Short Communication
Defensin from the ornate sheep tick Dermacentor marginatus and its effect on Lyme borreliosis spirochetes

https://doi.org/10.1016/j.dci.2014.04.005Get rights and content

Highlights

  • Tick D. marginatus expresses defensin (defDM) in several organs after blood feeding.

  • Mature defDM was purified from tick hemolymph and its full sequence was determined.

  • Synthetic and well-folded defDM possesses anti-Gram-positive bacterial activities.

  • DefDM reveals low hemolytic properties.

  • Borrelia afzelii, the Lyme disease agent, was inhibited by synthetic defDM.

Abstract

Expression of the previously reported defensin of the tick Dermacentor marginatus (defDM) was analysed in different organs by RT-PCR. mRNA of the defDM gene was detected in the hemolymph, midgut and salivary glands. Moreover defDM was isolated from the tick hemolymph using RP-HPLC and its sequence was determined by mass spectrometry and Edman degradation. Synthetic peptide was used for determining biological activities. The results showed an anti-Gram-positive bacterial role for the defensin. As D. marginatus ticks appear not to be vectors of the Lyme disease agent of the complex Borrelia burgdorferi sensu lato, we tested the influence of defDM on Borrelia afzelii. There is a very clear borrelicidal activity of the defensin, which is concentration dependent and suggests a possible role in the clearing of Borrelia ingested by D. marginatus ticks.

Introduction

Ticks are generally known as obligate blood sucking ectoparasites. Their impact on men and animals may be different: from unpleasant itching to severe anaemia or transmission of various pathogens. The European ornate sheep tick Dermacentor marginatus is a common vector of Ricketsiia slovaca, the causative agent of tick-borne lymphadenopathy (TIBOLA) in southern Europe (Masala et al., 2012). D. marginatus may also be playing a role as a vector of tick-borne encephalitis virus (TBEV), Coxiella burnetii, and the protozoan parasite Babesia canis (Kocianová et al., 2001, Nosek and Kožuch, 1985, Pietrobelli et al., 2007).

Ticks of the genus Dermacentor are found throughout various geographical regions that overlap areas where there is an incidence of human Lyme disease (LD). Studies revealed, that in contrast to Ixodes scapularis ticks, which are vectors of LD in North America, Dermacentor variabilis ticks are not able to successfully maintain and transmit Borrelia burgdorferi sensu lato, the causative agent of LD (Piesman and Sinsky, 1988). Many tick species of the genera Dermacentor and Amblyomma were examined to prove their incompatibility to maintain or transmit B. burgdorferi (Piesman and Happ, 1997, Piesman and Sinsky, 1988, Ryder et al., 1992). D. variabilis was shown to possess a 5.3 kDa peptide (defensin) with borrelicidal activity within the tick hemolymph (Johns et al., 2001a, Johns et al., 2001b). Also salivary gland (SGE) and midgut extracts (MGE) from the tick Dermacentor reticulatus had an impact on Borellia garinii in vitro. Both SGE and MGE affected the spirochaetal motility; moreover the SGE had changed borrelial morphology (Rudolf and Hubálek, 2003).

Defensins are a well studied family of antimicrobial peptides (AMPs) in ticks (Kopáček et al., 2010). Defensins identified within arthropods share sequence similarities and are produced as pre- pro-peptides. The signal sequence is cleaved out after transporting the peptide to the target site. The pro-segment includes a conserved region, the furin motif RVRR that separates the pro- part from the mature peptide. Mature defensins consist of 38–39 amino acid residues including six cysteine residues which are engaged in a characteristic conserved motif of three intramolecular disulfide bridges connected in a Cys1–Cys4, Cys2–Cys5 and Cys3–Cys6 pattern. This specific cysteine pairing forms a common structural element typical for arthropod defensins, known as the cysteine-stabilised αβ (CSαβ) motif that is essential for their antimicrobial activity (Bulet and Stöcklin, 2005). The importance of proper defensin folding was demonstrated by Isogai and colleagues (2011) where the linear form of the tick defensin Persulcatusin exhibited only low antibacterial activity in comparison to the same but well folded, three-dimensional peptide. Besides the previously mentioned anti-borrelial activity of D. variabilis defensin, tick defensins are known to be active primarily against Gram-positive bacteria with some studies also proving them to have antifungal or antiparasitic effects (Chrudimská et al., 2011, Saito et al., 2009, Tsuji et al., 2007).

In this study, we have focussed on characterizing defensin isolated from the tick D. marginatus with the main aim of revealing its anti-microbial properties including anti-borrelial activity.

Section snippets

Tick samples

D. marginatus tick colony is maintained at the Institute of Parasitology, Biological Centre, Academy of Sciences of the Czech Republic. Uninfected adult females were fed on adult guinea pigs (infection-free animals treated in accordance with the Animal Protection Law of the Czech Republic No. 246/1992 Sb; ethics approval number 137/2008). Ticks were collected after drop- off following blood feeding (fully fed ticks) or 4–5 days post-attachment (removed forcibly using forceps; semi-fed ticks).

Identification of defDM at the mRNA level and its expression profile in tick organs using RT-PCR

In the previous study on tick defensin isoforms, we had obtained a D. marginatus defensin using degenerative primer pairs (Chrudimská et al., 2010). To verify the 3′end of the mRNA defensin transcript, important for prediction of the mature protein sequence, 3′ RACE was performed. The mRNA defDM sequence (mature part), obtained by this method, was identical to the one previously published (accession number FJ222583; Supplemental Fig. 5).

Two step RT-PCR was used for determination of the

Discussion

Like most other arthropods, the tick immune system consists of only innate immune pathways and is able to utilize them effectively as a defence against certain pathogens. In the last two decades, the tick hemolymph as well as tick hemocytes have become a rich source for the identification and characterization of many antimicrobial proteins/peptides (AMPs). These include defensins, histidine rich AMPs, lysozymes and pattern recognition molecules (Chrudimská et al., 2011, Fogaça et al., 2004, Lai

Conclusions

In conclusion, a recently identified defensin from the hard tick D. marginatus was detected in different tick organs, purified from the tick hemolymph, synthesized and biologically characterized. Its low hemolytic activity as well as effective anti-Gram-positive bacterial properties may indicate a potential for future research. Anti-borrelial activities were confirmed suggesting a possible role in clearance of ingested Borrelia from a blood meal. It provides another layer in our understanding

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

We are grateful to Jan Erhart and Gabriela Loosová for their help during the work with the ticks and to Dr. Daniel Růžek for his help during TBEV testing. We also thank Lenka Monincová for performing the MS analyses and Vladimír Fučík for culturing the bacteria and yeast. This work was supported by the grant of the Czech Science Foundation P302/11/1901, institutional research grant Z60220518 (Biology Centre, Institute of Parasitology, Academy of Sciences of the Czech Republic), research project

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