Biology subjects

Raoux-Barbot, D.

Publications and source records attributed to Raoux-Barbot, D..

6 recordsLinked to original sources

bioRxiv2026

Covalently linked peptides and membrane potential enable CyaA segment translocation

The adenylate cyclase toxin (CyaA) from Bordetella pertussis intoxicates host cells by directly translocating its N-terminal catalytic domain across the plasma membrane; however, the forces driving this unique process remain poorly defined. Here, we dissect the membrane translocation mechanisms of two peptide segments derived from CyaA: P233 and P454 from the catalytic domain and the translocation region, respectively. Both P454 and P233 are calmodulin-binding segments that are sequentially involved in the translocation and activation of the catalytic domain. Using a newly developed Droplet Interface Bilayer (DIB) approach, called DIB-Pipette, which enables direct visualization of peptide transport under controlled membrane potentials, we show that P454 translocates across membranes independently of membrane potential, whereas P233 translocation requires a negative electric membrane potential. Strikingly, covalent coupling of P233 and P454 enables efficient translocation of the resulting peptide even in the absence of a membrane potential. Together, these results suggest that two distinct membrane-active segments within CyaA act cooperatively to promote translocation at the peptide level, revealing an intrinsic mechanism that may contribute to membrane potential-dependent translocation. These findings provide new mechanistic insights into CyaA cell intoxication process and reveal a multifunctional strategy for protein delivery across membranes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/716334v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@750ab3org.highwire.dtl.DTLVardef@11a980org.highwire.dtl.DTLVardef@18f2b27org.highwire.dtl.DTLVardef@5a3a59_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Post-translational acylation drives folding and activity of the CyaA bacterial toxin.

Post-translational modifications critically shape protein conformation and function, yet how they regulate bacterial toxins remains elusive. The adenylate cyclase (CyaA) toxin is a major virulence factor of Bordetella pertussis, the causative agent of whooping cough. CyaA is produced as an inactive precursor, proCyaA, which is activated by acylation of two lysine residues within the bacterium. Once acylated and secreted, CyaA invades innate immune cells and disrupts their phagocytic functions. High-resolution structural characterization of CyaA has remained elusive due to its size, multi-domain organization, flexibility, and aggregation propensity. Here, we overcome these challenges and generate the first structural ensembles of both non-acylated and acylated CyaA in solution by combining experimental data with integrative modeling. Coarse-grained molecular dynamics simulations reveal that acylation is critical to stabilize the native fold and to favorably orient CyaA on the target membrane. Overall, our findings reveal how post-translational acylation triggers native folding and provide mechanistic insights into the early steps of host cell intoxication.

biophysics↗

bioRxiv2025

Interplay between T3SS effectors, ExoY activation, and cGMP signaling in Pseudomonas aeruginosa infection

ExoY is a nucleotidyl cyclase, secreted by the Pseudomonas aeruginosa type 3 secretion system (T3SS), which generates various cyclic nucleotides within host cells, predominantly cGMP. How ExoY-catalyzed cNMPs production contributes to the infection process remains unclear. Using recombinant P. aeruginosa strains expressing ExoY variants with different substrate specificities, we identified that ExoY-derived cGMP modulates the cytotoxicity of ExoT, a co-injected T3SS exotoxin. Our results demonstrated that dephosphorylation of the host CrkII adaptor protein, a downstream effect of the ADP-ribosyltransferase activity of ExoT, is limited by ExoY-derived cGMP, therefore antagonizing ExoT-induced cell retraction. We also revealed a reciprocal interaction between T3SS effectors, showing that ExoT and ExoS, can in turn, suppress ExoY activity and its production of cGMP in certain cell types, thereby limiting their regulation by ExoY-synthesized cGMP. These findings highlight the intricate interplay between T3SS effectors and uncover a novel host cell-dependent regulation within P. aeruginosa infections.

microbiology↗

bioRxiv2023

Genome sequences of equine influenza A subtype H3N8 viruses by long read sequencing and functional characterization of the PB1-F2 virulence factor of A/equine/Paris/1/2018

