Decorating the surface of Escherichia coli with bacterial lipoproteins: a comparative analysis of different display systems.

BACKGROUND: The display of recombinant proteins on cell surfaces has a plethora of applications including vaccine development, screening of peptide libraries, whole-cell biocatalysts and biosensor development for diagnostic, industrial or environmental purposes. In the last decades, a wide variety of surface display systems have been developed for the exposure of recombinant proteins on the surface of Escherichia coli, such as autotransporters and outer membrane proteins. RESULTS: In this study, we assess three approaches for the surface display of a panel of heterologous and homologous mature lipoproteins in E. coli: four from Neisseria meningitidis and four from the host strain that are known to be localised in the inner leaflet of the outer membrane. Constructs were made carrying the sequences coding for eight mature lipoproteins, each fused to the delivery portion of three different systems: the autotransporter adhesin involved in diffuse adherence-I (AIDA-I) from enteropathogenic E. coli, the Lpp'OmpA chimaera and a truncated form of the ice nucleation protein (INP), InaK-NC (N-terminal domain fused with C-terminal one) from Pseudomonas syringae. In contrast to what was observed for the INP constructs, when fused to the AIDA-I or Lpp'OmpA, most of the mature lipoproteins were displayed on the bacterial surface both at 37 and 25 °C as demonstrated by FACS analysis, confocal and transmission electron microscopy. CONCLUSIONS: To our knowledge this is the first study that compares surface display systems using a number of passenger proteins. We have shown that the experimental conditions, including the choice of the carrier protein and the growth temperature, play an important role in the translocation of mature lipoproteins onto the bacterial surface. Despite all the optimization steps performed with the InaK-NC anchor motif, surface exposure of the passenger proteins used in this study was not achieved. For our experimental conditions, Lpp'OmpA chimaera has proved to be an efficient surface display system for the homologous passenger proteins although cell lysis and phenotype heterogeneity were observed. Finally, AIDA-I was found to be the best surface display system for mature lipoproteins (especially heterologous ones) in the E. coli host strain with no inhibition of growth and only limited phenotype heterogeneity.

Efficacy, immunogenicity, and safety of a parenteral vaccine against Helicobacter pylori in healthy volunteers challenged with a Cag-positive strain: a randomised, placebo-controlled phase 1/2 study.

BACKGROUND: Intramuscular immunisation with a vaccine composed of three recombinant Helicobacter pylori antigens-vacuolating cytotoxin A (VacA), cytotoxin-associated antigen (CagA), and neutrophil-activating protein (NAP)-prevented infection in animal models and was well tolerated and highly immunogenic in healthy adults. We aimed to assess the efficacy of the vaccine in prevention of a H pylori infection after challenge with a CagA-positive strain (BCM 300) in healthy volunteers. METHODS: In this randomised phase 1/2, observer-blind, placebo-controlled, single-centre study, healthy non-pregnant adults aged 18-40 years who were confirmed negative for H pylori infection were randomly assigned (3:4) to three intramuscular doses of either placebo or vaccine at 0, 1, and 2 months. Randomisation was via a computer-generated list with study numbers ensuring the correct ratio within a block size of seven. Participants were consecutively assigned in a double-blind manner to existing study numbers of the study protocol. Investigators and participants were blinded to allocation throughout the study. One month after the third immunisation, participants underwent challenge with a CagA-positive H pylori strain, which, for safety reasons, was initially administered in a subset of participants. The primary efficacy outcome was the efficacy of the vaccine as measured by the proportion of participants infected with H pylori 12 weeks after the challenge. At the end of the study, participants infected with H pylori were treated for 14 days with combination therapy consisting of a proton pump inhibitor and two antibiotics twice daily. Safety and immunogenicity were monitored at pre-established visits. This trial is registered with ClinicalTrials.gov, number NCT00736476, and is completed. FINDINGS: 63 patients were randomly assigned, 27 to placebo and 36 to the vaccine. 34 participants (19 in the vaccinated group and 15 in the placebo group) underwent infectious challenge, all but one of whom experienced transient mild-to-moderate epigastric symptoms. 12 weeks after infectious challenge, six (32%) of 19 people in the vaccinated group and six (40%) of 15 people in the placebo group remained positive for H pylori. Eradication was successful in everyone who remained infected at 12 weeks. The geometric mean concentrations of antibodies specific to CagA (202 [95% CI 69-588] vs 4·73 [95% CI 1·41-16]; p=0·001), VacA (1469 [838-2577] vs 73 [39-138]; p=0·001), and NAP (208 [139-313] vs 8·01 [5·05-13]; p=0·001) were significantly higher in the vaccine group than in the placebo group 12 weeks after infectious challenge. INTERPRETATION: Compared with placebo, the vaccine did not confer additional protection against H pylori infection after challenge with a CagA-positive strain, despite increased systemic humoral responses to key H pylori antigens. The finding of spontaneous clearance of H pylori infection in more than half the participants in the placebo group is remarkable and suggests important immune protection in the healthy adult population. FUNDING: Novartis Vaccine and Diagnostics.

