Phage display-derived antibody fragments against conserved regions of VacA toxin of Helicobacter pylori

Applied Microbiology and Biotechnology - Infection with Helicobacter pylori may result in the emergence of gastric adenocarcinoma. Among various toxins assisting pathogenesis of H. pylori,...     

Intoxication strategy of Helicobacter pylori VacA toxin

VacA toxin from the cancer-inducing bacterium Helicobacter pylori is currently classified as a pore-forming toxin but is also considered a multifunctional toxin, apparently causing many pleiotropic cell effects. However, an increasing body of evidence suggests that VacA could be the prototype of a new class of monofunctional A-B toxins in which the A subunit exhibits pore-forming instead of enzymatic activity. Thus, VacA may use a peculiar mechanism of action, allowing it to intoxicate the human stomach. By combining the action of a cell-binding domain, a specific intracellular trafficking pathway and a novel mitochondrion-targeting sequence, the VacA pore-forming domain is selectively delivered to the inner mitochondrial membrane to efficiently kill target epithelial cells by apoptosis.     

VacA from Helicobacter pylori: a hexameric chloride channel.

VacA is a unique protein toxin secreted by the human pathogen Helicobacter pylori. At a neutral pH, the cytotoxin self-associates into predominantly dodecameric complexes. In this report, we show that at an acidic pH, VacA forms anion selective channels in planar phospholipid bilayers. Similar to several other chloride channels, the VacA channel exhibits a moderate selectivity for anions over cations (P(Cl):P(Na) = 4.2:1), inhibition by the blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and a permeability sequence, SCN- > I- > Br- > Cl- > F, consistent with a 'weak field strength' binding site for the permeant anion. Single channel recordings reveal rapid transitions (486 s(-1)) between the closed state and a single open state of 24 pS (+60 mV, 1.5 M NaCl). Evaluation of the rate of increase in macroscopic current as well as atomic force microscopy suggest that this VacA channel is a hexamer, formed by the assembly of membrane-bound monomers. Not only are these VacA channels likely to play an important role in the pathological activity of this toxin, but they may also serve as a model system to further investigate the mechanism of anion selectivity in general.     

Structure and interaction of VacA of Helicobacter pylori with a lipid membrane.

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.     

幽门螺杆菌VacA重组蛋白表达、纯化及鉴定

目的研究幽门螺杆菌空泡毒素（VacA）编码基因在大肠埃希菌中的表达及纯化重组蛋白的抗原性。方法将PET32a-vacA-E.coli BE21（DE3）工程菌株常规培养,碱裂解法小量提取重组质粒DNA,琼脂糖凝胶电泳进行酶切鉴定,基因测序法进行插入基因序列分析。重组蛋白采用IPTG诱导表达,镍亲和层析原理提纯,ELISA法检测其抗原性。结果经酶切鉴定表明,插入的基因片段全长约2240bp,测序分析及与Genebank比较,可以肯定插入片段为vacA基因,ELISA法检测重组蛋白具有良好的抗原性。结论VacA重组蛋白在大肠埃希菌中成功表达,重组蛋白具有良好的抗原性。     

Inhibition of the vacuolating and anion channel activities of the VacA toxin of Helicobacter pylori.

VacA, the vacuolating cytotoxin secreted by Helicobacter pylori, is believed to be a major causative factor in the development of gastroduodenal ulcers. This toxin causes vacuolation of cultured cells and it has recently been found to form anion-selective channels upon insertion into planar bilayers as well as in the plasma membrane of HeLa cells. Here, we identify a series of inhibitors of VacA channels and we compare their effectiveness as channel blockers and as inhibitors of VacA-induced vacuolation, confirming that the two phenomena are linked. This characterization opens the way to studies in other experimental systems and to the search for a specific inhibitor of VacA action.     

Presumptive mechanisms of peptic ulceration by Helicobacter pylori VacA involving mucoprotease and CagA.

Helicobacter pylori vacuolating toxin (VacA) appears to be unusually stable, not only against extreme pH conditions or high temperatures, but also against common organic solvents or detergents. Under acidic conditions, its activity was markedly increased in the manner of temperature-independent, suggesting a spontaneous activation. A similar finding was also observed under alkaline conditions, however, it should have an appropriate temperature. From these observations, the mechanisms of VacA activation were suggested to be so redundant that either the case of acidic or basic amino acid residues could be involved in the VacA activation. Separately, we also found that the VacA production by H. pylori was pH-dependent: Its production was increased at a low pH region with a broad range (1.0-5.0), and at a high pH region with a narrow range (8.0-9.0). Astonishingly, a highly immunogenic CagA did not appear to be expressed under the acidic conditions. Its expression, however, was shown to be enhanced when the surrounding pH of this bacterium was raised. In contrast, mucoproteolytic activity in the H. pylori membrane was found to be increased at acidic conditions. Considering these observations, together with the stomach and duodenal pH of humans, two presumptive mechanisms of H. pylori VacA-associated ulceration may be deduced; namely, an acid- and an alkali-dependent type, involving mucoprotease and CagA, respectively.     

