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Spatial and temporal coordination of expression of immune response genes during Pseudomonas infection of horseshoe crab, Carcinoscorpius rotundicauda

Genes & Immunity volume 6, pages 557–574 (2005)Cite this article

Abstract

Knowledge on how genes are turned on/off during infection and immunity is lacking. Here, we report the coregulation of diverse clusters of functionally related immune response genes in a horseshoe crab, Carcinoscorpius rotundicauda. Expressed sequence tag (EST) clusters for frontline immune defense, cell signalling, apoptosis and stress response genes were expressed or repressed spatio-temporally during the acute phase of Pseudomonas infection. An infection time course monitored by virtual Northern evaluation indicates upregulation of genes in blood cells (amebocytes) at 3-h postinfection, whereas most of the hepatopancreas genes remained downregulated over 72 h of infection. Thus, the two tissues orchestrate a coordinated and timely response to infection. The hepatopancreas probably immunomodulates the expression of other genes and serves as a reservoir for later response, if/when chronic infection ensues. On the other hand, being the first to encounter pathogens, we reasoned that amebocytes would respond acutely to infection. Besides acute transactivation of the immune genes, the amebocytes maintained morphological integrity, indicating their ability to synthesise and store/secrete the immune proteins and effectors to sustain the frontline innate immune defense, while simultaneously elicit complement-mediated phagocytosis of the invading pathogen. Our results show that the immune response against Pseudomonas infection is spatially and temporally coordinated.

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References

  1. Beutler B . Innate immunity: an overview. Mol Immunol 2004; 40: 845–859.

    Article  CAS  PubMed  Google Scholar 

  2. Medzhitov R, Janeway Jr CA . How does the immune system distinguish self from nonself? Semin Immunol 2000; 12: 185–188; discussion 257–344.

    Article  CAS  PubMed  Google Scholar 

  3. Engstrom Y . Induction and regulation of antimicrobial peptides in Drosophila. Dev Comp Immunol 1999; 23: 345–358.

    Article  CAS  PubMed  Google Scholar 

  4. Lemaitre B, Reichhart JM, Hoffmann JA . Drosophila host defense: differential induction of antimicrobial peptide genes after infection by various classes of microorganisms. Proc Natl Acad Sci USA 1997; 94: 14614–14619.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Irving P, Troxler L, Heuer TS, Belvin M, Kopczynski C, Reichhart JM et al. A genome-wide analysis of immune responses in Drosophila. Proc Natl Acad Sci USA 2001; 98: 15119–15124.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. De Gregorio E, Spellman PT, Rubin GM, Lemaitre B . Genome-wide analysis of the Drosophila immune response by using oligonucleotide microarrays. Proc Natl Acad Sci USA 2001; 98: 12590–12595.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. De Gregorio E, Spellman PT, Tzou P, Rubin GM, Lemaitre B . The Toll and Imd pathways are the major regulators of the immune response in Drosophila. EMBO J 2002; 21: 2568–2579.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Dimopoulos G, Christophides GK, Meister S, Schultz J, White KP, Barillas-Mury C et al. Genome expression analysis of Anopheles gambiae: responses to injury, bacterial challenge, and malaria infection. Proc Natl Acad Sci USA 2002; 99: 8814–8819.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Ding JL, Navas MAr, Ho B . Two forms of factor C from the amoebocytes of Carcinoscorpius rotundicauda: purification and characterisation. Biochim Biophys Acta 1993; 1202: 149–156.

    Article  CAS  PubMed  Google Scholar 

  10. Ding JL, Navas III MA, Ho B . Molecular cloning and sequence analysis of factor C cDNA from the Singapore horseshoe crab, Carcinoscorpius rotundicauda. Mol Mar Biol Biotechnol 1995; 4: 90–103.

    CAS  PubMed  Google Scholar 

  11. Ding JL, Wang LH, Ho B . Current genome-wide analysis on serine proteases in innate immunity. Curr Genomics 2004; 5: 147–155.

    Article  CAS  Google Scholar 

  12. Iwanaga S, Kawabata S, Muta T . New types of clotting factors and defense molecules found in horseshoe crab hemolymph: their structures and functions. J Biochem (Tokyo) 1998; 123: 1–15.