Deciphering the Pathological Role of Staphylococcal α-Toxin and Panton-Valentine Leukocidin Using a Novel Ex Vivo Human Skin Model.

Staphylococcus aureus alpha-toxin and Panton-Valentine leukocidin (PVL) have been reported to play critical roles in different animal models of skin infection. These models, however, do not completely recapitulate the human disease due to the host specificity of these toxins as well as the intrinsic anatomical and immunological differences between animals and humans. Human skin explants represent a valid alternative to animal models for studying skin infections. Herein, we developed a human skin explant wound model to study the pathogenic role of alpha-toxin and PVL; inflammatory responses elicited by these toxins; and the neutralizing ability of antibodies to mitigate skin damage. Different concentrations of alpha-toxin and/PVL were applied to superficial wounds on human skin explants. Treatment with alpha-toxin resulted in high tissue toxicity and loss of skin epithelial integrity. PVL induced a milder but significant toxicity with no loss of skin structural integrity. The combination of both toxins resulted in increased tissue toxicity as compared with the individual toxins alone. Treatment of the skin with these toxins also resulted in a decrease of CD45-positive cells in the epidermis. In addition, both toxins induced the release of pro-inflammatory cytokines and chemokines. Finally, antibodies raised against alpha-toxin were able to mitigate tissue toxicity in a concentration-dependent manner. Results from this study confirm the key role of α-toxin in staphylococcal infection of the human skin and suggest a possible cooperation of the two toxins in tissue pathology.

CagL from Helicobacter pylori has ADP-ribosylation activity and exerts partial protective efficacy in mice.

Mono ADP-ribosyltransferases are a class of functionally conserved enzymes present in prokaryotic and eukaryotic organisms. In prokaryotes, mono ADP-ribose transfer enzymes often represent a family of exotoxins that display activity in a variety of bacteria responsible for causing disease in plants and animals. A bioinformatic approach has allowed us to identify that CagL gene from some Helicobacter pylori strains shares a sequence pattern with ADP-ribosylating toxins of the CT-group. In this manuscript we show that recombinant CagL from Shi470 is catalytically active showing ADP-ribosyltransferase, NAD-glycohydrolase, and auto-ADP-ribosylation activities. This is the first time that a catalytically active member of the ADP-ribosyltransferase family is identified in Helicobacter pylori. This observation may lead to the discovery of novel functions exerted by CagL in the pathogenesis of Helicobacter pylori. Indeed, we have shown that vaccination with CagL has protective efficacy in mice indicating that CagL may be considered as potential component of a Helicobacter pylori vaccine.

Innate Response Activator (IRA) B Cells Reside in Human Tonsils and Internalize Bacteria In Vitro.

Innate response activator (IRA) B cells have been described in mice as a subset of B-1a B cells that produce granulocyte/macrophage colony-stimulating factor (GM-CSF) and have been found in the spleen upon activation. In humans, identification, tissue localization and functionality of these lymphocytes are poorly understood. We hypothesized that IRA B cells could reside in human palatine tonsils, which are a first line of defense from infection of the upper respiratory tract. In the present work, we used flow cytometry and confocal microscopy to identify and characterize human IRA (hIRA) B cells in tonsils. We show that CD19⁺CD20⁺GM-CSF⁺ B cells are present in the tonsils of all the subjects studied at a frequency ranging between ~0.2% and ~0.4% of the conventional CD19⁺CD20⁺GM-CSF⁻ B cells. These cells reside within the B cell follicles, are mostly IgM⁺IgD⁺, express CD5 and show phagocytic activity. Our results support a role for hIRA B cells in the effector immune response to infections in tonsils.

Expression of the Streptococcus pneumoniae pilus-1 undergoes on and off switching during colonization in mice.

Streptococcus pneumoniae pili contribute to adherence and virulence. The regulation of pilus-1 expression is bistable, thus piliated strains contain a variable proportion of pilus-1-non-expressing bacteria. We investigated whether such proportion changes during colonization. Pilus-1-expressing bacteria were quantified in nasopharyngeal washes and pharyngeal tissues from mice that received intranasally bacterial populations with high (H), medium (M) or low (L) pilus-1 expression rates. In nasopharyngeal washes, at early colonization stages, pilus-1 expression rates decreased in H population, while increased in L and M; at later stages, expression rates decreased or remained low. Similar trends were observed in pharyngeal tissues, where, however, at late stages the expression rates were medium-high. In conclusion, pilus-1 is preferentially expressed at early colonization stages, consistently with its role in adhesion, while at later stages the expression is partially switched off. Pilus-1 expression rates observed in clinical isolates in vitro may not reflect the actual rates during colonization/infection.

