The neutrophil-activating protein (HP-NAP) of Helicobacter pylori is a protective antigen and a major virulence factor.

Helicobacter pylori infection induces the appearance of inflammatory infiltrates, consisting mainly of neutrophils and monocytes, in the human gastric mucosa. A bacterial protein with neutrophil activating activity (HP-NAP) has been previously identified, but its role in infection and immune response is still largely unknown. Here, we show that vaccination of mice with HP-NAP induces protection against H. pylori challenge, and that the majority of infected patients produce antibodies specific for HP-NAP, suggesting an important role of this factor in immunity. We also show that HP-NAP is chemotactic for human leukocytes and that it activates their NADPH oxidase to produce reactive oxygen intermediates, as demonstrated by the translocation of its cytosolic subunits to the plasma membrane, and by the lack of activity on chronic granulomatous disease leukocytes. This stimulating effect is strongly potentiated by tumor necrosis factor alpha and interferon gamma and is mediated by a rapid increase of the cytosolic calcium concentration. The activation of leukocytes induced by HP-NAP is completely inhibited by pertussis toxin, wortmannin, and PP1. On the basis of these results, we conclude that HP-NAP is a virulence factor important for the H. pylori pathogenic effects at the site of infection and a candidate antigen for vaccine development.

The Helicobacter pylori neutrophil-activating protein is an iron-binding protein with dodecameric structure.

The neutrophil-activating protein (HP-NAP) of Helicobacter pylori is a major 17 kDa antigen of the immune response of infected individuals. Amino acid sequence comparison indicated a high similarity between HP-NAP and both bacterial DNA-protecting proteins (Dps) and ferritins. The structure prediction and spectroscopic analysis presented here indicate a close similarity between HP-NAP and Dps. Electron microscopy revealed that HP-NAP forms hexagonal rings of 9-10 nm diameter with a hollow central core as seen in Dps proteins, clearly different from the 12 nm icositetrameric (24 subunits) ferritins. However, HP-NAP is resistant to thermal and chemical denaturation similar to the ferritin family of proteins. In addition, HP-NAP binds up to 40 atoms of iron per monomer and does not bind DNA. We therefore conclude that HP-NAP is an unusual, small, ferritin that folds into a four-helix bundle that oligomerizes into dodecamers with a central hole capable of binding up to 500 iron atoms per oligomer.

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.

Intranuclear delivery of an antiviral peptide mediated by the B subunit of Escherichia coli heat-labile enterotoxin.

We report an intracellular peptide delivery system capable of targeting specific cellular compartments. In the model system we constructed a chimeric protein consisting of the nontoxic B subunit of Escherichia coli heat-labile enterotoxin (EtxB) fused to a 27-mer peptide derived from the DNA polymerase of herpes simplex virus 1. Viral DNA synthesis takes places in the nucleus and requires the interaction with an accessory factor, UL42, encoded by the virus. The peptide, designated Pol, is able to dissociate this interaction. The chimeric protein, EtxB-Pol, retained the functional properties of both EtxB and peptide components and was shown to inhibit viral DNA polymerase activity in vitro via disruption of the polymerase-UL42 complex. When added to virally infected cells, EtxB-Pol had no effect on adenovirus replication but specifically interfered with herpes simplex virus 1 replication. Further studies showed that the antiviral peptide localized in the nucleus, whereas the EtxB component remained associated with vesicular compartments. The results indicate that the chimeric protein entered through endosomal acidic compartments and that the Pol peptide was cleaved from the chimeric protein before being translocated into the nucleus. The system we describe is suitable for delivery of peptides that specifically disrupt protein-protein interactions and may be developed to target specific cellular compartments.

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.

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.

Effect of helicobacter pylori vacuolating toxin on maturation and extracellular release of procathepsin D and on epidermal growth factor degradation.

The effect of vacuolating toxin (VacA) from Helicobacter pylori on endosomal and lysosomal functions was studied by following procathepsin D maturation and epidermal growth factor (EGF) degradation in HeLa cells exposed to the toxin. VacA inhibited the conversion of procathepsin D (53 kDa) into both the intermediate (47 kDa) and the mature (31 kDa) form. Nonprocessed cathepsin D was partly retained inside cells and partly secreted in the extracellular medium via the constitutive secretion pathway. Intracellular degradation of EGF was also inhibited by VacA with a similar dose-response curve. VacA did not alter endocytosis, cell surface recycling, and retrograde transport from plasma membrane to trans-Golgi network and endoplasmic reticulum, as estimated by using transferrin, diphtheria toxin, and ricin as tracers. Subcellular fractionation of intoxicated cells showed that procathepsin D and nondegraded EGF accumulate in lysosomes. Measurements of intracellular acidification with fluorescein isothiocyanate-dextran revealed a partial neutralization of the lumen of endosomes and lysosomes, sufficient to account for both mistargeting of procathepsin D outside the cell and the decreased activity of lysosomal proteases.

