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 [...

Protein-Protein Interaction: Bacterial Two Hybrid.

The bacterial two-hybrid (BACTH, for "Bacterial Adenylate Cyclase-based Two-Hybrid") system is a simple and fast genetic approach to detect and characterize protein-protein interactions in vivo. This system is based on the interaction-mediated reconstitution of a cAMP signaling cascade in Escherichia coli. As BACTH uses a diffusible cAMP messenger molecule, the physical association between the two interacting chimeric proteins can be spatially separated from the transcription activation readout, and therefore, it is possible to analyze protein-protein interactions that occur either in the cytosol or at the inner membrane level as well as those that involve DNA-binding proteins.

Defining Membrane Protein Topology Using pho-lac Reporter Fusions.

Experimental determination of membrane protein topology can be achieved using various techniques. Here, we present the pho-lac dual reporter system, a simple, convenient, and reliable tool to analyze the topology of membrane protein in vivo. The system is based on the use of two topological markers with complementary properties: The Escherichia coli β-galactosidase, LacZ, which is active in the cytoplasm, and the E.

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.

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.

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.

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.

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

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

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.

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 or the edema factor, EF, from . 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.

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.

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.

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.

Prevalence of ExoY Activity in 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 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 .

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 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.

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

The adenylate cyclase toxin, CyaA, is one of the key virulent factors produced by , 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.

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.

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

The adenylate cyclase (CyaA) toxin is a major virulence factor of , 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.

The Adenylate Cyclase (CyaA) Toxin from Has No Detectable Phospholipase A (PLA) Activity In Vitro.

The adenylate cyclase (CyaA) toxin produced in 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.

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.

Differential regulation of actin-activated nucleotidyl cyclase virulence factors by filamentous and globular actin.

Several bacterial pathogens produce nucleotidyl cyclase toxins to manipulate eukaryotic host cells. Inside host cells they are activated by endogenous cofactors to produce high levels of cyclic nucleotides (cNMPs). The ExoY toxin from Pseudomonas aeruginosa (PaExoY) and the ExoY-like module (VnExoY) found in the MARTX (Multifunctional-Autoprocessing Repeats-in-ToXin) toxin of Vibrio nigripulchritudo share modest sequence similarity (~38%) but were both recently shown to be activated by actin after their delivery to the eukaryotic host cell.

The extreme C terminus of the effector ExoY is crucial for binding to its eukaryotic activator, F-actin.

Bacterial nucleotidyl cyclase toxins are potent virulence factors that upon entry into eukaryotic cells are stimulated by endogenous cofactors to catalyze the production of large amounts of 3'5'-cyclic nucleoside monophosphates. The activity of the effector ExoY from is stimulated by the filamentous form of actin (F-actin). Utilizing yeast phenotype analysis, site-directed mutagenesis, functional biochemical assays, and confocal microscopy, we demonstrate that the last nine amino acids of the C terminus of ExoY are crucial for the interaction with F-actin and, consequently, for ExoY's enzymatic activity and toxicity in a yeast model.

Bioengineering of Adenylate Cyclase Toxin for Antigen-Delivery and Immunotherapy.

The adenylate cyclase toxin (CyaA) is one of the major virulence factors of , the causative agent of whooping cough. CyaA is able to invade eukaryotic cells where, upon activation by endogenous calmodulin, it synthesizes massive amounts of cAMP that alters cellular physiology. The CyaA toxin is a 1706 residues-long bifunctional protein: the catalytic domain is located in the 400 amino-proximal residues, whereas the carboxy-terminal 1306 residues are implicated in toxin binding to the cellular receptor, the αβ₂ (CD11b/CD18) integrin, and subsequently in the translocation of the catalytic domain across the cytoplasmic membrane of the target cells.