Lipid interaction of the 37-kDa and 58-kDa fragments of the Helicobacter pylori cytotoxin.
Helicobacter pylori cytotoxin vacA (95 kDa) causes a vacuolar degeneration of epithelial cells. There is evidence that this protein toxin acts inside cells, and hence has to cross a cell membrane. This cytotoxin is frequently obtained as two fragments of 58 kDa (p58) and 37 kDa (p37) and it is available only in minute amounts. Here, its membrane interaction was studied with the two fragments, produced in Escherichia coli. Light scattering and energy transfer experiments show that p37 and p58 cause aggregation and fusion of small unilamellar lipid vesicles; only a reversible aggregation is induced at neutral pH, whereas at acid pH fusion also takes place. p58, but not p37, causes potassium efflux from liposomes and this occurs only at acid pH. Hydrophobic photolabelling with photoactivatable phosphatidylcholines inserted into liposomes shows that both fragments are labelled at neutral pH. The amount of labelling of the two fragments is much higher at acid pH, consistent with a further penetration into the hydrophobic core of the lipid bilayer. Tryptophan fluorescence measurements indicate that the two fragments undergo a pH-driven conformational change. These data are consistent with cytotoxin entry in the cell cytosol via an intracellular acidic compartment.
Moll
G
G
Centre CNR Biomembrane, Università di Padova, Italy.
Papini
E
E
Colonna
R
R
Burroni
D
D
Telford
J
J
Rappuoli
R
R
Montecucco
C
C
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D003603:Cytotoxins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005453:Fluorescence; D006096:Gramicidin / Q000494:pharmacology; D016480:Helicobacter pylori / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000378:metabolism*; D008561:Membrane Fusion; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism*; D010777:Photochemistry; D011188:Potassium / Q000378:metabolism; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012542:Scattering, Radiation; D014364:Tryptophan
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003603:Cytotoxins; D008081:Liposomes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C074689:VacA protein, Helicobacter pylori; D006096:Gramicidin; D014364:Tryptophan; D011188:Potassium