Formation of anion-selective channels in the cell plasma membrane by the toxin VacA of Helicobacter pylori is required for its biological activity.

The vacuolating toxin VacA, a major determinant of Helicobacter pylori-associated gastric diseases, forms anion-selective channels in artificial planar lipid bilayers. Here we show that VacA increases the anion permeability of the HeLa cell plasma membrane and determines membrane depolarization. Electrophysiological and pharmacological approaches indicated that this effect is due to the formation of low-conductance VacA pores in the cell plasma membrane and not to the opening of Ca(2+)- or volume-activated chloride channels. VacA-dependent increase of current conduction both in artificial planar lipid bilayers and in the cellular system was effectively inhibited by the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), while2-[(2-cyclopentenyl-6,7dichloro-2, 3-dihydro-2-methyl-1-oxo-1H-inden-5-yl)oxy]acetic acid (IAA-94) was less effective. NPPB inhibited and partially reversed the vacuolation of HeLa cells and the increase of ion conductivity of polarized Madine Darby canine kidney cell monolayers induced by VacA, while IAA-94 had a weaker effect. We conclude that pore formation by VacA accounts for plasma membrane permeabilization and is required for both cell vacuolation and increase of trans-epithelial conductivity.     

Cryo-EM Analysis Reveals Structural Basis of Helicobacter pylori VacA Toxin Oligomerization

Helicobacter pylori colonizes the human stomach and contributes to the development of gastric cancer and peptic ulcer disease. H. pylori secretes a pore-forming toxin called vacuolating cytotoxin A (VacA), which contains two domains (p33 and p55) and assembles into oligomeric structures. Using single-particle cryo-electron microscopy, we have determined low-resolution structures of a VacA dodecamer and heptamer, as well as a 3.8-Å structure of the VacA hexamer. These analyses show that VacA p88 consists predominantly of a right-handed beta-helix that extends from the p55 domain into the p33 domain. We map the regions of p33 and p55 involved in hexamer assembly, model how interactions between protomers support heptamer formation, and identify surfaces of VacA that likely contact membrane. This work provides structural insights into the process of VacA oligomerization and identifies regions of VacA protomers that are predicted to contact the host cell surface during channel formation.     

RACK1 protein interacts with Helicobacter pylori VacA cytotoxin: the yeast two-hybrid approach.

The VacA toxin is the major virulence factor of Helicobacter pylori. The studies on VacA intracellular expression suggest that it interacts with cytosolic proteins and that this interaction contributes significantly to vacuolization. The aim of this study was to identify the host protein(s) that interacts with the VacA protein. We used the fragments of VacA protein fused with GAL4-BD as the baits in the yeast two-hybrid approach. The yeast transformed with plasmids encoding bait proteins were screened with human gastric mucosa cDNA library, encoded C-terminal fusion proteins with GAL4-AD. Three independent His-beta-Gal-positive clones were identified in VacA-b1 screen; they matched two different lengths of cDNA encoding RACK1 protein. The specific activity of beta-galactosidase found in the yeast expressing both VacA-b1 and RACK1 fusion proteins was 12-19 times higher compared to all negative controls used. VacA is capable of binding the RACK1 in vitro as was confirmed by the pull-down assay with GST fusion VacA protein and [(35)S]Met-labeled RACK1 protein fragments.     

Glycosylphosphatidylinositol-anchored proteins and actin cytoskeleton modulate chloride transport by channels formed by the Helicobacter pylori vacuolating cytotoxin VacA in HeLa cells

