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Clostridium difficile is the leading cause of antibiotic-associated diarrhoea and pseudomembranous colitis. While the role of toxins in pathogenesis has been extensively described, the contribution of surface determinants to intestinal colonization is still poorly understood. We focused our study on a novel member of the MSCRAMM family, named CbpA (Collagen binding protein A), for its adhesive properties towards collagen. We demonstrate that CbpA, which carries an LPXTG-like cell wall anchoring domain, is expressed on the bacterial surface of C. difficile and that the recombinant protein binds at high affinity to collagens I and V (apparent Kd in the order of 10(-9 ) M). These findings were validated by confocal microscopy studies showing the colocalization of the protein with type I and V collagen fibres produced by human fibroblasts and mouse intestinal tissues. However, the collagen binding activity of the wild-type C. difficile 630 strain was indistinguishable to the cbpA knock-out strain. To overcome this apparent clostridial adherence redundancy, we engineered a Lactococcus lactis strain for the heterologous expression of CbpA. When exposed on the surface of L. lactis, CbpA significantly enhances the ability of the bacterium to interact with collagen and to adhere to ECM-producing cells. The binding activity of L. lactis-CbpA strain was prevented by an antiserum raised against CbpA, demonstrating the specificity of the interaction. These results suggest that CbpA is a newsurface-exposed adhesin contributing to the C. difficile interaction with the host.

Human neutrophils activatd by PMA were found to induced the formation of a nitroxide radical from DFO. The presence of SOD was necessary to permit the formation of the DFO radical. The inactive phorbol ester did not induce DFO radical, and dl _sphinganine suppressed the radical produced by the active phorbol ester. Other cell stimuli (Zymocel and the chemotactic peptide) also induced the formation of the DFO radical, although radical concentration was very much lower than with PMA. Participation of .NO, ,OH or 1O2 was ruled out by the inability of NG-methyl-L-arginine, NG-nitro-L-arginine, DMSO, mannitol, histidine, and methionine to inhibit the formation of DFO radical produced by PMA-activated cells. Furthermore, PMA-activated cells dod not produce detectable levels of NO2−, as a stable oxidation product of .NO, and D2, which enhances the lifetime of singlet oxygen, did not modify the intensity or the lifetime of DFO radical. The involvement of cell MPO was suggested by the inhibition of the DFO radical observed after treatment with catalase or with antihuman MPO antibodies. Also, HOCI was found to induce the DFO radical in cell-free reactions, but our data indicate that the reaction leading to DFO radical formation by neutrophils involves the reduction of MPO compound II back to active enzyme (ferric-MPO). Anti-inflammatory drugs strongly increased the DFO radical produced by activated neutrophils. On the contrary, none of these drugs was able to increase the DFO radical produced by HOCl. Histidine and methionine that inhibited the DFO radical intensity in cell-free reactions, were shown to act directly onm HOCl. Experiments with MPO-H2O2 in SOD- and Cl−-free conditions showed the formation of DFO radical and confirmed the hypothesis of the involvement of compound II. The conversion of compound II to ferric MPO by DFO optimized the enzymatic activity of neurophils, and in the presence of monochlorodimedon (compound II promoting agent) we measured an increased HOCl production. When DFO was modified by conjugation with hydroxyethyl starch, it lost the ability to produce the radical either by neutrophils or by MPO-H2O2 and did not increase HOCl production. The inability of these DFO derivatives to produce potentially toxic species migh explain their reported lower toxicity in vivo.

We present an interdisciplinary approach that, by incorporating a range of experimental and computational techniques, allows the identification and characterization of functional/immunogenic domains. This approach has been applied to ArtJ, an arginine-binding protein whose orthologs in Chlamydiae trachomatis (CT ArtJ) and pneumoniae (CPn ArtJ) are shown to have different immunogenic properties despite a high sequence similarity (60% identity). We have solved the crystallographic structures of CT ArtJ and CPn ArtJ, which are found to display a type II transporter fold organized in two α-β domains with the arginine-binding region at their interface. Although ArtJ is considered to belong to the periplasm, we found that both domains contain regions exposed on the bacterial surface. Moreover, we show that recombinant ArtJ binds to epithelial cells in vitro, suggesting a role for ArtJ in host-cell adhesion during Chlamydia infection. Experimental epitope mapping and computational analysis of physicochemical determinants of antibody recognition revealed that immunogenic epitopes reside mainly in the terminal (D1) domain of both CPn and CT ArtJ, whereas the surface properties of the respective binding-prone regions appear sufficiently different to assume divergent immunogenic behavior. Neutralization assays revealed that sera raised against CPn ArtJ D1 partially reduce both CPn and CT infectivity in vitro, suggesting that functional antibodies directed against this domain may potentially impair chlamydial infectivity. These findings suggest that the approach presented here, combining functional and structure-based analyses of evolutionary-related antigens can be a valuable tool for the identification of cross-species immunogenic epitopes for 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.

