in situ. In response to infection, the predominant cells recruited were neutrophils, monocytes and eosinophils. Concomitantly, the chemokines KC, LIX, and MIP-2 in situ were drastically increased in mice infected with the SpyCEP knockout strain, and growth of this mutant strain was reduced compared to the wild type. SpyCEP has been described as a potential vaccine candidate against S. pyogenes, and we showed that surface-associated SpyCEP was recognized by specific antibodies. In vitro, such antibodies also counteracted the inhibitory effects of SpyCEP on chemokine mediated PMN recruitment. Thus, α-SpyCEP antibodies may benefit the host both directly by enabling opsonophagocytosis, and indirectly, by neutralizing an important virulence factor. The animal model we employed shows promise for broad application in the study of bacterial pathogenesis.

Exogenous sialic acid transport contributes to group B streptococcus infection of mucosal surfaces.

By sequence analysis of available group B streptococcus (GBS) genomes, we discovered a conserved putative operon involved in the catabolism of sialic acid, containing a tripartite transporter formed by two integral membrane components and a sugar-binding unit, named SAL0039. Expression analysis in the presence of different substrates revealed that SAL0039 was specifically upregulated by the presence of sialic acid and downregulated when bacteria were grown in human blood or in the presence of a high concentration of glucose. The role of SAL0039 in sugar transport was supported by the inability of the sal0039 deletion mutant strain to import exogenous sialic acid and to grow in semidefined medium supplemented with this sugar. Furthermore, in vivo evidence showed that the presence of exogenous sialic acid significantly increased the capacity of GBS to infect mice at the mucosal level. These findings suggest that transport of sialic acid may also contribute to GBS infections.

Multi high-throughput approach for highly selective identification of vaccine candidates: the Group A Streptococcus case.

We propose an experimental strategy for highly accurate selection of candidates for bacterial vaccines without using in vitro and/or in vivo protection assays. Starting from the observation that efficacious vaccines are constituted by conserved, surface-associated and/or secreted components, the strategy contemplates the parallel application of three high throughput technologies, i.e. mass spectrometry-based proteomics, protein array, and flow-cytometry analysis, to identify this category of proteins, and is based on the assumption that the antigens identified by all three technologies are the protective ones. When we tested this strategy for Group A Streptococcus, we selected a total of 40 proteins, of which only six identified by all three approaches. When the 40 proteins were tested in a mouse model, only six were found to be protective and five of these belonged to the group of antigens in common to the three technologies. Finally, a combination of three protective antigens conferred broad protection against a panel of four different Group A Streptococcus strains. This approach may find general application as an accelerated and highly accurate path to bacterial vaccine discovery.

New insights into the role of the glutamic acid of the E-box motif in group B Streptococcus pilus 2a assembly.

Group B Streptococcus pili are covalently linked structures assembled via a sortase-catalyzed transpeptidation mechanism involving specific residues and motifs. A sequence element containing a conserved glutamic acid, called the E-box, has been described to be involved in pilus formation. Although it is known that the glutamic acid is involved in stabilizing the internal isopeptide bonds, its role in pilus assembly still needs to be investigated. Using site-specific mutagenesis and complementation studies of knockout strains, we found that the E-box glutamic residue of the backbone and the major ancillary proteins is essential for pilus protein polymerization. NMR analysis revealed that the mutation of this residue seriously affected the folding of the protein. By contrast, the mutation of the lysine involved in the same isopeptide bond did not engender a structural destabilization, and the native fold was preserved. Moreover, molecular dynamics simulations on the E-box-containing domain of the backbone protein showed that the E-box glutamic acid is necessary to maintain the appropriate dryness of the domain core and that its mutation favors an unfolded state. The data provide the first direct evidence that the E-box has an additional and key role in maintaining the correct protein fold independently of isopeptide bond formation.

Sortase A substrate specificity in GBS pilus 2a cell wall anchoring.

Streptococcus agalactiae, also referred to as Group B Streptococcus (GBS), is one of the most common causes of life-threatening bacterial infections in infants. In recent years cell surface pili have been identified in several Gram-positive bacteria, including GBS, as important virulence factors and promising vaccine candidates. In GBS, three structurally distinct types of pili have been discovered (pilus 1, 2a and 2b), whose structural subunits are assembled in high-molecular weight polymers by specific class C sortases. In addition, the highly conserved housekeeping sortase A (SrtA), whose main role is to link surface proteins to bacterial cell wall peptidoglycan by a transpeptidation reaction, is also involved in pili cell wall anchoring in many bacteria. Through in vivo mutagenesis, we demonstrate that the LPXTG sorting signal of the minor ancillary protein (AP2) is essential for pilus 2a anchoring. We successfully produced a highly purified recombinant SrtA (SrtA(ΔN40)) able to specifically hydrolyze the sorting signal of pilus 2a minor ancillary protein (AP2-2a) and catalyze in vitro the transpeptidation reaction between peptidoglycan analogues and the LPXTG motif, using both synthetic fluorescent peptides and recombinant proteins. By contrast, SrtA(ΔN40) does not catalyze the transpeptidation reaction with substrate-peptides mimicking sorting signals of the other pilus 2a subunits (the backbone protein and the major ancillary protein). Thus, our results add further insight into the proposed model of GBS pilus 2a assembly, in which SrtA is required for pili cell wall covalent attachment, acting exclusively on the minor accessory pilin, representing the terminal subunit located at the base of the pilus.

Structure-based approach to rationally design a chimeric protein for an effective vaccine against Group B Streptococcus infections.

