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 α and interferon γ 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.

SummaryThe 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.

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 ⍀ ϫ

into heptamers and hexamers (Cover et al., 1994;Phadnis John L.Telford 3 , Roberto Manetti 3 , Schmitt and Haas, 1994; Telford et al., 1994b; Rino Rappuoli 3 , Marino Zerial 4 and Lupetti et al., 1996). In addition, VacA can be nicked by Cesare Montecuccobacterial proteinases into two fragments with no change of biological activity (de Bernard et al., 1995 . VacA is activated by a short exposure to low pH and becomes resistant to strong 1 Corresponding author acidification and pepsin degradation, conditions which mimic the intragastric environment (de Bernard et al., The VacA cytotoxin, produced by toxigenic strains of 1995). Helicobacter pylori, induces the formation of largeThe lumen of the large vacuoles induced by VacA in vacuoles highly enriched in the small GTPase rab7. To cultured cells (Cover and Blaser, 1992) as well as in vivo probe the role of rab7 in vacuolization, HeLa cells (Telford et al., 1994b) is acidified by the proton pumping were transfected with a series of rab mutants and activity of a membrane-bound vacuolar-type ATPase exposed to VacA. Dominant-negative mutants of rab7 (Cover et al., 1993; Papini et al., 1993aPapini et al., ,b, 1996. The effectively prevented vacuolization, whereas homo-H.pylori-induced vacuoles contain fluid phase markers logous rab5 and rab9 mutants were only partially and their membrane is highly enriched in rab7 (Papini inhibitory or ineffective, respectively. Expression of et al., 1994), a small GTP binding protein previously wild-type or GTPase-deficient rab mutants synergized shown to be associated with late endosomal compartments with VacA in inducing vacuolization. In vitro fusion of (Chavrier et al., 1990). The identification of the molecular late endosomes was enhanced by active rab7 and targets of bacterial toxins has contributed greatly to the inhibited by inactive rab7, consistent with vacuole present understanding of cell physiology. Very recently, formation by merging of late endosomes in a process clostridial neurotoxins were shown to block neuroexothat requires functional rab7. Taken together, the cytosis, a highly regulated vesicular trafficking process, effects of overexpressed rab proteins described here by cleaving specifically three proteins involved in synaptic indicate that continuous membrane flow along the vesicle docking and fusion (Montecucco and Schiavo, endocytic pathway is necessary for vacuole growth.1995). It is expected that the elucidation of the mechanism Keywords: cytotoxin/gastric ulcer/Helicobacter pylori/ late endosomes/rab7 of action of VacA will also contribute to the understanding of membrane traffic in eukaryotic cells. Available evidence favours the possibility that vacuoles derive from late endosomes and that VacA cytotoxin alters

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