1887

Abstract

The bacterium , a fish pathogen, uses the type three secretion system (TTSS) to inject effector proteins into host cells to promote the infection. The study of the genome of has revealed the existence of Ati2, a potential TTSS effector protein. In the present study, a structure–function analysis of Ati2 has been done to determine its role in the virulence of . Biochemical assays revealed that Ati2 is secreted into the medium in a TTSS-dependent manner. Protein sequence analyses, molecular modelling and biochemical assays demonstrated that Ati2 is an inositol polyphosphate 5-phosphatase, which hydrolyses PtdIns(4,5)P and PtdIns(3,4,5)P in a way similar to VPA0450, a protein from having high sequence similarity with Ati2. Mutants of Ati2 with altered amino acids at two different locations in the catalytic site displayed no phosphatase activity. Wild-type and mutant forms of Ati2 were cloned into expression systems for , a soil amoeba used as an alternative host to study virulence. Expression tests allowed us to demonstrate that Ati2 is toxic for the host cell in a catalytic-dependent manner. Finally, this study demonstrated the existence of a new TTSS effector protein in .

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.067959-0
2013-09-01
2019-10-18
Loading full text...

Full text loading...

/deliver/fulltext/micro/159/9/1937.html?itemId=/content/journal/micro/10.1099/mic.0.067959-0&mimeType=html&fmt=ahah

