1887

Abstract

The aquatic zoonotic pathogen represents a threat to the worldwide aquaculture industry and poses a risk to humans who handle raw fish. Because little is known about the mechanisms of pathogenesis or virulence factors, we established a high-throughput system combining whole-genome pyrosequencing and transposon mutagenesis that allowed us to identify virulence proteins, including Pdi, the polysaccharide deacetylase of , that we describe here. Using bioinformatics tools, we identified a highly conserved signature motif in Pdi that is also conserved in the peptidoglycan deacetylase PgdA protein family. A Δ mutant was attenuated for virulence in the hybrid striped bass model and for survival in whole fish blood. Moreover, Pdi was found to promote bacterial resistance to lysozyme killing and the ability to adhere to and invade epithelial cells. On the other hand, there was no difference in the autolytic potential, resistance to oxidative killing or resistance to cationic antimicrobial peptides between wild-type and Δ. In conclusion, we have demonstrated that is involved in adherence and invasion, lysozyme resistance and survival in fish blood, and have shown that plays a role in the pathogenesis of . Identification of Pdi and other virulence proteins is a necessary initial step towards the development of appropriate preventive and therapeutic measures against diseases and economic losses caused by this pathogen.

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2010-02-01
2019-10-15
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References

  1. Agnew, W. & Barnes, A. C. ( 2007; ). Streptococcus iniae: an aquatic pathogen of global veterinary significance and a challenging candidate for reliable vaccination. Vet Microbiol 122, 1–15.[CrossRef]
    [Google Scholar]
  2. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. ( 1997; ). Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402.[CrossRef]
    [Google Scholar]
  3. Apweiler, R., Attwood, T. K., Bairoch, A., Bateman, A., Birney, E., Biswas, M., Bucher, P., Cerutti, L., Corpet, F. & other authors ( 2001; ). The InterPro database, an integrated documentation resource for protein families, domains and functional sites. Nucleic Acids Res 29, 37–40.[CrossRef]
    [Google Scholar]
  4. Arancibia, S. A., Beltran, C. J., Aguirre, I. M., Silva, P., Peralta, A. L., Malinarich, F. & Hermoso, M. A. ( 2007; ). Toll-like receptors are key participants in innate immune responses. Biol Res 40, 97–112.
    [Google Scholar]
  5. Aziz, R. K., Bartels, D., Best, A. A., DeJongh, M., Disz, T., Edwards, R. A., Formsma, K., Gerdes, S., Glass, E. M. & other authors ( 2008; ). The RAST server: rapid annotations using subsystems technology. BMC Genomics 9, 75 [CrossRef]
    [Google Scholar]
  6. Baiano, J. C., Tumbol, R. A., Umapathy, A. & Barnes, A. C. ( 2008; ). Identification and molecular characterisation of a fibrinogen binding protein from Streptococcus iniae. BMC Microbiol 8, 67 [CrossRef]
    [Google Scholar]
  7. Barnes, A. C., Young, F. M., Horne, M. T. & Ellis, A. E. ( 2003; ). Streptococcus iniae: serological differences, presence of capsule and resistance to immune serum killing. Dis Aquat Organ 53, 241–247.[CrossRef]
    [Google Scholar]
  8. Bendtsen, J. D., Nielsen, H., von Heijne, G. & Brunak, S. ( 2004; ). Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340, 783–795.[CrossRef]
    [Google Scholar]
