Cerebrospinal fluid T-regulatory cells recognize Borrelia burgdorferi NAPA in chronic Lyme borreliosis.

The NapA protein of B. burgdorferi is essential for the persistence of spirochetes in ticks. One of the most intriguing aspects of NapA is its potential to interfere with the host immune system. Here, we investigated the role of the acquired immune responses induced by NapA in the cerebrospinal fluids (CSF) of patients with chronic Lyme borreliosis. We evaluated the cytokine profile induced in microglia cells and CSF T cells following NapA stimulation. We report here that NapA induced a regulatory T (Treg) response in the CSF of patients with chronic Lyme borreliosis and it is able to expand this suppressive response by promoting the production of TGF-beta and IL-10 by microglia cells. Collectively, these data strongly support a central role of NapA in promoting both Treg response and immune suppression in the CSF of patients with chronic Lyme borreliosis and suggest that NapA and the Treg pathway may represent novel therapeutic targets for the prevention and treatment of the disease.

Autoimmune gastritis: histology phenotype and OLGA staging.

BACKGROUND: Among Western populations, the declining incidence of Helicobacter pylori infection coincides with a growing clinical impact of autoimmune gastritis. AIMS: To describe the histological phenotype of autoimmune gastritis, also to test the prognostic impact of OLGA staging in the autoimmune setting. METHODS: A single-institutional series (spanning the years 2003-2011) of 562 consecutive patients (M:F ratio: 1:3.7; mean age = 57.6 ± 14.4 years) with serologically confirmed autoimmune gastritis underwent histology review and OLGA staging. RESULTS: Helicobacter pylori infection was ascertained histologically in 44/562 cases (7.8%). Forty six biopsy sets (8.2%) featured OLGA stages III-IV; they included all four cases of incidental epithelial neoplasia (three intraepithelial and one invasive; three of these four cases had concomitant H. pylori infection). There were 230 (40.9%) and 139 (24.7%) cases, respectively, of linear and micro-nodular enterochromaffin-like cell hyperplasia; 19 (3.4%) type I carcinoids were detected. The series included 116 patients who underwent repeated endoscopy/biopsy sampling (mean time elapsing between the two procedures = 54 months; range 24-108). Paired histology showed a significant (P = 0.009) trend towards a stage progression [the stage increased in 25/116 cases (22%); it remained unchanged in 87/116 cases (75%)]. CONCLUSIONS: In autoimmune gastritis, the cancer risk is restricted to high-risk gastritis stages (III-IV), and is associated mainly with concomitant H. pylori infection. OLGA staging consistently depicts the time-dependent organic progression of the autoimmune disease and provides key information for secondary gastric cancer prevention strategies.

Stimulation of TH1 response by Helicobacter pylori neutrophil activating protein decreases the protective role of IgE and eosinophils in experimental trichinellosis.

Th2 responses seem to play an important role in defence against Trichinella spiralis (Ts). The neutrophil Activating protein of Helicobacter pylori (HP-NAP), that induces IL-12, and IL-23 expression and shifts to Th1 allergen-specific Th2 cells in vitro was used as an anti-Th2 agent in BALB/c mice infected with T. spiralis. The muscle larvae (ML) burden was lower (p < 0.02) in untreated infected animals than those infected treated with HP-NAP. In both groups there was an inverse relationship between ML burden of each animal and total IgE level (controls: r -0.617, p = 0.0013 and HP-NAP-treated: r -0.678, p = 0.0001) or eosinophil count, evaluated in the same mouse on day 42 (r -0.390, p = 0.0592 and r -0.803, p = 0.0001, respectively). Inflammatory response around the nurse cell-parasite complex was significantly higher in HP-NAP-treated infected animals than in those untreated infected, on the contrary the number of eosinophils, counted around each complex was significantly lower in the first animal group. This study provides evidence of a powerful anti-Th2 activity in vivo by HP-NAP and for the partial protective effect of Th2 responses in T. spiralis infection.

Endosome-mitochondria juxtaposition during apoptosis induced by H. pylori VacA.

