Skip to main content
SpringerLink
Log in

The structure and function of Iristatin, a novel immunosuppressive tick salivary cystatin

  • Original Article
  • Published:
Cellular and Molecular Life Sciences Aims and scope Submit manuscript

Abstract

To successfully feed, ticks inject pharmacoactive molecules into the vertebrate host including cystatin cysteine protease inhibitors. However, the molecular and cellular events modulated by tick saliva remain largely unknown. Here, we describe and characterize a novel immunomodulatory cystatin, Iristatin, which is upregulated in the salivary glands of feeding Ixodes ricinus ticks. We present the crystal structure of Iristatin at 1.76 Å resolution. Purified recombinant Iristatin inhibited the proteolytic activity of cathepsins L and C and diminished IL-2, IL-4, IL-9, and IFN-γ production by different T-cell populations, IL-6 and IL-9 production by mast cells, and nitric oxide production by macrophages. Furthermore, Iristatin inhibited OVA antigen-induced CD4+ T-cell proliferation and leukocyte recruitment in vivo and in vitro. Our results indicate that Iristatin affects wide range of anti-tick immune responses in the vertebrate host and may be exploitable as an immunotherapeutic.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Data availability

The data set supporting the conclusions of this article is available in GenBank, accession number KY348759; PDB code 5O46.

References

  1. Francischetti IM, Sa-Nunes A, Mans BJ, Santos IM, Ribeiro JM (2009) The role of saliva in tick feeding. Front Biosci (Landmark Ed) 14:2051–2088

    Article  CAS  Google Scholar 

  2. Kotal J, Langhansova H, Lieskovska J, Andersen JF, Francischetti IM, Chavakis T, Kopecky J, Pedra JH, Kotsyfakis M, Chmelar J (2015) Modulation of host immunity by tick saliva. J Proteomics 128:58–68. https://doi.org/10.1016/j.jprot.2015.07.005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Chmelar J, Kotal J, Langhansova H, Kotsyfakis M (2017) Protease inhibitors in tick saliva: the role of serpins and cystatins in tick–host–pathogen interaction. Front Cell Infect Microbiol 7:216. https://doi.org/10.3389/fcimb.2017.00216

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Turk V, Bode W (1991) The cystatins: protein inhibitors of cysteine proteinases. FEBS Lett 285(2):213–219

    Article  CAS  PubMed  Google Scholar 

  5. Rawlings ND, Waller M, Barrett AJ, Bateman A (2014) MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res 42(Database issue):D503–D509. https://doi.org/10.1093/nar/gkt953

    Article  CAS  PubMed  Google Scholar 

  6. Schwarz A, Valdes JJ, Kotsyfakis M (2012) The role of cystatins in tick physiology and blood feeding. Ticks Tick Borne Dis 3(3):117–127. https://doi.org/10.1016/j.ttbdis.2012.03.004

    Article  PubMed  PubMed Central  Google Scholar 

  7. Kotsyfakis M, Sa-Nunes A, Francischetti IM, Mather TN, Andersen JF, Ribeiro JM (2006) Antiinflammatory and immunosuppressive activity of sialostatin L, a salivary cystatin from the tick Ixodes scapularis. J Biol Chem 281(36):26298–26307. https://doi.org/10.1074/jbc.M513010200

    Article  CAS  PubMed  Google Scholar 

  8. Sa-Nunes A, Bafica A, Antonelli LR, Choi EY, Francischetti IM, Andersen JF, Shi GP, Chavakis T, Ribeiro JM, Kotsyfakis M (2009) The immunomodulatory action of sialostatin L on dendritic cells reveals its potential to interfere with autoimmunity. J Immunol 182(12):7422–7429. https://doi.org/10.4049/jimmunol.0900075

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Kotsyfakis M, Karim S, Andersen JF, Mather TN, Ribeiro JM (2007) Selective cysteine protease inhibition contributes to blood-feeding success of the tick Ixodes scapularis. J Biol Chem 282(40):29256–29263. https://doi.org/10.1074/jbc.M703143200

    Article  CAS  PubMed  Google Scholar 

  10. Chen G, Wang X, Severo MS, Sakhon OS, Sohail M, Brown LJ, Sircar M, Snyder GA, Sundberg EJ, Ulland TK, Olivier AK, Andersen JF, Zhou Y, Shi GP, Sutterwala FS, Kotsyfakis M, Pedra JH (2014) The tick salivary protein sialostatin L2 inhibits caspase-1-mediated inflammation during Anaplasma phagocytophilum infection. Infect Immun 82(6):2553–2564. https://doi.org/10.1128/IAI.01679-14

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Lieskovska J, Palenikova J, Langhansova H, Campos Chagas A, Calvo E, Kotsyfakis M, Kopecky J (2015) Tick sialostatins L and L2 differentially influence dendritic cell responses to Borrelia spirochetes. Parasit Vectors 8:275. https://doi.org/10.1186/s13071-015-0887-1