  9. Blair, D. E., Schuttelkopf, A. W., MacRae, J. I. & van Aalten, D. M. ( 2005; ). Structure and metal-dependent mechanism of peptidoglycan deacetylase, a streptococcal virulence factor. Proc Natl Acad Sci U S A 102, 15429–15434.[CrossRef]
    [Google Scholar]
  10. Boneca, I. G., Dussurget, O., Cabanes, D., Nahori, M. A., Sousa, S., Lecuit, M., Psylinakis, E., Bouriotis, V., Hugot, J. P. & other authors ( 2007; ). A critical role for peptidoglycan N-deacetylation in Listeria evasion from the host innate immune system. Proc Natl Acad Sci U S A 104, 997–1002.[CrossRef]
    [Google Scholar]
  11. Buchanan, J. T., Stannard, J. A., Lauth, X., Ostland, V. E., Powell, H. C., Westerman, M. E. & Nizet, V. ( 2005; ). Streptococcus iniae phosphoglucomutase is a virulence factor and a target for vaccine development. Infect Immun 73, 6935–6944.[CrossRef]
    [Google Scholar]
  12. Buchanan, J. T., Simpson, A. J., Aziz, R. K., Liu, G. Y., Kristian, S. A., Kotb, M., Feramisco, J. & Nizet, V. ( 2006; ). DNase expression allows the pathogen group A Streptococcus to escape killing in neutrophil extracellular traps. Curr Biol 16, 396–400.[CrossRef]
    [Google Scholar]
  13. Datta, V., Myskowski, S. M., Kwinn, L. A., Chiem, D. N., Varki, N., Kansal, R. G., Kotb, M. & Nizet, V. ( 2005; ). Mutational analysis of the group A streptococcal operon encoding streptolysin S and its virulence role in invasive infection. Mol Microbiol 56, 681–695.[CrossRef]
    [Google Scholar]
  14. Dereeper, A., Guignon, V., Blanc, G., Audic, S., Buffet, S., Chevenet, F., Dufayard, J. F., Guindon, S., Lefort, V. & other authors ( 2008; ). Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res 36, W465–W469.[CrossRef]
    [Google Scholar]
  15. Eyngor, M., Chilmonczyk, S., Zlotkin, A., Manuali, E., Lahav, D., Ghittino, C., Shapira, R., Hurvitz, A. & Eldar, A. ( 2007; ). Transcytosis of Streptococcus iniae through skin epithelial barriers: an in vitro study. FEMS Microbiol Lett 277, 238–248.[CrossRef]
    [Google Scholar]
  16. Facklam, R., Elliott, J., Shewmaker, L. & Reingold, A. ( 2005; ). Identification and characterization of sporadic isolates of Streptococcus iniae isolated from humans. J Clin Microbiol 43, 933–937.[CrossRef]
    [Google Scholar]
  17. Felsenstein, J. ( 1997; ). An alternating least squares approach to inferring phylogenies from pairwise distances. Syst Biol 46, 101–111.[CrossRef]
    [Google Scholar]
  18. Finn, R. D., Tate, J., Mistry, J., Coggill, P. C., Sammut, S. J., Hotz, H. R., Ceric, G., Forslund, K., Eddy, S. R. & other authors ( 2008; ). The Pfam protein families database. Nucleic Acids Res 36, D281–D288.[CrossRef]
    [Google Scholar]
  19. Fuller, J. D., Camus, A. C., Duncan, C. L., Nizet, V., Bast, D. J., Thune, R. L., Low, D. E. & De Azavedo, J. C. ( 2002; ). Identification of a streptolysin S-associated gene cluster and its role in the pathogenesis of Streptococcus iniae disease. Infect Immun 70, 5730–5739.[CrossRef]
    [Google Scholar]
  20. Gallo, R. L. & Nizet, V. ( 2003; ). Endogenous production of antimicrobial peptides in innate immunity and human disease. Curr Allergy Asthma Rep 3, 402–409.[CrossRef]
    [Google Scholar]
  21. Gordon, D., Abajian, C. & Green, P. ( 1998; ). Consed: a graphical tool for sequence finishing. Genome Res 8, 195–202.[CrossRef]
    [Google Scholar]
  22. Higgins, D. G., Thompson, J. D. & Gibson, T. J. ( 1996; ). Using clustal for multiple sequence alignments. Methods Enzymol 266, 383–402.