The vacuolating cytotoxin VacA is an important virulence factor of Helicobacter pylori. Removing glycosylphosphatidylinositol-anchored proteins (GPI-Ps) from the cell surface by phosphatidylinositol-phospholipase C or disrupting the cell actin cytoskeleton by cytochalasin D reduced VacA-induced vacuolation of cells. Using the fluorescent dye 6-methoxy-N-ethylquinolinium chloride, an indicator for cytosolic chloride, we have investigated the role of either GPI-Ps or actin cytoskeleton in the activity of the selective anionic channel formed by VacA at the plasma membrane level. Removal of GPI-Ps from HeLa cell surfaces did not impair VacA localization into lipid rafts but strongly reduced VacA channel-mediated cell influx and efflux of chloride. Disruption of the actin cytoskeleton of HeLa cells by cytochalasin D did not affect VacA localization in lipid rafts but blocked VacA cell internalization and inhibited cell vacuolation while increasing the overall chloride transport by the toxin channel at the cell surface. Specific enlargement of Rab7-positive compartments induced by VacA could be mimicked by the weak base chloroquine alone, and the vacuolating activities of either chloroquine alone or VacA were blocked with the same potency by the anion channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid shown to inhibit VacA channel activity. We suggest that formation of functional VacA channels at the cell surface required GPI-Ps and that endocytosis of these channels by an actin-dependent process increases the chloride content of late endosomes that accumulate weak bases, provoking their enlargement by osmotic swelling.     

Sphingomyelin is important for the cellular entry and intracellular localization of Helicobacter pylori VacA

Plasma membrane sphingomyelin (SM) binds the Helicobacter pylori vacuolating toxin (VacA) to the surface of epithelial cells. To evaluate the importance of SM for VacA cellular entry, we characterized toxin uptake and trafficking within cells enriched with synthetic variants of SM, whose intracellular trafficking properties are strictly dependent on the acyl chain lengths of their sphingolipid backbones. While toxin binding to the surface of cells was independent of acyl chain length, cells enriched with 12‐ or 18‐carbon acyl chain variants of SM (e.g. C12‐SM or C18‐SM) were more sensitive to VacA, as indicated by toxin‐induced cellular vacuolation, than those enriched with shorter 2‐ or 6‐carbon variants (e.g. C2‐SM or C6‐SM). In C18‐SM‐enriched cells, VacA was taken into cells by a previously described Cdc42‐dependent pinocytic mechanism, localized initially to GPI‐enriched vesicles, and ultimately trafficked to Rab7/Lamp1 compartments. In contrast, within C2‐SM‐enriched cells, VacA was taken up at a slower rate by a Cdc42‐independent mechanism and trafficked to Rab11 compartments. VacA‐associated predominantly with detergent‐resistant membranes (DRMs) in cells enriched with C18‐SM, but predominantly with non‐DRMs in C2‐SM‐enriched cells. These results suggest that SM is required for targeting VacA to membrane rafts important for subsequent Cdc42‐dependent pinocytic cellular entry.     

Inhibitory effects of polyphenols on gastric injury by Helicobacter pylori VacA toxin

Background. Helicobacter pylori induces gastric damage and may be involved in the pathogenesis of gastric cancer. H. pylori‐vacuolating cytotoxin, VacA, is one of the important virulence factors, and is responsible for H. pylori‐induced gastritis and ulceration. The aim of this study is to assess whether several naturally occurring polyphenols inhibit VacA activities in vitro and in vivo. Materials and Methods. Effects of polyphenols on VacA were quantified by the inhibition of: 1, vacuolation; 2, VacA binding to AZ‐521 or G401 cells or its receptors; 3, VacA internalization. Effects of hop bract extract (HBT) containing high molecular weight polymerized catechin on VacA in vivo were investigated by quantifying gastric damage after oral administration of toxins to mice. Results. HBT had the strongest inhibitory activity among the polyphenols investigated. HBT inhibited, in a concentration‐dependent manner: 1, VacA binding to its receptors, RPTPα and RPTPβ; 2, VacA uptake; 3, VacA‐induced vacuolation in susceptible cells. In addition, oral administration of HBT with VacA to mice reduced VacA‐induced gastric damage at 48 hours. In vitro, VacA formed a complex with HBT. Conclusions. HBT may suppress the development of inflammation and ulceration caused by H. pylori VacA, suggesting that HBT may be useful as a new type of therapeutic agent for the prevention of gastric ulcer and inflammation caused by VacA.     

