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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.

The fourth EMBO workshop on “Lymphocyte antigen receptor and coreceptor signaling” gathered immunologists to share key findings, new questions and emerging trends in the field of cell signaling.

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.

We have established an in vitro 3D system which recapitulates the human tracheo-bronchial mucosa comprehensive of the pseudostratified epithelium and the underlying stromal tissue. In particular, we reported that the mature model, entirely constituted of primary cells of human origin, develops key markers proper of the native tissue such as the mucociliary differentiation of the epithelial sheet and the formation of the basement membrane. The infection of the pseudo-tissue with a strain of NonTypeable Haemophilus influenzae results in bacteria association and crossing of the mucus layer leading to an apparent targeting of the stromal space where they release large amounts of vesicles and form macro-structures. In summary, we propose our in vitro model as a reliable and potentially customizable system to study mid/long term host-pathogen processes.

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.

The purpose of this paper was to present the current knowledge on the prevention of group B streptococcus (GBS) neonatal infections and the status of prevention policies in European countries and to present the DEVANI pan-European program, launched in 2008. The aim of this program was to assess the GBS neonatal infection burden in Europe, to design a new vaccine to immunize neonates against GBS infections, to improve the laboratory performance for the diagnosis of GBS colonization and infection, and to improve the methods for the typing of GBS strains. The current guidelines for GBS prevention in different countries were ascertained and a picture of the burden before and after the instauration of prevention policies has been drawn. After the issue of the Centers for Disease Control and Prevention (CDC) guidelines, many European countries have adopted universal screening for the GBS colonization of pregnant women and intrapartum prophylaxis to colonized mothers. Nevertheless, some European countries continue advocating the risk factor approach to GBS prevention. Most European countries have implemented policies to prevent GBS neonatal infections and the burden of the disease has decreased during the last several years. Nevertheless, further steps are necessary in order to develop new strategies of prevention, to improve microbiological techniques to detect GBS colonization and infection, and to coordinate the prevention policies in the EU.

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.

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 β-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 (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.

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.

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.

The majority of currently available successful vaccines induce host responses against antigens that are highly conserved in the targeted pathogens. The diphtheria, tetanus, and pertussis vaccines confer protection by inducing neutralizing antibodies to the conserved bacterial toxins that are the major virulence factors. The Hemophilus influenzae B vaccine induces responses to conserved epitopes in the sugar structure of the bacterial capsular polysaccharide. However, the efficacy of more recently developed vaccines is limited by antigen variation, which also presents a challenge for future vaccine development. This review will explore bacterial genome variability and its impact on vaccine development.

CsrRS (or CovRS) is a two-component system implicated in the control of multiple virulence determinants in the important human pathogen, group A Streptococcus (GAS). Earlier studies suggested that extracellular Mg(2+) signalled through the presumed sensor histidine kinase, CsrS. We now confirm those findings, as complementation of a csrS mutant restored Mg(2+)-dependent gene regulation. Moreover, we present strong evidence that Mg(2+) signals through CsrS to regulate an extensive and previously undefined repertoire of GAS genes. The effect of Mg(2+) on regulation of global gene expression was evaluated using genomic microarrays in an M-type 3 strain of GAS and in an isogenic csrS mutant. Unexpectedly, of the 72 genes identified in the Mg(2+)-stimulated CsrRS regulon, 42 were absent from the CsrR regulon (the latter being defined by comparison of wild-type and CsrR mutant transcriptomes at low Mg(2+)). We observed CsrS-dependent regulation of 72 of the 73 genes whose expression changed in response to elevated extracellular Mg(2+) in wild-type bacteria, a result that identifies CsrS as the principal, if not exclusive, sensor for extracellular Mg(2+) in GAS. To our knowledge, this study is the first to characterize global gene regulation by a GAS two-component system in response to a specific environmental stimulus.

