Many pathogens manipulate host cell cAMP signaling pathways to promote their survival and proliferation. Bacterial Exoenzyme Y (ExoY) toxins belong to a family of invasive, structurally-related bacterial nucleotidyl cyclases (NC). Inactive in bacteria, they use proteins that are uniquely and abundantly present in eukaryotic cells to become potent, unregulated NC enzymes in host cells. Other well-known members of the family include Bacillus anthracis Edema Factor (EF) and Bordetella pertussis CyaA. Once bound to their eukaryotic protein cofactor, they can catalyze supra-physiological levels of various cyclic nucleotide monophosphates in infected cells. Originally identified in Pseudomonas aeruginosa, ExoY-related NC toxins appear now to be more widely distributed among various γ- and β-proteobacteria. ExoY-like toxins represent atypical, poorly characterized members within the NC toxin family. While the NC catalytic domains of EF and CyaA toxins use both calmodulin as cofactor, their counterparts in ExoY-like members from pathogens of the genus Pseudomonas or Vibrio use actin as a potent cofactor, in either its monomeric or polymerized form. This is an original subversion of actin for cytoskeleton-targeting toxins. Here, we review recent advances on the different members of the NC toxin family to highlight their common and distinct functional characteristics at the molecular, cytotoxic and enzymatic levels, and important aspects that need further characterizations.

The Type III Secretion System (T3SS) of Pseudomonas aeruginosa injects effector proteins into host cells to subvert cellular processes and promote infection. While the roles of several T3SS effectors in virulence are well established, the function of the nucleotidyl cyclase ExoY has remained elusive and debated. Here, we show that ExoY-produced cyclic GMP (cGMP) regulates the cytotoxic activity of the co-injected effector ExoT, thereby modulating host cell damage. Using engineered P. aeruginosa strains expressing ExoY variants with distinct substrate preferences, we demonstrate that cGMP production by ExoY limits ExoT-mediated dephosphorylation of the host adaptor protein CrkII, a downstream consequence of ExoT’s ADP-ribosyltransferase activity. This attenuation reduces ExoT-induced cell retraction and decreases bacterial virulence. In contrast, we find that ExoT and another T3SS effector, ExoS, can inhibit ExoY activity and cGMP production in certain cell types, thus limiting ExoY’s regulatory influence. These findings highlight the intricate interplay between T3SS effectors within host cells and reveal an unrecognized layer of ExoY-based complexity in P. aeruginosa ’s pathogenic strategy.

ExlA is a highly virulent pore-forming toxin that has been recently discovered in outlier strains from Pseudomonas aeruginosa. ExlA is part of a two-partner secretion system, in which ExlA is the secreted passenger protein and ExlB the transporter embedded in the bacterial outer membrane. In previous work, we observed that ExlA toxicity in a host cell was contact-dependent. Here, we show that ExlA accumulates at specific points of the outer membrane, is likely entrapped within ExlB pore, and is pointing outside. We further demonstrate that ExlA is maintained at the membrane in conditions where the intracellular content of second messenger cyclic-di-GMP is high; lowering c-di-GMP levels enhances ExlB-dependent ExlA secretion. In addition, we set up an ELISA to detect ExlA, and we show that ExlA is poorly secreted in liquid culture, while it is highly detectable in broncho-alveolar lavage fluids of mice infected with an exlA+ strain. We conclude that ExlA translocation is halted at mid-length in the outer membrane and its secretion is regulated by c-di-GMP. In addition, we developed an immunological test able to quantify ExlA in biological samples.

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.

Pseudomonas aeruginosa est une bactérie Gram-négative opportuniste, responsable de différents types d'infections, et notamment de maladies nosocomiales. Les infections aiguës causées par ce pathogène sont dues à un large panel de facteurs de virulence, dont le plus agressif est un système de sécrétion de type III. Parmi les quatre effecteurs injectés par ce système dans le cytoplasme des cellules cibles, ExoU est le plus cytotoxique. Les souches ExoU+ sont associées aux pathologies les plus sévères et à une multirésistance aux antibiotiques plus élevée. La toxine possède une activité phospholipase qui induit une nécrose cellulaire après rupture de la membrane plasmique. Cependant, le trafic de la toxine dans les cellules hôtes pour atteindre la membrane plasmique n’est pas connu.Dans ce travail, j’ai recherché des partenaires eucaryotes d’ExoU en réalisant un criblage génétique sur l’ensemble du génome humain, mettant en oeuvre le système CRISPR-Cas9. J’ai ainsi identifié le gène codant pour une protéine co-chaperone appelée DNAJC5. Cette protéine, localisée à la surface des endosomes tardifs, est impliquée dans un système de sécrétion non-conventionnel permettant le relargage de protéines cytosoliques mal repliées. Nous démontrons dans des modèles cellulaires et chez la drosophile, que l'absence de DNAJC5 confère une résistance à la nécrose induite par des souches ExoU+. Dans le cytoplasme, la toxine est localisée à la membrane des vésicules DNAJC5+. Des mutations dans le gène DNAJC5, empêchant le trafic des endosomes tardifs DNAJC5+ vers la membrane plasmique, entravent également la localisation d’ExoU à ce niveau et inhibent sa cytotoxicité. Nous concluons, qu'une fois administrée dans le cytoplasme de la cellule hôte, ExoU utilise des vésicules DNAJC5+, pour être transportée à la membrane plasmique, un processus strictement nécessaire à la nécrose cellulaire induite par la toxine.

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.