olate dispersal behavior to the landscape level (17, 21,27,28). Explicit tests of such models are needed (18). The tight fit between observed and predicted patterns of seed rain in our habitat patches provides strong support for the key assumption that small-scale behavioral responses can drive landscape-scale distributional patterns. From a conservation perspective, impacts of corridors can be predicted on the basis of behaviors that are relatively simple to measure (29).

SummaryWe have recently reported the presence of covalently linked pilus-like structures in the human pathogen, Group B Streptococcus (GBS). The pilus operon codes for three proteins which contain the conserved amino acid motif, LPXTG, associated with cell wall-anchored proteins together with two genes coding for sortase enzymes. Analysis of the eight sequenced genomes of GBS has led to the identification of a second, related genomic island of which there are two variants, each containing genes coding for proteins with LPXTG motifs and sortases. Here we show that both variant islands also code for pilus-like structures. Furthermore, we provide a thorough description and characterization of the genomic organization of the islands and the role of each protein in the assembly of the pili. For each pilus, polymerization of one of the three component proteins is essential for incorporation of the other two proteins into the pilus structure. In addition, two sortases are required for complete pilus assembly, each with specificity for one of the pilus components. A component protein of one of the newly identified pili is also a previously identified protective antigen and a second component of this pilus is shown to confer protection against GBS challenge. We propose that pilus-like structures are important virulence factors and potential vaccine candidates.

Pili are essential virulence factors in many Gram-negative bacteria; however, they have not been described in most important Gram-positive pathogens. While screening the sequence of multiple genomes of Group B Streptococcus , we identified protective antigens that formed high molecular weight polymers. Immunogold electron microscopy revealed that the structures have a pilus-like form. These large structures have gone unrecognized in decades of studies of Group B Streptococcus .

We recently described the presence of 3 pilus variants in the human pathogen group B streptococcus (GBS; also known as Streptococcus agalactiae), each encoded by a distinct pathogenicity island, as well as the ability of pilus components to elicit protection in mice against homologous challenge. To determine whether a vaccine containing a combination of proteins from the 3 pilus types could provide broad protection, we analyzed pili distribution and conservation in 289 clinical isolates. We found that pilus sequences in each island are conserved, all strains carried at least 1 of the 3 islands, and a combination of the 3 pilus components conferred protection against all tested GBS challenge strains. These data are the first to indicate that a vaccine exclusively constituted by pilus components can be effective in preventing infections caused by GBS, and they pave the way for the use of a similar approach against other pathogenic streptococci.

Both active and passive immunization strategies against Staphylococcus aureus have thus far failed to show efficacy in humans. With the attempt to develop an effective S. aureus vaccine, we selected five conserved antigens known to have different roles in S. aureus pathogenesis. They include the secreted factors α-hemolysin (Hla), ess extracellular A (EsxA), and ess extracellular B (EsxB) and the two surface proteins ferric hydroxamate uptake D2 and conserved staphylococcal antigen 1A. The combined vaccine antigens formulated with aluminum hydroxide induced antibodies with opsonophagocytic and functional activities and provided consistent protection in four mouse models when challenged with a panel of epidemiologically relevant S. aureus strains. The importance of antibodies in protection was demonstrated by passive transfer experiments. Furthermore, when formulated with a toll-like receptor 7-dependent (TLR7) agonist recently designed and developed in our laboratories (SMIP.7-10) adsorbed to alum, the five antigens provided close to 100% protection against four different staphylococcal strains. The new formulation induced not only high antibody titers but also a Th1 skewed immune response as judged by antibody isotype and cytokine profiles. In addition, low frequencies of IL-17-secreting T cells were also observed. Altogether, our data demonstrate that the rational selection of mixtures of conserved antigens combined with Th1/Th17 adjuvants can lead to promising vaccine formulations against S. aureus.Staphylococcus aureus | vaccine | TLR7 | adjuvant | Hla C urrent antibiotics are not efficacious against emerging multidrug-resistant strains of Staphylococcus aureus, a major human pathogen. Therefore, there is an urgent need to develop vaccines to target this pathogen. Two prophylactic vaccines have been tested recently for efficacy in humans: StaphVAX, which contained capsular polysaccharides type 5 and 8 (CP5 and CP8), and V710, based on a single protein antigen (IsdB) (1, 2). Both vaccines failed in phase III efficacy trials (3, 4). On the basis of these disappointing results and taking into account that S. aureus produces a plethora of virulence and immune evasion factors, different vaccine candidates, constituted by multiple components, are currently in phase I/II trials, but efficacy data are not available yet (5). In line with the multicomponent strategy, our laboratory has undertaken a vaccine discovery project aiming at the identification of conserved antigens, which play important roles in S. aureus virulence and pathogenicity. The main objective of the study was to combine the selected antigens in the presence of appropriate adjuvants and to demonstrate protective efficacy against a panel of genetically different S. aureus clinical isolates in different mouse models. ResultsAntigen Selection. The antigens included in our candidate combination vaccine were selected among surface and secreted factors previously shown to be protective and involved in S. aureus virulence. Two of them, the ferric hydroxamat...

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.backbone protein | isopeptide bonds | homology modeling

Vaccination has played a significant role in controlling and eliminating life-threatening infectious diseases throughout the world, and yet currently licensed vaccines represent only the tip of the iceberg in terms of controlling human pathogens. However, as we discuss in this Review, the arrival of the genome era has revolutionized vaccine development and catalyzed a shift from conventional culture-based approaches to genome-based vaccinology. The availability of complete bacterial genomes has led to the development and application of high-throughput analyses that enable rapid targeted identification of novel vaccine antigens. Furthermore, structural vaccinology is emerging as a powerful tool for the rational design or modification of vaccine antigens to improve their immunogenicity and safety.

Pili are putative virulence factors and promising vaccine candidates in Streptococcus agalactiae (group B Streptococcus [GBS]) infection, a leading cause of neonatal sepsis and meningitis. The genes necessary for pilus synthesis and assembly are clustered in pilus islands (PI). Each gene encodes three structural subunits (a backbone and two ancillary proteins) bearing a C-terminal LPXTG motif and two subfamily C sortases (SrtC) involved in covalent polymerization of the subunits. GBS strains also possess the conserved "housekeeping" sortase A (SrtA), but its role in pilus assembly is unclear. To address this issue, pilus expression and cell wall anchoring were analyzed in srtA deletion mutants. Loss of SrtA did not affect pilus polymerization. However, pilus expression on the cell surface was reduced, and pili accumulated in the culture supernatant. Furthermore, cell-associated pili could be readily released by detergent treatment, indicating that SrtA is involved in covalent anchoring of pili to the cell wall. When each of the genes comprising PI-2a was systematically deleted, only the absence of ancillary subunit GBS150 or the SrtC required for incorporation of GBS150 into pili mimicked the srtA mutant phenotype. Thus, from these data a model for GBS pilus assembly can be proposed in which PI sortases are responsible for polymerization of the pilus structure, while SrtA is required to covalently attach it to the cell wall, utilizing ancillary pilus subunit GBS150 as the anchor protein.