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

International audienceVarious 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

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

Posted December 16, 2022 on bioRxiv.Amphitropic proteins and peptides reversibly partition from solution to membrane, a key process that regulates their functions. Experimental approaches, such as fluorescence and circular dichroism, are classically used to measure the partitioning of amphitropic peptides and proteins into lipid bilayers, yet hardly usable when the peptides or proteins do not exhibit significant polarity and/or conformational changes upon membrane binding. Here, we describe B2LiVe ( i . e ., Binding to Lipid Vesicles), a simple, robust, and widely applicable NMR method to determine the solution-to-membrane partitioning of unlabeled proteins or peptides. The experimental strategy proposed here relies on previously described proton 1D NMR fast pulsing techniques with selective adiabatic pulses. Membrane partitioning induces a large line broadening leading to a progressive loss of protein signals, and therefore, the decrease of the NMR signal directly measures the fraction of membrane-bound protein. The B2LiVe method uses low polypeptide concentrations and has been validated on several membrane-interacting peptides and proteins, ranging from 3 to 54 kDa, with membrane vesicles of different sizes and various lipid compositions. Motivation Characterization of the interaction of peptides and proteins with lipid membranes is involved in various biological processes and is often challenging for polypeptides which do not possess intrinsic fluorophores, do not exhibit significant structural content changes, as well as for those characterized by low affinities for membranes. To meet these challenges, we have developed a simple and robust label-free NMR-based experimental approach, named B2LiVe, to measure the binding of polypeptides to lipid vesicles. The experimental strategy relies on previously described proton 1D NMR fast pulsing techniques with selective adiabatic pulses to excite the amide resonances. B2LiVe is a label-free method based on the observation of amide hydrogen nuclei which are naturally present in all protein and peptide backbones. Our results validate the B2LiVe method and indicate that it compares well with established technics to quantify polypeptide-membrane interactions. Overall, B2LiVe should efficiently complement the arsenal of label-free biophysical assays available to characterize protein-membrane interactions. In brief We describe a robust label-free NMR-based experimental approach (B2LiVe) to measure interactions between peptides or proteins with membranes. The validity of this approach has been established on several polypeptides and on various membrane vesicles. The B2LiVe method efficiently complements the arsenal of label-free biophysical techniques to characterize protein-membrane interactions. Highlights B2LiVe is a simple and robust NMR-based method to quantify affinity of proteins and peptides for membranes B2LiVe is a label-free approach that relies on proton 1D NMR fast pulsing techniques with selective excitation of amide resonances B2LiVe has been validated on several membrane-interacting peptides and proteins B2LiVe coupled to DOSY can pinpoint the presence within a membrane-bound protein of polypeptide segments remaining in solutio

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

EGA protects mammalian cells from clostridiumdifficile CDT, clostridium perfringens iota toxin clostridium botulinum C2 toxin

The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxin

Revisiting an old antibiotic: bacitracin neutralizes binary bacterial toxins and protects cells from intoxication

International audienceThe antibiotic bacitracin (Bac) inhibits cell wall synthesis of gram-positive bacteria. Here, we discovered a totally different activity of Bac: the neutralization of bacterial exotoxins. Bac prevented intoxication of mammalian cells with the binary enterotoxins Clostridium botulinum C2, C. perfringens ι, C. difficile transferase (CDT), and Bacillus anthracis lethal toxin. The transport (B) subunits of these toxins deliver their respective enzyme (A) subunits into cells. Following endocytosis, the B subunits form pores in membranes of endosomes, which mediate translocation of the A subunits into the cytosol. Bac inhibited formation of such B pores in lipid bilayers in vitro and in living cells, thereby preventing translocation of the A subunit into the cytosol. Bac preserved the epithelial integrity of toxin-treated CaCo-2 monolayers, a model for the human gut epithelium. In conclusion, Bac should be discussed as a therapeutic option against infections with medically relevant toxin-producing bacteria, including C. difficile and B. anthracis, because it inhibits bacterial growth and neutralizes the secreted toxins.-Schnell, L., Felix, I., Müller, B., Sadi, M., von Bank, F., Papatheodorou, P., Popoff, M. R., Aktories, K., Waltenberger, E., Benz, R., Weichbrodt, C., Fauler, M., Frick, M., Barth, H. Revisiting an old antibiotic: bacitracin neutralizes binary bacterial toxins and protects cells from intoxication

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

International audienceBackground 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. Graphical abstract 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

A High‐Affinity Calmodulin‐Binding Site in the CyaA Toxin Translocation Domain is Essential for Invasion of Eukaryotic Cells

This article also appears in:Hot Topic: MembranesInternational audienceThe 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