    Article  CAS  Google Scholar 

  13. Tan NS, Ho B, Ding JL . High-affinity LPS binding domain(s) in recombinant factor C of a horseshoe crab neutralizes LPS-induced lethality. FASEB J 2000; 14: 859–870.

    Article  CAS  PubMed  Google Scholar 

  14. Ng ML, Tan SH, Ho B, Ding JL . The C-reactive protein: a predominant LPS-binding acute phase protein responsive to Pseudomonas infection. J Endotoxin Res 2004; 10: 163–174.

    Article  CAS  PubMed  Google Scholar 

  15. Yau YH, Ho B, Tan NS, Ng ML, Ding JL . High therapeutic index of factor C Sushi peptides: potent antimicrobials against Pseudomonas aeruginosa. Antimicrob Agents Chemother 2001; 45: 2820–2825.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Zhu Y, Thangamani S, Ho B, Ding JL . The ancient origin of the complement system. EMBO J 2005; 24: 382–394.

    Article  CAS  PubMed  Google Scholar 

  17. Kawabata S, Iwanaga S . Role of lectins in the innate immunity of horseshoe crab. Dev Comp Immunol 1999; 23: 391–400.

    Article  CAS  PubMed  Google Scholar 

  18. Armstrong PB . The contribution of proteinase inhibitors to immune defense. Trends Immunol 2001; 22: 47–52.

    Article  CAS  PubMed  Google Scholar 

  19. Iwanaga S . The molecular basis of innate immunity in the horseshoe crab. Curr Opin Immunol 2002; 14: 87–95.

    Article  CAS  PubMed  Google Scholar 

  20. Aaronson JS, Eckman B, Blevins RA, Borkowski JA, Myerson J, Imran S et al. Toward the development of a gene index to the human genome: an assessment of the nature of high-throughput EST sequence data. Genome Res 1996; 6: 829–845.

    Article  CAS  PubMed  Google Scholar 

  21. Mita K, Morimyo M, Okano K, Koike Y, Nohata J, Kawasaki H et al. The construction of an EST database for Bombyx mori and its application. Proc Natl Acad Sci USA 2003; 100: 14121–14126.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Seitz V, Clermont A, Wedde M, Hummel M, Vilcinskas A, Schlatterer K et al. Identification of immunorelevant genes from greater wax moth (Galleria mellonella) by a subtractive hybridization approach. Dev Comp Immunol 2003; 27: 207–215.

    Article  CAS  PubMed  Google Scholar 

  23. White O, Kerlavage AR . TDB: new databases for biological discovery. Methods Enzymol 1996; 266: 27–40.

    Article  CAS  PubMed  Google Scholar 

  24. Iwanaga S . The limulus clotting reaction. Curr Opin Immunol 1993; 5: 74–82.

    Article  CAS  PubMed  Google Scholar 

  25. Osaki T, Kawabata S . Structure and function of coagulogen, a clottable protein in horseshoe crabs. Cell Mol Life Sci 2004; 61: 1257–1265.

    Article  CAS  PubMed  Google Scholar 

  26. Volokhina EB, Hulshof R, Haanen C, Vermes I . Tissue transglutaminase mRNA expression in apoptotic cell death. Apoptosis 2003; 8: 673–679.

    Article  CAS  PubMed  Google Scholar 

  27. Melino G, Piacentini M . ‘Tissue’ transglutaminase in cell death: a downstream or a multifunctional upstream effector? FEBS Lett 1998; 430: 59–63.

    Article  CAS  PubMed  Google Scholar 

  28. Tokunaga F, Yamada M, Miyata T, Ding YL, Hiranaga-Kawabata M, Muta T et al. Limulus hemocyte transglutaminase. Its purification and characterization, and identification of the intracellular substrates. J Biol Chem 1993; 268: 252–261.

    CAS  PubMed  Google Scholar 

  29. Nagai T, Osaki T, Kawabata S . Functional conversion of hemocyanin to phenoloxidase by horseshoe crab antimicrobial peptides. J Biol Chem 2001; 276: 27166–27170.

    Article  CAS  PubMed  Google Scholar 

  30. Lee SY, Lee BL, Soderhall K . Processing of an antibacterial peptide from hemocyanin of the freshwater crayfish Pacifastacus leniusculus. J Biol Chem 2003; 278: 7927–7933.

