The extreme C terminus of the Pseudomonas aeruginosa effector ExoY is crucial for binding to its eukaryotic activator, F-actin
| Autor: | Daniel Ladant, Ignacio Santecchia, Blandine Bourigault, Alexander Belyy, Undine Mechold, Louis Renault |
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| Přispěvatelé: | Biochimie des Interactions Macromoléculaires / Biochemistry of Macromolecular Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Biologie et Génétique de la Paroi bactérienne - Biology and Genetics of Bacterial Cell Wall (BGPB), Institut Pasteur [Paris] (IP), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] |
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
[SDV]Life Sciences [q-bio] Virulence GTPase medicine.disease_cause confocal microscopy Biochemistry type III secretion system (T3SS) protein-protein interaction 03 medical and health sciences protein cross-linking cyclic nucleotide ExoY pertussis adenylyl-cyclase medicine bacterial toxin Pseudomonas aeruginosa (P. aeruginosa) toxin genes Molecular Biology Gene filaments Actin domains aeruginosa) Activator (genetics) Pseudomonas aeruginosa Effector Chemistry gtpases C-terminus Pseudomonas aeruginosa (P structural basis Cell Biology 030104 developmental biology yeast actin protein |
| Zdroj: | Journal of Biological Chemistry Journal of Biological Chemistry, 2018, 293 (51), pp.19785--19796. ⟨10.1074/jbc.RA118.003784⟩ Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2018, 293 (51), pp.19785--19796. ⟨10.1074/jbc.RA118.003784⟩ |
| ISSN: | 0021-9258 1083-351X |
| Popis: | International audience; 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 35-cyclic nucleoside monophosphates. The activity of the effector ExoY from Pseudomonas aeruginosa 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 in vitro and toxicity in a yeast model. We observed that isolated C-terminal sequences of P. aeruginosa ExoY that had been fused to a carrier protein bind to F-actin and that synthetic peptides corresponding to the extreme ExoY C terminus inhibit ExoY enzymatic activity in vitro and compete with the full-length enzyme for F-actin binding. Interestingly, we noted that various P. aeruginosa isolates of the PA14 family, including highly virulent strains, harbor ExoY variants with a mutation altering the C terminus of this effector. We found that these naturally occurring ExoY variants display drastically reduced enzymatic activity and toxicity. Our findings shed light on the molecular basis of the ExoY-F-actin interaction, revealing that the extreme C terminus of ExoY is critical for binding to F-actin in target cells and that some P. aeruginosa isolates carry C-terminally mutated, low-activity ExoY variants. |
| Databáze: | OpenAIRE |
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