Many bacterial toxins utilize cell surface glycoconjugate receptors for attachment to target cells. In the present study the potential carbohydrate binding of Helicobacter pylori vacuolating cytotoxin VacA was investigated by binding to human gastric glycosphingolipids on thin-layer chromatograms. Thereby a distinct binding of the toxin to two compounds in the non-acid glycosphingolipid fraction was detected. The VacA-binding glycosphingolipids were isolated and characterized by mass spectrometry and proton NMR as galactosylceramide (Galbeta1Cer) and galabiosylceramide (Galalpha4Galbeta1Cer). Comparison of the binding preferences of the protein to reference glycosphingolipids from other sources showed an additional recognition of glucosylceramide (Glcbeta1Cer), lactosylceramide (Galbeta4Glcbeta1Cer) and globotriaosylceramide (Galalpha4Galbeta4Glcbeta1Cer). No binding to the glycosphingolipids recognized by the VacA holotoxin was obtained with a mutant toxin with deletion of the 37 kDa fragment of VacA (P58 molecule). Collectively our data show that the VacA cytotoxin is a glycosphingolipid binding protein, where the 37 kDa moiety is required for carbohydrate recognition. The ability to bind to short chain glycosphingolipids will position the toxin close to the cell membrane, which may facilitate toxin internalization.

Group B Streptococcus (GBS) is an opportunistic organism that can harmlessly colonize the human gut, vagina, and rectum but can also cause pneumonia, sepsis, and meningitis in neonates born to colonized mothers. We have shown previously that growth rate and oxygen level regulate the ability of GBS to invade eukaryotic cells in vitro. Herein we extend and expand on these observations to show that GBS type V, an emergent serotype, grown in a chemostat at a cell mass-doubling time ( t d ) of 1.8 h with oxygen invaded human ME-180 cervical epithelial cells in large numbers compared with those grown at the same t d without oxygen or at a slower t d of 11.0 h. The fact that several GBS type V cell wall-associated and membrane proteins were expressed exclusively under the invasive growth condition prompted an investigation, using genomics and proteomics, of all upregulated genes and proteins. Several proteins with potential roles in adherence were identified, including an undefined surface antigen (SAG1350), a lipoprotein (SAG0971), penicillin-binding protein 2b (SAG0765), glyceraldehyde-3-phosphate dehydrogenase (SAG0823), and an iron-binding protein (SAG1007). Mouse antisera to these five proteins inhibited binding of GBS type V to ME-180 cells by ≥85%. Recombinant undefined surface antigen (SAG1350), lipoprotein (SAG0971), and penicillin-binding protein 2b (SAG0765) each bound to ME-180 cells in a dose-dependent fashion, confirming their ability to act as ligands. Collectively, these data increase the number of potential GBS adherence factors and also suggest a role for these surface-associated proteins in initial pathogenic events.

Nature Communications 5: Article number:4544 (2014); Published 4 Aug 2014; Updated 28 Jan 2015 The original version of this Article contained an error in the spelling of a member of the DEVANI Consortium, Lucilla Baldassarri, which was incorrectly given as Lucilia Baldassari. This has now been corrected in both the PDF and HTML versions of the Article.

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.

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.

In a single decade, the complete genome sequence of ∼ 300 bacterial pathogens has been determined. The potential for acquisition of intellectual property in this time has been enormous. Two schools of thought pervade. In one, mainly associated with academic researchers, the new sequence is immediately made available in public databases to prevent monopolisation. In the other, mainly industry-based, patent applications are filed on the new sequence in order to support possible commercial development of products based on the data. In the end, due to limitations in patent duration and applicability, in this first wave of genome sequence publications it is likely that both strategies will have similar consequences and, in many instances, will have impact primarily as prior art. In order to extend and create value from any future genome-based intellectual property, a new generation of patents derived from functional analyses of the genome sequences will be required.

Colonization of the colon and vagina is thought to be important in the pathogenesis of group B Streptococcus (GBS) infection. However, little is known about the strategies used by GBS to translocate through the epithelial barrier during the onset of disease. We used differentiated epithelial cells grown on transwell inserts as a model of the epithelial barrier. Bacterial translocation occurred without a detectable decrease in transepithelial resistance. Whereas acapsular GBS was better able to adhere to and invade epithelial cells, the percentage of bacteria translocating across the epithelial monolayer was independent of the presence of the capsule. Transmission electron microscopy showed the intimate association of GBS with intercellular junctions and the capacity to cross the monolayer by a paracellular mechanism. This process consisted of an active and transient opening of cell junctions. Indeed, GBS was preferentially found along the cell perimeter, where it colocalized with junctional protein complexes.

