Many bacteria pathogenic for plants or animals, including Shigella spp., which is responsible for shigellosis in humans, use a type III secretion apparatus to inject effector proteins into host cells. Effectors alter cell signaling and host responses induced upon infection; however, their precise biochemical activities have been elucidated in very few cases. Utilizing Saccharomyces cerevisiae as a surrogate host, we show that the Shigella effector IpaH9.8 interrupts pheromone response signaling by promoting the proteasome-dependent destruction of the MAPKK Ste7. In vitro, IpaH9.8 displayed ubiquitin ligase activity toward ubiquitin and Ste7. Replacement of a Cys residue that is invariant among IpaH homologs of plant and animal pathogens abolished the ubiquitin ligase activity of IpaH9.8. We also present evidence that the IpaH homolog SspH1 from Salmonella enterica can ubiquitinate ubiquitin and PKN1, a previously identified SspH1 interaction partner. This study assigns a function for IpaH family members as E3 ubiquitin ligases.

The Repeat in Toxin (RTX) motif is a tandemly repeated calcium-binding nonapeptide sequence present in proteins that are secreted by the type I secretion system (T1SS) of Gram-negative bacteria. Here, we have characterized the structural and hydrodynamic properties of the RTX Repeat Domain (RD) of the CyaA toxin from Bordetella pertussis. This 701-amino acid long domain contains about 40 RTX motifs. We showed that, in the absence of calcium, RD was natively disordered, weakly stable, and highly hydrated. Calcium binding induced compaction and dehydration of RD, along with the formation of stable secondary and tertiary structures. The calcium-induced conformational switch between unfolded conformations of apo-RD and stable structures of holo-RD is likely to be a key property for the biological function of the CyaA toxin: in the low calcium environment of the bacterial cytosol, the intrinsically disordered character of the protein may facilitate its secretion through the secretion machinery. In the extracellular medium, calcium binding can then trigger the folding of the polypeptide into its functional state. The intrinsic disorder of RTX-containing proteins in the absence of calcium may thus be directly involved in the efficient secretion of proteins through T1SS.

The study of the membrane insertion of the translocation domain of diphtheria toxin deepens our insight into the interactions between proteins and membranes. During cell intoxication, this domain undergoes a change from a soluble and folded state at alkaline pH to a functional membrane-inserted state at acid pH. We found that hydrophobic and electrostatic interactions occur in a sequential manner between the domain and the membrane during the insertion. The first step involves hydrophobic interactions by the C-terminal region. This is because of the pH-induced formation of a molten globule specialized for binding to the membrane. Accumulation of this molten globule follows a precise molecular mechanism adapted to the toxin function. The second step, as the pH decreases, leads to the functional inserted state. It arises from the changes in the balance of electrostatic attractions and repulsions between the N-terminal part and the membrane. Our study shows how the structural changes and the interaction with membranes of the translocation domain are finely tuned by pH changes to take advantage of the cellular uptake system.Folding and insertion of membrane proteins (1, 2), binding of hormones to membrane receptors (3), action of antibiotic peptides (4, 5), protein translocation, and internalization of toxins (6) are examples of phenomena that require the interactions of polypeptide chains with membranes. Because of the anisotropic nature of membranes, the initial steps of the association and the final structure and localization of polypeptide chains within membranes depend on a combination of hydrophobic and electrostatic interactions (7). Hydrophobic interactions are dominant for the insertion of transmembrane polypeptides. Electrostatic interactions are important for the binding of antibiotic peptides (5,8), the association of proteins with the surface of the membrane (9 -11), and as determinants of the topology of integral membrane proteins after biosynthesis (12). In most cases, electrostatic interactions are the result of the attraction between anionic phospholipid head groups and basic amino acid side chains (13,14). However, there are examples where electrostatic repulsions are involved in the membrane association of peptides, particularly when their structure and localization within the membrane is regulated by the pH (15-19). The interplay of hydrophobicity and electrostatics and their distribution within the polypeptide sequence have only been studied in detail for small peptides (3-5, 7, 13-15, 17-20). In the case of proteins, the role of these effects on the association with and the insertion into membranes are still poorly understood (2,(21)(22)(23)(24).The study of the membrane insertion process of the translocation (T) 1 domain of diphtheria toxin (25) can provide precious insight into the interactions between proteins and membranes and the refolding mechanisms of membrane proteins. During intoxication of cells (25), the toxin reaches the early endosomes through the clathrin-coated pathway (26). Beca...

