Designed Ankyrin Repeat Proteins provide insights into the structure and function of CagI and are potent inhibitors of CagA translocation by the Helicobacter pylori type IV secretion system

The bacterial human pathogen Helicobacter pylori produces a type IV secretion system ( cag T4SS) to inject the oncoprotein CagA into gastric cells. The cag T4SS external pilus mediates attachment of the apparatus to the target cell and the delivery of CagA. While the composition of the pilus is unclear, CagI is present at the surface of the bacterium and required for pilus formation. Here, we have investigated the properties of CagI by an integrative structural biology approach. Using Alpha Fold 2 and Small Angle X-ray scattering, it was found that CagI forms elongated dimers mediated by rod-shape N-terminal domains (CagI N ) prolonged by globular C-terminal domains (CagI C ). Three Designed Ankyrin Repeat Proteins (DARPins) K2, K5 and K8 selected against CagI interacted with CagI C with subnanomolar affinities. The crystal structures of the CagI:K2 and CagI:K5 complexes were solved and identified the interfaces between the molecules, thereby providing a structural explanation for the difference in affinity between the two binders. Purified CagI and CagI C were found to interact with adenocarcinoma gastric (AGS) cells, induced cell spreading and the interaction was inhibited by K2. The same DARPin inhibited CagA translocation by up to 65% in AGS cells while inhibition levels were 40% and 30% with K8 and K5, respectively. Our study suggests that CagI C plays a key role in cag T4SS-mediated CagA translocation and that DARPins targeting CagI represent potent inhibitors of the cag T4SS, a crucial risk factor for gastric cancer development.Bases structurale du système de secretion de type IV d'Helicobacter pyloriBases structurales et moléculaires de l'exploitation de l'integrin a5ß1 par le système de sécrétion de type IV d'Helicobacter pylor

Helicobacter pylori binds human Annexins via Lipopolysaccharide to interfere with Toll-like Receptor 4 signaling

Author summaryH. pylori is very well adapted to its natural habitat, the human gastric mucosa. For this purpose, the bacterium has evolved a number of highly specific virulence factors, such as the cag-type IV secretion system, vacuolating cytotoxin A (VacA) or secreted gamma-glutamyl transpeptidase. An important function of these bacterial factors is to manipulate the host immune response to enable a chronic H. pylori infection. The present work identifies a new player in this process. Here, we have discovered that H. pylori, as well as several other bacterial species, can bind human annexins (ANX), suggesting a more widespread phenomenon. We show that H. pylori specifically binds ANXA5 via lipid A. The interaction is strictly dependent on calcium and modulated by the phosphorylation status of lipid A. Notably, ANXA5 binding strongly inhibits LPS-mediated Toll-like receptor 4 (TLR4) signal transduction, suggesting that H. pylori exploits ANXs binding to avoid its recognition by this important receptor of the innate immune system. The study thus provides novel molecular and mechanistic insights into a further strategy of H. pylori to successfully evade recognition by the host. Helicobacter pylori colonizes half of the global population and causes gastritis, peptic ulcer disease or gastric cancer. In this study, we were interested in human annexin (ANX), which comprises a protein family with diverse and partly unknown physiological functions, but with a potential role in microbial infections and possible involvement in gastric cancer. We demonstrate here for the first time that H. pylori is able to specifically bind ANXs. Binding studies with purified H. pylori LPS and specific H. pylori LPS mutant strains indicated binding of ANXA5 to lipid A, which was dependent on the lipid A phosphorylation status. Remarkably, ANXA5 binding almost completely inhibited LPS-mediated Toll-like receptor 4- (TLR4) signaling in a TLR4-specific reporter cell line. Furthermore, the interaction is relevant for gastric colonization, as a mouse-adapted H. pylori increased its ANXA5 binding capacity after gastric passage and its ANXA5 incubation in vitro interfered with TLR4 signaling. Moreover, both ANXA2 and ANXA5 levels were upregulated in H. pylori-infected human gastric tissue, and H. pylori can be found in close association with ANXs in the human stomach. Furthermore, an inhibitory effect of ANXA5 binding for CagA translocation could be confirmed. Taken together, our results highlight an adaptive ability of H. pylori to interact with the host cell factor ANX potentially dampening innate immune recognition

Inhibition of Type IV Secretion Activity and Growth of by Cisplatin and Other Platinum Complexes.

