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

Voegele, Alexis; Subrini, Orso; Sapay, Nicolas; Ladant, Daniel; Chenal, Alexandre

doi:10.3390/toxins9110369

Cited by 24 publications

(45 citation statements)

References 78 publications

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“…Reverse phase evaporation was used to prepare small unilamellar vesicles (SUVs). A 10 mM lipid solution made of an 8:2 mixture of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoylglycero-3-phosphoglycerol (POPG) (Avanti Polar Lipids) was prepared as described 68 . Chloroform was removed by evaporation under vacuum conditions and the dried phospholipid film was resuspended in a mixture of diethyl ether and buffer (25 mM Hepes pH 7.4, 10 mM MgCl 2 and 150 mM KCl).…”

Section: Methodsmentioning

confidence: 99%

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Essential dynamic interdependence of FtsZ and SepF for Z-ring and septum formation in Corynebacterium glutamicum

et al. 2020

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The mechanisms of Z-ring assembly and regulation in bacteria are poorly understood, particularly in non-model organisms. Actinobacteria, a large bacterial phylum that includes the pathogen Mycobacterium tuberculosis, lack the canonical FtsZ-membrane anchors and Z-ring regulators described for E. coli. Here we investigate the physiological function of Corynebacterium glutamicum SepF, the only cell division-associated protein from Actinobacteria known to interact with the conserved C-terminal tail of FtsZ. We show an essential interdependence of FtsZ and SepF for formation of a functional Z-ring in C. glutamicum. The crystal structure of the SepF-FtsZ complex reveals a hydrophobic FtsZ-binding pocket, which defines the SepF homodimer as the functional unit, and suggests a reversible oligomerization interface. FtsZ filaments and lipid membranes have opposing effects on SepF polymerization, indicating that SepF has multiple roles at the cell division site, involving FtsZ bundling, Z-ring tethering and membrane reshaping activities that are needed for proper Z-ring assembly and function.

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Section: Methodsmentioning

confidence: 99%

“…The KaleidaGraph software was used to fit the K x to the experimental data (for details, see ref. 68 ).…”

Section: Methodsmentioning

confidence: 99%

Essential dynamic interdependence of FtsZ and SepF for Z-ring and septum formation in Corynebacterium glutamicum

et al. 2020

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“…The preferential binding of P454 to calmodulin over membrane was confirmed by a lipid vesicle permeabilization assay 63,65 . In this set up, addition of P454 peptide to ANTS:DPX loaded vesicles induces membrane permeabilization leading to a dye efflux that is monitored by fluorescence increase as a result of ANTS dequenching.…”

Section: -The P454 Peptide From the Cyaa Toxin Binds To Calmodulinmentioning

confidence: 94%

“…However, the molecular mechanism and forces involved in the translocation of the CyaA catalytic domain across the plasma membrane have thus far remained elusive. We have previously shown that a peptide, corresponding to the C-terminus of the translocation region TR of CyaA, P454 (residues 454-484, Figure S1), exhibits membrane-active properties related to antimicrobial peptides (AMPs) 17,63 : this peptide adopts a helical conformation upon membrane interaction and induces a local destabilization of the lipid bilayer [63][64][65][66] . This property is likely essential for CyaA as deletion of the TR region (residues 384-489) encompassing the P454 segment, selectively abrogates the ability of the modified toxin to intoxicate target cells 17 .…”

Section: Introductionmentioning

confidence: 99%

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

Voegele

Sadi

O’Brien

et al. 2020

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The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells have thus far remained elusive. 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. We have previously identified a translocation region in CyaA that contains a segment, P454 (residues 454-484), exhibiting membrane-active properties related to antimicrobial peptides. Herein, we show that this peptide is able to translocate across membranes and interact with calmodulin. Structural and biophysical analyses have revealed the key residues of P454 involved in membrane destabilization and calmodulin binding. Mutational analysis demonstrated that these residues play a crucial role in CyaA translocation into target cells. We have also shown that calmidazolium, a calmodulin inhibitor, efficiently blocks CyaA internalization. We propose that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin. Trapping of the CyaA polypeptide chain by the CaM:P454 interaction in the cytosol may assist the entry of the N-terminal catalytic domain by converting the stochastic process of protein translocation into an efficient vectorial chain transfer into host cells.

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“…CyaA is a large, multidomain protein of 1706 amino-acid residues, with AC (residues 1-364) (16), whereas the C-terminal hemolysin region (residues 365-1706) contains several domains responsible for AC membrane translocation and pore-forming activity (19)(20)(21)(22)(23)(24). The translocation region (TR; residues 365-527) is involved in AC translocation across target cell membranes (24) and membrane permeabilization (25) and exhibits features similar to membrane-active peptides (24,26,27). The hydrophobic region (HR) corresponds to residues 528-710, whereas the acylation region (AR) extends from residues 711 through 1005.…”

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

et al. 2019

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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. FASEB J. 33, 10065–10076 (2019). http://www.fasebj.org

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

Cited by 24 publications

References 78 publications

Essential dynamic interdependence of FtsZ and SepF for Z-ring and septum formation in Corynebacterium glutamicum

Essential dynamic interdependence of FtsZ and SepF for Z-ring and septum formation in Corynebacterium glutamicum

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

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

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