We recently characterized the function of BibA, an immunogenic surface-associated antigen expressed by group B Streptococcus (GBS) that is involved in virulence. In this study, we performed a bibA gene variability analysis on a panel of 72 clinical isolate strains. The bibA gene was present in all the strains analyzed, and 4 allelic variants, correlating with serotype, were identified. Moreover, although the BibA protein was expressed in all strains tested, only 54% of strains demonstrated surface exposure of the antigen in vitro. Importantly, expression of BibA on the bacterial surface was correlated with protection, because mice immunized with BibA were protected against challenge by a GBS strain with high levels of surface exposure of the antigen. In agreement with these findings, serum samples from mice immunized with recombinant BibA induced neutrophil-mediated in vitro opsonophagocytic killing of GBS. In conclusion, we propose BibA as a novel GBS vaccine candidate.

To identify factors involved in the response of group B streptococci (GBS) to environmental pH, we performed a comparative global gene expression analysis of GBS at acidic and neutral pHs. We found that the transcription of 317 genes was increased at pH 5.5 relative to that at pH 7.0, while 61 genes were downregulated. The global response to acid stress included the differential expression of genes involved in transport, metabolism, stress response, and virulence. Known vaccine candidates, such as BibA and pilus components, were also regulated by pH. We observed that many of the genes involved in the GBS response to pH are known to be controlled by the CsrRS two-component system. Comparison of the regulon of wild-type strain 2603 V/R with that of a csrRS deletion mutant strain revealed that the pH-dependent regulation of 90% of the downregulated genes and 59.3% of the up-regulated genes in strain 2603 V/R was CsrRS dependent and that many virulence factors were overexpressed at high pH. Beta-hemolysin regulation was abrogated by selective inactivation of csrS , suggesting the implication of the CsrS protein in pH sensing. These results imply that the translocation of GBS from the acidic milieu of the vagina to the neutral pH of the neonatal lung signals the up-regulation of GBS virulence factors and conversion from a colonizing to an invasive phenotype. In addition, the fact that increased exposure of BibA on the bacterial surface at pH 7.0 induced opsonophagocytic killing of GBS in immune serum highlights the importance of pH regulation in the protective efficacy of specific antibodies to surface-exposed GBS proteins.

We have recently shown that group B Streptococcus (GBS) crosses the epithelial barrier by a paracellular route. Here, we show that, although deletion of the pilus backbone protein did not affect GBS adhesiveness, it reduced the pathogen's capacity to transcytose through differentiated human epithelial cells. In addition, contrary to our expectation, a strain with a mutant pilus ancillary protein and reduced adhesiveness translocated through the epithelial monolayer in a fashion identical to that of the isogenic wild-type strain. To monitor the localization of pili during GBS paracytosis, we performed 3-dimensional confocal experiments. By this approach, we observed that pili located in the intercellular space ahead of translocating bacteria. These results were also confirmed by a novel in vitro model of GBS infection in which bacteria bind to epithelial surfaces against the action of gravitation. These findings suggest a dual role for pilus components during the critical steps leading to GBS dissemination in the host.

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.

The B subunit of Escherichia coli heat-labile enterotoxin (EtxB) is a potent immunomodulatory molecule capable of treating and preventing autoimmune disease. These properties result from its ability to bind to glycolipid receptors, principally G(M1) ganglioside, and modulate immune cell function. EtxB receptor binding causes B cell activation, modulates monocyte cytokine secretion and triggers apoptosis of CD8+ T cells. These wide-ranging effects suggest that B subunit receptor interaction triggers signaling events affecting cellular differentiation. We have investigated the processes by which EtxB induces CD8+ T cell apoptosis. We show that receptor interaction by EtxB activates caspase-3 in CD8+ but not in CD4+ T cells. Inhibition of caspase-3 blocks the apoptotic process. EtxB induces the activation of NF-kappaB in both CD8+ and CD4+ T cells. The findings that (i) SN50, a peptide inhibitor of NF-kappaB nuclear translocation, prevents caspase-3 activation and subsequent apoptosis, and (ii) CD8+CD4- thymocytes from transgenic mice expressing a dominant-negative form of the IkappaBalpha protein were markedly less susceptible to EtxB-induced apoptosis than cells from wild-type mice, indicate that NF-kappaB is important in the induction of the apoptotic pathway. Further investigations revealed that while caspase-8 activity is detected concomitant to caspase-3, caspase-9 activation, following mitochondrial cytochrome c release, is detectable later on. These observations are consistent with death receptor-mediated signaling, however, experiments using lpr/lpr and p55 TNFR -/- mice rule out the involvement of Fas and the p55 TNF receptor, respectively. The data therefore indicate that EtxB-mediated apoptosis occurs via a novel pathway involving NF-kappaB.