    Article  CAS  PubMed  Google Scholar 

  31. Furie B, Furie BC . Molecular and cellular biology of blood coagulation. N Engl J Med 1992; 326: 800–806.

    Article  CAS  PubMed  Google Scholar 

  32. Reid KB, Porter RR . The proteolytic activation systems of complement. Annu Rev Biochem 1981; 50: 433–464.

    Article  CAS  PubMed  Google Scholar 

  33. Cohn Z . The role of proteases in macrophage physiology. In: Reich E, Rifkin DB, Shaw E (eds). Proteases and Biological Control. Cold Spring Harbr Press: Cold Spring Harbor, 1975.

    Google Scholar 

  34. Werb Z . Proteases and matrix degradation. In: Kelly WN, Harris ED, Sledge RS (eds). Textbook of Rheumatology. Saunders: Philadelphia, 1993.

    Google Scholar 

  35. Wang J, Tan NS, Ho B, Ding JL . Modular arrangement and secretion of a multidomain serine protease. Evidence for involvement of proline-rich region and N-glycans in the secretion pathway. J Biol Chem 2002; 277: 36363–36372.

    Article  CAS  PubMed  Google Scholar 

  36. Wang LH, Ho B, Ding JL . Transcriptional regulation of limulus Factor C: repression of an NF B motif modulates its responsiveness to bacterial lipopolysaccharide. J Biol Chem 2003; 278: 49428–49437.

    Article  CAS  PubMed  Google Scholar 

  37. Saravanan T, Weise C, Sojka D, Kopacek P . Molecular cloning, structure and bait region splice variants of alpha2-macroglobulin from the soft tick Ornithodoros moubata. Insect Biochem Mol Biol 2003; 33: 841–851.

    Article  CAS  PubMed  Google Scholar 

  38. Iwaki D, Kawabata S, Miura Y, Kato A, Armstrong PB, Quigley JP et al. Molecular cloning of Limulus alpha 2-macroglobulin. Eur J Biochem 1996; 242: 822–831.

    Article  CAS  PubMed  Google Scholar 

  39. Armstrong PB, Quigley JP . Proteinase inhibitory activity released from the horseshoe crab blood cell during exocytosis. Biochim Biophys Acta 1985; 827: 453–459.

    Article  CAS  PubMed  Google Scholar 

  40. Nakamura T, Tokunaga F, Morita T, Iwanaga S . Interaction between lipopolysaccharide and intracellular serine protease zymogen, factor C, from horseshoe crab (Tachypleus tridentatus) hemocytes. J Biochem (Tokyo) 1988; 103: 370–374.

    Article  CAS  Google Scholar 

  41. Donovan MA, Laue TM . A novel trypsin inhibitor from the hemolymph of the horseshoe crab Limulus polyphemus. J Biol Chem 1991; 266: 2121–2125.

    CAS  PubMed  Google Scholar 

  42. Golino P . The inhibitors of the tissue factor: factor VII pathway. Thromb Res 2002; 106: V257–V265.

    Article  CAS  PubMed  Google Scholar 

  43. Miura Y, Kawabata S, Wakamiya Y, Nakamura T, Iwanaga S . A limulus intracellular coagulation inhibitor type 2. Purification, characterization, cDNA cloning, and tissue localization. J Biol Chem 1995; 270: 558–565.

    Article  CAS  PubMed  Google Scholar 

  44. Richards RC, O'Neil DB, Thibault P, Ewart KV . Histone H1: an antimicrobial protein of Atlantic salmon (Salmo salar). Biochem Biophys Res Commun 2001; 284: 549–555.

    Article  CAS  PubMed  Google Scholar 

  45. Augusto LA, Decottignies P, Synguelakis M, Nicaise M, Le Marechal P, Chaby R . Histones: a novel class of lipopolysaccharide-binding molecules. Biochemistry 2003; 42: 3929–3938.

    Article  CAS  PubMed  Google Scholar 

  46. Arai H, Koizumi H, Aoki J, Inoue K . Platelet-activating factor acetylhydrolase (PAF-AH). J Biochem (Tokyo) 2002; 131: 635–640.

    Article  CAS  Google Scholar 

  47. Tjoelker LW, Stafforini DM . Platelet-activating factor acetylhydrolases in health and disease. Biochim Biophys Acta 2000; 1488: 102–123.

