Helicobacter pyloriVacA Cytotoxin: A Probe for a Clathrin-independent and Cdc42-dependent Pinocytic Pathway Routed to Late Endosomes

Abstract

The vacuolating cytotoxin VacA is a major virulence factor of Helicobacter pylori, a bacterium responsible for gastroduodenal ulcers and cancer. VacA associates with lipid rafts, is endocytosed, and reaches the late endocytic compartment where it induces vacuolation. We have investigated the endocytic and intracellular trafficking pathways used by VacA, in HeLa and gastric AGS cells. We report here that VacA was first bound to plasma-membrane domains localized above F-actin structures that were controlled by the Rac1 GTPase. VacA was subsequently pinocytosed by a clathrin-independent mechanism into cell peripheral early endocytic compartments lacking caveolin 1, the Rab5 effector early endosomes antigen-1 (EEA1) and transferrin. These compartments took up fluid-phase (as evidenced by the accumulation of fluorescent dextran) and glycosylphosphatidylinositol-anchored proteins (GPI-APs). VacA pinocytosis was controlled by Cdc42 and did not require cellular tyrosine kinases, dynamin 2, ADP-ribosylating factor 6, or RhoA GTPase activities. VacA was subsequently routed to EEA1-sorting endosomes and then sorted to late endosomes. During all these different endocytic steps, VacA was continuously associated with detergent resistant membrane domains. From these results we propose that VacA might be a valuable probe to study raft-associated molecules, pinocytosed by a clathrin-independent mechanism, and routed to the degradative compartment.

Helicobacter pylori CagA inhibits endocytosis of cytotoxin VacA in host cells

“…. S5), consistent with a previous report (Gauthier et al, 2005) and our finding that CagA suppresses endocytosis of dextran. These observations indicate that CagA in AGS cells inhibits pinocytic endocytosis of VacA without affecting VacA attachment to the plasma membrane.…”

Pleiotropic Actions of Helicobacter pylori Vacuolating Cytotoxin, VacA

“…These results suggest that VacA-induced vacuolization is a result of a toxin-induced alteration of intracellular vesicle trafficking. Interestingly, it was demonstrated that CD2-associated protein (CD2AP), an adaptor protein implicated in intracellular trafficking, which regulates filamentous actin (F-actin) structures, was required to transfer VacA from early to late endosomes (Gauthier et al 2007) after VacA was internalized by a pinocytic mechanism that involves F-actin and Cdc42 but was independent of clathrin, dynamin, and ARF6 GTPase (Gauthier et al 2005). They also suggested that sorting of VacA in these compartments requires dynamic F-actin structures on early endosomes, which is characterized as being enriched with GPI-anchored proteins Gauthier et al 2005).…”

Dual effects of Helicobacter pylori vacuolating cytotoxin on human eosinophil apoptosis in early and late periods of stimulation

“…The purification of VacA protein was kindly supported by Professor Patrice Boquet at Laboratoire de Bacteriologie, l'Hopital de l' 0 Archet 2, France [42]. The purified protein was activated immediately before use on cells; 250 mM HCl was added to the purified VacA until a pH of 2.0 was reached [43]. NH 4 Cl (5 mM) was also added to the medium to enhance the VacA activity [22].…”