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.

Brucella effectors NyxA and NyxB target SENP3 to modulate the subcellular localisation of nucleolar proteins.

The cell nucleus is a primary target for intracellular bacterial pathogens to counteract immune responses and hijack host signalling pathways to cause disease. Here we identify two Brucella abortus effectors, NyxA and NyxB, that interfere with host protease SENP3, and this facilitates intracellular replication of the pathogen. The translocated Nyx effectors directly interact with SENP3 via a defined acidic patch (identified from the crystal structure of NyxB), preventing nucleolar localisation of SENP3 at late stages of infection. By sequestering SENP3, the effectors promote cytoplasmic accumulation of nucleolar AAA-ATPase NVL and ribosomal protein L5 (RPL5) in effector-enriched structures in the vicinity of replicating bacteria. The shuttling of ribosomal biogenesis-associated nucleolar proteins is inhibited by SENP3 and requires the autophagy-initiation protein Beclin1 and the SUMO-E3 ligase PIAS3. Our results highlight a nucleomodulatory function of two Brucella effectors and reveal that SENP3 is a crucial regulator of the subcellular localisation of nucleolar proteins during Brucella infection, promoting intracellular replication of the pathogen.

Biochemical characterization of the Helicobacter pylori Cag Type 4 Secretion System protein CagN and its interaction partner CagM.

Highly virulent Helicobacter pylori strains contain the cag pathogenicity island (cagPAI). It codes for about 30 proteins forming a type IV secretion system (T4SS) which translocates the pro-inflammatory protein CagA into epithelial host cells. While CagA and various other Cag proteins have been extensively studied, several cagPAI proteins are poorly characterized or of unknown function. CagN (HP0538) is of unknown function but highly conserved in the cagPAI suggesting an important role. cagM (HP0537) is the first gene of the cagMN operon and its product is part of the CagT4SS core complex. Both proteins do not have detectable homologs in other type IV secretion systems. We have characterized the biochemical and structural properties of CagN and CagM and their interaction. We demonstrate by circular dichroism, Multi-Angle Light Scattering (MALS) and small angle X-ray scattering (SAXS) that CagN is a folded, predominantly monomeric protein with an elongated shape in solution. CagM is folded and forms predominantly dimers that are also elongated in solution. We found by various in vivo and in vitro methods that CagN and CagM directly interact with each other. CagM self-interacts stably with a low nanomolar KD and can form stable multimers. Finally, in vivo experiments show that deletion of CagM reduces the amounts of CagN and other outer CagPAI proteins in H. pylori cells.

Molecular dissection of protein-protein interactions between integrin α5ß1 and the Helicobacter pylori Cag type IV secretion system.

The more severe strains of the bacterial human pathogen Helicobacter pylori produce a type IV secretion system (cagT4SS) to inject the oncoprotein cytotoxin-associated gene A (CagA) into gastric cells. This syringe-like molecular apparatus is prolonged by an external pilus that exploits integrins as receptors to mediate the injection of CagA. The molecular determinants of the interaction of the cagT4SS pilus with the integrin ectodomain are still poorly understood. In this study, we have used surface plasmon resonance (SPR) to generate a comprehensive analysis of the protein-protein interactions between purified CagA, CagL, CagI, CagY repeat domain II (CagYRRII ), CagY C-terminal domain (CagYB10 ) and integrin α5ß1 ectodomain (α5ß1E ) or headpiece domain (α5ß1HP ). We found that CagI, CagA, CagL and CagYB10 but not CagYRRII were able to interact with α5ß1E with affinities similar to the one observed for α5ß1E interaction with its physiological ligand fibronectin. We further showed that integrin activation and its associated conformational change increased CagA, CagL and CagYB10 affinities for the receptor. Furthermore, CagI did not interact with integrin unless the receptor was in open conformation. CagI, CagA but not CagL and CagYB10 interacted with the α5ß1HP . Our SPR study also revealed novel interactions between CagA and CagL, CagA and CagYB10 , and CagA and CagI. Altogether, our data map the network of interactions between host-cell α5ß1 integrin and the cagT4SS proteins and suggest that activation of the receptor promotes interactions with the secretion apparatus and possibly CagA injection.

