Skip to main content
SpringerLink
Log in

Functional analysis of a novel cysteine-rich antimicrobial peptide from the salivary glands of the tick Rhipicephalus haemaphysaloides

  • Original Paper
  • Published:
Parasitology Research Aims and scope Submit manuscript

Abstract

Ticks encounter various microbes while sucking blood from an infected host and carrying these pathogens in themselves. Ticks can then transmit these pathogens to vertebrate hosts. The immune system of ticks can be stimulated to produce many bioactive molecules during feeding and pathogen invasion. Antimicrobial peptides (AMPs) are key effector molecules of a tick’s immune response, as they can kill invading pathogenic microorganisms. In this study, we identified a novel cysteine-rich AMP, designated Rhamp1, in the salivary glands of unfed and fed female ticks (Rhipicephalus haemaphysaloides). Rhamp1 is encoded by a gene with an open reading frame of 333 bp, which in turn encodes a peptide of 12 kDa with a 22 amino acid residue signal peptide. The Rhamp1 protein had a pI of 8.6 and contained six conserved cysteine residues at the C-terminus. Rhamp1 shared 43 % amino acid identity with a secreted cysteine-rich protein of another tick species, Ixodes scapularis. We cloned the Rhamp1 gene and attempted to express a recombinant protein using prokaryotic and eukaryotic systems, to determine its biological significance. Recombinant Rhamp1 was successfully expressed in both systems, yielding a glutathione S-transferase (GST)-tagged protein (36 kDa) from the prokaryotic system, and a polyhistidine-tagged Rhamp1 protein (14 kDa) from the eukaryotic system. Rhamp1 inhibited the activities of chymotrypsin (16 %) and elastase (22 %) and exerted low hemolytic activity. It also inhibited the growth of Gram-negative bacteria, including Pseudomonas aeruginosa (49 %), Salmonella typhimurium (50 %), and Escherichia coli (52 %). Our findings suggest that Rhamp1 is a novel AMP in R. haemaphysaloides with the ability to inhibit proteinase activity.

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
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Armstrong PB (2001) The contribution of proteinase inhibitors to immune defense. Trends Immunol 22:47–52

    Article  CAS  PubMed  Google Scholar 

  • Assenga SP, You M, Shy CH, Yamagishi J, Sakaguchi T, Zhou J, Kibe MK, Xuan X, Fujisaki K (2006) The use of a recombinant baculovirus expressing a chitinase from the hard tick Haemaphysalis longicornis and its potential application as a bioacaricide for tick control. Parasitol Res 98:111–118

    Article  PubMed  Google Scholar 

  • Belmonte R, Cruz CE, Pires JR, Daffre S (2012) Purification and characterization of Hb 98-114: a novel hemoglobin-derived antimicrobial peptide from the midgut of Rhipicephalus (Boophilus) microplus. Peptides 37:120–127

    Article  CAS  PubMed  Google Scholar 

  • Bignami GS (1993) A rapid and sensitive hemolysis neutralization assay for palytoxin. Toxicon 31:817–820

    Article  CAS  PubMed  Google Scholar 

  • Brogden NK, Brogden KA (2011) Will new generations of modified antimicrobial peptides improve their potential as pharmaceuticals? Int J Antimicrob Agents 38:217–225

    CAS  PubMed Central  PubMed  Google Scholar 

  • Bulet P, Hetru C, Dimarcq JL, Hoffmann D (1999) Antimicrobial peptides in insects; structure and function. Dev Comp Immunol 23:329–344

    Article  CAS  PubMed  Google Scholar 

  • Chrudimská T, Slaninová J, Rudenko N, Růžek D, Grubhoffer L (2011) Functional characterization of two defensin isoforms of the hard tick Ixodes ricinus. Parasit Vectors 4:63

    Article  PubMed Central  PubMed  Google Scholar 

  • Chrudimská T, Čeřovský V, Slaninová J, Rego RO, Grubhoffer L (2014) Defensin from the ornate sheep tick Dermacentor marginatus and its effect on Lyme borreliosis spirochetes. Dev Comp Immunol 46:165–170

