Infection of Helicobacter pylori cagA-positive strains is associated with gastritis, ulcerations and gastric cancer. CagA is translocated into infected epithelial cells by type IV secretion system and can be tyrosine phosphorylated, inducing signal transduction and motogenic responses in epithelial cells. Cellular cholesterol, a vital component of membrane, contributes to membrane dynamics and functions and is important in VacA intoxication and phagocyte evasion during H. pylori infection. In this investigation, we showed that cholesterol extraction by methyl-beta-cyclodextrin reduced the level of CagA translocation and phosphorylation. Confocal microscope visualization revealed that a significant portion of translocated CagA was colocalized with rafts marker GM1 and c-Src during infection. Moreover, CagA and VacA were co-fractionated with detergent-resistant membranes (DRMs), suggesting that distribution of CagA and VacA was associated with rafts in infected cells. Upon cholesterol depletion, their distribution shifted to non-DRMs. Accordingly, CagA-induced hummingbird phenotype and IL-8 induction was blocked by cholesterol depletion. We showed that raft-disrupting agents did not influence bacterial adherence but did significantly reduce internalization activity in AGS cells. These results together suggest that delivery of CagA into epithelial cells by the bacterial type IV secretion system is mediated via a cholesterol-dependent manner.