Structural vaccinology is an emerging strategy for the rational design of vaccine candidates. We successfully applied structural vaccinology to design a fully synthetic protein with multivalent protection activity. In Group B Streptococcus, cell-surface pili have aroused great interest because of their direct roles in virulence and importance as protective antigens. The backbone subunit of type 2a pilus (BP-2a) is present in six immunogenically different but structurally similar variants. We determined the 3D structure of one of the variants, and experimentally demonstrated that protective antibodies specifically recognize one of the four domains that comprise the protein. We therefore constructed a synthetic protein constituted by the protective domain of each one of the six variants and showed that the chimeric protein protects mice against the challenge with all of the type 2a pilus-carrying strains. This work demonstrates the power of structural vaccinology and will facilitate the development of an optimized, broadly protective pilus-based vaccine against Group B Streptococcus by combining the uniquely generated chimeric protein with protective pilin subunits from two other previously identified pilus types. In addition, this work describes a template procedure that can be followed to develop vaccines against other bacterial pathogens.

Shigella targets T cells.

Using a syringe-like device, Shigella delivers an array of virulence factors into host cells to facilitate bacterial colonization and disable the host's innate immune defense. In this issue of Cell Host & Microbe, Konradt and colleagues (Konradt et al., 2011) show that Shigella also subverts adaptive immunity by targeting T cells through a mechanism involving PIP(2) breakdown.

Structure analysis and site-directed mutagenesis of defined key residues and motives for pilus-related sortase C1 in group B Streptococcus.

In group B Streptococcus (GBS), 3 structurally distinct types of pili have been discovered as potential virulence factors and vaccine candidates. The pilus-forming proteins are assembled into high-molecular-weight polymers via a transpeptidation mechanism mediated by specific class C sortases. Using a multidisciplinary approach including bioinformatics, structural and biochemical studies, and in vivo mutagenesis, we performed a broad characterization of GBS sortase C1 of pilus island 2a. The high-resolution X-ray structure of the enzyme revealed that the active site, into the ß-barrel core of the enzyme, is made of the catalytic triad His157-Cys219-Arg228 and covered by a loop, known as the "lid." We show that the catalytic triad and the predicted N- and C-terminal transmembrane regions are required for the enzyme activity. Interestingly, by in vivo complementation mutagenesis studies, we found that the deletion of the entire lid loop or mutations in specific lid key residues had no effect on catalytic activity of the enzyme. In addition, kinetic characterizations of recombinant enzymes indicate that the lid mutants can still recognize and cleave the substrate-mimicking peptide at least as well as the wild-type protein.

Simple sequence repeats and genome plasticity in Streptococcus agalactiae.

Simple sequence repeats (SSRs) and their role in phase variation have been extensively studied in Gram-negative organisms, where they have been associated with antigenic variation and other adaptation strategies. In this study, we apply comparative genomics in order to find evidence of slipped-strand mispairing in the human Gram-positive pathogen Streptococcus agalactiae. In two consecutive screenings, 2,233 (650 + 1,583) SSRs were identified in our reference genome 2603V/R, and these loci were examined in seven other S. agalactiae genomes. A total of 56 SSR loci were found to exhibit variation, where gain or loss of repeat units was observed in at least one other genome, resulting in aberrant genotypes. Homopolymeric adenine tracts predominated among the repeats that varied. Positional analysis revealed that long polyadenine tracts were overrepresented in the 5' ends of open reading frames (ORFs) and underrepresented in the 3' ends. Repeat clustering in ORFs was also examined, and the highest degree of clustering was observed for a capsule biosynthesis gene and a pilus sortase. A statistical analysis of observed over expected ratios suggested a selective pressure against long homopolymeric tracts. Altered phenotypes were verified for three genes encoding surface-attached proteins, in which frameshifts or fusions led to truncation of proteins and/or affected surface localization through loss or gain of the cell wall sorting signal. The data suggest that SSRs contributes to genome plasticity in S. agalactiae but that the bet-hedging strategy is different from Gram-negative organisms.

Relevance of pili in pathogenic streptococci pathogenesis and vaccine development.

A common mechanism used by bacteria to initiate adhesion to host tissues during colonization is the expression of long filamentous structures extending from their surface. These structures, known as pili or fimbriae, were initially identified in Gram-negative bacteria, and are typically formed by noncovalent interactions between pilin subunits. Pili have only recently been described in Gram-positive bacteria. In particular, in pathogenic streptococci the proteinaceous components of pili are covalently polymerized by the action of sortase enzymes similar to those involved in the covalent attachment of Gram-positive surface proteins to the peptidoglycan cell wall. With great relevance to the development of strategies to combat Gram-positive-associated infections, pilus components from pathogenic streptococci have been shown to induce protective immunity in mouse models of streptococcal disease. In addition, recent papers have created new perspectives on the role of such organelles in streptococcal pathogenesis, from the involvement in colonization and biofilm formation to translocation of tissue barriers. All this information makes the characterization of pili a hot scientific issue that we believe will lead to important future developments in understanding bacterial dynamics that lead to successful occupation of microbial niches.

Genome-based approaches to vaccine development.

The hundreds of bacterial genome sequences available together with advances in bioinformatics and the development of new experimental proteomic tools are revolutionizing the vaccinology field. The merge of stringent in silico criteria and different experimental approaches is allowing a more targeted strategy to obtain a restricted and prioritized list of potential antigens for testing in immunogenicity assays, reducing the time and the cost of novel protein vaccine formulations.