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1. Actin activates Pseudomonas aeruginosa ExoY nucleotidyl cyclase toxin and ExoY-like effector domains from MARTX toxins

Author

Undine Mechold, Abdelkader Namane, Cosmin Saveanu, Vasily Ogryzko, Alexander Belyy, Daniel Ladant, Lina Worpenberg, Souad Fellous, Dorothée Raoux-Barbot, Christien Merrifield, Elodie Assayag, Violaine David, Louis Renault, Véronique Henriot, Biochimie des Interactions Macromoléculaires / Biochemistry of Macromolecular Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Gamaleya Research Center, Génétique des Interactions Macromoléculaires, Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), 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), This project was funded by the Institut Pasteur under #PTR425 to U.M. and C.S. and by CNRS. A.B. was supported by a stipend from the Pasteur—Paris University (PPU) International PhD Program. We acknowledge the core facilities of Imagerie-Gif,(http://www.i2bc.paris-saclay.fr), supported by ‘France-BioImaging’ (ANR-10-INBS-04-01). This paper is dedicated to the memory of Vasily Ogryzko (1960–2016)., ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Biochimie des Interactions Macromoléculaires, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique ( CNRS ), Signalisation, noyaux et innovations en cancérologie ( UMR8126 ), Université Paris-Sud - Paris 11 ( UP11 ) -Institut Gustave Roussy ( IGR ) -Centre National de la Recherche Scientifique ( CNRS ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), and ANR-10-INBS-04-01/10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée ( 2010 )

Subjects
0301 basic medicine, Science, Bacterial Toxins, General Physics and Astronomy, macromolecular substances, Saccharomyces cerevisiae, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, [ SDV.MP.BAC ] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Filamentous actin, Cyclase, Article, General Biochemistry, Genetics and Molecular Biology, Type three secretion system, Adenylyl cyclase, 03 medical and health sciences, Cyclic nucleotide, chemistry.chemical_compound, Bacterial Proteins, ExoY, Type III Secretion Systems, Adenylate cyclase, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Actin, Multidisciplinary, Virulence, Mass spectrometry, [ SDV.BC ] Life Sciences [q-bio]/Cellular Biology, Chemistry, Effector, General Chemistry, Transfection, Guanylate cyclase, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Actins, 3. Good health, Cell biology, 030104 developmental biology, Glucosyltransferases, Mutation, Pseudomonas aeruginosa, Protein Binding
Abstract

The nucleotidyl cyclase toxin ExoY is one of the virulence factors injected by the Pseudomonas aeruginosa type III secretion system into host cells. Inside cells, it is activated by an unknown eukaryotic cofactor to synthesize various cyclic nucleotide monophosphates. ExoY-like adenylate cyclases are also found in Multifunctional-Autoprocessing Repeats-in-ToXin (MARTX) toxins produced by various Gram-negative pathogens. Here we demonstrate that filamentous actin (F-actin) is the hitherto unknown cofactor of ExoY. Association with F-actin stimulates ExoY activity more than 10,000 fold in vitro and results in stabilization of actin filaments. ExoY is recruited to actin filaments in transfected cells and alters F-actin turnover. Actin also activates an ExoY-like adenylate cyclase MARTX effector domain from Vibrio nigripulchritudo. Finally, using a yeast genetic screen, we identify actin mutants that no longer activate ExoY. Our results thus reveal a new sub-group within the class II adenylyl cyclase family, namely actin-activated nucleotidyl cyclase (AA-NC) toxins., The ExoY toxin is injected by Pseudomonas aeruginosa into host cells, where it is activated by an unknown host factor. Here the authors identify such factor as filamentous actin.

Published
2016

2. Mus spretus SEG/Pas mice resist virulent Yersinia pestis, under multigenic control

Author

C. Fayolle, Elisabeth Carniel, Marek Szatanik, Geneviève Milon, Charlène Blanchet, Jean-Jacques Panthier, Xavier Montagutelli, Jean Jaubert, Génétique Fonctionnelle de la Souris, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Yersinia, Institut Pasteur [Paris] (IP), Immunophysiologie et Parasitisme Intracellulaire, CB is a recipient of a DGA (Délégation Générale pour l’Armement) fellowship. This work was supported by a grant from Aventis Pharma (Sanofi-Aventis group) and Bayer Pharma as part of a multi-organism call for proposal. The Mouse functional Genetics Unit is supported by Merck Serono., We thank Jean-Louis Guénet for his constant interest and challenging discussions. We are grateful to Colin Crist for critical reading and suggestions on the paper, to Isabelle Lanctin for careful breeding of the SEG/Pas colony, and to the CNG (Centre National de Génotypage) for the SNP genotyping of BC mice., Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris]