Equine influenza virus (EIV) remains a persistent threat to equines, despite the availability of vaccines. Currently, strategies to monitor the virus and prevent any potential vaccine failure revolve around serological assays, RT-qPCR amplification, and sequencing the viral hemagglutinin (HA) and neuraminidase (NA) genes. These approaches overlook the contribution of other viral proteins in driving virulence. This study assesses the potential of long-read nanopore sequencing for swift and precise sequencing of circulating equine influenza viruses. To this end, two French Florida Clade 1 strains, including the one circulating in winter 2018-2019 exhibiting more pronounced pathogenicity than usual, as well as the two currently used OIE-recommended vaccine strains, were sequenced. Our results demonstrated the reliability of this sequencing method in generating accurate sequences. Sequence analysis of HA revealed a subtle antigenic drift in the French EIV strains, with specific substitutions, such as T163I in A/equine/Paris/1/2018 and the N188T mutation in post-2015 strains; both substitutions were located in antigenic site B. Antigenic site E exhibited modifications in post-2018 strains, with the N63D substitution. Segment 2 sequencing also revealed that the A/equine/Paris/1/2018 strain encodes a longer variant of the PB1-F2 protein when compared to other Florida clade 1 strains (90 amino acids long versus 81 amino acids long). Further biological and biochemistry assays demonstrated that this PB1-F2 variant has enhanced abilities to abolish the mitochondrial membrane potential {Delta}{Psi}m and permeabilize synthetic membranes. Altogether, our results highlight the interest in rapidly characterizing the complete genome of circulating strains with next-generation sequencing technologies to adapt vaccines and identify specific virulence markers of EIV.

microbiology↗

bioRxiv2023

Functional and structural insights into the interaction and multi-step activation of bacterial ExoY nucleotidyl cyclases toxins by profilin-bound G-actin

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 are actin-activated NC toxins. Here, we investigate 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) rather than filamentous (F-actin) actin. Vn-ExoY binds with only modest affinity to free or profilin-bound G-actin, but can capture the G-actin:profilin complex for its own activation by preventing the spontaneous or VASP- or formin-mediated assembly of G-actin:profilin at the barbed ends of F-actin in vitro. This may prolong the lifetime of the cofactor-bound state of Vn-ExoY at sites of active actin cytoskeleton remodelling. 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 show that the cofactor only partially stabilises the nucleotide-binding pocket (NBP) of all NC toxins. Substrate binding promotes a large, previously-unidentified, closure of their NBP. This confines catalytically important residues of the NC toxins and metal cofactors around the substrate and promotes the recruitment of two metal ions to tightly coordinate the triphosphate moiety of purine or pyrimidine nucleotide substrates. Residues that play an important role in both the purinyl and pyrimidinyl cyclase activity of NC toxins are validated in Vn-ExoY and the distantly-related ExoY from Pseudomonas aeruginosa that interact with F-actin. The data conclusively demonstrate that NC toxins employ a similar two-metal-ion mechanism for catalysing the cyclisation reaction 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 open up new perspectives for identifying ways to specifically inhibit these bacterial NC enzymes. Author SummaryExoY toxins belong to a family of bacterial nucleotidyl cyclases (NCs) that are injected into eukaryotic cells and bind to specific host cofactors to trigger their toxic, potent NC enzymatic activity. They alter host cell signalling by overproducing purine and pyrimidine cyclic nucleotides, which act as canonical and non-canonical intracellular messengers, respectively. The molecular and mechanistic details underlying the activation and catalytic specificities of NC toxins are only partially understood. Here, we investigate ExoY-type members that are unable to interact with actin filaments for their activation. We show in vitro that such ExoYs capture the actin:profilin complex for activation by disrupting its association with the most dynamic ends of actin filaments. We have captured several structural snapshots along the Vn-ExoY activation pathway by G-actin or G-actin-profilin without or with purine or pyrimidine nucleotide analogues. Our structural data reveal unprecedented mechanistic details of how the active site of all NC toxins is sequentially remodelled by cofactor and substrate binding, how they can accommodate nucleotides of different sizes as substrates, and elucidate important features of their catalytic reaction. These structural insights into the multi-step activation of NC toxins provide new perspectives for identifying ways to specifically inhibit this class of NC enzymes.

biophysics↗

bioRxiv2020

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

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.

biophysics↗