Immaculada Margarit

Complete genome sequence and comparative genomic analysis of an emerging human pathogen, serotype V Streptococcus agalactiae

The 2,160,267 bp genome sequence of Streptococcus agalactiae, the leading cause of bacterial sepsis, pneumonia, and meningitis in neonates in the U.S. and Europe, is predicted to encode 2,175 genes. Genome comparisons among S. agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, and the other completely sequenced genomes identified genes specific to the streptococci and to S. agalactiae. These in silico analyses, combined with comparative genome hybridization experiments between the sequenced serotype V strain 2603 V/R and 19 S. agalactiae strains from several serotypes using whole-genome microarrays, revealed the genetic heterogeneity among S. agalactiae strains, even of the same serotype, and provided insights into the evolution of virulence mechanisms.

Pili in gram-positive pathogens.

Most bacterial pathogens have long filamentous structures known as pili or fimbriae extending from their surface. These structures are often involved in the initial adhesion of the bacteria to host tissues during colonization. In Gram-negative bacteria, pili are typically formed by non-covalent interactions between pilin subunits. By contrast, the recently discovered pili in Gram-positive pathogens are formed by covalent polymerization of adhesive pilin subunits. Evidence from studies of pili in the three principal streptococcal pathogens of humans indicates that the genes that encode the pilin subunits and the enzymes that are required for the assembly of these subunits into pili have been acquired en bloc by the horizontal transfer of a pathogenicity island.

Identification of novel genomic islands coding for antigenic pilus‐like structures in Streptococcus agalactiae

We 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.

Preventing Bacterial Infections with Pilus-Based Vaccines: the Group B Streptococcus Paradigm

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.

Streptococcus pyogenes pili promote pharyngeal cell adhesion and biofilm formation

Group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram‐positive human pathogen responsible for several acute diseases and autoimmune sequelae that account for half a million deaths worldwide every year. GAS infections require the capacity of the pathogen to adhere to host tissues and assemble in cell aggregates. Furthermore, a role for biofilms in GAS pathogenesis has recently been proposed. Here we investigated the role of GAS pili in biofilm formation. We demonstrated that GAS pilus‐negative mutants, in which the genes encoding either the pilus backbone structural protein or the sortase C1 have been deleted, showed an impaired capacity to attach to a pharyngeal cell line. The same mutants were much less efficient in forming cellular aggregates in liquid culture and microcolonies on human cells. Furthermore, mutant strains were incapable of producing the typical three‐dimensional layer with bacterial microcolonies embedded in a carbohydrate polymeric matrix. Complemented mutants had an adhesion and aggregation phenotype similar to the wild‐type strain. Finally, in vivo expression of pili was indirectly confirmed by demonstrating that most of the sera from human patients affected by GAS‐mediated pharyngitis recognized recombinant pili proteins. These data support the role of pili in GAS adherence and colonization and suggest a general role of pili in all pathogenic streptococci.

Emergence and Global Dissemination of Host-Specific Streptococcus agalactiae Clones

ABSTRACT To examine the global diversity of Streptococcus agalactiae (group B streptococci [GBS]) and to elucidate the evolutionary processes that determine its population genetics structure and the reported changes in host tropism and infection epidemiology, we examined a collection of 238 bovine and human isolates from nine countries on five continents. Phylogenetic analysis based on the sequences of 15 housekeeping genes combined with patterns of virulence-associated traits identified a genetically heterogeneous core population from which virulent lineages occasionally emerge as a result of recombination affecting major segments of the genome. Such lineages, like clonal complex 17 (CC17) and two distinct clusters of CC23, are exclusively adapted to either humans or cattle and successfully spread globally. The recent emergence and expansion of the human-associated and highly virulent sequence type 17 (ST17) could conceivably account, in part, for the increased prevalence of neonatal GBS infections after 1960. The composite structure of the S. agalactiae genome invalidates phylogenetic inferences exclusively based on multilocus sequence typing (MLST) data and thereby the previously reported conclusion that the human-associated CC17 emerged from the bovine-associated CC67. IMPORTANCE Group B streptococci (GBS) (Streptococcus agalactiae) have long been recognized as important causes of mastitis in cattle. After 1960, GBS also became the most prevalent cause of invasive and often fatal infections in newborns. At the same time, GBS are carried by a substantial proportion of healthy individuals. The aims of this study were to elucidate the genetic mechanisms that lead to diversification of the GBS population and to examine the relationship between virulence and host preference of evolutionary lineages of GBS. Genetic analysis of GBS isolates from worldwide sources demonstrated epidemic clones adapted specifically to either the human or bovine host. Such clones seem to emerge from a genetically heterogeneous core population as a result of recombination affecting major segments of the genome. Emergence and global spread of certain clones explain, in part, the change in epidemiology of GBS disease and may have implications for prevention. Group B streptococci (GBS) (Streptococcus agalactiae) have long been recognized as important causes of mastitis in cattle. After 1960, GBS also became the most prevalent cause of invasive and often fatal infections in newborns. At the same time, GBS are carried by a substantial proportion of healthy individuals. The aims of this study were to elucidate the genetic mechanisms that lead to diversification of the GBS population and to examine the relationship between virulence and host preference of evolutionary lineages of GBS. Genetic analysis of GBS isolates from worldwide sources demonstrated epidemic clones adapted specifically to either the human or bovine host. Such clones seem to emerge from a genetically heterogeneous core population as a result of recombination affecting major segments of the genome. Emergence and global spread of certain clones explain, in part, the change in epidemiology of GBS disease and may have implications for prevention.

