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

  • Jan Kotál
  • Natascha Stergiou
  • Michal Buša
  • Adéla Chlastáková
  • Zuzana Beránková
  • Pavlína Řezáčová
  • Helena Langhansová
  • Alexandra Schwarz
  • Eric Calvo
  • Jan Kopecký
  • Michael Mareš
  • Edgar Schmitt
  • Jindřich Chmelař
  • Michail KotsyfakisEmail author
Original Article


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.


Cathepsin Crystal structure Immune responses Ixodes ricinus Saliva 



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.

Author 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.


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.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

18_2019_3034_MOESM1_ESM.docx (252 kb)
Supplementary material 1 (DOCX 252 kb)


  1. 1.
    Francischetti IM, Sa-Nunes A, Mans BJ, Santos IM, Ribeiro JM (2009) The role of saliva in tick feeding. Front Biosci (Landmark Ed) 14:2051–2088CrossRefGoogle Scholar
  2. 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. CrossRefGoogle Scholar
  3. 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. CrossRefGoogle Scholar
  4. 4.
    Turk V, Bode W (1991) The cystatins: protein inhibitors of cysteine proteinases. FEBS Lett 285(2):213–219CrossRefGoogle Scholar
  5. 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. CrossRefGoogle Scholar
  6. 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. CrossRefGoogle Scholar
  7. 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. CrossRefGoogle Scholar
  8. 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. CrossRefGoogle Scholar
  9. 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. CrossRefGoogle Scholar
  10. 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. CrossRefGoogle Scholar
  11. 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. CrossRefGoogle Scholar
  12. 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. CrossRefGoogle Scholar
  13. 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):5209–5212CrossRefGoogle Scholar
  14. 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. CrossRefGoogle Scholar
  15. 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):103–113CrossRefGoogle Scholar
  16. 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. CrossRefGoogle Scholar
  17. 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. CrossRefGoogle Scholar
  18. 18.
    Vray B, Hartmann S, Hoebeke J (2002) Immunomodulatory properties of cystatins. Cell Mol Life Sci 59(9):1503–1512CrossRefGoogle Scholar
  19. 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):3989–3996Google Scholar
  20. 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. CrossRefGoogle Scholar
  21. 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. CrossRefGoogle Scholar
  22. 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. CrossRefGoogle Scholar
  23. 23.
    Kyckova K, Kopecky J (2006) Effect of tick saliva on mechanisms of innate immune response against Borrelia afzelii. J Med Entomol 43(6):1208–1214CrossRefGoogle Scholar
  24. 24.
    Bode W, Huber R (2000) Structural basis of the endoproteinase-protein inhibitor interaction. Biochim Biophys Acta 1477(1–2):241–252CrossRefGoogle Scholar
  25. 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. CrossRefGoogle Scholar
  26. 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. CrossRefGoogle Scholar
  27. 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):6186–6192CrossRefGoogle Scholar
  28. 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. CrossRefGoogle Scholar
  29. 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. CrossRefGoogle Scholar
  30. 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. CrossRefGoogle Scholar
  31. 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. CrossRefGoogle Scholar
  32. 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. CrossRefGoogle Scholar
  33. 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):28077–28081CrossRefGoogle Scholar
  34. 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. CrossRefGoogle Scholar
  35. 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. CrossRefGoogle Scholar
  36. 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. Google Scholar
  37. 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. CrossRefGoogle Scholar
  38. 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. CrossRefGoogle Scholar
  39. 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):4265–4272CrossRefGoogle Scholar
  40. 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. CrossRefGoogle Scholar
  41. 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. CrossRefGoogle Scholar
  42. 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. CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Jan Kotál
    • 1
    • 2
  • Natascha Stergiou
    • 3
  • Michal Buša
    • 4
  • Adéla Chlastáková
    • 2
  • Zuzana Beránková
    • 2
  • Pavlína Řezáčová
    • 4
  • Helena Langhansová
    • 2
  • Alexandra Schwarz
    • 1
  • Eric Calvo
    • 5
  • Jan Kopecký
    • 2
  • Michael Mareš
    • 4
  • Edgar Schmitt
    • 3
  • Jindřich Chmelař
    • 2
  • Michail Kotsyfakis
    • 1
    Email author
  1. 1.Laboratory of Genomics and Proteomics of Disease VectorsBiology Centre CAS, Institute of ParasitologyČeské BudějoviceCzech Republic
  2. 2.Department of Medical Biology, Faculty of ScienceUniversity of South Bohemia in České BudějoviceČeské BudějoviceCzech Republic
  3. 3.Institute for ImmunologyUniversity Medical Center of the Johannes Gutenberg-University MainzMainzGermany
  4. 4.Institute of Organic Chemistry and BiochemistryAcademy of Sciences of the Czech RepublicPragueCzech Republic
  5. 5.Laboratory of Malaria and Vector ResearchNational Institute of Allergy and Infectious Diseases, National Institutes of HealthRockvilleUSA

Personalised recommendations