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The bacterial human pathogenHelicobacter pyloriproduces a type IV secretion system (cagT4SS) to inject the oncoprotein CagA into gastric cells. ThecagT4SS 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-shaped N-terminal domains (CagIN) and prolonged by globular C-terminal domains (CagIC). Three Designed Ankyrin Repeat Proteins (DARPins) K2, K5 and K8 selected against CagI interacted with CagICwith 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 CagICwere 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 CagICplays a key role incagT4SS-mediated CagA translocation and that DARPins targeting CagI represent potent inhibitors of thecagT4SS, a crucial risk factor for gastric cancer development.Author summaryHelicobacter pyloriis a bacterial pathogen that colonises the human stomach in half of the world’s population. The most virulent strains use the cag- type IV secretion system (cagT4SS), a molecular nanomachine capable of injecting the oncoprotein CagA into gastric cells. How CagA is delivered is unknown, but thecagT4SS produces an external appendage referred to as pilus, which interacts with host cell receptors, mediating CagA translocation from the cytoplasm of the bacteria to the inner membrane of the host cell. In this study we have investigated the structural and functional properties of CagI, a protein long-thought to be associated with thecagT4SS pilus but with yet unknown function. We found that CagI displays a unique dimeric structure and that its C-terminal domain is involved in interaction with the host cell. Designed Ankyrin Repeat Proteins were selected against CagI and found to interact with its C-terminal moiety with high affinity. DARPin binding was able to prevent CagI interaction with the host cell and inhibited CagA translocation byH. pylori. Our study reveals the role of CagI incagT4SS interaction with gastric cells and provides a first example of a small protein binder inhibiting thecagT4SS activity.

Helicobacter pylori displays a worldwide infection rate of about 50 %. The Gram-negative bacterium is the main reason for gastric cancer and other severe diseases. Despite considerable knowledge about the metabolic inventory of H. pylori, carbon fluxes through the citrate cycle (TCA cycle) remained enigmatic. In this study, different 13 C-labeled substrates were supplied as carbon sources to H. pylori during microaerophilic growth in a complex medium. After growth, 13 C-excess and 13 C-distribution were determined in multiple metabolites using GC-MS analysis. [U-13 C6 ]Glucose was efficiently converted into glyceraldehyde but only less into TCA cycle-related metabolites. In contrast, [U-13 C5 ]glutamate, [U-13 C4 ]succinate, and [U-13 C4 ]aspartate were incorporated at high levels into intermediates of the TCA cycle. The comparative analysis of the 13 C-distributions indicated an adaptive TCA cycle fully operating in the closed oxidative direction with rapid equilibrium fluxes between oxaloacetate - succinate and α-ketoglutarate - citrate. 13 C-Profiles of the four-carbon intermediates in the TCA cycle, especially of malate, together with the observation of an isocitrate lyase activity by in vitro assays, suggested carbon fluxes via a glyoxylate bypass. In conjunction with the lack of enzymes for anaplerotic CO2 fixation, the glyoxylate bypass could be relevant to fill up the TCA cycle with carbon atoms derived from acetyl-CoA.

Type IV secretion of effector proteins is an important principle for interaction of human pathogens with their target cells. The corresponding secretion systems may transport a multitude of effector proteins that have to be deployed in the respective spatiotemporal context, or only a single translocated protein, as in the case of the CagA effector protein produced by the human gastric pathogen Helicobacter pylori. For a more detailed analysis of the kinetics and mode of action of CagA type IV secretion by H. pylori, we describe here, a novel, highly sensitive split luciferase-based translocation reporter which can be easily adapted to different end-point or real-time measurements. Using this reporter, we showed that H. pylori cells are able to rapidly inject a limited amount of their CagA supply into cultured gastric epithelial cells. We have further employed the reporter system to address the question whether CagA has to be unfolded prior to translocation by the type IV secretion system. We showed that protein domains co-translocated with CagA as protein fusions are more readily tolerated as substrates than in other secretion systems, but also provide evidence that unfolding of effector proteins is a prerequisite for their transport.

Pathogenic bacteria often utilize type IV secretion systems to interact with host cells and to modify their microenvironment in a favourable way. The human pathogen Helicobacter pylori produces such a system to inject only a single protein, CagA, into gastric cells, but this injection represents a major risk factor for gastric cancer development. Here, we discuss the unusual structure of the Cag secretion nanomachine and other features that make it unique among bacterial protein transporters.

The production of anti-Zika virus (ZIKV) therapeutics has become increasingly important as the propagation of the devastating virus continues largely unchecked. Notably, a causal relationship between ZIKV infection and neurodevelopmental abnormalities has been widely reported, yet a specific mechanism underlying impaired neurological development has not been identified. Here, we report on the design of several synthetic competitive inhibitory peptides against key pathogenic ZIKV proteins through the prediction of protein-protein interactions (PPIs). Often, PPIs between host and viral proteins are crucial for infection and pathogenesis, making them attractive targets for therapeutics. Using two complementary sequence-based PPI prediction tools, we first produced a comprehensive map of predicted human-ZIKV PPIs (involving 209 human protein candidates). We then designed several peptides intended to disrupt the corresponding host-pathogen interactions thereby acting as anti-ZIKV therapeutics. The data generated in this study constitute a foundational resource to aid in the multi-disciplinary effort to combat ZIKV infection, including the design of additional synthetic proteins.

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. YeastURE2encodes for a protein that has glutathione peroxidase activity and is homologous to mammalian glutathione S-transferases. TheURE2expression is critical to cell survival under heavy metal stress. Here, we report on the finding of two genes,ITT1, an inhibitor of translation termination, andRPS1A, a small ribosomal protein, that when deleted yeast cells exhibit similar metal sensitivity phenotypes to gene deletion strain forURE2. Neither of these genes were previously linked to metal toxicity. Our gene expression analysis illustrates that these two genes affectURE2mRNA expression at the level of translation.Summary statementWe identified two yeast genes,ITT1andRPS1A, that when deleted, results in yeast cells sensitivity to heavy metals and metalloids. Further investigation indicated that they influence the expression ofURE2gene, a key player in metal detoxification, by upregulating its translation. Our findings suggest thatITT1andRPS1Aplay an indirect role in responding to toxic metal stress.