There are two alleles, m1 and m2, of the midregion of the vacuolating cytotoxin gene ( vacA ) of Helicobacter pylori which code for toxins with different cell specificities. Here we describe the construction of five chimeric strains in which regions of vacA were exchanged between the two genotypes. By analyzing the toxicity of these strains for HeLa and RK13 cells we have confirmed that a 148-amino-acid region determines the phenotypic differences between the two forms of the protein and that this entire region is important for cytotoxicity. Furthermore, we have used our chimeric strains to investigate whether variations in the midregion of VacA have an effect on phorbol 12-myristate 13-acetate (PMA)-induced VacA sensitivity in HL-60 cells. The PMA-induced VacA sensitivity of HL-60 cells has been previously associated with the appearance of the cell surface receptor protein tyrosine phosphatase beta (RPTPβ). Our data indicate that both the m1 and m2 forms of VacA are able to utilize RPTPβ, and the cell-specific phenotype of the midregion is independent of the presence of RPTPβ. It appears that another as-yet-unidentified receptor exists in HL-60 cells that accounts for the m2 phenotype in this cell line. Also, by studying the effect of PMA on levels of RPTPβ in other cell lines and toxicity of VacA in these cell lines we have shown that RPTPβ does not play a major role in the vacuolation of HeLa cells.

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.

Common variable immunodeficiency (CVID) is a primary immune disorder characterized by impaired antibody production, which is in many instances secondary to defective T-cell function (T-CVID). We have previously identified a subset of patients with T-CVID characterized by defective T-cell receptor (TCR)-dependent protein tyrosine phosphorylation. In these patients, ZAP-70 fails to be recruited to the TCR as the result of impaired CD3ζ phosphorylation, which is, however, not dependent on defective Lck expression or activity. Here we show that neither Fyn nor CD45 is affected in these patients. On the other hand, T-CVID T cells show dramatic defects in the Vav/Rac pathway controlling F-actin dynamics. A significant deficiency in Vav protein was indeed observed; in 3 of 4 patients with T-CVID, it was associated with reduced VAV1 mRNA levels. The impairment in Vav expression correlated with defective F-actin reorganization in response to TCR/CD28 coengagement. Furthermore, TCR/CD28-dependent up-regulation of lipid rafts at the cell surface, which requires F-actin dynamics, was impaired in these patients. The actin cytoskeleton defect could be reversed by reconstitution of Vav1 expression in the patients' T cells. Results demonstrate an essential role of Vav in human T cells and strongly suggest Vav insufficiency in T-CVID. (Blood. 2005;106:626-634)

Summary Helicobacter pylori is the causative agent of peptic ulcer disease. A major virulence factor of H. pylori is VacA, a toxin that causes massive vacuolization of epithelial cell lines in vitro and gastric epithelial erosion in vivo. Although VacA is exported over the outer membrane and is released from the bacteria, a portion of the toxin remains associated with the bacterial surface. We have found surface-associated toxin to be biologically active and spatially organized into distinct toxin-rich domains on the bacterial surface. Upon bacterial contact with host cells, toxin clusters are transferred directly from the bacterial surface to the host cell surface at the bacteria–cell interface, followed by uptake and intoxication. This contact-dependent transfer of VacA represents a cost-efficient route for delivery of VacA and potentially other bacterial effector molecules to target cells.

Helicobacter pylori toxin, VacA, damages the gastric epithelium by erosion and loosening of tight junctions. Here we report that VacA also interferes with T cell activation by two different mechanisms. Formation of anion-specific channels by VacA prevents calcium influx from the extracellular milieu. The transcription factor NF-AT thus fails to translocate to the nucleus and activate key cytokine genes. A second, channel-independent mechanism involves activation of intracellular signaling through the mitogen-activated protein kinases MKK3/6 and p38 and the Rac-specific nucleotide exchange factor, Vav. As a consequence of aberrant Rac activation, disordered actin polymerization is stimulated. The resulting defects in T cell activation may help H. pylori to prevent an effective immune response leading to chronic colonization of its gastric niche.