Sequence Variation in Group A Streptococcus Pili and Association of Pilus Backbone Types with Lancefield T Serotypes

BACKGROUND We previously reported that group A Streptococcus (GAS) pili are the T antigens described by Rebecca Lancefield. We also showed that these pili, constituted by backbone, ancillary 1, and ancillary 2 proteins, confer protection against GAS challenge in a mouse model. METHODS We evaluated pilus distribution and conservation by sequencing the subunits of 39 new GAS isolates and used immunoblot analysis and agglutination assays to define the specificity of T sera to pilus subunits. RESULTS GAS pili are encoded by 9 different islands within which backbone protein, ancillary protein 1, and ancillary protein 2 cluster in 15, 16, and 5 variants, respectively. Immunoblot and agglutination assays revealed that T type is determined by the backbone variant. This observation enabled us to set up a simple polymerase chain reaction assay to define the T type of GAS isolates. CONCLUSIONS We propose the use of a tee gene sequence typing, analogous to the emm gene typing, as a valuable molecular tool that could substitute for the serological T classification of GAS strains. From our sequence analysis and from recent epidemiological data, we estimate that a vaccine comprising a combination of 12 backbone variants would protect against > 90% of currently circulating strains.

Surfome Analysis as a Fast Track to Vaccine Discovery IDENTIFICATION OF A NOVEL PROTECTIVE ANTIGEN FOR GROUP B STREPTOCOCCUS HYPERVIRULENT STRAIN COH1

Safe recombinant vaccines, based on a small number of antigenic proteins, are emerging as the most attractive, cost-effective solution against infectious diseases. In the present work, we confirmed previous data from our laboratory showing that whole viable bacterial cell treatment with proteases followed by the identification of released peptides by mass spectrometry is the method of choice for the rapid and reliable identification of vaccine candidates in Gram-positive bacteria. When applied to the Group B Streptococcus COH1 strain, 43 surface-associated proteins were identified, including all the protective antigens described in the literature as well as a new protective antigen, the cell wall-anchored protein SAN_1485 belonging to the serine-rich repeat protein family. This strategy overcomes the difficulties so far encountered in the identification of novel vaccine candidates and speeds up the entire vaccine discovery process by reducing the number of recombinant proteins to be tested in the animal model.

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.

Use of Lactococcus lactis Expressing Pili from Group B Streptococcus as a Broad-Coverage Vaccine against Streptococcal Disease

Recent data indicate that the human pathogen group B Streptococcus (GBS) produces pilus-like structures encoded in genomic islands with similar organization to pathogenicity islands. On the basis of the amino acid sequence of their protein components, 3 different types of pili have been identified in GBS, at least 1 of which is present in all isolates. We recently demonstrated that recombinant pilus proteins protect mice from lethal challenge with GBS and are thus potential vaccine candidates. Here, we show that GBS pilin island 1, transferred into the nonpathogenic microorganism Lactococcus lactis, leads to pilus assembly. We also show that systemically or mucosally delivered Lactococcus expressing pilin island 1 protects mice from challenge with GBS isolates carrying pilus 1. Furthermore, lactococci engineered to express hybrid pili containing GBS pilus 1 and pilus 2 components confer protection against strains expressing either of the 2 pilus types. These data pave the way to the design of pilus-based, multivalent live vaccines against streptococcal pathogens.