Toxins, Pathogenicity, Anti-Toxins, a Bicentennial Contribution.

The bicentenary of Louis Pasteur's birth raises the opportunity to revisit the activity and influence of L [...].

B2LiVe, a label-free 1D-NMR method to quantify the binding of amphitropic peptides or proteins to membrane vesicles.

Amphitropic proteins and peptides reversibly partition from solution to membrane, a key process that regulates their functions. Experimental approaches classically used to measure protein partitioning into lipid bilayers, such as fluorescence and circular dichroism, are hardly usable when the peptides or proteins do not exhibit significant polarity and/or conformational changes upon membrane binding. Here, we describe binding to lipid vesicles (B2LiVe), a simple, robust, and widely applicable nuclear magnetic resonance (NMR) method to determine the solution-to-membrane partitioning of unlabeled proteins or peptides. B2LiVe relies on previously described proton 1D-NMR fast-pulsing techniques. Membrane partitioning induces a large line broadening, leading to a loss of protein signals; therefore, the decrease of the NMR signal directly measures the fraction of membrane-bound protein. The method uses low polypeptide concentrations and has been validated on several membrane-interacting polypeptides, ranging from 3 to 54 kDa, with membrane vesicles of different sizes and various lipid compositions.

Functional and structural insights into the multi-step activation and catalytic mechanism of bacterial ExoY nucleotidyl cyclase toxins bound to actin-profilin.

ExoY virulence factors are members of a family of bacterial nucleotidyl cyclases (NCs) that are activated by specific eukaryotic cofactors and overproduce cyclic purine and pyrimidine nucleotides in host cells. ExoYs act as actin-activated NC toxins. Here, we explore the Vibrio nigripulchritudo Multifunctional-Autoprocessing Repeats-in-ToXin (MARTX) ExoY effector domain (Vn-ExoY) as a model for ExoY-type members that interact with monomeric (G-actin) instead of filamentous (F-actin) actin. Vn-ExoY exhibits moderate binding affinity to free or profilin-bound G-actin but can capture the G-actin:profilin complex, preventing its spontaneous or VASP- or formin-mediated assembly at F-actin barbed ends in vitro. This mechanism may prolong the activated cofactor-bound state of Vn-ExoY at sites of active actin cytoskeleton remodelling. We present a series of high-resolution crystal structures of nucleotide-free, 3'-deoxy-ATP- or 3'-deoxy-CTP-bound Vn-ExoY, activated by free or profilin-bound G-actin-ATP/-ADP, revealing that the cofactor only partially stabilises the nucleotide-binding pocket (NBP) of NC toxins. Substrate binding induces a large, previously-unidentified, closure of their NBP, confining catalytically important residues and metal cofactors around the substrate, and facilitating the recruitment of two metal ions to tightly coordinate the triphosphate moiety of purine or pyrimidine nucleotide substrates. We validate critical residues for both the purinyl and pyrimidinyl cyclase activity of NC toxins in Vn-ExoY and its distantly-related ExoY from Pseudomonas aeruginosa, which specifically interacts with F-actin. The data conclusively demonstrate that NC toxins employ a similar two-metal-ion mechanism for catalysing the cyclisation of nucleotides of different sizes. These structural insights into the dynamics of the actin-binding interface of actin-activated ExoYs and the multi-step activation of all NC toxins offer new perspectives for the specific inhibition of class II bacterial NC enzymes.

Animal Toxins: A Historical Outlook at the Institut Pasteur of Paris.

Humans have faced poisonous animals since the most ancient times. It is recognized that certain animals, like specific plants, produce toxic substances that can be lethal, but that can also have therapeutic or psychoactive effects. The use of the term "venom", which initially designated a poison, remedy, or magic drug, is now confined to animal poisons delivered by biting. Following Louis Pasteur's work on pathogenic microorganisms, it was hypothesized that venoms could be related to bacterial toxins and that the process of pathogenicity attenuation could be applied to venoms for the prevention and treatment of envenomation. Cesaire Phisalix and Gabriel Bertrand from the National Museum of Natural History as well as Albert Calmette from the Institut Pasteur in Paris were pioneers in the development of antivenomous serotherapy. Gaston Ramon refined the process of venom attenuation for the immunization of horses using a formalin treatment method that was successful for diphtheria and tetanus toxins. This paved the way for the production of antivenomous sera at the Institut Pasteur, as well as for research on venom constituents and the characterization of their biological activities. The specific activities of certain venom components, such as those involved in blood coagulation or the regulation of chloride ion channels, raises the possibility of developing novel therapeutic drugs that could serve as anticoagulants or as a treatment for cystic fibrosis, for example. Scientists of the Institut Pasteur of Paris have significantly contributed to the study of snake venoms, a topic that is reported in this review.

