Helicobacter pylori vacuolating cytotoxin A exploits human endosomes for intracellular activation

Helicobacter pylori infection is the main cause of gastric cancer. Vacuolating cytotoxin A (VacA) is a H. pylori pore-forming toxin and a key determinant of gastric cancer risk. VacA is secreted as an 88-kDa polypeptide (p88) that upon interaction with host cells induces cytotoxic effects, including cell vacuolation and mitochondrial dysfunction. These effects are currently believed to be due to VacA p88 accumulating inside host cells and forming oligomeric anion-specific channels in membranes of intracellular compartments. However, the molecular nature of intracellular VacA channels in host cells remains undefined. Here we show that VacA p88 does not accumulate inside human epithelial cells, but instead is rapidly processed in endosomes into smaller p31/p28 and p37 products in a manner that precedes VacA-induced vacuolation. VacA processing requires endosomal acidification and concerted cleavage by multiple endo-lysosomal proteases including cathepsins. In situ structural mapping reveals that upon processing, the toxin’s central hydrophilic linker and globular C-terminus are excised, whereas oligomerization determinants are retained. Congruently, the processed products are constituents of a high-molecular-weight complex inside the host cell ─ which we propose is the intracellular, mature and active VacA pore. These findings suggest that VacA exploits human endosomes for proteolytic processing and intracellular activation. Significance Statement Helicobacter pylori is a cancer-causing bacterium that infects the stomach of billions of people worldwide. Vacuolating cytotoxin A (VacA) is an important H. pylori virulence factor and its activity directly correlates with gastric carcinogenesis. Yet despite decades of intense research, the mechanisms underlying VacA activity in human cells remain incompletely understood. Here, we present evidence suggesting that VacA is activated inside human cells by multi-step proteolytic processing involving endo-lysosomal proteases including cathepsins. We also track and identify the functional processed VacA isoforms in host cells. These results revolutionize our understanding of the mechanism of VacA activation in human cells, whilst expanding our knowledge of the diversity of microbial virulence factors that exploit human endo-lysosomes for pathogenesis. ### Competing Interest Statement The authors have declared no competing interest.