The vacuolating cytotoxin (VacA) is an important virulence factor of Helicobacter pylori with pleiotropic effects on mammalian cells, including the ability to trigger mitochondria-dependent apoptosis. However, the mechanism by which VacA exerts its apoptotic function is unclear. Using a genetic approach, in this study we show that killing by VacA requires the proapoptotic Bcl-2 family members BAX and BAK at the mitochondrial level, but not adequate endoplasmic reticulum Ca²(+) levels, similarly controlled by BAX and BAK. A combination of subcellular fractionation and imaging shows that wild-type VacA, but not mutants in its channel-forming region, induces the accumulation of BAX on endosomes and endosome-mitochondria juxtaposition that precedes the retrieval of active BAX on mitochondria. It is noteworthy that in Bax- and Bak-deficient cells, VacA is unable to cause endosome-mitochondria juxtaposition and is not retrieved in mitochondria. Thus, VacA causes BAX/BAK-dependent juxtaposition of endosomes and mitochondria early in the process of cell death, revealing a new function for these proapoptotic proteins in the regulation of relative position of organelles.

Cell vacuolization induced by Helicobacter pylori VacA cytotoxin does not depend on late endosomal SNAREs.

Cellular vacuoles induced by the Helicobacter pylori vacuolating cytotoxin VacA originate from late endosomal compartments. Their biogenesis requires the activity of both rab7 GTPase and the ATPase proton pump. The toxin has been suggested to cause an increased luminal osmotic pressure via its anion-specific channel activity localized on late endosomal compartments after endocytosis. Here, we show that the extensive membrane fusion that takes place in the transition from the small late endosomal compartments to the large vacuoles does not depend on soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) proteins. The process of vacuolization leads to disappearance of the large array of internal membranes of late endosomes. We suggest that most of the vacuole-limiting membrane derives from internal membranes.

Virulence factors of Helicobacter pylori.

To date a number of virulence factors have been identified and characterised from the gastric pathogen Helicobacter pylori. The vacuolating toxin (VacA) is a major determinant of H. pylori-associated gastric disease. In non-polarised cells, VacA alters the endocytic pathway, resulting in the release of acid hydrolases and the reduction of both extracellular ligand degradation and antigen processing. The toxin forms trans-membrane anion-specific channels and reduces the transepithelial electrical resistance of polarized monolayers. Localization of the VacA channels in acidic intracellular compartments causes osmotic swelling which, together with membrane fusion, leads to vacuole formation. The neutrophil-activating protein of H. pylori (HP-NAP) induces the production of oxygen radicals in human neutrophils via a cascade of intracellular activation events which may contribute to the damage of the stomach mucosa. This protein has recently been shown to be an important antigen in the human immune response to H. pylori infection. In addition, mice vaccinated with recombinant HP-NAP were protected against H. pylori challenge. H. pylori strains that are associated with severe tissue damage and inflammation possess the cag pathogenicity island that contains several genes encoding factors involved in the induction of proinflammatory cytokines/chemokines and of a type IV secretion system involved in the delivery of a highly immunogenic protein, CagA, into eukaryotic cells. Recent advances in our understanding of the involvement of VacA, HP-NAP and the CagA/Type IV secretion system in the H. pylori-associated disease process are discussed in this review.

Bacterial toxins with intracellular protease activity.

The recent determination of their primary sequence has lead to the discovery of the metallo-proteolytic activity of the bacterial toxins responsible for tetanus, botulism and anthrax. The protease domain of these toxins enters into the cytosol where it displays a zinc-dependent endopeptidase activity of remarkable specificity. Tetanus neurotoxin and botulinum neurotoxins type B, D, F and G cleave VAMP, an integral protein of the neurotransmitter containing synaptic vesicles. Botulinum neurotoxins type A and E cleave SNAP-25, while the type C neurotoxin cleaves both SNAP-25 and syntaxin, two proteins located on the cytosolic face of the presynaptic membrane. Such specific proteolysis leads to an impaired function of the neuroexocytosis machinery with blockade of neurotransmitter release and consequent paralysis. The lethal factor of Bacillus anthracis is specific for the MAPkinase-kinases which are cleaved within their amino terminus. In this case, however, such specific biochemical lesion could not be correlated with the pathogenesis of anthrax. The recently determined sequence of the vacuolating cytotoxin of Helicobacter pylori contains within its amino terminal domain elements related to serine-proteases, but such an activity as well as its cytosolic target remains to be detected.