Association of Helicobacter pylori vacuolating toxin (VacA) with lipid rafts

A variety of extracellular ligands and pathogens interact with raft domains in the plasma membrane of eukaryotic cells. In this study, we examined the role of lipid rafts and raft-associated glycosylphosphatidylinositol (GPI)-anchored proteins in the process by which Helicobacter pylori vacuolating toxin (VacA) intoxicates cells. We first investigated whether GPI-anchored proteins are required for VacA toxicity by analyzing wild-type Chinese hamster ovary (CHO) cells and CHO-LA1 mutant cells that are defective in production of GPI-anchored proteins. Whereas wild-type and mutant cells differed markedly in susceptibility to aerolysin (a bacterial toxin that binds to GPI-anchored proteins), they were equally susceptible to VacA. We next determined whether VacA physically associates with lipid rafts. CHO or HeLa cells were incubated with VacA, and Triton-insoluble membranes then were separated by sucrose density gradient centrifugation. Immunoblot analysis revealed that a substantial proportion of cell-associated toxin was associated with detergent-resistant membranes (DRMs). DRM association required acid activation of the purified toxin prior to contact with cells, and acid activation also was required for VacA cytotoxicity. Treatment of cells with methyl-beta-cyclodextrin (a cholesterol-depleting agent) did not inhibit VacA-induced depolarization of the plasma membrane, but interfered with the internalization or intracellular localization of VacA and inhibited the capacity of the toxin to induce cell vacuolation. Treatment of cells with nystatin also inhibited VacA-induced cell vacuolation. These data indicate that VacA associates with lipid raft microdomains in the absence of GPI-anchored proteins and suggest that association of the toxin with lipid rafts is important for VacA cytotoxicity.     

Aneurysm and Helicobacter pylori relationship: the seropositivity of CagA, VacA and other antigens of Helicobacter pylori in abdominal and ascending aortic aneurysms

Helicobacter pylori is thought to be related to atherosclerosis and aneurysm development. We aimed to detect virulance factors of H. pylori and examine the potential etiopathogenetic relationship between aortic aneurysm and H. pylori, 58 abdominal aortic aneurysm (AAA) and 38 ascending aortic aneurysm (AsAA) cases and 57 Healty control group (HCG) were included. We investigated H. pylori IgG by ELISA and virulance factors by Western-Blot (WB) method. No difference was found between AAA (67.24%), AsAA (73.68%) and HCG (57.89%) for H. pylori IgG (p > 0.05). A significant difference was found between AsAA (78.95%) and HCG (57.89%) for H.pylori IgG (p < 0.05) by ELISA and a significant difference was found only between AsAA (100%) and HCG (37.5%) for H. pylori IgG in the 45-55 age group by WB. A statistically significant difference was found between AAA and AsAA for VacA and CagA + VacA and CagA + VacA + UreA antigens and also a significant difference was found between AsAA and HCG for CagA + UreA antigens (p < 0.05). Finally, we suggest that H. pylori VacA has a more important role than CagA in the development of two aneurysms especially in ruptured AAA. New extended studies detecting H. pylori DNA are needed to detect the aetiopathogenesis between aneurysm types and H. pylori.     

Helicobacter pylori toxin VacA induces vacuole formation by acting in the cell cytosol

Cells exposed to Helicobacter pylori toxin VacA develop large vacuoles that originate from massive swelling of membranous compartments of late stages of the endocytic pathway. To determine if the toxin is active from the cell cytosol, cells were either microinjected with toxin or transfected with plasmids encoding VacA. Both procedures cause formation of intracellular vacuoles. Cytosolic localization of the toxin was assessed by indirect immunofluorescence with specific antibodies and by expression of an active green fluorescence protein (GFP)–VacA chimera. Vacuoles induced by internally produced VacA are morphologically and functionally identical to those induced by externally added toxin. It is concluded that VacA is a toxin acting intracellularly by altering a cytosol-exposed target, possibly involved in the control of membrane trafficking.     

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.     

抗重组VacA IgY体内抗幽门螺杆菌感染的实验研究

目的在构建H.pylori的基因工程菌pQE30-v-DH5a的基础上,诱导表达VacA重组蛋白,以此为抗原,制备抗VacA的蛋黄抗体（VacA IgY）。通过小鼠口服试验,证实VacA IgY治疗H.pylori感染的作用,为进一步制备抗H．pylori感染的IgY制剂提供实验依据。方法用重组H.pylori VacA蛋白免疫母鸡,水稀释结合氯仿有机沉淀法提取IgY,ELISA法测定其针对VacA的效价。建立H.pylori感染的Balb／c小鼠动物模型,治疗组在小鼠灌喂菌液后灌喂不同剂量的VacA IgY。以H.pylori培养和病理切片观察胃黏膜H.pylori定植和炎症反应程度。结果制备了高效价的IgY（1：12800）。动物实验阳性对照组H.pylori的总感染率为70．4％,12周后的感染率为88．9％。治疗组的感染率与同期阳性对照组相似,胃黏膜的炎症反应程度比阳性对照组弱,随IgY剂量的增加,炎症减弱明显,IgY剂量为4mg／ml时,能达到较理想的治疗效果。结论成功制备了高效价的特异性VacA IgV,小鼠体内实验证实了口服VacA IgY具有治疗H.pylori感染的作用,可用于制备口服制剂。