The development of efficient and inexpensive genome sequencing methods has revolutionized the study of human bacterial pathogens and improved vaccine design. Unfortunately, the sequence of a single genome does not reflect how genetic variability drives pathogenesis within a bacterial species and also limits genome-wide screens for vaccine candidates or for antimicrobial targets. We have generated the genomic sequence of six strains representing the five major disease-causing serotypes of Streptococcus agalactiae , the main cause of neonatal infection in humans. Analysis of these genomes and those available in databases showed that the S. agalactiae species can be described by a pan-genome consisting of a core genome shared by all isolates, accounting for ≈80% of any single genome, plus a dispensable genome consisting of partially shared and strain-specific genes. Mathematical extrapolation of the data suggests that the gene reservoir available for inclusion in the S. agalactiae pan-genome is vast and that unique genes will continue to be identified even after sequencing hundreds of genomes.

Isolated for the first time in 1982 from human gastric biopsy, Helicobacter pylori is responsible for gastritis, peptic ulcer, and gastric cancer. A pathogenicity island acquired by horizontal transfer, coding for a type IV secretion system, is a major determinant of virulence. The infection is now treated with antibiotics, and vaccines are in preparation. The geographic distribution suggests coevolution of man and Helicobacter pylori.

The gram negative, microaerophilic bacterium Helicobacter I~Iori colonizes the human gastric mucosa and establishes a chronic infection that is tightly associated with atrophic gastritis, peptic ulcer, and gastric carcinoma. Cloning of the H. pylori cytotoxin gene shows that the protein is synthesized as a 140-kD precursor that is processed to a 94-kD fully active toxin. Oral administration to mice of the purified 94-kD protein caused ulceration and gastric lesions that bear some similarities to the pathology observed in humans. The cloning of the cytotoxin gene and the development of a mouse model of human gastric disease will provide the basis for the understanding of H. ~lori pathogenesis and the development of therapeutics and vaccines. The recently discovered, gram negative, microaerophilic bacterium Helicobacter pylori colonizes the human gastric mucosa and establishes a chronic infection that is tightly associated with atrophic gastritis, peptic ulcer, and gastric carcinoma (1-5). H. pylori infection is a worldwide problem, since in developing countries it affects over 80% of the population older than 20. Also in developed countries the infection is present in 20% of the population by the age of 30 rising to over 50% by the age of 60. Clinical isolates of H. pylori can be classified into two groups based on the presence or absence of the vacuolating cytotoxin (6, 7) whose expression is linked to a surface exposed immunodominant antigen (CagA) (8, 9). Since high titers of serum antibodies to the CagA protein are detected in all patients with duodenal ulcer (8) and most of those with gastric carcinoma (10, 11), it has been proposed that disease development requires infection with cytotoxin-producing strains.The cytotoxin causes massive vacuolation in several mammalian cell lines (6), and similar vacuoles have also been observed in the gastric epithelia of patients with active chronic gastritis associated with H. pylori infection (12), indicating that the cytotoxin can contribute significantly to the pathogenesis of gastritis. Cell vacuolation in vitro can be blocked and reversed by inhibitors of V-type ATPases and potentiated by inhibitors of the Na+-K + ATPase (13,14), suggesting that the mechanism of action of the toxin is due to aberrant cation transport within the target cells. The purified toxin has been described as a protein of ~87 kD that is found in the bacterial culture supernatants, and the sequence of the NH2-terminal 23 amino acids has been determined (7).Despite the epidemiological correlation between infection with cytotoxic strains and disease (8) and the in vitro evidence for the presence of a cytotoxin, the in vivo roles of infection and cytotoxin have not been established due to the lack of a suitable animal model. H. ~lori does not colonize the gastric mucosa of mice or other small laboratory animals.To overcome this limitation, we administered H. pylori extracts and purified cytotoxin orally to mice. Using this model, extracts from cytotoxic H. Ioylori strains and purified cytotoxin ind...