The VacA toxin of Helicobacter pylori identifies a new intermediate filament-interacting protein.

The VacA toxin produced by Helicobacter pylori acts inside cells and induces the formation of vacuoles arising from late endosomal/lysosomal compartments. Using VacA as bait in a yeast two-hybrid screening of a HeLa cell library, we have identified a novel protein of 54 kDa (VIP54), which interacts specifically with VacA, as indicated by co-immunoprecipitation and binding experiments. VIP54 is expressed in cultured cells and many tissues, with higher expression in the brain, muscle, kidney and liver. Confocal immunofluorescence microscopy with anti-VIP54 affinity- purified antibodies shows a fibrous pattern typical of intermediate filaments. Double label immunofluorescence performed on various cell lines with antibodies specific to different intermediate filament proteins revealed that VIP54 largely co-distributes with vimentin. In contrast to known intermediate filament proteins, VIP54 is predicted to contain approximately 50% of helical segments, but no extended coiled-coil regions. The possible involvement of this novel protein in interactions between intermediate filaments and late endosomal compartments is discussed.

3D imaging of the 58 kDa cell binding subunit of the Helicobacter pylori cytotoxin.

Pathogenic strains of Helicobacter pylori produce a potent exotoxin, VacA, which intoxicates gastric epithelial cells and leads to peptic ulcer. The toxin is released from the bacteria as a high molecular mass homo-oligomer of a 95 kDa polypeptide which undergoes specific proteolytic cleavage to 37 kDa and 58 kDa subunits. We have engineered a strain of H. pylori to delete the gene sequence coding for the 37 kDa subunit. The remaining 58 kDa subunit is expressed efficiently and exported as a soluble dimer that is non-toxic but binds target cells in a manner similar to the holotoxin. A 3D reconstruction of the molecule from electron micrographs of quick-freeze, deep-etched preparations reveals the contribution of each building block to the structure and permits the reconstruction of the oligomeric holotoxin starting from individual subunits. In this model P58 subunits are assembled in a ring structure with P37 subunits laying on the top. The data indicate that the 58 kDa subunit is capable of folding autonomously into a discrete structure recognizable within the holotoxin and containing the cell binding domain.

Molecular and cellular activities of Helicobacter pylori pathogenic factors.

Stomach infection with pathogenic strains of Helicobacter pylori causes in some patients severe gastroduodenal diseases. These bacteria produce various virulence factors and, here, we review the recent acquisition on the biochemical mode of action of three major factors. We discuss the role of urease both as buffer of the stomach pH and as source of ammonia. The vacuolating toxin alters the endocytic pathway of non-polarized cells, inducing the release of acid hydrolases, the depression of extracellular ligand degradation and of antigen processing and, in the presence of ammonia, swelling of late-prelysosomal compartments. In polarized epithelial monolayers, vacuolating toxin induces an increase of the paracellular permeability, independent of vacuolation. The neutrophil activating protein induces the production of oxygen radicals in human neutrophils and could contribute to the damage of the stomach mucosa. The activities of these factors are discussed in terms of the need of the bacterium of increasing the supply of nutrients from the stomach lumen and from the mucosa.

Helicobacter pylori vacuolating toxin forms anion-selective channels in planar lipid bilayers: possible implications for the mechanism of cellular vacuolation.