    Article  CAS  PubMed  Google Scholar 

  48. Montague JW, Hughes Jr FM, Cidlowski JA . Native recombinant cyclophilins A, B, and C degrade DNA independently of peptidylprolyl cis–trans-isomerase activity. Potential roles of cyclophilins in apoptosis. J Biol Chem 1997; 272: 6677–6684.

    Article  CAS  PubMed  Google Scholar 

  49. Matsuda S, Koyasu S . Mechanisms of action of cyclosporine. Immunopharmacology 2000; 47: 119–125.

    Article  CAS  PubMed  Google Scholar 

  50. Clow LA, Gross PS, Shih CS, Smith LC . Expression of SpC3, the sea urchin complement component, in response to lipopolysaccharide. Immunogenetics 2000; 51: 1021–1033.

    Article  CAS  PubMed  Google Scholar 

  51. Underhill DM, Ozinsky A . Phagocytosis of microbes: complexity in action. Annu Rev Immunol 2002; 20: 825–852.

    Article  CAS  PubMed  Google Scholar 

  52. Nauta AJ, Daha MR, van Kooten C, Roos A . Recognition and clearance of apoptotic cells: a role for complement and pentraxins. Trends Immunol 2003; 24: 148–154.

    Article  CAS  PubMed  Google Scholar 

  53. Khush RS, Leulier F, Lemaitre B . Drosophila immunity: two paths to NF-kappaB. Trends Immunol 2001; 22: 260–264.

    Article  CAS  PubMed  Google Scholar 

  54. Kim DH, Feinbaum R, Alloing G, Emerson FE, Garsin DA, Inoue H et al. A conserved p38 MAP kinase pathway in Caenorhabditis elegans innate immunity. Science 2002; 297: 623–626.

    Article  CAS  PubMed  Google Scholar 

  55. Solon E, Gupta AP, Gaugler R . Signal transduction during exocytosis in Limulus polyphemus granulocytes. Dev Comp Immunol 1996; 20: 307–321.

    Article  CAS  PubMed  Google Scholar 

  56. Ariki S, Koori K, Osaki T, Motoyama K, Inamori K, Kawabata S . A serine protease zymogen functions as a pattern-recognition receptor for lipopolysaccharides. Proc Natl Acad Sci USA 2004; 101: 953–958.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Gruszynski AE, DeMaster A, Hooper NM, Bangs JD . Surface coat remodeling during differentiation of Trypanosoma brucei. J Biol Chem 2003; 278: 24665–24672.

    Article  CAS  PubMed  Google Scholar 

  58. Crabtree GR, Clipstone NA . Signal transmission between the plasma membrane and nucleus of T lymphocytes. Annu Rev Biochem 1994; 63: 1045–1083.

    Article  CAS  PubMed  Google Scholar 

  59. Bergner A, Muta T, Iwanaga S, Beisel HG, Delotto R, Bode W . Horseshoe crab coagulogen is an invertebrate protein with a nerve growth factor-like domain. Biol Chem 1997; 378: 283–287.

    Article  CAS  PubMed  Google Scholar 

  60. Mizuguchi K, Parker JS, Blundell TL, Gay NJ . Getting knotted: a model for the structure and activation of Spatzle. Trends Biochem Sci 1998; 23: 239–242.

    Article  CAS  PubMed  Google Scholar 

  61. Grassme H, Jendrossek V, Gulbins E . Molecular mechanisms of bacteria induced apoptosis. Apoptosis 2001; 6: 441–445.

    Article  CAS  PubMed  Google Scholar 

  62. Newmeyer DD, Ferguson-Miller S . Mitochondria: releasing power for life and unleashing the machineries of death. Cell 2003; 112: 481–490.

    Article  CAS  PubMed  Google Scholar 

  63. Dussmann H, Kogel D, Rehm M, Prehn JH . Mitochondrial membrane permeabilization and superoxide production during apoptosis. A single-cell analysis. J Biol Chem 2003; 278: 12645–12649.

    Article  PubMed  Google Scholar 

  64. Brunori M, Wilson MT . Electron transfer and proton pumping in cytochrome oxidase. Biochimie 1995; 77: 668–676.

    Article  CAS  PubMed  Google Scholar 

  65. Lavrov DV, Boore JL, Brown WM . The complete mitochondrial DNA sequence of the horseshoe crab Limulus polyphemus. Mol Biol Evol 2000; 17: 813–824.