The adenylate cyclase toxin (CyaA) is one of the major virulence factors of Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic cells by a unique mechanism that consists in a calcium-dependent, direct translocation of the CyaA catalytic domain across the plasma membrane of the target cells. CyaA possesses a series of a glycine-and aspartate-rich nonapeptide repeats (residues 1006 -1613) of the prototype GGXG(N/D)DX(L/I/F)X (where X represents any amino acid) that are characteristic of the RTX (repeat in toxin) family of bacterial cytolysins. These repeats are arranged in a tandem fashion and may fold into a characteristic parallel ␤-helix or ␤-roll motif that constitutes a novel type of calcium binding structure, as revealed by the three-dimensional structure of the Pseudomonas aeruginosa alkaline protease. Here we have characterized the structure-function relationships of various fragments from the CyaA RTX subdomain. Our results indicate that the RTX functional unit includes both the tandem repeated nonapeptide motifs and the adjacent polypeptide segments, which are essential for the folding and calcium responsiveness of the RTX module. Upon calcium binding to the RTX repeats, a conformational rearrangement of the adjacent non-RTX sequences may act as a critical molecular switch to trigger the CyaA entry into target cells.The adenylate cyclase toxin (CyaA) 2 is one of the major virulence factors of Bordetella pertussis, the causative agent of whooping cough (1-3). The 1706 residue-long CyaA is a bi-functional protein endowed with both catalytic (adenylate cyclase) and hemolytic activities (2, 4, 5). Synthesized as an inactive precursor, it is converted to the active toxin by a post translational palmitoylation of two internal lysine residues (Lys 860 and Lys 983 ) (6, 7). This active CyaA toxin is then able to deliver its catalytic domain directly across the plasma membrane of a variety of eukaryotic cells and disrupts their physiological functions by uncontrolled synthesis of cAMP (5, 8 -11), leading to the cell death by apoptosis (12)(13)(14). CyaA is constructed in a modular fashion; the calmodulinactivated catalytic domain is located in the 400-amino-proximal residues, whereas the C-terminal moiety (residues 400 -1706) is endowed with hemolytic activity (4, 5, 15, 16), which results from its ability to form cation-selective channels in membranes (17,18). It also mediates the binding and internalization of the toxin into eukaryotic cells (5,11,19). The hemolytic and the RTX domains display structural characteristics that link CyaA to the RTX (repeat in toxin) family of bacterial toxins (20, 21). Indeed, it contains a pore-forming domain (from residues 500 -700) with four hydrophobic segments (17,18,22,23), the target site for the post-translational palmitoylation (7, 24), 30 -40 copies of a characteristic glycine-and aspartate-rich nonapeptide repeats (residues 1006 -1613) of the prototype GGXG(N/ D)DX(U)X (X represents any amino acid, and U represents any large ...

The adenylate cyclase toxin (CyaA) is one of the major virulence factors of Bordetella pertussis, the causative agent of whooping cough. Its C-terminal region, the receptor-binding domain (RD), contains ∼40 calcium-binding Repeat in ToXin (RTX) motifs, which are characteristic of many virulence factors of pathogenic bacteria. We previously showed that RD is intrinsically disordered in the absence of calcium and acquires its functional three-dimensional structure upon calcium binding. To gain further insight into the physicochemical properties of RD, we characterized its calcium-induced conformational and stability changes by combining spectroscopic approaches. We show that RD, in the absence of calcium, adopts premolten globule conformations, due in part to the strong internal electrostatic repulsions between the negative charges of the aspartate-rich polypeptide sequence. Accordingly, sodium is able to screen these electrostatic repulsions, allowing a partial compaction of the polypeptide, whereas calcium triggers a strong compaction as well as the acquisition of secondary and tertiary structures in a highly cooperative manner. The differential sensitivity of the calcium-loaded state to guanidinium- and urea-induced denaturations provides further evidence that electrostatic interactions play a critical role in the folding and stability of RD. These results provide new insights into the folding/function relationship of the RTX motifs.

Secondary structure refolding is a key event in biology as it modulates the conformation of many proteins in the cell, generating functional or aberrant states. The crystal structures of mannosyltransferase PimA reveal an exceptional flexibility of the protein along the catalytic cycle, including β-strand-to-α-helix and α-helix-to-β-strand transitions. These structural changes modulate catalysis and are promoted by interactions of the protein with anionic phospholipids in the membrane.

Background:The translocation of the Bordetella pertussis CyaA toxin across membrane is still poorly understood. Results: A membrane-active peptide isolated from the CyaA toxin is characterized by biophysical approaches. Conclusion:The ␣-helical peptide is inserted in plane and induces membrane permeabilization. Significance: The membrane-destabilizing activity of this peptide may assist the initial steps of the CyaA translocation process.

Background: Translocation of the CyaA toxin across plasma membrane is still poorly understood. Results: The region 375-485 is involved in membrane destabilization in vitro and required for cell intoxication. Conclusion:The region 375-485 is crucial for membrane insertion and translocation of the catalytic domain of CyaA. Significance: These results provide new insights on the early stages of the cell intoxication process.