The Helicobacter pylori VacA toxin plays a major role in the gastric pathologies associated with this bacterium. When added to cultured cells, VacA induces vacuolation, an effect potentiated by preexposure of the toxin to low pH. Its mechanism of action is unknown. We report here that VacA forms anion-selective, voltage-dependent pores in artificial membranes. Channel formation was greatly potentiated by acidic conditions or by pretreatment of VacA at low pH. No requirement for particular lipid(s) was identified. Selectivity studies showed that anion selectivity was maintained over the pH range 4.8-12, with the following permeability sequence: Cl- approximately HCO3- > pyruvate > gluconate > K+ approximately Li+ approximately Ba2+ > NH4+. Membrane permeabilization was due to the incorporation of channels with a voltage-dependent conductance in the 10-30 pS range (2 M KCl), displaying a voltage-independent high open probability. Deletion of the NH2 terminus domain (p37) or chemical modification of VacA by diethylpyrocarbonate inhibited both channel activity and vacuolation of HeLa cells without affecting toxin internalization by the cells. Collectively, these observations strongly suggest that VacA channel formation is needed to induce cellular vacuolation, possibly by inducing an osmotic imbalance of intracellular acidic compartments.

Identification of the Helicobacter pylori VacA toxin domain active in the cell cytosol.

Cells exposed to Helicobacter pylori toxin VacA develop large vacuoles which originate from massive swelling of membranous compartments at late stages of the endocytic pathway. When expressed in the cytosol, VacA induces vacuolization as it does when added from outside. This and other evidence indicate that VacA is a toxin capable of entering the cell cytosol, where it displays its activity. In this study, we have used cytosolic expression to identify the portion of the toxin molecule responsible for the vacuolating activity. VacA mutants with deletions at the C and N termini were generated, and their activity was analyzed upon expression in HeLa cells. We found that the vacuolating activity of VacA resides in the amino-terminal region, the whole of which is required for its intracellular activity.

Cell vacuolization induced by Helicobacter pylori VacA toxin: cell line sensitivity and quantitative estimation.

A major virulence factor released by Helicobacter pylori is a protein toxin, termed VacA, which induces the formation of large intracellular vacuoles characterised by a lumenal acidic pH. Consequently they accumulate membrane permeable weak bases. The increase in neutral red uptake by intoxicated cells is the only known in vitro procedure to estimate quantitatively the activity of VacA. With the goal to standardize this assay, several parameters were evaluated: cell type, serum concentration, cell density and toxin concentration. Among the different cell types tested, HeLa cells were found to be the most sensitive to VacA. Results show that several factors contribute to VacA activity and that optimal vacuolation is achieved at non-confluent cell density, in the presence of low serum concentrations.

TPA and butyrate increase cell sensitivity to the vacuolating toxin of Helicobacter pylori.

The Helicobacter pylori toxin VacA induces large membrane-bound vacuolar compartments of late endosomal/lysosomal origin. Pre-treatment of cells with TPA and butyrate enhances the toxin induced vacuolisation up to 20 times, depending on the cell line, whereas other differentiating factors such as DMSO, EGF, valeric and retinoic acid have no effect. The higher toxin sensitivity induced by TPA does not result from an increased surface binding or endocytosis. The effect of TPA is apparent after several hours from addition and is inhibited by a PKC specific inhibitor. These data suggest that expression of cellular proteins, other than the toxin receptor(s), influences the vacuolating activity of VacA and may contribute to the sensitivity of different cell lines. The present findings define the most sensitive in vitro assay of the activity of VacA.

The m2 form of the Helicobacter pylori cytotoxin has cell type-specific vacuolating activity.

The Helicobacter pylori toxin VacA causes vacuolar degeneration in mammalian cell lines in vitro and plays a key role in peptic ulcer disease. Two alleles, m1 and m2, of the mid-region of the vacA gene have been described, and the m2 cytotoxin always has been described as inactive in the in vitro HeLa cell assay. However, the m2 allele is associated with peptic ulcer and is prevalent in populations in which peptic ulcer and gastric cancer have high incidence. In this paper, we show that, despite the absence of toxicity on HeLa cells, the m2 cytotoxin is able to induce vacuolization in primary gastric cells and in other cell lines such as RK-13. The absence of Hela cell activity is due to an inability to interact with the cell surface, suggesting a receptor-mediated interaction. This result is consistent with the observation that the m2 allele is found in a population that has a high prevalence of peptic ulcer disease and gastric cancer. VacA is the first bacterial toxin described for which the same active subunit can be delivered by different receptor binding domains.