    Article  PubMed  PubMed Central  Google Scholar 

  12. Kotsyfakis M, Horka H, Salat J, Andersen JF (2010) The crystal structures of two salivary cystatins from the tick Ixodes scapularis and the effect of these inhibitors on the establishment of Borrelia burgdorferi infection in a murine model. Mol Microbiol 77(2):456–470. https://doi.org/10.1111/j.1365-2958.2010.07220.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Kotsyfakis M, Anderson JM, Andersen JF, Calvo E, Francischetti IM, Mather TN, Valenzuela JG, Ribeiro JM (2008) Cutting edge: immunity against a “silent” salivary antigen of the Lyme vector Ixodes scapularis impairs its ability to feed. J Immunol 181(8):5209–5212

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Cramaro WJ, Revets D, Hunewald OE, Sinner R, Reye AL, Muller CP (2015) Integration of Ixodes ricinus genome sequencing with transcriptome and proteome annotation of the naive midgut. BMC Genomics 16:871. https://doi.org/10.1186/s12864-015-1981-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Kopacek P, Zdychova J, Yoshiga T, Weise C, Rudenko N, Law JH (2003) Molecular cloning, expression and isolation of ferritins from two tick species—Ornithodoros moubata and Ixodes ricinus. Insect Biochem Mol Biol 33(1):103–113

    Article  CAS  PubMed  Google Scholar 

  16. Sojka D, Hajdusek O, Dvorak J, Sajid M, Franta Z, Schneider EL, Craik CS, Vancova M, Buresova V, Bogyo M, Sexton KB, McKerrow JH, Caffrey CR, Kopacek P (2007) IrAE: an asparaginyl endopeptidase (legumain) in the gut of the hard tick Ixodes ricinus. Int J Parasitol 37(7):713–724. https://doi.org/10.1016/j.ijpara.2006.12.020

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Cramaro WJ, Hunewald OE, Bell-Sakyi L, Muller CP (2017) Genome scaffolding and annotation for the pathogen vector Ixodes ricinus by ultra-long single molecule sequencing. Parasites Vectors 10(1):71. https://doi.org/10.1186/s13071-017-2008-9

    Article  PubMed  PubMed Central  Google Scholar 

  18. Vray B, Hartmann S, Hoebeke J (2002) Immunomodulatory properties of cystatins. Cell Mol Life Sci 59(9):1503–1512

    Article  CAS  PubMed  Google Scholar 

  19. Schmitt E, Germann T, Goedert S, Hoehn P, Huels C, Koelsch S, Kuhn R, Muller W, Palm N, Rude E (1994) IL-9 production of naive CD4+ T cells depends on IL-2, is synergistically enhanced by a combination of TGF-beta and IL-4, and is inhibited by IFN-gamma. J Immunol 153(9):3989–3996

    CAS  PubMed  Google Scholar 

  20. Klein M, Bruhl TJ, Staudt V, Reuter S, Grebe N, Gerlitzki B, Hoffmann M, Bohn T, Ulges A, Stergiou N, de Graaf J, Lower M, Taube C, Becker M, Hain T, Dietzen S, Stassen M, Huber M, Lohoff M, Campos Chagas A, Andersen J, Kotal J, Langhansova H, Kopecky J, Schild H, Kotsyfakis M, Schmitt E, Bopp T (2015) Tick salivary sialostatin L represses the initiation of immune responses by targeting IRF4-dependent transcription in murine mast cells. J Immunol 195(2):621–631. https://doi.org/10.4049/jimmunol.1401823

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Salat J, Paesen GC, Rezacova P, Kotsyfakis M, Kovarova Z, Sanda M, Majtan J, Grunclova L, Horka H, Andersen JF, Brynda J, Horn M, Nunn MA, Kopacek P, Kopecky J, Mares M (2010) Crystal structure and functional characterization of an immunomodulatory salivary cystatin from the soft tick Ornithodoros moubata. Biochem J 429(1):103–112. https://doi.org/10.1042/BJ20100280

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Laroux FS, Pavlick KP, Hines IN, Kawachi S, Harada H, Bharwani S, Hoffman JM, Grisham MB (2001) Role of nitric oxide in inflammation. Acta Physiol Scand 173(1):113–118. https://doi.org/10.1046/j.1365-201X.2001.00891.x

    Article  CAS  PubMed  Google Scholar 

  23. Kyckova K, Kopecky J (2006) Effect of tick saliva on mechanisms of innate immune response against Borrelia afzelii. J Med Entomol 43(6):1208–1214

