The small GTP binding protein rab 7 is essential for cellular vacuolation induced by Helicobacter pylori cytotoxin C-terminal domain and released into the extracellular

C-terminal domain and released into the extracellular Emanuele Papini1, Barbara Satin, medium as a 95 kDa mature protein which oligomerizes Cecilia Bucci2, Marina de Bernard, into heptamers and hexamers (Cover et al., 1994; Phadnis John L.Telford3, Roberto Manetti3, et al., 1994; Schmitt and Haas, 1994; Telford et al., 1994b; Rino Rappuoli3, Marino Zerial4 and Lupetti et al., 1996). In addition, VacA can be nicked by Cesare Montecucco bacterial proteinases into two fragments with no change of biological activity (de Bernard et al., 1995; Lupetti Centro CNR Biomembrane and Dipartimento di Scienze Biomediche, Università di Padova, Via Trieste 75, 35121 Padova, 2Dipartimento di et al., 1996). The C-terminal 58 kDa fragment is able to Biologia e Patologia Molecolare e Cellulare ‘L.Califano’, Università di increase the permeability of liposomes to monovalent Napoli Federico II and Centro CNR di Endocrinologia ed Oncologia cations at low pH values (Moll et al., 1995), similarly to Sperimentale, Via S.Pansini 5, 80131 Napoli, 3IRIS, Via Fiorentina 1, several bacterial protein toxins with intracellular targets 53100 Siena, Italy and 4European Molecular Biology Laboratory, Postfach 10.2209, D-69012 Heidelberg, Germany (Montecucco et al., 1994). VacA is activated by a short exposure to low pH and becomes resistant to strong 1Corresponding 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 large The 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., 1993a,b, 1996). The effectively prevented vacuolization, whereas homoH.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