A high-affinity calmodulin-binding site in the CyaA toxin translocation domain is essential for invasion of eukaryotic cells

The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells are still a matter of intense research. The adenylate cyclase (CyaA) toxin from Bordetella pertussis displays a unique intoxication pathway in which its catalytic domain is directly translocated across target cell membranes. The CyaA translocation region contains a segment, P454 (residues 454-484), which exhibits membrane-active properties related to antimicrobial peptides. Herein, the results show that this peptide is able to translocate across membranes and to interact with calmodulin (CaM). Structural and biophysical analyses reveal the key residues of P454 involved in membrane destabilization and calmodulin binding. Mutational analysis demonstrates that these residues play a crucial role in CyaA translocation into target cells. In addition, calmidazolium, a calmodulin inhibitor, efficiently blocks CyaA internalization. It is proposed that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin. Trapping of CyaA by the CaM:P454 interaction in the cytosol may assist the entry of the N-terminal catalytic domain by converting the stochastic motion of the polypeptide chain through the membrane into an efficient vectorial chain translocation into host cells

Etude du mécanisme de translocation de la toxine CyaA de bordetella pertussis

La toxine adénylcyclase (CyaA) est un des principaux facteurs de virulence produite par Bordetella pertussis, l’agent de la coqueluche. CyaA a l’unique capacité de transloquer son domaine catalytique directement à travers la membrane plasmique. Puis le domaine catalytique lie la calmoduline (CaM) pour produire de grandes quantités d’AMPc, conduisant à l’intoxication de la cellule. Bien que plusieurs modèles aient été proposés, le mécanisme moléculaire et les forces impliquées dans la translocation de CyaA restent peu connus. Un gradient de calcium, un potentiel de membrane et des acylations post-traductionnelles sont requis pour la translocation de CyaA. Pendant mon doctorat, je me suis principalement intéressé au processus de translocation. Il a été montré précédemment que la suppression de la région de translocation abolit le passage du domaine catalytique. Dans cette région, le peptide P454 (résidus 454 à 484 de CyaA) a été identifié et montre des propriétés membranaires, i.e interaction avec la membrane, repliement en hélice α au contact de la membrane et perméabilisation membranaire. Nous avons étudié le rôle de P454 dans le processus de translocation. Nous avons observé que des lipides fluides et chargés négativement favorisent l’insertion de P454 dans les membranes. Le peptide possède deux arginines qui sont impliquées dans ses activités membranaires. P454 possède aussi la capacité de transloquer à travers la membrane et de former un complexe avec la CaM. Nous avons identifié plusieurs résidus de P454 impliqués dans la liaison à la membrane et la CaM. Dans le contexte de la toxine entière, ces résidus sont essentiels pour la translocation du domaine catalytique et la production d’AMPc. On propose un modèle de translocation dans lequel le segment P454 de la région de translocation déstabilise la membrane, favorisant sa translocation. Dans le cytosol, le segment P454 est piégé par la CaM et le complexe pourrait agir comme une force tirant le domaine catalytique à travers la membrane. Nous avons aussi montré que la liaison à la CaM du peptide liant la CaM dans le domaine catalytique induit des effets allostériques qui stabilisent le site catalytique, permettant la catalyse rapide d’ATP en AMPc. La pertinence de ces résultats pour la translocation et l’activation de CyaA sont discutées.The adenylate cyclase toxin (CyaA) is one of the major virulence factor produced by Bordetella pertussis, the causative agent of whopping cough. CyaA has the unique capacity to translocate its catalytic domain directly across the plasma membrane. Then, the catalytic domain binds to calmodulin (CaM) to produce high levels of cAMP, leading to cell intoxication. Although several models have been proposed, the molecular mechanism and the forces involved in the translocation of CyaA remain elusive. The calcium gradient, the membrane potential across the plasma membrane and post-translational acylation are required for an efficient CyaA translocation. During my PhD, I mainly investigated the translocation process. It has been previously shown that deletion of the translocation region abolishes the delivery of the catalytic domain into the cytosol of target cells. In this region, the peptide P454 (residues 454 to 484 of CyaA) was identified and exhibits membrane-active properties related to antimicrobial peptides, i.e membrane interaction, α-helical folding upon membrane insertion and membrane permeabilization. We have investigated the role of P454 on the translocation process. We observed that negatively charged and fluidic membrane favor P454 membrane insertion. The peptide contains two arginine residues that are critically involved in its membrane-active properties. We further identified that P454 exhibits the intrinsic propensity to translocate across lipid bilayers and forms a stable complex with CaM. We identified several residues from P454 involved in both membrane interaction and CaM binding. We showed in the context of the full-length CyaA toxin that these residues are essential for the efficient translocation of the catalytic domain into the cell and production of cAMP. We propose a translocation model in which the membrane-active P454 segment from the translocation region destabilizes the membrane, favoring its translocation. In the cytosol, the P454 segment is trapped by CaM and the formation of the complex may act as a driving force pulling the catalytic domain across the plasma membrane. We further showed that CaM binding to the main CaM-binding site in the catalytic domain induces local and long-range allosteric effects that stabilize the enzymatic site, allowing fast ATP catalysis to cAMP, leading to host subversion. The relevance of these results for the translocation and activation of CyaA are discussed