    Article  CAS  PubMed  Google Scholar 

  24. Bode W, Huber R (2000) Structural basis of the endoproteinase-protein inhibitor interaction. Biochim Biophys Acta 1477(1–2):241–252

    Article  CAS  PubMed  Google Scholar 

  25. Bjork I, Brieditis I, Raub-Segall E, Pol E, Hakansson K, Abrahamson M (1996) The importance of the second hairpin loop of cystatin C for proteinase binding. Characterization of the interaction of Trp-106 variants of the inhibitor with cysteine proteinases. Biochemistry 35(33):10720–10726. https://doi.org/10.1021/bi960420u

    Article  CAS  PubMed  Google Scholar 

  26. Sun T, Wang F, Pan W, Wu Q, Wang J, Dai J (2018) An immunosuppressive tick salivary gland protein DsCystatin interferes with toll-like receptor signaling by downregulating TRAF6. Front Immunol 9:1245. https://doi.org/10.3389/fimmu.2018.01245

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Skallova A, Iezzi G, Ampenberger F, Kopf M, Kopecky J (2008) Tick saliva inhibits dendritic cell migration, maturation, and function while promoting development of Th2 responses. J Immunol 180(9):6186–6192

    Article  CAS  PubMed  Google Scholar 

  28. Slamova M, Skallova A, Palenikova J, Kopecky J (2011) Effect of tick saliva on immune interactions between Borrelia afzelii and murine dendritic cells. Parasite Immunol 33(12):654–660. https://doi.org/10.1111/j.1365-3024.2011.01332.x

    Article  CAS  PubMed  Google Scholar 

  29. Horka H, Staudt V, Klein M, Taube C, Reuter S, Dehzad N, Andersen JF, Kopecky J, Schild H, Kotsyfakis M, Hoffmann M, Gerlitzki B, Stassen M, Bopp T, Schmitt E (2012) The tick salivary protein sialostatin L inhibits the Th9-derived production of the asthma-promoting cytokine IL-9 and is effective in the prevention of experimental asthma. J Immunol 188(6):2669–2676. https://doi.org/10.4049/jimmunol.1100529

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Wasserman HA, Singh S, Champagne DE (2004) Saliva of the Yellow Fever mosquito, Aedes aegypti, modulates murine lymphocyte function. Parasite Immunol 26(6–7):295–306. https://doi.org/10.1111/j.0141-9838.2004.00712.x

    Article  CAS  PubMed  Google Scholar 

  31. Boppana VD, Thangamani S, Adler AJ, Wikel SK (2009) SAAG-4 is a novel mosquito salivary protein that programmes host CD4 T cells to express IL-4. Parasite Immunol 31(6):287–295. https://doi.org/10.1111/j.1365-3024.2009.01096.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Chen L, He B, Hou W, He L (2017) Cysteine protease inhibitor of Schistosoma japonicum—a parasite-derived negative immunoregulatory factor. Parasitol Res 116(3):901–908. https://doi.org/10.1007/s00436-016-5363-0

    Article  PubMed  Google Scholar 

  33. Verdot L, Lalmanach G, Vercruysse V, Hartmann S, Lucius R, Hoebeke J, Gauthier F, Vray B (1996) Cystatins up-regulate nitric oxide release from interferon-gamma-activated mouse peritoneal macrophages. J Biol Chem 271(45):28077–28081

    Article  CAS  PubMed  Google Scholar 

  34. Yang X, Liu J, Yue Y, Chen W, Song M, Zhan X, Wu Z (2014) Cloning, expression and characterisation of a type II cystatin from Schistosoma japonicum, which could regulate macrophage activation. Parasitol Res 113(11):3985–3992. https://doi.org/10.1007/s00436-014-4064-9

    Article  PubMed  Google Scholar 

  35. Sato K, Ozaki K, Oh I, Meguro A, Hatanaka K, Nagai T, Muroi K, Ozawa K (2007) Nitric oxide plays a critical role in suppression of T-cell proliferation by mesenchymal stem cells. Blood 109(1):228–234. https://doi.org/10.1182/blood-2006-02-002246

    Article  CAS  PubMed  Google Scholar 

  36. Niedbala W, Cai B, Liew FY (2006) Role of nitric oxide in the regulation of T cell functions. Ann Rheum Dis 65(Suppl 3):iii37–iii40. https://doi.org/10.1136/ard.2006.058446

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Schuijs MJ, Hartmann S, Selkirk ME, Roberts LB, Openshaw PJ, Schnoeller C (2016) The helminth-derived immunomodulator AvCystatin reduces virus enhanced inflammation by induction of regulatory IL-10+ T cells. PLoS One 11(8):e0161885. https://doi.org/10.1371/journal.pone.0161885