    [Google Scholar]
  23. Hooper, S. D. & Berg, O. G. ( 2003; ). On the nature of gene innovation: duplication patterns in microbial genomes. Mol Biol Evol 20, 945–954.[CrossRef]
    [Google Scholar]
  24. Karlyshev, A. V., Pallen, M. J. & Wren, B. W. ( 2000; ). Single-primer PCR procedure for rapid identification of transposon insertion sites. Biotechniques 28, 1078, 1080, 1082
    [Google Scholar]
  25. Kristian, S. A., Datta, V., Weidenmaier, C., Kansal, R., Fedtke, I., Peschel, A., Gallo, R. L. & Nizet, V. ( 2005; ). d-Alanylation of teichoic acids promotes group A Streptococcus antimicrobial peptide resistance, neutrophil survival, and epithelial cell invasion. J Bacteriol 187, 6719–6725.[CrossRef]
    [Google Scholar]
  26. Lau, S. K., Woo, P. C., Tse, H., Leung, K. W., Wong, S. S. & Yuen, K. Y. ( 2003; ). Invasive Streptococcus iniae infections outside North America. J Clin Microbiol 41, 1004–1009.[CrossRef]
    [Google Scholar]
  27. Lau, S. K., Woo, P. C., Luk, W. K., Fung, A. M., Hui, W. T., Fong, A. H., Chow, C. W., Wong, S. S. & Yuen, K. Y. ( 2006; ). Clinical isolates of Streptococcus iniae from Asia are more mucoid and β-hemolytic than those from North America. Diagn Microbiol Infect Dis 54, 177–181.[CrossRef]
    [Google Scholar]
  28. Lauth, X., Shike, H., Burns, J. C., Westerman, M. E., Ostland, V. E., Carlberg, J. M., Van Olst, J. C., Nizet, V., Taylor, S. W. & other authors ( 2002; ). Discovery and characterization of two isoforms of moronecidin, a novel antimicrobial peptide from hybrid striped bass. J Biol Chem 277, 5030–5039.[CrossRef]
    [Google Scholar]
  29. Liu, G. Y., Doran, K. S., Lawrence, T., Turkson, N., Puliti, M., Tissi, L. & Nizet, V. ( 2004; ). Sword and shield: linked group B streptococcal β-hemolysin/cytolysin and carotenoid pigment function to subvert host phagocyte defense. Proc Natl Acad Sci U S A 101, 14491–14496.[CrossRef]
    [Google Scholar]
  30. Locke, J. B., Colvin, K. M., Datta, A. K., Patel, S. K., Naidu, N. N., Neely, M. N., Nizet, V. & Buchanan, J. T. ( 2007a; ). Streptococcus iniae capsule impairs phagocytic clearance and contributes to virulence in fish. J Bacteriol 189, 1279–1287.[CrossRef]
    [Google Scholar]
  31. Locke, J. B., Colvin, K. M., Varki, N., Vicknair, M. R., Nizet, V. & Buchanan, J. T. ( 2007b; ). Streptococcus iniae β-hemolysin streptolysin S is a virulence factor in fish infection. Dis Aquat Organ 76, 17–26.[CrossRef]
    [Google Scholar]
  32. Locke, J. B., Aziz, R. K., Vicknair, M. R., Nizet, V. & Buchanan, J. T. ( 2008; ). Streptococcus iniae M-like protein contributes to virulence in fish and is a target for live attenuated vaccine development. PLoS One 3, e2824 [CrossRef]
    [Google Scholar]
  33. Lowe, B. A., Miller, J. D. & Neely, M. N. ( 2007; ). Analysis of the polysaccharide capsule of the systemic pathogen Streptococcus iniae and its implications in virulence. Infect Immun 75, 1255–1264.[CrossRef]
    [Google Scholar]
  34. McNeil, L. K., Reich, C., Aziz, R. K., Bartels, D., Cohoon, M., Disz, T., Edwards, R. A., Gerdes, S., Hwang, K. & other authors ( 2007; ). The National Microbial Pathogen Database Resource (NMPDR): a genomics platform based on subsystem annotation. Nucleic Acids Res 35, D347–D353.[CrossRef]
    [Google Scholar]
  35. Meyrand, M., Boughammoura, A., Courtin, P., Mezange, C., Guillot, A. & Chapot-Chartier, M. P. ( 2007; ). Peptidoglycan N-acetylglucosamine deacetylation decreases autolysis in Lactococcus lactis. Microbiology 153, 3275–3285.[CrossRef]
    [Google Scholar]
  36. Miller, J. D. & Neely, M. N. ( 2005; ). Large-scale screen highlights the importance of capsule for virulence in the zoonotic pathogen Streptococcus iniae. Infect Immun 73, 921–934.[CrossRef]
    [Google Scholar]
  37. Nakano, Y., Yoshida, Y., Yamashita, Y. & Koga, T. ( 1995; ). Construction of a series of pACYC-derived plasmid vectors. Gene 162, 157–158.[CrossRef]