Atherosclerotic lesions are infiltrated by macrophages and T lymphocytes, potentially reactive to pathogens. We studiedin vivoactivated T lymphocytes that infiltrate atherosclerotic plaques ofHelicobacter pylori-infected patients with or without anti-Chlamydia pneumoniaeantibodies. In all atherosclerotic lesions, T helper type 1 (Th1) cells were predominant.C. pneumoniae-specific T cells were detected only in the plaques of anti-C. pneumoniaeseropositive patients, whereasH. pylori-specific T cells were found in the gastric mucosa but not in the plaques of the same patients. Plaque-derived Th1 cells expressed cytotoxicity, proapoptotic activity, and help for monocyte tissue factor production. Although multifactorial, atherosclerosis can be regarded as a Th1-driven immunopathological condition.

VacA is a pore-forming cytotoxin produced by Helicobacter pylori in several strain-specific isoforms, which have been classified in two main families, m1 and m2, according to the sequence of a variable “midregion.” Both forms are associated with gastric pathologies and can induce vacuolation of cultured cells. The comparison of two representative toxins, m1 17874 and m2 9554, has indicated that the m2 form is less powerful in vacuolation assays and that its effects are more strongly cell type dependent. To rationalize these differences and to investigate structure-function relationships in this toxin, we have compared the properties of the channels formed by these two variants and by a construct derived from 17874 by deleting a loop that connects the two toxin domains, which is shorter in 9554 than in 17874. Although the channels formed by all three proteins are similar, m2 9554 channels have, on average, a lower conductance and are less anion-selective and more voltage-dependent than the m1 pores. Furthermore, the rate of incorporation of 9554 VacA into planar bilayers depends on lipid composition much more strongly than that of 17874. The comparison with the behavior of the loop deletion mutant indicates that this latter property, as well as a portion of the conductance decrease, may be attributed to the reduction in loop length. The differences in pore properties are proposed to account in part for the different cytotoxicity exhibited by the two toxin isoforms. We furthermore present evidence suggesting that the conformation of the membrane-embedded toxin may be influenced by the lipid composition of the membrane itself.

The interaction of VacA with membranes involves: (i) a low pH activation that induces VacA monomerization in solution, (ii) binding of the monomers to the membrane, (iii) oligomerization and (iv) channel formation. To better understand the structure–activity relationship of VacA, we determined its topology in a lipid membrane by a combination of proteolytic, structural and fluorescence techniques. Residues 40–66, 111–169, 205–266, 548–574 and 723–767 were protected from proteolysis because of their interaction with the membrane. This last peptide was shown to most probably adopt a surface orientation. Both α-helices and β-sheets were found in the structure of the protected peptides.

We have previously identified a subset of common variable immunodeficiency (CVID) patients with defective T cell function associated with impaired activation of the TCR-dependent tyrosine phosphorylation cascade. Here we have assessed the structural and functional integrity of the principal components involved in coupling the TCR/CD3 complex to intracellular tyrosine kinases in two of these patients. We show that ZAP-70 fails to bind the signaling-competent CD3zeta tyrosine phosphorylation isoform and to become activated following TCR engagement, suggesting that defective recruitment of ZAP-70 might underlie the TCR signaling dysfunction in these patients. Determination of the nucleotide sequences encoding the intracellular domains of the CD3/zeta subunits and ZAP-70 did not reveal any mutation. Furthermore, ZAP-70 from these patients could interact in vitro with recombinant phospho-zeta, ruling out genetic defects at the immunoreceptor tyrosine-based activation motif/SH2 domain interface responsible for ZAP-70 recruitment to the activated TCR. No defect was found in expression, activity or subcellular localization of Lck, which is thought to be primarily responsible for CD3zeta phosphorylation. Hence, while the T cell defect in these CVID patients can be pinpointed to the interaction between ZAP-70 and CD3zeta, the integrity in the components of the signaling machinery involved in this process suggests that additional components might be required for completion of this step.