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Danilowicz-Luebert E, Steinfelder S, Kuhl AA, Drozdenko G, Lucius R, Worm M, Hamelmann E, Hartmann S (2013) A nematode immunomodulator suppresses grass pollen-specific allergic responses by controlling excessive Th2 inflammation. Int J Parasitol 43(3–4):201–210. https://doi.org/10.1016/j.ijpara.2012.10.014

    Article  CAS  PubMed  Google Scholar 

  39. Schnoeller C, Rausch S, Pillai S, Avagyan A, Wittig BM, Loddenkemper C, Hamann A, Hamelmann E, Lucius R, Hartmann S (2008) A helminth immunomodulator reduces allergic and inflammatory responses by induction of IL-10-producing macrophages. J Immunol 180(6):4265–4272

    Article  CAS  PubMed  Google Scholar 

  40. Staun-Ram E, Miller A (2011) Cathepsins (S and B) and their inhibitor Cystatin C in immune cells: modulation by interferon-beta and role played in cell migration. J Neuroimmunol 232(1–2):200–206. https://doi.org/10.1016/j.jneuroim.2010.10.015

    Article  CAS  PubMed  Google Scholar 

  41. Wei N, Lin Z, Xu Z, Cao J, Zhou Y, Zhang H, Gong H, Zhou J, Li G (2018) A tick cysteine protease inhibitor RHcyst-1 exhibits antitumor potential. Cell Physiol Biochem 46(6):2385–2400. https://doi.org/10.1159/000489645

    Article  CAS  PubMed  Google Scholar 

  42. Chmelar J, Kotal J, Kopecky J, Pedra JHF, Kotsyfakis M (2016) All for one and one for all on the tick-host battlefield. Trends Parasitol 32(5):368–377. https://doi.org/10.1016/j.pt.2016.01.004

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank Professor Jose MC Ribeiro and Drs. Petr Kopáček and Daniel Sojka for constructive discussions, Dr. Daniel Sojka for kindly providing published reagent, ARVYS Proteins for service provision, Dr. Andrezza Campos-Chagas and Mr. Jan Erhart for technical assistance and Nextgenediting for editorial assistance. We also thank the anonymous reviewers for constructive comments about the draft.

Funding

This work was supported by the Grant Agency of the Czech Republic (Grant 19-07247S to MK and Grant 16-07117Y to JC), by the Grant Agency of the University of South Bohemia (Grant 038/2016/P to JKot), and by ERD Funds, project CePaViP OPVVV (No. CZ.02.1.01/0.0/0.0/16_019/0000759 to MK) and institutional project RVO 60077344 to MK. MB, MM, and PŘ were supported by project ChemBioDrug (No. CZ.02.1.01/0.0/0.0/16_019/0000729) from ERD Funds and institutional project RVO 61388963.

Author information

Authors and Affiliations

  1. Laboratory of Genomics and Proteomics of Disease Vectors, Biology Centre CAS, Institute of Parasitology, Branišovská 1160/31, 37005, České Budějovice, Czech Republic

    Jan Kotál, Alexandra Schwarz & Michail Kotsyfakis

  2. Department of Medical Biology, Faculty of Science, University of South Bohemia in České Budějovice, Branišovská 1760c, 37005, České Budějovice, Czech Republic

    Jan Kotál, Adéla Chlastáková, Zuzana Beránková, Helena Langhansová, Jan Kopecký & Jindřich Chmelař

  3. Institute for Immunology, University Medical Center of the Johannes Gutenberg-University Mainz, Langenbeckstrasse 1, Mainz, 55131, Germany

    Natascha Stergiou & Edgar Schmitt

  4. Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 16610, Prague, Czech Republic

    Michal Buša, Pavlína Řezáčová & Michael Mareš

  5. Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12735 Twinbrook Parkway, Rockville, MD, 20852, USA

    Eric Calvo

Authors
  1. Jan Kotál
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Natascha Stergiou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michal Buša
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Adéla Chlastáková
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zuzana Beránková
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Pavlína Řezáčová
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Helena Langhansová
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Alexandra Schwarz
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Eric Calvo
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jan Kopecký
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Michael Mareš
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Edgar Schmitt
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Jindřich Chmelař
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Michail Kotsyfakis
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

JKot designed and performed experiments, performed the analyses, and wrote the manuscript; NS, MB, AC, ZB, PŘ, HL, and AS designed and performed experiments; JC and MK designed experiments, performed analyses, and edited the manuscript; MM and ES designed experiments and revised the manuscript; EC and JKop revised the manuscript.

Corresponding author

Correspondence to Michail Kotsyfakis.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 252 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kotál, J., Stergiou, N., Buša, M. et al. The structure and function of Iristatin, a novel immunosuppressive tick salivary cystatin. Cell. Mol. Life Sci. 76, 2003–2013 (2019). https://doi.org/10.1007/s00018-019-03034-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00018-019-03034-3

Keywords

Search

Navigation