RrgB321, a fusion protein of the three variants of the pneumococcal pilus backbone RrgB, is protective in vivo and elicits opsonic antibodies.

Streptococcus pneumoniae pilus 1 is present in 30 to 50% of invasive disease-causing strains and is composed of three subunits: the adhesin RrgA, the major backbone subunit RrgB, and the minor ancillary protein RrgC. RrgB exists in three distinct genetic variants and, when used to immunize mice, induces an immune response specific for each variant. To generate an antigen able to protect against the infection caused by all pilus-positive S. pneumoniae strains, we engineered a fusion protein containing the three RrgB variants (RrgB321). RrgB321 elicited antibodies against proteins from organisms in the three clades and protected mice against challenge with piliated pneumococcal strains. RrgB321 antisera mediated complement-dependent opsonophagocytosis of piliated strains at levels comparable to those achieved with the PCV7 glycoconjugate vaccine. These results suggest that a vaccine composed of RrgB321 has the potential to cover 30% or more of all pneumococcal strains and support the inclusion of this fusion protein in a multicomponent vaccine against S. pneumoniae.

Helicobacter pylori induces activation of human peripheral γδ+ T lymphocytes.

Helicobacter pylori is a gram-negative bacterium that causes gastric and duodenal diseases in humans. Despite a robust antibody and cellular immune response, H. pylori infection persists chronically. To understand if and how H. pylori could modulate T cell activation, in the present study we investigated in vitro the interaction between H. pylori and human T lymphocytes freshly isolated from peripheral blood of H. pylori-negative donors. A direct interaction of live, but not killed bacteria with purified CD3+ T lymphocytes was observed by microscopy and confirmed by flow cytometry. Live H. pylori activated CD3+ T lymphocytes and predominantly γδ+ T cells bearing the TCR chain Vδ2. Upon interaction with H. pylori, these cells up-regulated the activation molecule CD69 and produced cytokines (such as TNFα, IFNγ) and chemokines (such as MIP-1ß, RANTES) in a non-antigen-specific manner. This activation required viable H. pylori and was not exhibited by other gram-negative bacteria. The cytotoxin-associated antigen-A (CagA), was at least partially responsible of this activation. Our results suggest that H. pylori can directly interact with T cells and modulate the response of γδ+ T cells, thereby favouring an inflammatory environment which can contribute to the chronic persistence of the bacteria and eventually to the gastric pathology.

Structural and functional characterization of the Streptococcus pneumoniae RrgB pilus backbone D1 domain.

Streptococcus pneumoniae expresses on its surface adhesive pili, involved in bacterial attachment to epithelial cells and virulence. The pneumococcal pilus is composed of three proteins, RrgA, RrgB, and RrgC, each stabilized by intramolecular isopeptide bonds and covalently polymerized by means of intermolecular isopeptide bonds to form an extended fiber. RrgB is the pilus scaffold subunit and is protective in vivo in mouse models of sepsis and pneumonia, thus representing a potential vaccine candidate. The crystal structure of a major RrgB C-terminal portion featured an organization into three independently folded protein domains (D2-D4), whereas the N-terminal D1 domain (D1) remained unsolved. We have tested the four single recombinant RrgB domains in active and passive immunization studies and show that D1 is the most effective, providing a level of protection comparable with that of the full-length protein. To elucidate the structural features of D1, we solved the solution structure of the recombinant domain by NMR spectroscopy. The spectra analysis revealed that D1 has many flexible regions, does not contain any intramolecular isopeptide bond, and shares with the other domains an Ig-like fold. In addition, we demonstrated, by site-directed mutagenesis and complementation in S. pneumoniae, that the D1 domain contains the Lys residue (Lys-183) involved in the formation of the intermolecular isopeptide bonds and pilus polymerization. Finally, we present a model of the RrgB protein architecture along with the mapping of two surface-exposed linear epitopes recognized by protective antisera.

Sortase A confers protection against Streptococcus pneumoniae in mice.

Streptococcus pneumoniae sortase A (SrtA) is a transpeptidase that is highly conserved among pneumococcal strains, whose involvement in adhesion/colonization has been reported. We found that intraperitoneal immunization with recombinant SrtA conferred to mice protection against S. pneumoniae intraperitoneal challenge and that the passive transfer of immune serum before intraperitoneal challenge was also protective. Moreover, by using the intranasal challenge model, we observed a significant reduction of bacteremia when mice were intraperitoneally immunized with SrtA, while a moderate decrease of lung infection was achieved by intranasal immunization, even though no influence on nasopharynx colonization was seen. Taken together, our results suggest that SrtA is a good candidate for inclusion in a multicomponent, protein-based, pneumococcal vaccine.

