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
2024-03-28
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 [View Article][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 [View Article][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 [View Article][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 [View Article][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 [View Article][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 [View Article][PubMed]
    [Google Scholar]
  8. Cardelli J. ( 2001). Phagocytosis and macropinocytosis in Dictyostelium: phosphoinositide-based processes, biochemically distinct. Traffic 2:311–320 [View Article][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 [View Article][PubMed]
    [Google Scholar]
  10. Cornelis G. R. ( 2006). The type III secretion injectisome. Nat Rev Microbiol 4:811–825 [View Article][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 [View Article][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 [View Article][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 [View Article][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 [View Article][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 [View Article][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 [View Article][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 [View Article][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 [View Article][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 [View Article][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 [View Article][PubMed]
    [Google Scholar]
  22. Hiney M., Olivier G. ( 1999). Furunculosis (Aeromonas salmonicida). Fish Diseases and Disorders III: Viral, Bacterial and Fungal Infections341–425 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 [View Article][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 [View Article]
    [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 [View Article][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 [View Article][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 [View Article][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 [View Article][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 [View Article][PubMed]
    [Google Scholar]
  31. Preston G. M. ( 2007). Metropolitan microbes: type III secretion in multihost symbionts. Cell Host Microbe 2:291–294 [View Article][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 [View Article][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 [View Article][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 [View Article][PubMed]
    [Google Scholar]
  35. UniProt Consortium ( 2012). Reorganizing the protein space at the Universal Protein Resource (UniProt). Nucleic Acids Res 40:Database issueD71–D75 [View Article][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 [View Article][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 [View Article][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 [View Article][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 [View Article][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 [View Article][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 [View Article][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