Membrane-Active Properties of an Amphitropic Peptide from the CyaA Toxin Translocation Region

International audienceThe adenylate cyclase toxin CyaA is involved in the early stages of infection by Bordetella pertussis, the causative agent of whooping cough. CyaA intoxicates target cells by a direct translocation of its catalytic domain (AC) across the plasma membrane and produces supraphysiological levels of cAMP, leading to cell death. The molecular process of AC translocation remains largely unknown, however. We have previously shown that deletion of residues 375-485 of CyaA selectively abrogates AC translocation into eukaryotic cells. We further identified within this "translocation region" (TR), P454 (residues 454-484), a peptide that exhibits membrane-active properties, i.e., is able to bind and permeabilize lipid vesicles. Here, we analyze various sequences from CyaA predicted to be amphipatic and show that although several of these peptides can bind membranes and adopt a helical conformation, only the P454 peptide is able to permeabilize membranes. We further characterize the contributions of the two arginine residues of P454 to membrane partitioning and permeabilization by analyzing the peptide variants in which these residues are substituted by different amino acids (e.g., A, K, Q, and E). Our data shows that both arginine residues significantly contribute, although diversely, to the membrane-active properties of P454, i.e., interactions with both neutral and anionic lipids, helix formation in membranes, and disruption of lipid bilayer integrity. These results are discussed in the context of the translocation process of the full-length CyaA toxin

The Adenylate Cyclase (CyaA) Toxin from <i>Bordetella pertussis</i> Has No Detectable Phospholipase A (PLA) Activity In Vitro

The adenylate cyclase (CyaA) toxin produced in Bordetella pertussis is the causative agent of whooping cough. CyaA exhibits the remarkable capacity to translocate its N-terminal adenyl cyclase domain (ACD) directly across the plasma membrane into the cytosol of eukaryotic cells. Once translocated, calmodulin binds and activates ACD, leading to a burst of cAMP that intoxicates the target cell. Previously, Gonzalez-Bullon et al. reported that CyaA exhibits a phospholipase A activity that could destabilize the membrane to facilitate ACD membrane translocation. However, Bumba and collaborators lately reported that they could not replicate these results. To clarify this controversy, we assayed the putative PLA activity of two CyaA samples purified in two different laboratories by using two distinct fluorescent probes reporting either PLA2 or both PLA1 and PLA2 activities, as well as in various experimental conditions (i.e., neutral or negatively charged membranes in different buffers.) However, we could not detect any PLA activity in these CyaA batches. Thus, our data independently confirm that CyaA does not possess any PLA activity

Gallocin A, an Atypical Two-Peptide Bacteriocin with Intramolecular Disulfide Bonds Required for Activity

International audienceStreptococcus gallolyticus subsp. gallolyticus (SGG) is an opportunistic gut pathogen associated with colorectal cancer. We previously showed that colonization of the murine colon by SGG in tumoral conditions was strongly enhanced by the production of gallocin A, a two-peptide bacteriocin. Here, we aimed to characterize the mechanisms of its action and resistance. Using a genetic approach, we demonstrated that gallocin A is composed of two peptides, GllA1 and GllA2, which are inactive alone and act together to kill "target" bacteria. We showed that gallocin A can kill phylogenetically close relatives of the pathogen. Importantly, we demonstrated that gallocin A peptides can insert themselves into membranes and permeabilize lipid bilayer vesicles. Next, we showed that the third gene of the gallocin A operon, gip, is necessary and sufficient to confer immunity to gallocin A. Structural modeling of GllA1 and GllA2 mature peptides suggested that both peptides form alpha-helical hairpins stabilized by intramolecular disulfide bridges. The presence of a disulfide bond in GllA1 and GllA2 was confirmed experimentally. Addition of disulfide-reducing agents abrogated gallocin A activity. Likewise, deletion of a gene encoding a surface protein with a thioredoxin-like domain impaired the ability of gallocin A to kill Enterococcus faecalis. Structural modeling of GIP revealed a hairpin-like structure strongly resembling those of the GllA1 and GllA2 mature peptides, suggesting a mechanism of immunity by competition with GllA1/2. Finally, identification of other class IIb bacteriocins exhibiting a similar alpha-helical hairpin fold stabilized with an intramolecular disulfide bridge suggests the existence of a new subclass of class IIb bacteriocins. IMPORTANCE Streptococcus gallolyticus subsp. gallolyticus (SGG), previously named Streptococcus bovis biotype I, is an opportunistic pathogen responsible for invasive infections (septicemia, endocarditis) in elderly people and is often associated with colon tumors. SGG is one of the first bacteria to be associated with the occurrence of colorectal cancer in humans. Previously, we showed that tumor-associated conditions in the colon provide SGG with an ideal environment to proliferate at the expense of phylogenetically and metabolically closely related commensal bacteria such as enterococci (1). SGG takes advantage of CRC-associated conditions to outcompete and substitute commensal members of the gut microbiota using a specific bacteriocin named gallocin, recently renamed gallocin A following the discovery of gallocin D in a peculiar SGG isolate. Here, we showed that gallocin A is a two-peptide bacteriocin and that both GllA1 and GllA2 peptides are required for antimicrobial activity. Gallocin A was shown to permeabilize bacterial membranes and kill phylogenetically closely related bacteria such as most streptococci, lactococci, and enterococci, probably through membrane pore formation. GllA1 and GllA2 secreted peptides are unusually long (42 and 60 amino acids long) and have very few charged amino acids compared to well-known class IIb bacteriocins. In silico modeling revealed that both GllA1 and GllA2 exhibit a similar hairpin-like conformation stabilized by an intramolecular disulfide bond. We also showed that the GIP immunity peptide forms a hairpin-like structure similar to GllA1/GllA2. Thus, we hypothesize that GIP blocks the formation of the GllA1/GllA2 complex by interacting with GllA1 or GllA2. Gallocin A may constitute the first class IIb bacteriocin which displays disulfide bridges important for its structure and activity and might be the founding member of a subtype of class IIb bacteriocins