    [Google Scholar]
  38. Nigro, G., Fazio, L. L., Martino, M. C., Rossi, G., Tattoli, I., Liparoti, V., De Castro, C., Molinaro, A., Philpott, D. J. & Bernardini, M. L. ( 2008; ). Muramylpeptide shedding modulates cell sensing of Shigella flexneri. Cell Microbiol 10, 682–695.[CrossRef]
    [Google Scholar]
  39. Overbeek, R., Begley, T., Butler, R. M., Choudhuri, J. V., Chuang, H. Y., Cohoon, M., de Crécy-Lagard, V., Diaz, N., Disz, T. & other authors ( 2005; ). The subsystems approach to genome annotation and its use in the project to annotate 1000 genomes. Nucleic Acids Res 33, 5691–5702.[CrossRef]
    [Google Scholar]
  40. Psylinakis, E., Boneca, I. G., Mavromatis, K., Deli, A., Hayhurst, E., Foster, S. J., Varum, K. M. & Bouriotis, V. ( 2005; ). Peptidoglycan N-acetylglucosamine deacetylases from Bacillus cereus, highly conserved proteins in Bacillus anthracis. J Biol Chem 280, 30856–30863.[CrossRef]
    [Google Scholar]
  41. Rajam, G., Phillips, D. J., White, E., Anderton, J., Hooper, C. W., Sampson, J. S., Carlone, G. M., Ades, E. W. & Romero-Steiner, S. ( 2008; ). A functional epitope of the pneumococcal surface adhesin A activates nasopharyngeal cells and increases bacterial internalization. Microb Pathog 44, 186–196.[CrossRef]
    [Google Scholar]
  42. Shimizu, C., Shike, H., Malicki, D. M., Breisch, E., Westerman, M., Buchanan, J., Ligman, H. R., Phillips, R. B., Carlberg, J. M. & other authors ( 2003; ). Characterization of a white bass (Morone chrysops) embryonic cell line with epithelial features. In Vitro Cell Dev Biol Anim 39, 29–35.[CrossRef]
    [Google Scholar]
  43. Soehnlein, O., Kenne, E., Rotzius, P., Eriksson, E. E. & Lindbom, L. ( 2008; ). Neutrophil secretion products regulate anti-bacterial activity in monocytes and macrophages. Clin Exp Immunol 151, 139–145.
    [Google Scholar]
  44. Subramaniam, S. ( 1998; ). The Biology Workbench – a seamless database and analysis environment for the biologist. Proteins 32, 1–2.[CrossRef]
    [Google Scholar]
  45. Sun, J. R., Yan, J. C., Yeh, C. Y., Lee, S. Y. & Lu, J. J. ( 2007; ). Invasive infection with Streptococcus iniae in Taiwan. J Med Microbiol 56, 1246–1249.[CrossRef]
    [Google Scholar]
  46. Thompson, J. D., Higgins, D. G. & Gibson, T. J. ( 1994; ). clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.[CrossRef]
    [Google Scholar]
  47. Vollmer, W. & Tomasz, A. ( 2000; ). The pgdA gene encodes for a peptidoglycan N-acetylglucosamine deacetylase in Streptococcus pneumoniae. J Biol Chem 275, 20496–20501.[CrossRef]
    [Google Scholar]
  48. Vollmer, W. & Tomasz, A. ( 2002; ). Peptidoglycan N-acetylglucosamine deacetylase, a putative virulence factor in Streptococcus pneumoniae. Infect Immun 70, 7176–7178.[CrossRef]
    [Google Scholar]
  49. Vuong, C., Kocianova, S., Voyich, J. M., Yao, Y., Fischer, E. R., DeLeo, F. R. & Otto, M. ( 2004; ). A crucial role for exopolysaccharide modification in bacterial biofilm formation, immune evasion, and virulence. J Biol Chem 279, 54881–54886.[CrossRef]
    [Google Scholar]
  50. Weinstein, M. R., Litt, M., Kertesz, D. A., Wyper, P., Rose, D., Coulter, M., McGeer, A., Facklam, R., Ostach, C. & other authors ( 1997; ). Invasive infections due to a fish pathogen, Streptococcus iniae. N Engl J Med 337, 589–594.[CrossRef]
    [Google Scholar]
  51. Williamson, Y. M., Gowrisankar, R., Longo, D. L., Facklam, R., Gipson, I. K., Ades, E. P., Carlone, G. M. & Sampson, J. S. ( 2008; ). Adherence of nontypeable Streptococcus pneumoniae to human conjunctival epithelial cells. Microb Pathog 44, 175–185.[CrossRef]
    [Google Scholar]
  52. Zlotkin, A., Chilmonczyk, S., Eyngor, M., Hurvitz, A., Ghittino, C. & Eldar, A. ( 2003; ). Trojan horse effect: phagocyte-mediated Streptococcus iniae infection of fish. Infect Immun 71, 2318–2325.[CrossRef]
    [Google Scholar]
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