The small GTP binding protein rab7 is essential for cellular vacuolation induced by Helicobacter pylori cytotoxin.

The VacA cytotoxin, produced by toxigenic strains of Helicobacter pylori, induces the formation of large vacuoles highly enriched in the small GTPase rab7. To probe the role of rab7 in vacuolization, HeLa cells were transfected with a series of rab mutants and exposed to VacA. Dominant-negative mutants of rab7 effectively prevented vacuolization, whereas homologous rab5 and rab9 mutants were only partially inhibitory or ineffective, respectively. Expression of wild-type or GTPase-deficient rab mutants synergized with VacA in inducing vacuolization. In vitro fusion of late endosomes was enhanced by active rab7 and inhibited by inactive rab7, consistent with vacuole formation by merging of late endosomes in a process that requires functional rab7. Taken together, the effects of overexpressed rab proteins described here indicate that continuous membrane flow along the endocytic pathway is necessary for vacuole growth.

The vacuolar ATPase proton pump is present on intracellular vacuoles induced by Helicobacter pylori.

Cytotoxic strains of Helicobacter pylori cause an intense vacuolar degeneration of cells, due to the enlargement of late endosomes in the presence of membrane permeant weak bases. Bafilomycins, specific inhibitors of the vacuolar-type (V-) ATPase proton pump, prevent vacuole formation. The presence of the V-ATPase on vacuolar membranes was demonstrated by immunofluorescence with a monoclonal antibody (MAb) specific for the human 116-kDa regulatory subunit. The V-ATPase co-localised with the late endosomal marker rab7 on vacuolar membranes. In contrast, the early recycling endosomal compartment was not altered by the VacA cytotoxin, although it was endowed with the V-ATPase. Endocytosis of a MAb against the 116-kDa regulatory subunit of V-ATPase blocked endosomal acidification in HeLa cells and prevented VacA action. These results indicate that selective swelling of late endosomes, due to accumulation of osmotically active weak bases driven by the V-ATPase, is essential for vacuole formation.

[Immunohistochemical study of the accumulation of antibody-forming cells in the lungs following intrapulmonary and subcutaneous administration of apneumotropic antigen]. / Immunogistokhimicheskoe izuchenie nakopleniia antiteloobrazuiushchikh kletok v legkikh pri vnutrilegochnom i podkozhnom vvedenii apnevmotropnogo antigena.

Accumulation of the antibody-synthesizing cells in the lungs of guinea pigs in intratracheal and subcutaneous immunization was studied on a model of a complete typhoid bacilli antigen by means of Coons' method. Antibody-forming cells located in the interalveolar septi and in the peribronchial and perivascular connective tissue appeared with both method of the apneumotropic antigen administration. More of these cells accumulated in intrapulmonary application of the antigen. Repeated immunization by any method also led to the increase of their amount. The problem on the role played by the lungs in the immunogenesis is discussed.

[Enzymatic activity in the lung cells to the intrapulmonary administration of antigen]. / Izuchenie aktivnosti fermentov v kletkakh legkikh pri vnutrilegochnom vvedenii antigena.

Histochemical study of the activity of the redox and hydrolytic enzymes in the immunocompetent cells of the lungs of guinea pigs following intratracheal immunization with complete typhoid antigen demonstrated that following a single immunization at the initial periods elevation of the activity of AP, LDH, NAD- and NADP-diaphorases was seen in the macrophages; later GDH, G-6-PDH and AP was activated in the plasma cells. Double immunization was followed by an earlier and more intensive increase of the enzymatic activity in the cells under study.

[Morphological changes using live vaccine from the suppressor S. typhimurium revertant in model experiments]. / Morfologicheskie ismeneniia pri ispol'zovanii zhivoi vaktsiny iz supressornogo revertanta S. typhimurium v model'nykh opytakh

The work of was aimed at the morphological study of the harmlessness and protective activity of the live salmonella vaccine prepared form the suppressor S. typhimurium revertant intended for the prophylaxis and treatment of carrier state after the sustained food poisoning. It was shown to model experiments on mice that oral immunization caused no development of pathological changes, and at the same time prevented the appearance of the disease in infection of the animals by the virulent strain--S. typhimurium No. 415 in a dose of 100 LD50. A marked immunomorphological reaction developed in the small intestine and the lymphoid organs.