Cholesterol Depletion ReducesHelicobacter pyloriCagA Translocation and CagA-Induced Responses in AGS Cells

Abstract

Infection with Helicobacter pylori cagA-positive strains is associated with gastritis, ulcerations, and gastric cancer. CagA is translocated into infected epithelial cells by a type IV secretion system and can be tyrosine phosphorylated, inducing signal transduction and motogenic responses in epithelial cells. Cellular cholesterol, a vital component of the 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-␤-cyclodextrin reduced the level of CagA translocation and phosphorylation. Confocal microscope visualization revealed that a significant portion of translocated CagA was colocalized with the raft marker GM1 and c-Src during infection. Moreover, GM1 was rapidly recruited into sites of bacterial attachment by live-cell imaging analysis. CagA and VacA were cofractionated with detergent-resistant membranes (DRMs), suggesting that the distribution of CagA and VacA is associated with rafts in infected cells. Upon cholesterol depletion, the distribution shifted to non-DRMs. Accordingly, the CagA-induced hummingbird phenotype and interleukin-8 induction were blocked by cholesterol depletion. Raft-disrupting agents did not influence bacterial adherence but did significantly reduce internalization activity in AGS cells. Together, these results suggest that delivery of CagA into epithelial cells by the bacterial type IV secretion system is mediated in a cholesterol-dependent manner.

Invasion and Multiplication of Helicobacter pylori in Gastric Epithelial Cells and Implications for Antibiotic Resistance

“…The internalized H. pylori bacteria are found in LAMP1-containing vacuoles. The CagA protein can be translocated to the plasma membrane and form rafts with the invading H. pylori (12).…”

Helicobacter pylori Exploits Cholesterol-Rich Microdomains for Induction of NF-κB-Dependent Responses and Peptidoglycan Delivery in Epithelial Cells

“…It has recently been suggested that virulence factors of H. pylori associate with cholesterol-rich microdomains of the plasma membrane, commonly termed lipid rafts (34). These domains not only are enriched in cholesterol but also contain sphingolipids and proteins (53) and have been reported to be sites utilized by bacteria to interact with host cells (1) or as portals of entry by which bacteria enter these cells (29,33).…”

“…Furthermore, disruption of cholesterol-rich microdomains using cholesterol-depleting agents such as methyl-␤-cyclodextrin (M␤CD) was shown to significantly reduce the internalization of the vacuolating cytotoxin (VacA) into target cells (49) and to inhibit the ability of the toxin to induce cell vacuolation (31,48). Moreover, Lai et al reported that cholesterol-rich microdomains are also crucial for efficient TFSSmediated CagA translocation by H. pylori, as CagA-induced cellular responses, including the cell-scattering phenotype and IL-8 production, were found to be reduced in M␤CD-treated cells (34).…”

Colonize, evade, flourish

“…Host membrane cholesterol most likely contributes by providing a sturdy rigid membrane for docking of the needle-like apparatus, which can then inject toxin CagA into the cytoplasm of host cells. In support of this, Lai et al 86 showed that cholesterol depletion in host cells prevents translocation and phosphorylation of the CagA toxin, as well as its cellular functions including induction of the motile "hummingbird" phenotype and IL-8 secretion. Interestingly, the same phenotypes are seen with the encoded by the pgl locus, H. pylori only seems to possess a homolog to PglK, which it encodes in the wzk gene.…”

“…80 Further, infection with wild-type H. pylori is known to cause clustering of lipid rafts at sites of host-pathogen contact, thereby concentrating local cholesterol levels. 86 Some of this cholesterol is extracted from the host cell membrane and converted into cholesteryl-α-glucosides. However, the pathogen seems to maintain a balance with the host membrane, as host cell cholesterol-rich domains are also required for proper assembly and function of the bacterial T4SS.…”