Immune responses and protection against Streptococcus pneumoniae elicited by recombinant Bordetella pertussis adenylate cyclase (CyaA) carrying fragments of pneumococcal surface protein A, PspA.

Streptococcus pneumoniae is a common agent of important human diseases such as otitis media, pneumonia, meningitis and sepsis. Current available vaccines that target capsular polysaccharides induce protection against invasive disease and nasopharyngeal colonization in children, yet their efficacy is limited to the serotypes included in the formulations. The virulence factor Pneumococcal Surface Protein A (PspA) interacts with host immune system and helps the bacteria to evade phagocytosis. Due to its essential role in virulence, PspA is an important vaccine candidate. Here we have tested a delivery system based on the adenylate cyclase toxin of Bordetella pertussis (CyaA) to induce immune responses against PspA in mice. CyaA was engineered to express fragments of the N-terminal region of PspAs from clades 2 and 4 (A2 and A4) and the resulting proteins were used in immunization experiments in mice. The recombinant CyaA-A2 and CyaA-A4 proteins were able to induce high levels of anti-PspA antibodies that reacted with pneumococcal strains expressing either PspA2 or PspA4. Moreover, reactivity of the antibodies against pneumococcal strains that express PspAs from clades 3 and 5 (PspA3 and PspA5) was also observed. A formulation containing CyaA-A2 and CyaA-A4 was able to protect mice against invasive pneumococcal challenges with isolates that express PspA2, PspA4 or PspA5. Moreover, a CyaA-A2-A4 fusion protein induced antibodies at similar levels and with similar reactivity as the formulation containing both proteins, and protected mice against the invasive challenge. Our results indicate that CyaA-PspA proteins are good candidates to induce broad protection against pneumococcal isolates.

Interaction network among de novo purine nucleotide biosynthesis enzymes in Escherichia coli.

In human cells, de novo purine nucleotide biosynthesis is known to be regulated through the formation of a metabolon called purinosome. Here, we employed a bacterial two-hybrid approach to characterize the protein-protein interactions network among the corresponding enzymes of Escherichia coli. Our study revealed a dense network of binary interactions that connect most purine nucleotide biosynthesis enzymes. Notably, PurK, an exclusive prokaryotic enzyme, appears as one of the central hubs of this network. We further showed that modifications in PurK, which disrupted several interactions in the network, affected the purine nucleotide pools and altered the bacterial fitness. Our data suggest that the bacterial de novo purine nucleotide biosynthesis enzymes can assemble in a supramolecular complex and that proper interactions among the components of this complex can contribute to bacterial fitness.

Editorial of the Special Issue "Toxins: Mr Hyde or Dr Jekyll?"

The 27th Annual Meeting of the French Society of Toxinology (SFET, http://sfet [...].

A Robust and Sensitive Spectrophotometric Assay for the Enzymatic Activity of Bacterial Adenylate Cyclase Toxins.

Various bacterial pathogens are producing toxins that target the cyclic Nucleotide Monophosphate (cNMPs) signaling pathways in order to facilitate host colonization. Among them, several are exhibiting potent nucleotidyl cyclase activities that are activated by eukaryotic factors, such as the adenylate cyclase (AC) toxin, CyaA, from Bordetella pertussis or the edema factor, EF, from Bacillus anthracis. The characterization of these toxins frequently requires accurate measurements of their enzymatic activity in vitro, in particular for deciphering their structure-to-function relationships by protein engineering and site-directed mutagenesis. Here we describe a simple and robust in vitro assay for AC activity based on the spectrophotometric detection of cyclic AMP (cAMP) after chromatographic separation on aluminum oxide. This assay can accurately detect down to fmol amounts of B. pertussis CyaA and can even be used in complex media, such as cell extracts. The relative advantages and disadvantages of this assay in comparison with other currently available methods are briefly discussed.

A Bacterial Two-Hybrid System for In Vivo Assays of Protein-Protein Interactions and Drug Discovery.

