How cells keep going in the face of adversity: When cells experience stresses that affect their ability to process newly synthesized proteins, they turn down their rates of translation to help them survive the stress. They also turn on the translation of proteins that will help them cope with the misfolded proteins generated during stress. How do they turn down translation in general, but maintain or increase translation of specific proteins? Starck et al. developed an approach that allowed them to look at the translation of specific messenger RNAs that were not down-regulated by stress. They identified a motif that helped keep chaperone protein synthesis going. Science , this issue p. 10.1126/science.aad3867

Mucosal mast cells strategically located at the optimal site interact with invading bacteria. Presence of VacA, the virulent Helicobacter pylori cytotoxin, is correlated with the severity of H. pylori-induced gastritis. To examine the mechanisms of inflammation in H. pylori-induced gastritis, we administered VacA to the mice. Inoculation of VacA resulted in epithelium vacuolization and marked infiltrations of mast cells and mononuclear cells into the mucosal epithelium within 24 h. In an in vitro study using bone marrow-derived mast cells, VacA directly bound and showed a chemotactic activity to the mast cell. In addition, VacA induced bone marrow-derived mast cells to produce proinflammatory cytokines, TNF-α, macrophage-inflammatory protein-1α, IL-1β, IL-6, IL-10, and IL-13 in a dose-dependent manner without causing degranulation. The present study suggests that early activation of mast cells by VacA may be the host early response to clear the bacteria and also may contribute to the pathogenesis of H. pylori-induced gastritis.

In Helicobacter pylori infection, vacuolating cytotoxin (VacA)-induced mitochondrial damage leading to apoptosis is believed to be a major cause of cell death. It has also been proposed that VacA-induced autophagy serves as a host mechanism to limit toxin-induced cellular damage. Apoptosis and autophagy are two dynamic and opposing processes that must be balanced to regulate cell death and survival. Here we identify the low-density lipoprotein receptor-related protein-1 (LRP1) as the VacA receptor for toxin-induced autophagy in the gastric epithelial cell line AZ-521, and show that VacA internalization through binding to LRP1 regulates the autophagic process including generation of LC3-II from LC3-I, which is involved in formation of autophagosomes and autolysosomes. Knockdown of LRP1 and Atg5 inhibited generation of LC3-II as well as cleavage of PARP, a marker of apoptosis, in response to VacA, whereas caspase inhibitor, benzyloxycarbonyl-VAD-fluoromethylketone (Z-VAD-fmk), and necroptosis inhibitor, Necrostatin-1, did not inhibit VacA-induced autophagy, suggesting that VacA-induced autophagy via LRP1 binding precedes apoptosis. Other VacA receptors such as RPTPα, RPTPβ, and fibronectin did not affect VacA-induced autophagy or apoptosis. Therefore, we propose that the cell surface receptor, LRP1, mediates VacA-induced autophagy and apoptosis.

Helicobacter pylori, a Gram-negative gastric bacterium, secretes VacA, a cytotoxin that causes vacuolar degeneration of susceptible cells. Velocity sedimentation analysis showed that treatment of VacA at alkaline pH led to disassembly of VacA oligomers, an observation reported previously for acid-treated VacA. Exposure of VacA to acid or alkali increased its binding to AZ-521 cells, as shown by indirect immunofluorescence and flow cytometry. Moreover, immunoprecipitates with polyclonal antibodies against VacA from AZ-521 cells previously exposed to acid- or alkali-treated VacA had a 250-kDa glycoprotein containing galactose-β(1–3)-N-acetylgalactosamine and galactose-β(1–4)-N-acetylglucosamine. p250, purified by chromatography on peanut agglutinin affinity and Superose 6 columns, contained N-terminal and internal amino acid sequences of YRQQRKLVEEIGWSYT and LIIQDHILEATQDDY, respectively. These sequences are identical to those of a receptor protein-tyrosine phosphatase (RPTPβ/PTPζ); in agreement, p250 reacted with anti-human RPTPβ monoclonal antibody. Immunoprecipitation with anti-human RPTPβ antibody of solubilized membrane preparations previously incubated with VacA or heat-inactivated VacA demonstrated that RPTPβ bound native, but not denatured, VacA. Acidic and alkaline treatments were associated with activation of VacA and increased binding to the cell surface RPTPβ.

Helicobacter pylori VacA toxin contributes to the pathogenesis and severity of gastric injury. We found that incubation of AZ-521 cells with VacA resulted in phosphorylation of protein kinase B (Akt) and glycogen synthase kinase-3β (GSK3β) through a PI3K-dependent pathway. Following phosphorylation and inhibition of GSK3β,β-catenin was released from a GSK3β/β-catenin complex, with subsequent nuclear translocation. Methyl-β-cyclodextrin (MCD) and phosphatidylinositol-specific phospholipase C (PI-PLC), but not 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and bafilomycin A1, inhibited VacA-induced phosphorylation of Akt, indicating that it does not require VacA internalization and is independent of vacuolation. VacA treatment of AZ-521 cells transfected with TOPtkLuciferase reporter plasmid or control FOPtkLucifease reporter plasmid resulted in activation of TOPtkLuciferase, but not FOPtkLucifease. In addition, VacA transactivated the β-catenin-dependent cyclin D1 promoter in a luciferase reporter assay. Infection of AZ-521 cells by a vacA mutant strain of H. pylori failed to induce phosphorylation of Akt and GSK3β, or release of β-catenin from a GSK3β/β-catenin complex. Taken together, these results support the conclusion that VacA activates the PI3K/Akt signaling pathway, resulting in phosphorylation and inhibition of GSK3β, and subsequent translocation ofβ-catenin to the nucleus, consistent with effects of VacA on β-catenin-regulated transcriptional activity. These data introduce the possibility that Wnt-dependent signaling might play a role in the pathogenesis of H. pylori infection, including the development of gastric cancer.