    Article  PubMed  Google Scholar 

  • Esteves E, Fogaça AC, Maldonado R, Silva FD, Manso PP, Pelajo-Machado M, Valle D, Daffre S (2009) Antimicrobial activity in the tick Rhipicephalus (Boophilus) microplus eggs: Cellular localization and temporal expression of microplusin during oogenesis and embryogenesis. Dev Comp Immunol 33:913–919

    Article  CAS  PubMed  Google Scholar 

  • Fogaça AC, Lorenzini DM, Kaku LM, Esteves E, Bulet P, Daffre S (2004) Cysteine-rich antimicrobial peptides of the cattle tick Boophilus microplus: isolation, structural characterization and tissue expression profile. Dev Comp Immunol 28:191–200

    Article  PubMed  Google Scholar 

  • Fogaça AC, Almeida IC, Eberlin MN, Tanaka AS, Bulet P, Daffre S (2006) Ixodidin, a novel antimicrobial peptide from the hemocytes of the cattle tick Boophilus microplus with inhibitory activity against serine proteinases. Peptides 27:667–674

    Article  PubMed  Google Scholar 

  • Galay RL, Maeda H, Aung KM, Umemiya-Shirafuji R, Xuan X, Igarashi I, Tsuji N, Tanaka T, Fujisaki K (2012) Anti-babesial activity of a potent peptide fragment derived from longicin of Haemaphysalis longicornis. Trop Anim Health Prod 44:343–348

    Article  PubMed  Google Scholar 

  • Hajdušek O, Síma R, Ayllón N, Jalovecká M, Perner J, de la Fuente J, Kopáček P (2013) Interaction of the tick immune system with transmitted pathogens. Front Cell Infect Microbiol 3:26

    PubMed Central  PubMed  Google Scholar 

  • Hynes WL, Ceraul SM, Todd SM, Seguin KC, Sonenshine DE (2005) A defensin-like gene expressed in the black-legged tick, Ixodes scapularis. Med Vet Entomol 19:339–344

    Article  CAS  PubMed  Google Scholar 

  • Isogai E, Isogai H, Takahashi K, Kobayashi-Sakamoto M, Okumura K (2009) Antimicrobial activity of three tick defensins and four mammalian cathelicidin-derived synthetic peptides against Lyme disease spirochetes and bacteria isolated from the midgut. Exp Appl Acarol 49:221–228

    Article  CAS  PubMed  Google Scholar 

  • Kato S, Ohtoko K, Ohtake H, Kimura T (2005) Vector-capping: a simple method for preparing a high-quality full-length cDNA library. DNA Res 28:53–62

    Article  Google Scholar 

  • Kopacek P, Hajdusek O, Buresova V (2012) Tick as a model for the study of a primitive complement system. Adv Exp Med Biol 710:83–93

    Article  CAS  PubMed  Google Scholar 

  • Li J, Xu X, Xu C, Zhou W, Zhang K, Yu H, Zhang Y, Zheng Y, Rees HH, Lai R, Yang DWJ (2007) Anti-infection peptidomics of amphibian skin. Mol Cell Proteomics 6:882–994

    Article  CAS  PubMed  Google Scholar 

  • Lieskovská J, Kopecký J (2012) Tick saliva suppresses IFN signalling in dendritic cells upon Borrelia afzelii infection. Parasite Immunol 34:32–39

    Article  PubMed  Google Scholar 

  • Liu Z, Liu H, Liu X, Wu X (2008) Purification and cloning of a novel antimicrobial peptide from salivary glands of the hard tick, Ixodes sinensis. Comp Biochem Physiol B Biochem Mol Biol 149:557–561

    Article  PubMed  Google Scholar 

  • Liu L, Dai J, Zhao YO, Narasimhan S, Yang Y, Zhang L, Fikrig E (2012) Ixodes scapularis JAK-STAT pathway regulates tick antimicrobial peptides, thereby controlling the agent of human granulocytic anaplasmosis. J Infect Dis 206:1233–1241

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Loukas A, Bethony JM, Williamson AL, Goud GN, Mendez S, Zhan B, Hawdon JM, Elena Bottazzi M, Brindley PJ, Hotez PJ (2004) Vaccination of dogs with a recombinant cysteine protease from the intestine of canine hookworms diminishes the fecundity and growth of worms. J Infect Dis 189:1952–1961