There are two alleles of the vacuolating cytotoxin gene from Helicobacter pylori , which code for toxins with different cell specificities. By analyzing the phenotypes of natural and artificial chimeras between the two forms of the protein, we have delimited a short stretch of amino acids which determine the cell specificity.

In its mature form, the VacA toxin of Helicobacter pylori is a 95-kDa protein which is released from the bacteria as a low-activity complex. This complex can be activated by low-pH treatment that parallels the activity of the toxin on target cells. VacA has been previously shown to insert itself into lipid membranes and to induce anion-selective channels in planar lipid bilayers. Binding of VacA to lipid vesicles and its ability to induce calcein release from these vesicles were systematically compared as a function of pH. These two phenomena show a different pH-dependence, suggesting that the association with the lipid membrane may be a two-step mechanism. The secondary and tertiary structure of VacA as a function of pH and the presence of lipid vesicles were investigated by Fourier-transform infrared spectroscopy. The secondary structure of VacA is identical whatever the pH and the presence of a lipid membrane, but the tertiary structure in the presence of a lipid membrane is dependent on pH, as evidenced by H/D exchange.

The mechanisms by which Helicobacter pylori releases its virulence factors are poorly known. Active secretion has been proposed for some products, including a vacuolating toxin (VacA). Outer membrane vesicles represent another mechanism by which some Gram-negative bacteria may release virulence factors. This study sought to localize VacA by immunocytochemistry in H. pylori cells, to determine whether H. pylori produces outer membrane vesicles, and to investigate whether such vesicles might constitute a vehicle for the delivery of bacterial virulence factors to the gastric mucosa. Small (50-300 nm) membrane vesicles were found in H. pylori culture media from both H. pylori strain 60190 and strain CCUG 17874. These vesicles appeared to originate from blebs arising on the bacterial outer membrane. VacA was immunolocalized in the periplasm and outer membrane of intact bacteria and also in outer membrane blebs and vesicles. Both soluble secreted VacA and VacA-containing vesicles bound to, and were internalized by, MKN28 cells and were detectable in the gastric mucosa from H. pylori-infected humans. The release of outer membrane vesicles by H. pylori may represent a mechanism, additional to secretory pathways, for the delivery of bacterial toxins and antigens to the gastric mucosa.

Isolated for the first time in 1982 from human gastric biopsy,Helicobacter pyloriis 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 andHelicobacter pylori.

Preliminary clinical evidence suggests that Helicobacter pylori may be associated with diarrhea through its vacuolating toxin (VacA). To establish whether VacA induces intestinal secretion, epithelial damage, or both, purified pH-activated VacA was added to Caco-2 cell monolayers mounted in Ussing chambers, and electrical parameters were monitored. Mucosal addition of VacA induced an increase in short circuit current, consistent with enterotoxic effect. The effect was time- and dose-dependent and saturable. It was not found if the toxin was not pH-activated, added to the serosal side, or preheated. In cells preloaded with the Ca2+ buffering compound BAPTA/AM or with the Cl- channel inhibitor 5-nitro-2-3-(3-phenylpropylamino)benzoic acid, short circuit current did not change, indicating that VacA induces activation of Ca2+-dependent Cl- channels. VacA did not show cytopathic effects, as judged by tissue resistance. These results support the hypothesis that H. pylori may be associated with diarrhea through production of VacA.

The vacuolating cytotoxin (VacA) is a major virulence factor of Helicobacter pylori which is not yet well characterised and is difficult to obtain in large quantities. Here we describe the production of a monoclonal antibody that recognises the native but not the denatured form of VacA. The antibody can be efficiently used in affinity chromatography for one-step purification of large quantities of VacA from culture supernatants. Elution at acidic pH dissociates the oligomeric molecule into monomers that reanneal in a time-dependent fashion. The purified cytotoxin is able to bind, and to intoxicate HeLa cells.