The bacterial adenylate cyclase-based two-hybrid (BACTH) system is a robust and simple genetic assay used to monitor protein-protein interactions in vivo. This system is based on functional complementation between two fragments from the catalytic domain of Bordetella pertussis adenylate cyclase (AC) to reconstitute a cyclic AMP (cAMP)-signaling cascade in Escherichia coli. Interactions between two chimeric proteins result in the synthesis of cAMP, which activates the transcription of various catabolite operons, leading to selectable phenotypes. One advantageous feature of this signaling cascade is that the physical association between the two interacting hybrid proteins is spatially separated from the transcriptional activation readout. Consequently, the BACTH system can detect protein-protein interactions occurring at various subcellular localizations. The system has been used to characterize interactions between soluble or membrane proteins of prokaryotic, eukaryotic, or viral origin. The BACTH assay can be used to uncover the region(s), domain(s), or amino acid residue(s) of a protein involved in an interaction with a specific partner. The BACTH system can also be adapted for the high-throughput screening of small molecules able to interfere with protein-protein interactions.

Dynamics and structural changes of calmodulin upon interaction with the antagonist calmidazolium.

BACKGROUND: Calmodulin (CaM) is an evolutionarily conserved eukaryotic multifunctional protein that functions as the major sensor of intracellular calcium signaling. Its calcium-modulated function regulates the activity of numerous effector proteins involved in a variety of physiological processes in diverse organs, from proliferation and apoptosis, to memory and immune responses. Due to the pleiotropic roles of CaM in normal and pathological cell functions, CaM antagonists are needed for fundamental studies as well as for potential therapeutic applications. Calmidazolium (CDZ) is a potent small molecule antagonist of CaM and one the most widely used inhibitors of CaM in cell biology. Yet, CDZ, as all other CaM antagonists described thus far, also affects additional cellular targets and its lack of selectivity hinders its application for dissecting calcium/CaM signaling. A better understanding of CaM:CDZ interaction is key to design analogs with improved selectivity. Here, we report a molecular characterization of CaM:CDZ complexes using an integrative structural biology approach combining SEC-SAXS, X-ray crystallography, HDX-MS, and NMR. RESULTS: We provide evidence that binding of a single molecule of CDZ induces an open-to-closed conformational reorientation of the two domains of CaM and results in a strong stabilization of its structural elements associated with a reduction of protein dynamics over a large time range. These CDZ-triggered CaM changes mimic those induced by CaM-binding peptides derived from physiological protein targets, despite their distinct chemical natures. CaM residues in close contact with CDZ and involved in the stabilization of the CaM:CDZ complex have been identified. CONCLUSION: Our results provide molecular insights into CDZ-induced dynamics and structural changes of CaM leading to its inhibition and open the way to the rational design of more selective CaM antagonists. Calmidazolium is a potent and widely used inhibitor of calmodulin, a major mediator of calcium-signaling in eukaryotic cells. Structural characterization of calmidazolium-binding to calmodulin reveals that it triggers open-to-closed conformational changes similar to those induced by calmodulin-binding peptides derived from enzyme targets. These results provide molecular insights into CDZ-induced dynamics and structural changes of CaM leading to its inhibition and open the way to the rational design of more selective CaM antagonists.

Report from the 27th (Virtual) Meeting on Toxinology, "Toxins: Mr Hyde or Dr Jekyll?", Organized by the French Society of Toxinology, 9-10 December 2021.

The French Society of Toxinology (SFET) organized its 27th annual meeting on 9-10 December 2021 as a virtual meeting (e-RT27). The central theme of this meeting was "Toxins: Mr Hyde or Dr Jekyll?", emphasizing the latest findings on plant, fungal, algal, animal and bacterial toxins during 10 lectures, 15 oral communications (shorter lectures) and 20 posters shared by ca. 80 participants. The abstracts of lectures and posters, as well as the winners of the best oral communication and poster awards, are presented in this report.

Prevalence of ExoY Activity in Pseudomonas aeruginosa Reference Panel Strains and Impact on Cytotoxicity in Epithelial Cells.

ExoY is among the effectors that are injected by the type III secretion system (T3SS) of Pseudomonas aeruginosa into host cells. Inside eukaryotic cells, ExoY interacts with F-actin, which stimulates its potent nucleotidyl cyclase activity to produce cyclic nucleotide monophosphates (cNMPs). ExoY has broad substrate specificity with GTP as a preferential substrate in vitro. How ExoY contributes to the virulence of P. aeruginosa remains largely unknown. Here, we examined the prevalence of active ExoY among strains from the international P. aeruginosa reference panel, a collection of strains that includes environmental and clinical isolates, commonly used laboratory strains, and sequential clonal isolates from cystic fibrosis (CF) patients and thus represents the large diversity of this bacterial species. The ability to secrete active ExoY was determined by measuring the F-actin stimulated guanylate cyclase (GC) activity in bacterial culture supernatants. We found an overall ExoY activity prevalence of about 60% among the 40 examined strains with no significant difference between CF and non-CF isolates. In parallel, we used cellular infection models of human lung epithelial cells to compare the cytotoxic effects of isogenic reference strains expressing active ExoY or lacking the exoY gene. We found that P. aeruginosa strains lacking ExoY were in fact more cytotoxic to the epithelial cells than those secreting active ExoY. This suggests that under certain conditions, ExoY might partly alleviate the cytotoxic effects of other virulence factors of P. aeruginosa.