References

  1. Altschul S. F. , Gish W. , Miller W. , Myers E. W. , Lipman D. J. . ( 1990; ). Basic local alignment search tool. . J Mol Biol 215:, 403–410.[PubMed] [CrossRef]
    [Google Scholar]
  2. Beitz E. . ( 2000; ). TEXshade: shading and labeling of multiple sequence alignments using LATEX2 epsilon. . Bioinformatics 16:, 135–139. [CrossRef] [PubMed]
    [Google Scholar]
  3. Broberg C. A. , Zhang L. , Gonzalez H. , Laskowski-Arce M. A. , Orth K. . ( 2010; ). A Vibrio effector protein is an inositol phosphatase and disrupts host cell membrane integrity. . Science 329:, 1660–1662. [CrossRef] [PubMed]
    [Google Scholar]
  4. Buczynski G. , Grove B. , Nomura A. , Kleve M. , Bush J. , Firtel R. A. , Cardelli J. . ( 1997; ). Inactivation of two Dictyostelium discoideum genes, DdPIK1 and DdPIK2, encoding proteins related to mammalian phosphatidylinositide 3-kinases, results in defects in endocytosis, lysosome to postlysosome transport, and actin cytoskeleton organization. . J Cell Biol 136:, 1271–1286. [CrossRef] [PubMed]
    [Google Scholar]
  5. Burr S. E. , Stuber K. , Wahli T. , Frey J. . ( 2002; ). Evidence for a type III secretion system in Aeromonas salmonicida subsp. salmonicida. . J Bacteriol 184:, 5966–5970. [CrossRef] [PubMed]
    [Google Scholar]
  6. Burr S. E. , Stuber K. , Frey J. . ( 2003; ). The ADP-ribosylating toxin, AexT, from Aeromonas salmonicida subsp. salmonicida is translocated via a type III secretion pathway. . J Bacteriol 185:, 6583–6591. [CrossRef] [PubMed]
    [Google Scholar]
  7. Büttner D. . ( 2012; ). Protein export according to schedule: architecture, assembly, and regulation of type III secretion systems from plant- and animal-pathogenic bacteria. . Microbiol Mol Biol Rev 76:, 262–310. [CrossRef] [PubMed]
    [Google Scholar]
  8. Cardelli J. . ( 2001; ). Phagocytosis and macropinocytosis in Dictyostelium: phosphoinositide-based processes, biochemically distinct. . Traffic 2:, 311–320. [CrossRef] [PubMed]
    [Google Scholar]
  9. Casadaban M. J. , Cohen S. N. . ( 1980; ). Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. . J Mol Biol 138:, 179–207. [CrossRef] [PubMed]
    [Google Scholar]
  10. Cornelis G. R. . ( 2006; ). The type III secretion injectisome. . Nat Rev Microbiol 4:, 811–825. [CrossRef] [PubMed]
    [Google Scholar]
  11. Cornillon S. , Pech E. , Benghezal M. , Ravanel K. , Gaynor E. , Letourneur F. , Brückert F. , Cosson P. . ( 2000; ). Phg1p is a nine-transmembrane protein superfamily member involved in Dictyostelium adhesion and phagocytosis. . J Biol Chem 275:, 34287–34292. [CrossRef] [PubMed]
    [Google Scholar]
  12. Costa G. L. , Bauer J. C. , McGowan B. , Angert M. , Weiner M. P. . ( 1996; ). Site-directed mutagenesis using a rapid PCR-based method. . Methods Mol Biol 57:, 239–248.[PubMed]
    [Google Scholar]
  13. Dacanay A. , Knickle L. , Solanky K. S. , Boyd J. M. , Walter J. A. , Brown L. L. , Johnson S. C. , Reith M. . ( 2006; ). Contribution of the type III secretion system (TTSS) to virulence of Aeromonas salmonicida subsp. salmonicida. . Microbiology 152:, 1847–1856. [CrossRef] [PubMed]
    [Google Scholar]
  14. Daher R. K. , Filion G. , Tan S. G. , Dallaire-Dufresne S. , Paquet V. E. , Charette S. J. . ( 2011; ). Alteration of virulence factors and rearrangement of pAsa5 plasmid caused by the growth of Aeromonas salmonicida in stressful conditions. . Vet Microbiol 152:, 353–360. [CrossRef] [PubMed]
    [Google Scholar]
  15. Dallaire-Dufresne S. , Paquet V. E. , Charette S. J. . ( 2011; ). Dictyostelium discoideum: a model for the study of bacterial virulence. . Can J Microbiol 57:, 699–707 (in French). [CrossRef] [PubMed]
    [Google Scholar]
  16. Dean P. . ( 2011; ). Functional domains and motifs of bacterial type III effector proteins and their roles in infection. . FEMS Microbiol Rev 35:, 1100–1125. [CrossRef] [PubMed]
    [Google Scholar]
  17. Dormann D. , Weijer G. , Dowler S. , Weijer C. J. . ( 2004; ). In vivo analysis of 3-phosphoinositide dynamics during Dictyostelium phagocytosis and chemotaxis. . J Cell Sci 117:, 6497–6509. [CrossRef] [PubMed]
    [Google Scholar]
  18. Ebanks R. O. , Knickle L. C. , Goguen M. , Boyd J. M. , Pinto D. M. , Reith M. , Ross N. W. . ( 2006; ). Expression of and secretion through the Aeromonas salmonicida type III secretion system. . Microbiology 152:, 1275–1286. [CrossRef] [PubMed]
    [Google Scholar]
  19. Fast M. D. , Tse B. , Boyd J. M. , Johnson S. C. . ( 2009; ). Mutations in the Aeromonas salmonicida subsp. salmonicida type III secretion system affect Atlantic salmon leucocyte activation and downstream immune responses. . Fish Shellfish Immunol 27:, 721–728. [CrossRef] [PubMed]
    [Google Scholar]
  20. Forsberg A. , Bölin I. , Norlander L. , Wolf-Watz H. . ( 1987; ). Molecular cloning and expression of calcium-regulated, plasmid-coded proteins of Y. pseudotuberculosis. . Microb Pathog 2:, 123–137. [CrossRef] [PubMed]
    [Google Scholar]
  21. Froquet R. , Cherix N. , Burr S. E. , Frey J. , Vilches S. , Tomas J. M. , Cosson P. . ( 2007; ). Alternative host model to evaluate Aeromonas virulence. . Appl Environ Microbiol 73:, 5657–5659. [CrossRef] [PubMed]
    [Google Scholar]