The development of safer analogues of immunosuppressants such as cyclosporin A and FK506 is an important goal for a number of clinical applications ranging from transplantation to the treatment of autoimmune diseases. Here we show the generation and the characterization of Jurkat T cell lines stably transfected with a reporter construct containing the firefly luciferase gene under the control of NF-AT. These lines specifically respond in a cyclosporin A-sensitive manner to T cell antigen receptor-derived signals. Due to the high levels of luciferase activity expression fewer than 1000 cells are required for detection of luciferase. In addition, a simplified luciferase assay allows to reduce both the manipulations and the time required for the assay, making these lines potentially useful models for the automated screening of cyclosporin A and FK506 analogues.

We have previously shown that infection of mice with H. pylori can be prevented by oral immunization with H. pylori antigens given together with E. coli heat-labile enterotoxin (LT) as adjuvant. Since LT cannot be used in humans because of its unacceptable toxicity, we investigated whether protection of mice could be achieved by co-administration of antigens with non-toxic LT mutants. Here we show that CD1/SPF mice are protected against infection after oral vaccination with either purified H. pylori antigens (native and recombinant VacA, urease and CagA), or whole-cell vaccine formulations, given together with the non-toxic mutant LTK63 as a mucosal adjuvant. Furthermore we show that such protection is antigen-specific since immunization with recombinant or native VacA plus LTK63 conferred protection against infection by an H. pylori Type I strain, which expresses VacA, but not against challenge with a Type II strain which is not able to express this antigen. These results show that: (1) protection against H. pylori can be achieved in the mouse model of infection using subunit recombinant constructs plus non-toxic mucosal adjuvants; and (2) this mouse model is an useful tool in testing H. pylori vaccine formulations for eventual use in humans.

Helicobacter pylori is a 'slow' bacterial pathogen. While infection is usually acquired early in life, only decades later does severe pathology appear. During this long period of incubation, the host mounts a vigorous immune response against H. pylori which fails to resolve the infection and may in fact contribute to the severity of the disease. In the past year, evidence has accumulated indicating a role for a polarized T helper 1 cell response in the gastric pathology induced by H. pylori. Furthermore, a pathogenicity island in type I H. pylori strains has been shown to be responsible for H. pylori induced inflammation. Recent advances in animal models have provided the rationale for entering into human clinical trials of an H. pylori vaccine

Helicobacter pylori (Hp) infection almost invariably results in chronic antral gastritis, but only a proportion of patients develop peptic ulcer. Some Hp strains may be more ulcerogenic than others, but some ulcerogenic mechanisms may also depend on the type of the host immune response. In this study, the antigen specificity and the cytokine profile of 53 Hp-specific CD4+ T cell clones derived from the antral mucosa of five patients with Hp-induced uncomplicated chronic gastritis (CG) were assessed and compared with those of 34 Hp-specific CD4+ T cell clones derived from six Hp-infected patients with chronic gastritis and peptic ulcer (CG-PU). The majority (28/34; 82%) of gastric Hp-specific T cell clones from CG-PU patients expressed the Th1 profile and 17 (all Th1) of the 34 clones were specific for cytotoxin-associated protein (CagA). In contrast, 34 (64%) of the 53 Hp-specific gastric T cell clones derived from CG patients were able to secrete both Th1 and Th2 cytokines (Th0 profile) and only 36% expressed a polarized Th1 profile. The majority (85%) of Hp-specific clones from CG patients recognized Hp antigens other than CagA, since 13/53 (25%) were specific for urease, 6 (11%) for VacA, 6 (11%) for HSP and 20 (38%) for other undefined Hp antigens. Results provide evidence that the type of T helper cell response against Hp may vary according to the antigen involved and suggest that a polarized Th1 response may play a role in the genesis of peptic ulcer, whereas a local Th0 response, including interleukin-4 production, may represent an individual host factor which contributes to lower the degree of gastric inflammation and prevent ulcer complication.

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., PLoS ONE, 8 (4), ISSN:1932-6203

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.