Subjects
Male, Yersinia pestis, Mus spretus, Quantitative Trait Loci, Immunology, MESH: Yersinia pestis, Virulence, Quantitative trait locus, Mice, 03 medical and health sciences, Species Specificity, Genetics, Animals, MESH: Species Specificity, MESH: Animals, Allele, MESH: Mice, Genetics (clinical), 030304 developmental biology, 0303 health sciences, [SDV.GEN]Life Sciences [q-bio]/Genetics, biology, 030306 microbiology, Wild type, biology.organism_classification, Phenotype, Immunity, Innate, MESH: Male, MESH: Quantitative Trait Loci, 3. Good health, Backcrossing, Female, MESH: Immunity, Innate, MESH: Female
Abstract

International audience; Laboratory mice are well known to be highly susceptible to virulent strains of Yersinia pestis in experimental models of bubonic plague. We have found that Mus spretus-derived SEG/Pas (SEG) mice are exceptionally resistant to virulent CO92 and 6/69 wild type strains. Upon subcutaneous injection of 10(2) colony-forming units (CFU), 90% of females and 68% of males survived, compared with only an 8% survival rate for both male and female C57BL/6 mice. Furthermore, half of the SEG mice survived a challenge of up to 10(7) CFU. The time required for mortality was similar between B6 and SEG, suggesting that survival is dependent on early rather than late processes. The analysis of 322 backcross mice identified three significant quantitative trait loci (QTLs) on chromosomes 3, 4 and 6, with dominant SEG protective alleles. Each QTL increased the survival rate by approximately 20%. The three QTLs function additively, thereby accounting for 67% of the difference between the parental phenotypes. Mice heterozygous for the three QTLs were just as resistant as SEG mice to Y. pestis challenge. The SEG strain therefore offers an invaluable opportunity to unravel mechanisms and underlying genetic factors of resistance against Y. pestis infection.

Published
2011

3. Shigella flexneri 2a strain 2457T expresses three members of the H-NS-like protein family: characterization of the Sfh protein

Author

Christophe Beloin, Padraig Deighan, Charles J. Dorman, Marie Doyle, Génétique des Biofilms, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), University of Dublin, This work was supported by European Training and Mobility of Researchers Award ERBFMRXCT98-0164, Wellcome Trust Award 064284/Z01/Z and Enterprise Ireland Award SC/2002/22. P. Deighan was supported by a Wellcome Trust Prize Fellowship. The research described in this paper was performed in compliance with current laws governing genetic experimentation in the Republic of Ireland, We thank Philippe Bertin for the E. coli strains BE1414 and YK3421, Maria Mavris for S. flexneri strains, and Pablo Garcia Bravo and Antonio Cerqueira for help with protein purification., and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)

Subjects
MESH: Operon, MESH: Shigella flexneri, Protein family, [SDV]Life Sciences [q-bio], Virulence, Plasmid R27, Shigella flexneri, H-NS protein, 03 medical and health sciences, Plasmid, Bacterial Proteins, MESH: Plasmids, Operon, Genetics, Escherichia coli, MESH: Phylogeny, MESH: Bacterial Proteins, Molecular Biology, Gene, Phylogeny, 030304 developmental biology, Regulator gene, Regulation of gene expression, MESH: Gene Expression Regulation, Bacterial, 0303 health sciences, biology, MESH: Escherichia coli, 030306 microbiology, Structural gene, Sfh protein, General Medicine, Gene Expression Regulation, Bacterial, biology.organism_classification, Molecular biology, DNA-Binding Proteins, StpA protein, MESH: DNA-Binding Proteins, Plasmids
Abstract

Shigella flexneri 2a is known to express the H-NS nucleoid-structuring protein and the paralogous protein StpA. Using bioinformatic analysis we have now discovered a third member of the H-NS protein family, Sfh (S higella f lexneri H-NS-like protein), in strain 2457T. This protein is encoded by the sfh gene, which is located on a high-molecular-mass plasmid that is closely related to the self-transmissible plasmid R27. When expressed in Escherichia coli, the Sfh protein can complement an hns null mutation, restoring wild-type Bgl, porin protein, and mucoidy phenotypes, and wild-type expression of the fliC and proU genes. While a knockout mutation in the sfh gene alone had no effect on the expression of virulence genes in S. flexneri, an additive effect on virulence gene derepression was seen when the sfh lesion was combined with a mutation in hns. Over-expression of the sfh gene repressed expression of the VirB virulence regulatory protein and transcription of a VirB-dependent structural gene promoter. The purified Sfh protein bound specifically to DNA sequences containing the promoters of the virF and virB virulence regulatory genes. These findings show that Sfh has the ability to influence genetic events beyond the genetic element that encodes it, including the expression of the S. flexneri virulence genes. They raise the possibility of a triangular relationship among three closely related proteins with broad consequences for genetic events in the bacterium that harbours them.

Published
2003