    Article  CAS  PubMed  Google Scholar 

  66. Hasnain SE, Begum R, Ramaiah KV, Sahdev S, Shajil EM, Taneja TK et al. Host–pathogen interactions during apoptosis. J Biosci 2003; 28: 349–358.

    Article  CAS  PubMed  Google Scholar 

  67. Bogdan C, Rollinghoff M, Diefenbach A . Reactive oxygen and reactive nitrogen intermediates in innate and specific immunity. Curr Opin Immunol 2000; 12: 64–76.

    Article  CAS  PubMed  Google Scholar 

  68. Hauptmann N, Grimsby J, Shih JC, Cadenas E . The metabolism of tyramine by monoamine oxidase A/B causes oxidative damage to mitochondrial DNA. Arch Biochem Biophys 1996; 335: 295–304.

    Article  CAS  PubMed  Google Scholar 

  69. Malorni W, Giammarioli AM, Matarrese P, Pietrangeli P, Agostinelli E, Ciaccio A et al. Protection against apoptosis by monoamine oxidase A inhibitors. FEBS Lett 1998; 426: 155–159.

    Article  CAS  PubMed  Google Scholar 

  70. Duan H, Wang Y, Aviram M, Swaroop M, Loo JA, Bian J et al. SAG, a novel zinc RING finger protein that protects cells from apoptosis induced by redox agents. Mol Cell Biol 1999; 19: 3145–3155.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Nappi AJ, Vass E, Frey F, Carton Y . Superoxide anion generation in Drosophila during melanotic encapsulation of parasites. Eur J Cell Biol 1995; 68: 450–456.

    CAS  PubMed  Google Scholar 

  72. Wilce MC, Parker MW . Structure and function of glutathione S-transferases. Biochim Biophys Acta 1994; 1205: 1–18.

    Article  CAS  PubMed  Google Scholar 

  73. Chen J, Berry MJ . Selenium and selenoproteins in the brain and brain diseases. J Neurochem 2003; 86: 1–12.

    Article  CAS  PubMed  Google Scholar 

  74. Hirota K, Nakamura H, Masutani H, Yodoi J . Thioredoxin superfamily and thioredoxin-inducing agents. Ann NY Acad Sci 2002; 957: 189–199.

    Article  CAS  PubMed  Google Scholar 

  75. Nishinaka Y, Masutani H, Nakamura H, Yodoi J . Regulatory roles of thioredoxin in oxidative stress-induced cellular responses. Redox Rep 2001; 6: 289–295.

    Article  CAS  PubMed  Google Scholar 

  76. Soti C, Csermely P . Molecular chaperones and the aging process. Biogerontology 2000; 1: 225–233.

    Article  CAS  PubMed  Google Scholar 

  77. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997; 25: 3389–3402.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Anderson I, Brass A . Searching DNA databases for similarities to DNA sequences: when is a match significant? Bioinformatics 1998; 14: 349–356.

    Article  CAS  PubMed  Google Scholar 

  79. Endege WO, Steinmann KE, Boardman LA, Thibodeau SN, Schlegel R . Representative cDNA libraries and their utility in gene expression profiling. Biotechniques 1999; 26: 542–548, 550.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by a grant (03/1/21/17/227) from the Agency of Science, Technology and Research (A*STAR), Singapore. We thank Ms Kaitian Peng (an A*STAR-funded undergraduate scholar of the Imperial College, London, UK) for help with sequencing some ESTs.

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  1. K C Tan

    Present address: The Australian Centre For Necrotrophic Fungal Pathogens, Murdoch University, 6150, Australia

  2. J Wang

    Present address: Institute of Molecular Cell Biology, Singapore

Authors and Affiliations

  1. Department of Biological Sciences, National University of Singapore, Singapore, 117543, Singapore

    J L Ding, K C Tan, S Thangamani, N Kusuma, W K Seow, T H H Bui & J Wang

  2. Department of Microbiology, National University of Singapore, Singapore, 117543, Singapore

    B Ho

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Ding, J., Tan, K., Thangamani, S. et al. Spatial and temporal coordination of expression of immune response genes during Pseudomonas infection of horseshoe crab, Carcinoscorpius rotundicauda. Genes Immun 6, 557–574 (2005). https://doi.org/10.1038/sj.gene.6364240

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