Selective increase of the permeability of polarized epithelial cell monolayers by Helicobacter pylori vacuolating toxin.

The effects of the vacuolating toxin (VacA) released by pathogenic strains of Helicobacter pylori on several polarized epithelial monolayers were investigated. Trans-epithelial electric resistance (TER) of monolayers formed by canine kidney MDCK I, human gut T84, and murine mammary gland epH4, was lowered by acid-activated VacA. Independent of the cell type and of the starting TER value, VacA reduced it to a minimal value of 1,000-1,300 Omega x cm2. TER decrease was paralleled by a three- to fourfold increase of [14C]-mannitol (molecular weight 182.2) and a twofold increase of [14C]-sucrose (molecular weight 342.3) transmonolayer flux. On the contrary, transmembrane flux of the proinflammatory model tripeptide [14C]-N-formyl-Met-Leu-Phe (molecular weight 437.6), of [3H]-inuline (molecular weight 5,000) and of HRP (molecular weight 47,000) did not change. These data indicate that VacA increases paracellular epithelial permeability to molecules with molecular weight < 350-440. Accordingly, the epithelial permeability of Fe3+ and Ni2+ ions, essential for H. pylori survival in vivo, was also increased by VacA. High-resolution immunofluorescence and SDS-PAGE analysis failed to reveal alterations of junctional proteins ZO-1, occludin, cingulin, and E-cadherin. It is proposed that induction by VacA of a selective permeabilization of the epithelial paracellular route to low molecular weight molecules and ions may serve to supply nutrients, which favor H. pylori growth in vivo.

Characterisation of a monoclonal antibody and its use to purify the cytotoxin of Helicobacter pylori.

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.

Action site and cellular effects of cytotoxin VacA produced by Helicobacter pylori.

Cells treated with the VacA toxin from Helicobacter pylori develop large membrane-bound vacuoles that originate from the late endocytotic pathway. Using different experimental approaches, we showed that VacA can induce vacuoles by acting within the cell cytosol. Moreover, separation of VacA-induced vacuoles at an early stage of formation, using a novel isopycnic density ultracentrifugation method, allowed us to show that they resemble a hybrid compartment, containing elements of both late endosomes and lysosomes. Functional defects of the endocytotic pathway were also studied before any macroscopic vacuolation is evident. VacA-intoxicated cells degrade extracellular ligands with reduced efficiency and, at the same time, they secrete acidic hydrolases into the extracellular medium, normally sorted to lysosomes. All these findings indicate that VacA translocates into the cell cytosol where it causes a lesion of the late endosomal/lysosomal compartments, such that protein trafficking across this crucial cross-point is altered with consequences that may be relevant to the pathogenesis of gastroduodenal ulcers.

The acid activation of Helicobacter pylori toxin VacA: structural and membrane binding studies.

The cell vacuolating activity of the protein toxin VacA, released by Helicobacter pylori, is strongly increased in vitro by exposure to acidic pH followed by neutralization. This short acid exposure does not increase significantly the binding of VacA to cell or to lipid membranes. However, membrane photolabeling with photoactivatable radioactive phospholipids and ANS binding studies show that VacA transiently exposed to pH equal or lower than 5 changes conformation and exposes on its surface hydrophobic segments. Both the 32 and the 58 kDa subunits of the toxin insert in the lipid bilayer and interact with the fatty acid chains of phospholipids. Membrane binding and penetration are enhanced by incubating target cells or liposomes with the toxin at mild acidic pH values, similar to those present around H. pylori on the stomach mucosa. These findings are discussed with respect to the critical step in cell intoxication consisting in the translocation of the active toxin domain into the cell cytosol. We suggest that membrane translocation takes place at the plasma membrane level.