Structural Insights into Helicobacter pylori Cag Protein Interactions with Host Cell Factors.

The most virulent strains of Helicobacter pylori carry a genomic island (cagPAI) containing a set of 27-31 genes. The encoded proteins assemble a syringe-like apparatus to inject the cytotoxin-associated gene A (CagA) protein into gastric cells. This molecular device belongs to the type IV secretion system (T4SS) family albeit with unique characteristics. The cagPAI-encoded T4SS and its effector protein CagA have an intricate relationship with the host cell, with multiple interactions that only start to be deciphered from a structural point of view. On the one hand, the major roles of the interactions between CagL and CagA (and perhaps CagI and CagY) and host cell factors are to facilitate H. pylori adhesion and to mediate the injection of the CagA oncoprotein. On the other hand, CagA interactions with host cell partners interfere with cellular pathways to subvert cell defences and to promote H. pylori infection. Although a clear mechanism for CagA translocation is still lacking, the structural definition of CagA and CagL domains involved in interactions with signalling proteins are progressively coming to light. In this chapter, we will focus on the structural aspects of Cag protein interactions with host cell molecules, critical molecular events precluding H. pylori-mediated gastric cancer development.

Structural and Molecular Biology of Type IV Secretion Systems.

Type IV secretion systems (T4SSs) are nanomachines that Gram-negative, Gram-positive bacteria, and some archaea use to transport macromolecules across their membranes into bacterial or eukaryotic host targets or into the extracellular milieu. They are the most versatile secretion systems, being able to deliver both proteins and nucleoprotein complexes into targeted cells. By mediating conjugation and/or competence, T4SSs play important roles in determining bacterial genome plasticity and diversity; they also play a pivotal role in the spread of antibiotic resistance within bacterial populations. T4SSs are also used by human pathogens such as Legionella pneumophila, Bordetella pertussis, Brucella sp., or Helicobacter pylori to sustain infection. Since they are essential virulence factors for these important pathogens, T4SSs might represent attractive targets for vaccines and therapeutics. The best-characterized conjugative T4SSs of Gram-negative bacteria are composed of twelve components that are conserved across many T4SSs. In this chapter, we will review our current structural knowledge on the T4SSs by describing the structures of the individual components and how they assemble into large macromolecular assemblies. With the combined efforts of X-ray crystallography, nuclear magnetic resonance (NMR), and more recently electron microscopy, structural biology of the T4SS has made spectacular progress during the past fifteen years and has unraveled the properties of unique proteins and complexes that assemble dynamically in a highly sophisticated manner.

The structure of Legionella pneumophila LegK4 type four secretion system (T4SS) effector reveals a novel dimeric eukaryotic-like kinase.

Bacterial pathogens subvert signalling pathways to promote invasion and/or replication into the host. LegK1-4 proteins are eukaryotic-like serine/threonine kinases that are translocated by the Dot/Icm type IV secretion system (T4SS) of several Legionella pneumophila strains. We present the crystal structures of an active fragment of the LegK4 protein in apo and substrate-bound states. The structure of LegK4(1-445) reveals a eukaryotic-like kinase domain flanked by a novel cap domain and a four-helix bundle. The protein self-assembles through interactions mediated by helices αF and αG that generate a dimeric interface not previously observed in a protein kinase. The helix αG is displaced compared to previous kinase structures, and its role in stabilization of the activation loop is taken on by the dimerisation interface. The apo-form of the protein has an open conformation with a disordered P-loop but a structured activation segment in absence of targeted phosphorylation. The nucleotide-binding site of LegK4 contains an unusual set of residues that mediate non-canonical interactions with AMP-PNP. Nucleotide binding results in limited changes in the active site, suggesting that LegK4 constitutive kinase activity does not depend on phosphorylation of the activation loop but on the stabilizing effects of the dimer.