When epithelial cells first encounter cholera toxin (Ctx) produced by Vibrio cholerae they secrete not only chloride ions responsible for causing diarrhoea, but also a number of cytokines that may contribute to the toxin's potent immunomodulatory properties. Much less is known about the ability of the heat-labile enterotoxin of Escherichia coli (Etx), a close homologue of Ctx, to elicit cytokine secretion by epithelial cells. This study shows that treatment of human intestinal epithelial T84 cells with Etx induces expression of IL-6, IL-10, IL-1R antagonist, as well as IL-1alpha and IL-1beta and low levels of IL-8. Such induction was totally dependent on the intrinsic ADP-ribosylating activity of the toxin A-subunit, and could be mimicked by cAMP-elevating agents, such as forskolin and dibutyryl cAMP. By comparison, neither an enzymically inactive mutant of Etx nor EtxB was able to induce cytokine secretion. The behaviour of Ctx and CtxB was very similar to that of Etx and EtxB, respectively. The spectrum of cytokines released by Etx and Ctx indicates that the toxins may create a local microenvironment that strongly biases the immune response towards an anti-inflammatory and a polarized Th2 response.

Haemolytic uraemic syndrome (HUS), which is caused by Shiga toxin (Stx)-producing Escherichia coli, is the commonest cause of acute renal failure in childhood. It is widely believed that HUS develops following the release of Stx, an AB5 toxin that inhibits protein synthesis and has a direct toxic effect on the kidney endothelium. There remains, however, a mismatch between the current understanding of the pathogenesis of HUS and the evolution of the clinical signs of the disease. Our hypothesis is that Stx-mediated immune cell activation in the gut is the missing link in the pathogenesis of this condition, initiating the characteristic renal pathology of HUS either alone or in synergy with Stx. Validation of this hypothesis could lead to a targeted anti-inflammatory approach aimed at modulating immune cell function in HUS.

We have previously demonstrated that the oxidizing component of ultraviolet-A (UVA) plays a central role in the activation of the nuclear oncogene and transcription factor, c-fos, in cultured human skin fibroblasts. We have now shown that expression of both c-jun and c-fos (AP-1) family of transcription factors is modulated by short and long wavelength solar ultraviolet (UV) radiation in human fibroblasts and human KB cells. UVA radiation activated c-jun and c-fos in both fibroblasts and KB cells, whereas ultraviolet-B (UVB) radiation activates such oncogenes only in KB cells. Moreover, decreasing the intracellular levels of reducing equivalents in human fibroblasts by glutathione (GSH) depletion lowered the UVA dose threshold for c-jun and c-fos activation several-fold and greatly amplified the UVA-mediated activation of such genes. A more modest effect was observed in GSH-depleted KB cells. In both GSH-depleted fibroblasts and KB cells, UVB radiation failed to amplify c-jun and c-fos activation indicating that the oxidative component of UVB plays a minor role in the modulation of such oncogene expression. These findings clearly indicate that both c-jun and c-fos are activated by the oxidizing component of UVA radiation in human fibroblasts and KB cells, while UVB-mediated modulation seems to be restricted to human epithelial cells and does not involve oxidizing intermediates.

The molecular events between the second messenger-mediated triggering of regulated exocytosis and the subsequent fusion of the secretory granules with the apical plasma membrane are unclear. The glycoprotein GP-2, the most abundant of the very few proteins of the pancreatic zymogen granule membrane has been cloned and sequenced in dog and rat, but no (enzymatic) function has so far been ascribed to it. Nucleoside phosphatase activities associated with the pig zymogen granule membrane were recently assumed to be related to GP-2. To identify the protein(s) carrying these activities we have used a novel combination of native and denaturing one- and two-dimensional polyacrylamide gel electrophoresis in the detergents CHAPS, Triton X-100 or SDS. Histochemical examination on the gels and incubation with lectins and phosphatidylinositol phospholipase-C have allowed characterization of the protein with the nucleoside di- and tri-phosphatase activities. SDS-PAGE of the single protein spot with nucleoside phosphatase activity excised from Triton X-100 2-dimensional gels showed the presence of 92 kDa and 67 kDa glycoproteins. The isolated protein had an isoelectric point of 5.2, formed high molecular weight complexes, was shown to be glycosylphosphatidylinositol-anchored and contained complex carbohydrate structures. It hydrolyses di- and tri-phosphate nucleotides in dependence of the glycosylphosphatidylinositol anchor and is sensitive to non-mitochondrial diphosphohydrolase inhibitors. In summary, this paper identifies GP-2 as a nucleoside phosphatase within the zymogen granule membrane, suggesting it may be involved in energy-requiring processes on the cytosolic side of the granules.