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.

Bioengineering of Bordetella pertussis Adenylate Cyclase Toxin for Vaccine Development and Other Biotechnological Purposes.

The adenylate cyclase toxin, CyaA, is one of the key virulent factors produced by Bordetella pertussis, the causative agent of whooping cough. This toxin primarily targets innate immunity to facilitate bacterial colonization of the respiratory tract. CyaA exhibits several remarkable characteristics that have been exploited for various applications in vaccinology and other biotechnological purposes. CyaA has been engineered as a potent vaccine vehicle to deliver antigens into antigen-presenting cells, while the adenylate cyclase catalytic domain has been used to design a robust genetic assay for monitoring protein-protein interactions in bacteria. These two biotechnological applications are briefly summarized in this chapter.

Functional and structural consequences of epithelial cell invasion by Bordetella pertussis adenylate cyclase toxin.

Bordetella pertussis, the causative agent of whopping cough, produces an adenylate cyclase toxin (CyaA) that plays a key role in the host colonization by targeting innate immune cells which express CD11b/CD18, the cellular receptor of CyaA. CyaA is also able to invade non-phagocytic cells, via a unique entry pathway consisting in a direct translocation of its catalytic domain across the cytoplasmic membrane of the cells. Within the cells, CyaA is activated by calmodulin to produce high levels of cyclic adenosine monophosphate (cAMP) and alter cellular physiology. In this study, we explored the effects of CyaA toxin on the cellular and molecular structure remodeling of A549 alveolar epithelial cells. Using classical imaging techniques, biochemical and functional tests, as well as advanced cell mechanics method, we quantify the structural and functional consequences of the massive increase of intracellular cyclic AMP induced by the toxin: cell shape rounding associated to adhesion weakening process, actin structure remodeling for the cortical and dense components, increase in cytoskeleton stiffness, and inhibition of migration and repair. We also show that, at low concentrations (0.5 nM), CyaA could significantly impair the migration and wound healing capacities of the intoxicated alveolar epithelial cells. As such concentrations might be reached locally during B. pertussis infection, our results suggest that the CyaA, beyond its major role in disabling innate immune cells, might also contribute to the local alteration of the epithelial barrier of the respiratory tract, a hallmark of pertussis.

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

The 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.

The Adenylate Cyclase (CyaA) Toxin from Bordetella pertussis 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.

Translocation and calmodulin-activation of the adenylate cyclase toxin (CyaA) of Bordetella pertussis.

The adenylate cyclase toxin (CyaA) is a multi-domain protein secreted by Bordetella pertussis, the causative agent of whooping cough. CyaA is involved in the early stages of respiratory tract colonization by Bordetella pertussis. CyaA is produced and acylated in the bacteria, and secreted via a dedicated secretion system. The cell intoxication process involves a unique mechanism of transport of the CyaA toxin catalytic domain (ACD) across the plasma membrane of eukaryotic cells. Once translocated, ACD binds to and is activated by calmodulin and produces high amounts of cAMP, subverting the physiology of eukaryotic cells. Here, we review our work on the identification and characterization of a critical region of CyaA, the translocation region, required to deliver ACD into the cytosol of target cells. The translocation region contains a segment that exhibits membrane-active properties, i.e. is able to fold upon membrane interaction and permeabilize lipid bilayers. We proposed that this region is required to locally destabilize the membrane, decreasing the energy required for ACD translocation. To further study the translocation process, we developed a tethered bilayer lipid membrane (tBLM) design that recapitulate the ACD transport across a membrane separating two hermetic compartments. We showed that ACD translocation is critically dependent on calcium, membrane potential, CyaA acylation and on the presence of calmodulin in the trans compartment. Finally, we describe how calmodulin-binding triggers key conformational changes in ACD, leading to its activation and production of supraphysiological concentrations of cAMP.