Type IV secretion systems are protein secretion machineries that are frequently used by pathogenic bacteria to inject their virulence factors into target cells of their respective hosts. In the case of the human gastric pathogen Helicobacter pylori, the cytotoxin-associated gene (Cag) type IV secretion system is considered a major cause for severe disease, such as gastric cancer, and thus constitutes an attractive target for specific treatment options against H. pylori infections. Here, we have used a Cag type IV secretion reporter assay for screening a repurposing compound library for inhibitors targeting this system. We found that the antitumor agent cisplatin, a platinum coordination complex that kills target cells by formation of DNA crosslinks, is a potent inhibitor of the Cag type IV secretion system. Strikingly, we found that this inhibitory activity of cisplatin depends on a ligand exchange reaction which incorporates a solvent molecule (dimethylsulfoxide) into the complex, a modification which is known to be deleterious for DNA crosslinking, and for its anticancer activity. We extended our analysis to several analogous platinum complexes containing N-heterocyclic carbene, as well as DMSO or other ligands, and found varying inhibitory activities toward the Cag system which were not congruent with their DNA-binding properties, suggesting that protein interactions may cause the inhibitory effect. Inhibition experiments under varying conditions revealed effects on adherence and bacterial viability as well, and showed that the type IV secretion-inhibitory capacity of platinum complexes can be inactivated by sulfur-containing reagents and in complex bacterial growth media. Taken together, our results demonstrate DNA binding-independent inhibitory effects of cisplatin and other platinum complexes against different H. pylori processes including type IV secretion

Designed Ankyrin Repeat Proteins provide insights into the structure and function of CagI and are potent inhibitors of CagA translocation by the Helicobacter pylori type IV secretion system.

The bacterial human pathogen Helicobacter pylori produces a type IV secretion system (cagT4SS) to inject the oncoprotein CagA into gastric cells. The cagT4SS external pilus mediates attachment of the apparatus to the target cell and the delivery of CagA. While the composition of the pilus is unclear, CagI is present at the surface of the bacterium and required for pilus formation. Here, we have investigated the properties of CagI by an integrative structural biology approach. Using Alpha Fold 2 and Small Angle X-ray scattering, it was found that CagI forms elongated dimers mediated by rod-shape N-terminal domains (CagIN) prolonged by globular C-terminal domains (CagIC). Three Designed Ankyrin Repeat Proteins (DARPins) K2, K5 and K8 selected against CagI interacted with CagIC with subnanomolar affinities. The crystal structures of the CagI:K2 and CagI:K5 complexes were solved and identified the interfaces between the molecules, thereby providing a structural explanation for the difference in affinity between the two binders. Purified CagI and CagIC were found to interact with adenocarcinoma gastric (AGS) cells, induced cell spreading and the interaction was inhibited by K2. The same DARPin inhibited CagA translocation by up to 65% in AGS cells while inhibition levels were 40% and 30% with K8 and K5, respectively. Our study suggests that CagIC plays a key role in cagT4SS-mediated CagA translocation and that DARPins targeting CagI represent potent inhibitors of the cagT4SS, a crucial risk factor for gastric cancer development

Heavy metal sensitivities of gene deletion strains for ITT1 and RPS1A connect their activities to the expression of URE2, a key gene involved in metal detoxification in yeast.

Heavy metal and metalloid contaminations are among the most concerning types of pollutant in the environment. Consequently, it is important to investigate the molecular mechanisms of cellular responses and detoxification pathways for these compounds in living organisms. To date, a number of genes have been linked to the detoxification process. The expression of these genes can be controlled at both transcriptional and translational levels. In baker's yeast, Saccharomyces cerevisiae, resistance to a wide range of toxic metals is regulated by glutathione S-transferases. Yeast URE2 encodes for a protein that has glutathione peroxidase activity and is homologous to mammalian glutathione S-transferases. The URE2 expression is critical to cell survival under heavy metal stress. Here, we report on the finding of two genes, ITT1, an inhibitor of translation termination, and RPS1A, a small ribosomal protein, that when deleted yeast cells exhibit similar metal sensitivity phenotypes to gene deletion strain for URE2. Neither of these genes were previously linked to metal toxicity. Our gene expression analysis illustrates that these two genes affect URE2 mRNA expression at the level of translation

Designing anti-Zika virus peptides derived from predicted human-Zika virus protein-protein interactions

The production of anti-Zika virus (ZIKV) therapeutics has become increasingly important as the propagation of the devastating virus continues largely u