Very few studies have so far been dedicated to the systematic analysis of protein interactions occurring between surface and/or secreted proteins in bacteria. Such interactions are expected to play pivotal biological roles that deserve investigation. Taking advantage of the availability of a detailed map of surface and secreted proteins in Streptococcus pyogenes (group A Streptococcus (GAS)), we used protein array technology to define the "surface interactome" in this important human pathogen. Eighty-three proteins were spotted on glass slides in high density format, and each of the spotted proteins was probed for its capacity to interact with any of the immobilized proteins. A total of 146 interactions were identified, 25 of which classified as "reciprocal," namely, interactions that occur irrespective of which of the two partners was immobilized on the chip or in solution. Several of these interactions were validated by surface plasmon resonance and supported by confocal microscopy analysis of whole bacterial cells. By this approach, a number of interesting interactions have been discovered, including those occurring between OppA, DppA, PrsA, and TlpA, proteins known to be involved in protein folding and transport. These proteins, all localizing at the septum, might be part, together with HtrA, of the recently described ExPortal complex of GAS. Furthermore, SpeI was found to strongly interact with the metal transporters AdcA and Lmb. Because SpeI strictly requires zinc to exert its function, this finding provides evidence on how this superantigen, a major player in GAS pathogenesis, can acquire the metal in the host environment, where it is largely sequestered by carrier proteins. We believe that the approach proposed herein can lead to a deeper knowledge of the mechanisms underlying bacterial invasion, colonization, and pathogenesis.

Extracellular fluids contain low-molecular-weight antioxidants that are actively involved in the defense against reactive oxygen species. The antioxidant activity of these compounds is largely due to their ability to trap oxygen radicals. Less known is the ability of extracellular antioxidants to scavenge carbon-centered free radicals (C-radicals). These radicals can be involved in the damage under hypoxic/anoxic conditions as well as in ischemia/reperfusion injury. We studied the reactivity of some plasma antioxidants toward a water-soluble C-radical generated by the azocompound 2,2'-azobis(2-amidinopropane) hydrochloride (AAP) under anaerobic conditions. The AAP C-radical in plasma was trapped by the spin trap 3,5-dibromo-4-nitrosobenzene-sulfonic acid (DBNBS) and produced a DBNBS radical. The scavenging properties of urate, cysteine, glutathione, natural amino acids, and serum albumin were assessed by the inhibition of the intensity of DBNBS radical. The antioxidant activity of ascorbate and that of vitamin E was measured directly by the formation of their free radicals. Urate, vitamin E and non-SH amino acids were ineffective and ascorbate was a poor scavenger of AAP C-radical. At variance, cysteine and glutathione (0.1-1.0 mM) were effective scavengers of AAP C-radicals and, importantly, protected plasma ascorbate from oxidation under both aerobic or anaerobic conditions. Our data show that ascorbate in aerobic plasma can reduce vitamin E radical and the oxidized ascorbate may be recycled by a thiol antioxidant cycle. Low-molecular-weight antioxidants accounted only partially for plasma scavenging activity of C-radicals. Plasma strongly reduced the intensity of DBNBS radical and, after dialysis, its activity was reduced by approximately 10%. Serum albumin showed an antioxidant activity comparable to dialyzed plasma. Also the cysteine residue of serum albumin was an efficient scavenger of C-radicals as shown by approximately 20% decrease in the protein scavenging activity after thiol alkylation. These results suggest that elevation in the concentration of total reduced thiols in plasma may improve its antioxidant activity under hypoxic/anoxic conditions. This may be particularly useful since other important antioxidant mechanisms such as urate, ascorbate, and vitamin E appear to be inefficient.

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 .

