Helicobacter pylori CagA-mediated IL-8 induction in gastric epithelial cells is cholesterol-dependent and requires the C-terminal tyrosine phosphorylation-containing domain

Abstract

Upon infection of the gastric epithelial cells, the Helicobacter pylori cytotoxin-associated gene A (CagA) virulence protein is injected into the epithelial cells via the type IV secretion system (TFSS), which is dependent on cholesterol. Translocated CagA is targeted by the membrane-recruited c-Src family kinases in which a tyrosine residue in the Glu-Pro-Ile-Tyr-Ala (EPIYA)-repeat region, which can be phosphorylated, induces cellular responses, including interleukin-8 (IL-8) secretion and hummingbird phenotype formation. In this study, we explored the role of EPIYA-containing C-terminal domain (CTD) in CagA tethering to the membrane lipid rafts and in IL-8 activity. We found that disruption of the lipid rafts reduced the level of CagA translocation/phosphorylation as well as CagA-mediated IL-8 secretion. By CagA truncated mutagenesis, we identified that the CTD, rather than the N-terminal domain, was responsible for CagA tethering to the plasma membrane and association with detergent-resistant membranes, leading to CagA-induced IL-8 promoter activity. Our results suggest that CagA CTD-containing EPIYAs directly interact with cholesterol-rich microdomains that induce efficient IL-8 secretion in the epithelial cells.

Statin Decreases Helicobacter pylori Burden in Macrophages by Promoting Autophagy

“…In previous studies, we demonstrated that reduced cellular cholesterol resulted in reduced VacA activity, as well as attenuated CagA-induced inflammation and decreased bacterial survival, in H. pylori -infected gastric epithelial cells (Lai et al, 2008, 2011; Wang et al, 2012). Specifically, reductions in cholesterol disrupt the integrity of lipid rafts, resulting in decreased type IV secretion system-mediated translocation of CagA into host cells (Hutton et al, 2010), thereby reducing downstream signaling and attenuating the inflammatory response (Lai et al, 2011). In the current study, we demonstrate that statin treatment yielded enhanced autophagy and reduced bacterial burdens in macrophages, followed by decreased levels of H. pylori -induced IL-1β production, suggesting that statin may attenuate H. pylori -induced pathogenesis via multiple mechanisms.…”

“…In particular, cholesterol plays a crucial role in H. pylori cell invasion and virulence (Lai et al, 2013). Depletion of cellular cholesterol not only attenuates H. pylori -induced pathogenesis (Hutton et al, 2010; Lai et al, 2011) but also promotes autophagy (Cheng et al, 2006), which has been shown to contribute to immune defense against invading pathogens (Levine et al, 2011; Deretic et al, 2013; Lai et al, 2015). Statins, inhibitors of HMG-CoA reductase that are widely prescribed for lowering serum cholesterol, have also been employed to reduce the risk of certain bacterial infections (Chow et al, 2010; Nseir et al, 2010; Boyd et al, 2012; Motzkus-Feagans et al, 2012).…”

Ceramide and Toll-Like Receptor 4 Are Mobilized into Membrane Rafts in Response to Helicobacter pylori Infection in Gastric Epithelial Cells

“…With H. pylori, previous studies have shown that cholesterol-rich microdomains are required for VacA binding, delivery, and intoxication of cells (20,28,41,48). Our previous studies demonstrated that translocation of CagA and the internalization of H. pylori in gastric epithelial cells are reduced significantly upon treatment of cells with M␤CD, thereby decreasing the pathogenesis induced by this bacterium (30,32). Moreover, we recently reported that the glucosylation of cholesterol in H. pylori is crucial for efficient cag type 4 secretion system (TFSS)-associated activity, which may due to the reorganization of membrane architecture (58).…”

Zinc Oxide Nanoparticles Impair Bacterial Clearance by Macrophages

“…The threshold was set above the non-template control background and within the linear phase of target gene amplification in order to calculate the cycle number at which the transcript was detected (denoted as C T ). NF-kB reporter luciferase assay RAW264.7 cells were plated in a 12-well plate and then transfected with a NF-kB-luc reporter plasmid (1 µg) using Lipofectamine 2000 (Invitrogen), as described previously [36]. The transfected cells were lysed, and luciferase assays were performed with the Dual-Luciferase Reporter Assay System and normalized by cotransfection with a b-galactosidase expression vector (Promega, MA, USA).…”