    Article  CAS  PubMed  Google Scholar 

  • Mans BJ, Andersen JF, Francischetti IM, Valenzuela JG, Schwan TG, Pham VM, Garfield MK, Hammer CH, Ribeiro JM (2008) Comparative sialomics between hard and soft ticks: implications for the evolution of blood-feeding behavior. Insect Biochem Mol Biol 38:42–58

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Oldiges DP, Parizi LF, Zimmer KR, Lorenzini DM, Seixas A, Masuda A, Da Silva Vaz I Jr, Termignoni C (2012) A Rhipicephalus (Boophilus) microplus cathepsin with dual peptidase and antimicrobial activity. Int J Parasitol 42:635–645

    Article  CAS  PubMed  Google Scholar 

  • Reddy KV, Yedery RD, Aranha C (2004) Antimicrobial peptides: premises and promises. Int J Antimicrob Agents 24:536–547

    Article  CAS  PubMed  Google Scholar 

  • Saito Y, Konnai S, Yamada S, Imamura S, Nishikado H, Ito T, Onuma M, Ohashi K (2009) Identification and characterization of antimicrobial peptide, defensin, in the taiga tick, Ixodes persulcatus. Insect Mol Biol 18:531–539

    Article  CAS  PubMed  Google Scholar 

  • Silva FD, Rossi DC, Martinez LR, Frases S, Fonseca FL, Campos CB, Rodrigues ML, Nosanchuk JD, Daffre S (2011) Effects of microplusin, a copper-chelating antimicrobial peptide, against Cryptococcus neoformans. FEMS Microbiol Lett 324:64–72

    Article  CAS  PubMed  Google Scholar 

  • Steffen H, Rieg S, Wiedemann I, Kalbacher H, Deeg M, Sahl HG, Peschel A, Götz F, Garbe C, Schittek B (2006) Naturally processed dermcidin-derived peptides do not permeabilize bacterial membranes and kill microorganisms irrespective of their charge. Antimicrob Agents Chemother 50:2608–2620

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Tanaka T, Maeda H, Matsuo T, Boldbattar D, Umemiya-Shirafuji R, Kume A, Suzuki H, Xuan X, Tsuji N, Fujisaki K (2012) Parasiticidal activity of Haemaphysalis longicornis longicin P4 peptide against Toxoplasma gondii. Peptides 34:242–250

    Article  CAS  PubMed  Google Scholar 

  • Tirloni L, Reck J, Terra RM, Martins JR, Mulenga A, Sherman NE, Fox JW, Yates JR 3rd, Termignoni C, Pinto AF, Vaz Ida S Jr (2014) Proteomic analysis of cattle tick Rhipicephalus (Boophilus) microplus saliva: a comparison between partially and fully engorged females. PLoS One 9:e94831

    Article  PubMed Central  PubMed  Google Scholar 

  • Tonk M, Cabezas-Cruz A, Valdés JJ, Rego R, Chrudimská T, Strnad M, Íma R, Bell-Sakyi L, Franta ZK, Vilcinskas A, Grubhoffer L, Rahnamaeian M (2014a) Defensins from the tick Ixodes scapularis are effective against phytopathogenic fungi and the human bacterial pathogen Listeria grayi. Parasit Vectors 7:554

    Article  PubMed Central  PubMed  Google Scholar 

  • Tonk M, Cabezas-Cruz A, Valdés JJ, Rego RO, Rudenko N, Golovchenko M, Bell-Sakyi L, de la Fuente J, Grubhoffer L (2014b) Identification and partial characterisation of new members of the Ixodes ricinus defensin family. Gene 540:146–152

    Article  CAS  PubMed  Google Scholar 

  • Tsuji N, Battsetseg B, Boldbaatar D, Miyoshi T, Xuan X, Jr Oliver JH, Fujisaki K (2007) Babesial vector tick defensin against Babesia sp. parasites. Infect Immun 75:3633–3640

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Yang ST, Shin SY, Hahmb KS, Kim JI (2006) Design of perfectly symmetric Trp-rich peptides with potent and broad-spectrum antimicrobial activities. Int J Antimicrob Agents 27:325–330

    Article  CAS  PubMed  Google Scholar 

  • Yount NY, Bayer AS, Xiong YQ, Yeaman MR (2006) Advances in antimicrobial peptide immunobiology. Biopolymers 84:435–458