The B subunit of Escherichia coli heat-labile enterotoxin (EtxB) is a potent immunomodulatory molecule capable of treating and preventing autoimmune disease. These properties result from its ability to bind to glycolipid receptors, principally GM1 ganglioside, and modulate immune cell function. EtxB receptor binding causes B cell activation, modulates monocyte cytokine secretion and triggers apoptosis of CD8+ T cells. These wide-ranging effects suggest that B subunit receptor interaction triggers signaling events affecting cellular differentiation. We have investigated the processes by which EtxB induces CD8+ T cell apoptosis. We show that receptor interaction by EtxB activates caspase-3 in CD8+ but not in CD4+ T cells. Inhibition of caspase-3 blocks the apoptotic process. EtxB induces the activation of NF-κB in both CD8+ and CD4+ T cells. The findings that (i) SN50, a peptide inhibitor of NF-κB nuclear translocation, prevents caspase-3 activation and subsequent apoptosis, and (ii) CD8+CD4– thymocytes from transgenic mice expressing a dominant-negative form of the IκBα protein were markedly less susceptible to EtxB-induced apoptosis than cells from wild-type mice, indicate that NF-κB is important in the induction of the apoptotic pathway. Further investigations revealed that while caspase-8 activity is detected concomitant to caspase-3, caspase-9 activation, following mitochondrial cytochrome c release, is detectable later on. These observations are consistent with death receptor-mediated signaling, however, experiments using lpr/lpr and p55 TNFR –/– mice rule out the involvement of Fas and the p55 TNF receptor, respectively. The data therefore indicate that EtxB-mediated apoptosis occurs via a novel pathway involving NF-κB.

A study was made of the interaction of plasma ascorbate and ascorbate free radical (AFR) with exogenously added iron. The quantitative determination of AFR has the advantage that transient increases in ascorbate oxidation can be directly monitored by e.p.r. spectroscopy. An AFR signal was found in the plasma of all donors and was unaffected by superoxide dismutase, catalase and the strong iron chelator deferoxamine. These findings and the rapid decrease in AFR under a nitrogen atmosphere suggest that plasma AFR is probably a result of air auto-oxidation. Iron loading of plasma did not affect the intensity of the AFR signal until the iron concentration approached or exceeded the plasma latent iron-binding capacity. In iron-overloaded plasma, the intensity of the AFR signal increased to about 10 times the normal level before decreasing rapidly to undetectable levels after 15-20 min. Determination of plasma ascorbate showed that the disappearance of AFR was due to a complete loss of the vitamin. When 50 microM-ascorbate was loaded with iron in iso-osmotic phosphate buffer there was an increase in the AFR signal, independent of the iron concentration, which was stable at least for 15 min. Thus the rate of ascorbate loss in the iso-osmotic phosphate buffer was considerably lower than in iron-overloaded plasma. The addition of different iron chelators produced comparable effects on the intensity of the AFR signal in both iron-overloaded plasma and ascorbate solution. These results suggest that the characteristic behaviour of plasma AFR after iron loading is due to its specific iron-binding capacity and to plasma ferroxidase activity. The ferroxidase activity of plasma is important to promote the transfer of Fe2+ into transferrin without a transient ascorbate oxidation. Spin-trapping studies with 5,5-dimethyl-1-pyrroline N-oxide and N-t-butyl-alpha-phenylnitrone revealed that iron-overloaded plasma was unable to produce spin-trap adducts even in the presence of 50-300 microM-hydrogen peroxide or 100 microM-azide. Evidence of OH. radical formation was obtained only after the addition of EDTA. Therefore, iron-overloaded plasma itself does not produce a Fenton reaction and, if ascorbate does indeed have a free-radical-mediated pro-oxidant role, it is not detectable in plasma by spin-trapping experiments.

One of the major challenges in vaccine development is the identification of microbial components that give rise to a protective immune response. Over the last decade, genome and proteome-based methods have proven successful in discovering new vaccine candidates for many pathogens through the selection of secreted or surface-exposed protein antigens and their screening in proper biological assays. However, these approaches still require intensive research activities to single out, among the large number of secreted and surface exposed proteins those very few which are protective. The question of which structural properties render an antigen capable of eliciting the production of functional remains most challenging. In such scientific and methodological context, the EU-funded project BacAbs was set up in 2007 with the main objective of developing a knowledge-based protocol able to discern protective from non-protective protein antigens, based on their amino acid sequences and molecular properties. The successful BacAbs multidisciplinary approach is highlighted by the recent analysis of three antigens from two diverse pathogens. Here, we describe the work and results carried out so far by the BacAbs Consortium, and discuss its relevance with regards to accelerating antigen selection processes and state-of-the-art vaccine development.