Red wine and green tea reduce H pylori- or VacA-induced gastritis in a mouse model.

AIM: To investigate whether red wine and green tea could exert anti-H pylori or anti-VacA activity in vivo in a mouse model of experimental infection. METHODS: Ethanol-free red wine and green tea concentrates were administered orally as a mixture of the two beverages to H pylori infected mice, or separately to VacA-treated mice. Gastric colonization and gastric inflammation were quantified by microbiological, histopathological, and immunohistochemical analyses. RESULTS: In H pylori-infected mice, the red wine and green tea mixture significantly prevented gastritis and limited the localization of bacteria and VacA to the surface of the gastric epithelium. Similarly, both beverages significantly prevented gastric epithelium damage in VacA-treated mice; green tea, but not red wine, also altered the VacA localization in the gastric epithelium. CONCLUSION: Red wine and green tea are able to prevent H pylori-induced gastric epithelium damage, possibly involving VacA inhibition. This observation supports the possible relevance of diet on the pathological outcome of H pylori infection.

The Helicobacter pylori VacA cytotoxin activates RBL-2H3 cells by inducing cytosolic calcium oscillations.

Helicobacter pylori causes an acute inflammatory response followed by chronic infection of the human gastric mucosa. Identification of the bacterial molecules endowed with a pro-inflammatory activity is essential to a molecular understanding of the pathogenesis of H. pylori associated diseases. The vacuolating cytotoxin A (VacA) induces mast cells to release pro-inflammatory cytokines. Here, we show that VacA activates the mast cell line RBL-2H3 by rapidly inducing an oscillation of the level of cytosolic calcium with exocytosis of secretory granules. Cytosolic calcium derives mainly from intracellular stores. VacA also stimulates a calcium-dependent production of pro-inflammatory cytokines, including tumour necrosis factor alpha (TNF-alpha). These observations indicate that VacA may act as a pro-inflammatory factor of H. pylori at very early stages of the innate immune response.

Therapeutic vaccination against Helicobacter pylori in the beagle dog experimental model: safety, immunogenicity, and efficacy.

Helicobacter pylori is a gram-negative bacterium that colonizes the human gastric mucosa causing gastritis and peptic ulcer and increasing the risk of gastric cancer. The efficacy of current antibiotic-based therapies can be limited by problems of patient compliance and increasing antibiotic resistance; the vaccine approach can overcome these limits. The present study describes the therapeutic vaccination of experimentally H. pylori-infected beagle dogs, an animal model that reproduces several aspects of the human infection with H. pylori. The vaccine consisted of three recombinant H. pylori antigens, CagA, VacA, and NAP, formulated at different doses (10, 25, or 50 microg each) with alum and administered intramuscularly either weekly or monthly. No adverse effects were observed after vaccination and a good immunoglobulin G response was generated against each of the three antigens. Bacterial colonization and gastritis were decreased after the completion of the vaccination cycle, especially in the case of the monthly immunization schedule. In conclusion, therapeutic vaccination in the beagle dog model was safe and immunogenic and was able to limit H. pylori colonization and the related gastric pathology.

Helicobacter pylori infection negatively influences pregnancy outcome in a mouse model.

BACKGROUND: Helicobacter pylori infects the human stomach, causing gastritis, peptic ulcer, and gastric cancer. H. pylori infection has also been related to extra-gastric disorders. We investigated whether H. pylori infection can influence pregnancy in a murine model. METHODS: Female CD1 mice were infected with the H. pylori SPM326 strain before mating, and then assessed throughout pregnancy for embryo/fetus characteristics and histopathological changes of the endometrium. RESULTS: Infected mice showed higher numbers of resorption and lower fetal weights than noninfected controls. These pathological phenomena were accompanied by macrophage activation, and increases both of CD4+ and CD8+ lymphocytes and of interferon-gamma and major histocompatibility complex class II expression at the endometrial level, as evaluated by immunohistochemistry. DISCUSSION: During pregnancy, preferential induction of Th2-type cytokines downregulates Th1-type responses, allowing fetal survival. Our results suggest that H. pylori infection can induce activation of resident uterine immune cells and/or recruitment of cells at the endometrial level. It can be hypothesized that the local Th1-type response induced by H. pylori infection could alter the systemic Th1/Th2-type cytokine balance at sites under particular physiopathological conditions of active tissue and/or vascular formation, such as pregnancy. CONCLUSIONS: This is the first evidence in an animal model of the possible influence of H. pylori infection on pregnancy. Further work is required on its mechanism and its relevance for humans.