Bacterial kinesin light chain (Bklc) links the Btub cytoskeleton to membranes

International audienceBacterial kinesin light chain is a TPR domain-containing protein encoded by the bklc gene, which co-localizes with the bacterial tubulin (btub) genes in a conserved operon in Prosthecobacter. Btub heterodimers show high structural homology with eukaryotic tubulin and assemble into head-to-tail protofilaments. Intriguingly, Bklc is homologous to the light chain of the microtubule motor kinesin and could thus represent an additional eukaryotic-like cytoskeletal element in bacteria. Using biochemical characterization as well as cryo-electron tomography we show here that Bklc interacts specifically with Btub protofilaments, as well as lipid vesicles and could thus play a role in anchoring the Btub filaments to the membrane protrusions in Prosthecobacter where they specifically localize in vivo. This work sheds new light into possible ways in which the microtubule cytoskeleton may have evolved linking precursors of microtubules to the membrane via the kinesin moiety that in today's eukaryotic cytoskeleton links vesicle-packaged cargo to microtubules

Post-translational acylation controls the folding and functions of the CyaA RTX toxin

International audienceThe adenylate cyclase (CyaA) toxin is a major virulence factor of Bordetella pertussis, the causative agent of whooping cough. CyaA is synthetized as a pro-toxin, pro-CyaA, and converted into its cytotoxic form upon acylation of two lysines. After secretion, CyaA invades eukaryotic cells and produces cAMP, leading to host defense subversion. To gain further insights into the effect of acylation, we compared the functional and structural properties of pro-CyaA and CyaA proteins. HDX-MS results show that the refolding process of both proteins upon progressive urea removal is initiated by calcium binding to the C-terminal RTX domain. We further identified a critical hydrophobic segment, distal from the acylation region, that folds at higher urea concentration in CyaA than in pro-CyaA. Once refolded into monomers, CyaA is more compact and stable than pro-CyaA, due to a complex set of interactions between domains. Our HDX-MS data provide direct evidence that the presence of acyl chains in CyaA induces a significant stabilization of the apolar segments of the hydrophobic domain and of most of the acylation region. We propose a refolding model dependent on calcium and driven by local and distal acylation-dependent interactions within CyaA. Therefore, CyaA acylation is not only critical for cell intoxication, but also for protein refolding into its active conformation. Our data shed light on the complex relationship between post-translational modifications, structural disorder and protein folding. Coupling calcium-binding and acylation-driven folding is likely pertinent for other repeat-in-toxin cytolysins produced by many Gram-negative bacterial pathogens.-O'Brien, D. P., Cannella, S. E., Voegele, A., Raoux-Barbot, D., Davi, M., Douché, T., Matondo, M., Brier, S., Ladant, D., Chenal, A. Post-translational acylation controls the folding and functions of the CyaA RTX toxin

Calmodulin fishing with a structurally disordered bait triggers CyaA catalysis.

Once translocated into the cytosol of target cells, the catalytic domain (AC) of the adenylate cyclase toxin (CyaA), a major virulence factor of Bordetella pertussis, is potently activated by binding calmodulin (CaM) to produce supraphysiological levels of cAMP, inducing cell death. Using a combination of small-angle X-ray scattering (SAXS), hydrogen/deuterium exchange mass spectrometry (HDX-MS), and synchrotron radiation circular dichroism (SR-CD), we show that, in the absence of CaM, AC exhibits significant structural disorder, and a 75-residue-long stretch within AC undergoes a disorder-to-order transition upon CaM binding. Beyond this local folding, CaM binding induces long-range allosteric effects that stabilize the distant catalytic site, whilst preserving catalytic loop flexibility. We propose that the high enzymatic activity of AC is due to a tight balance between the CaM-induced decrease of structural flexibility around the catalytic site and the preservation of catalytic loop flexibility, allowing for fast substrate binding and product release. The CaM-induced dampening of AC conformational disorder is likely relevant to other CaM-activated enzymes