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.

We describe a proteomic approach for identifying bacterial surface-exposed proteins quickly and reliably for their use as vaccine candidates. Whole cells are treated with proteases to selectively digest protruding proteins that are subsequently identified by mass spectrometry analysis of the released peptides. When applied to the sequenced M1_SF370 group A Streptococcus strain, 68 PSORT-predicted surface-associated proteins were identified, including most of the protective antigens described in the literature. The number of surface-exposed proteins varied from strain to strain, most likely as a consequence of different capsule content. The surface-exposed proteins of the highly virulent M23_DSM2071 strain included 17 proteins, 15 in common with M1_SF370. When 14 of the 17 proteins were expressed in E. coli and tested in the mouse for their capacity to confer protection against a lethal dose of M23_DSM2071, one new protective antigen (Spy0416) was identified. This strategy overcomes the difficulties so far encountered in surface protein characterization and has great potential in vaccine discovery.

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).

Although pili have long been recognized in Gram-negative pathogens as important virulence factors involved in adhesion and invasion, very little is known about extended surface organelles in Gram-positive pathogens. Here we report that Group A Streptococcus (GAS), a Gram-positive human-specific pathogen that causes pharyngitis, impetigo, invasive disease, necrotizing fasciitis, and autoimmune sequelae has long, surface-exposed, pilus-like structures composed of members of a family of extracellular matrix-binding proteins. We describe four variant pili and show that each is recognized by a specific serum of the Lancefield T-typing system, which has been used for over five decades to characterize GAS isolates. Furthermore, we show that immunization of mice with a combination of recombinant pilus proteins confers protection against mucosal challenge with virulent GAS bacteria. The data indicate that induction of a protective immune response against these structures may be a useful strategy for development of a vaccine against disease caused by GAS infection.fibronectin-binding ͉ Gram-positive

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 V͞R 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. Streptococcus agalactiae, or group B Streptococcus, is the leading cause of bacterial sepsis, pneumonia, and meningitis in neonates in the U.S. and Europe. Although S. agalactiae usually behaves as a commensal organism that colonizes the gastrointestinal or genital tract of 25-40% of healthy women, it can cause life-threatening invasive infection in susceptible hosts: newborn infants, pregnant women, and nonpregnant adults with underlying chronic illnesses (1, 2). Since guidelines recommending intrapartum antibiotic prophylaxis for high-risk or colonized women were issued in 1996 (3), the incidence of neonatal infections has decreased, and invasive S. agalactiae infections in immunocompromised adults have become more common. Adult disease now accounts for the majority of serious S. agalactiae infections. First recognized as a pathogen in bovine mastitis, S. agalactiae is distinguished from other pathogenic streptococci by the cell wall-associated group B carbohydrate.Another polysaccharide constitutes the organism's capsule, an important S. agalactiae virulence determinant. S. agalactiae strains of capsular serotype V were rarely isolated before the mid-1980s but now account for approximately one-third of clinical isolates in the U.S. (4-6). Type V is the most common capsular serotype associated with invasive infection in nonpregnant adults, and the emergence of type V strains over the past decade has been temporally linked to an increase in S. agalactiae disease in this population (7). As a species, S. agalactiae shares certain features with other pathogenic streptococci; however, the precise repertoire of shared and unique attributes that account for the emergence of S. agalactiae as a major pathogen for specific human populations remains undefined. To elucidate the molecular basis for S. agalactiae virulence, we determined the complete genome sequence (8) of the recent clinical type V isolate 2603 V͞R (www.tigr.org) and performed comparative analyses with other S. agalactiae strains and with other species of pathogenic streptococci. Methods ORF Prediction and Gene Identification.ORFs were predicted by GLIMMER (9, 10) trained with ORFs larger than 600 bp from the genomic sequence and S. agalactiae genes available in GenBank. All predicted proteins la...