  22. Hiney M. , Olivier G. . ( 1999; ). Furunculosis (Aeromonas salmonicida). . In Fish Diseases and Disorders III: Viral, Bacterial and Fungal Infections, pp. 341–425. Edited by Woo P. , Bruno D. . . Oxford:: CAB Publishing;.
    [Google Scholar]
  23. Ibarra J. A. , Steele-Mortimer O. . ( 2009; ). Salmonella-the ultimate insider. Salmonella virulence factors that modulate intracellular survival. . Cell Microbiol 11:, 1579–1586. [CrossRef] [PubMed]
    [Google Scholar]
  24. Karlsson M. , Strid Å. , Sirsjö A. , Eriksson L. A. . ( 2008; ). Homology Models and molecular modeling of human retinoic acid metabolizing enzymes cytochrome P450 26A1 (CYP26A1) and P450 26B1 (CYP26B1). . J Chem Theory Comput 4:, 1021–1027. [CrossRef]
    [Google Scholar]
  25. Kortholt A. , King J. S. , Keizer-Gunnink I. , Harwood A. J. , Van Haastert P. J. . ( 2007; ). Phospholipase C regulation of phosphatidylinositol 3,4,5-trisphosphate-mediated chemotaxis. . Mol Biol Cell 18:, 4772–4779. [CrossRef] [PubMed]
    [Google Scholar]
  26. Lambert de Rouvroit C. , Sluiters C. , Cornelis G. R. . ( 1992; ). Role of the transcriptional activator, VirF, and temperature in the expression of the pYV plasmid genes of Yersinia enterocolitica. . Mol Microbiol 6:, 395–409. [CrossRef] [PubMed]
    [Google Scholar]
  27. Mercanti V. , Charette S. J. , Bennett N. , Ryckewaert J. J. , Letourneur F. , Cosson P. . ( 2006; ). Selective membrane exclusion in phagocytic and macropinocytic cups. . J Cell Sci 119:, 4079–4087. [CrossRef] [PubMed]
    [Google Scholar]
  28. Michel C. . ( 1979; ). Furunculosis of salmonids: vaccination attempts in rainbow trout (Salmo gairdneri) by formalin-killed germs. . Ann Rech Vet 10:, 33–40.[PubMed]
    [Google Scholar]
  29. Navarro L. , Alto N. M. , Dixon J. E. . ( 2005; ). Functions of the Yersinia effector proteins in inhibiting host immune responses. . Curr Opin Microbiol 8:, 21–27. [CrossRef] [PubMed]
    [Google Scholar]
  30. Notredame C. , Higgins D. G. , Heringa J. . ( 2000; ). T-Coffee: A novel method for fast and accurate multiple sequence alignment. . J Mol Biol 302:, 205–217. [CrossRef] [PubMed]
    [Google Scholar]
  31. Preston G. M. . ( 2007; ). Metropolitan microbes: type III secretion in multihost symbionts. . Cell Host Microbe 2:, 291–294. [CrossRef] [PubMed]
    [Google Scholar]
  32. Reith M. E. , Singh R. K. , Curtis B. , Boyd J. M. , Bouevitch A. , Kimball J. , Munholland J. , Murphy C. , Sarty D. . & other authors ( 2008; ). The genome of Aeromonas salmonicida subsp. salmonicida A449: insights into the evolution of a fish pathogen. . BMC Genomics 9:, 427. [CrossRef] [PubMed]
    [Google Scholar]
  33. Schmid A. C. , Wise H. M. , Mitchell C. A. , Nussbaum R. , Woscholski R. . ( 2004; ). Type II phosphoinositide 5-phosphatases have unique sensitivities towards fatty acid composition and head group phosphorylation. . FEBS Lett 576:, 9–13. [CrossRef] [PubMed]
    [Google Scholar]
  34. Stuber K. , Burr S. E. , Braun M. , Wahli T. , Frey J. . ( 2003; ). Type III secretion genes in Aeromonas salmonicida subsp salmonicida are located on a large thermolabile virulence plasmid. . J Clin Microbiol 41:, 3854–3856. [CrossRef] [PubMed]
    [Google Scholar]
  35. UniProt Consortium ( 2012; ). Reorganizing the protein space at the Universal Protein Resource (UniProt). . Nucleic Acids Res 40: (Database issue), D71–D75. [CrossRef] [PubMed]
    [Google Scholar]
  36. Veltman D. M. , Keizer-Gunnink I. , Haastert P. J. . ( 2009a; ). An extrachromosomal, inducible expression system for Dictyostelium discoideum. . Plasmid 61:, 119–125. [CrossRef] [PubMed]
    [Google Scholar]
  37. Veltman D. M. , Akar G. , Bosgraaf L. , Van Haastert P. J. . ( 2009b; ). A new set of small, extrachromosomal expression vectors for Dictyostelium discoideum. . Plasmid 61:, 110–118. [CrossRef] [PubMed]
    [Google Scholar]
  38. Vlahou G. , Schmidt O. , Wagner B. , Uenlue H. , Dersch P. , Rivero F. , Weissenmayer B. A. . ( 2009; ). Yersinia outer protein YopE affects the actin cytoskeleton in Dictyostelium discoideum through targeting of multiple Rho family GTPases. . BMC Microbiol 9:, 138. [CrossRef] [PubMed]
    [Google Scholar]
  39. Whisstock J. C. , Romero S. , Gurung R. , Nandurkar H. , Ooms L. M. , Bottomley S. P. , Mitchell C. A. . ( 2000; ). The inositol polyphosphate 5-phosphatases and the apurinic/apyrimidinic base excision repair endonucleases share a common mechanism for catalysis. . J Biol Chem 275:, 37055–37061. [CrossRef] [PubMed]
    [Google Scholar]
  40. Whisstock J. C. , Wiradjaja F. , Waters J. E. , Gurung R. . ( 2002; ). The structure and function of catalytic domains within inositol polyphosphate 5-phosphatases. . IUBMB Life 53:, 15–23. [CrossRef] [PubMed]
    [Google Scholar]
  41. Zouwail S. , Pettitt T. R. , Dove S. K. , Chibalina M. V. , Powner D. J. , Haynes L. , Wakelam M. J. , Insall R. H. . ( 2005; ). Phospholipase D activity is essential for actin localization and actin-based motility in Dictyostelium. . Biochem J 389:, 207–214. [CrossRef] [PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.067959-0
Loading
/content/journal/micro/10.1099/mic.0.067959-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error