    Article  CAS  PubMed  Google Scholar 

  • Yu D, Sheng Z, Xu X, Li J, Yang H, Liu Z, Rees HH, Lai R (2006) A novel antimicrobial peptide from salivary glands of the hard tick, Ixodes sinensis. Peptides 27:31–35

    Article  CAS  PubMed  Google Scholar 

  • Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature 415:389–395

    Article  CAS  PubMed  Google Scholar 

  • Zhang H, Lee EG, Liao M, Compaore MK, Zhang G, Kawase O, Fujisaki K, Sugimoto C, Nishikawa Y, Xuan X (2007) Identification of ribosomal phosphoprotein P0 of Neospora caninum as a potential common vaccine candidate for the control of both neosporosis and toxoplasmosis. Mol Biochem Parasitol 153:141–148

    Article  CAS  PubMed  Google Scholar 

  • Zhang H, Nishikawa Y, Yamagishi J, Zhou J, Ikehara Y, Kojima N, Yokoyama N, Xuan X (2010) Neospora caninum: application of apical membrane antigen 1 encapsulated in the oligomannose-coated liposomes for reduction of offspring mortality from infection in BALB/c mice. Exp Parasitol 125:130–136

    Article  CAS  PubMed  Google Scholar 

  • Zhang H, Zhang W, Wang X, Zhou Y, Wang N, Zhou J (2011) Identification of a cysteine-rich antimicrobial peptide from salivary glands of the tick Rhipicephalus haemaphysaloides. Peptides 32:441–446

    Article  PubMed  Google Scholar 

  • Zheng H, Zhou L, Yang X, Wang D, Liu J (2012) Cloning and characterization of a male-specific defensin-like antimicrobial peptide from the tick Haemaphysalis longicornis. Dev Comp Immunol 37:207–211

    Article  CAS  PubMed  Google Scholar 

  • Zhou J, Gong H, Zhou Y, Xuan X, Fujisaki K (2006a) Identification of a glycine-rich protein from the tick Rhipicephalus haemaphysaloides and evaluation of its vaccine potential against tick feeding. Parasitol Res 100:77–84

    Article  PubMed  Google Scholar 

  • Zhou J, Liao M, Hatta T, Tanaka M, Xuan X, Fujisaki K (2006b) Identification of a follistatin-related protein from the tick Haemaphysalis longicornis and its effect on tick oviposition. Gene 372:191–198

    Article  CAS  PubMed  Google Scholar 

  • Zhou J, Liao M, Ueda M, Gong H, Xuan X, Fujisaki K (2007) Sequence characterization and expression patterns of two defensin-like antimicrobial peptides from the tick Haemaphysalis longicornis. Peptides 28:1304–1310

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the Special Fund for Health Research in the Public Interest (grant No. 201202019), and the Agricultural Science and Technology Innovation Program (ASTIP) of China.

Author information

Author notes

    Authors and Affiliations

    1. Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518 Ziyue Road, Minhang District, Shanghai, 200241, China

      Houshuang Zhang, Siqi Yang, Haiyan Gong, Jie Cao, Yongzhi Zhou & Jinlin Zhou

    2. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, China

      Jinlin Zhou

    3. Natural History Research Center, Shanghai Natural History Museum, Shanghai Science & Technology Museum, Shanghai, 200127, China

      Siqi Yang

    Authors
    1. Houshuang Zhang
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Siqi Yang
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Haiyan Gong
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Jie Cao
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Yongzhi Zhou
      View author publications

      You can also search for this author in PubMed Google Scholar

    6. Jinlin Zhou
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to Jinlin Zhou.

    Additional information

    Houshuang Zhang and Siqi Yang contributed equally to this work.

    Rights and permissions

    Reprints and permissions

    About this article

    Check for updates. Verify currency and authenticity via CrossMark

    Cite this article

    Zhang, H., Yang, S., Gong, H. et al. Functional analysis of a novel cysteine-rich antimicrobial peptide from the salivary glands of the tick Rhipicephalus haemaphysaloides . Parasitol Res 114, 3855–3863 (2015). https://doi.org/10.1007/s00436-015-4615-8

    Download citation

    • Received:

    • Accepted:

    • Published:

    • Issue Date:

    • DOI: https://doi.org/10.1007/s00436-015-4615-8

    Keywords

    Search

    Navigation