Molecular and structural characterization of novel cystatins from the taiga tick Ixodes persulcatus

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Abstract

Cystatins are cysteine peptidase inhibitors that in ticks mediate processes such as blood feeding and digestion. The ixodid tick Ixodes persulcatus is endemic to the Eurasia, where it is the principal vector of Lyme borreliosis. To date, no I. persulcatus cystatin has been characterized. In the present work, we describe three novel cystatins from I. persulcatus, named JpIpcys2a, JpIpcys2b and JpIpcys2c. In addition, the potential of tick cystatins as cross-protective antigens was evaluated by vaccination of hamsters using BrBmcys2c, a cystatin from Rhipicephalus microplus, against I. persulcatus infestation. Sequence analysis showed that motifs that are characteristic of cystatins type 2 are fully conserved in JpIpcys2b, while mutations are present in both JpIpcys2a and JpIpcys2c. Protein-protein docking simulations further revealed that JpIpcys2a, JpIpcys2b and JpIpcys2c showed conserved binding sites to human cathepsins L, all of them covering the active site cleft. Cystatin transcripts were detected in different I. persulcatus tissues and instars, showing their ubiquitous expression during I. persulcatus development. Serological analysis showed that although hamsters immunized with BrBmcys2c developed a humoral immune response, this response was not adequate to protect against a heterologous challenge with I. persulcatus adult ticks. The lack of cross-protection provided by BrBmcys2c immunization is perhaps linked to the fact that cystatins cluster into multigene protein families that are expressed differentially and exhibit functional redundancy. How to target such small proteins that are secreted in low quantities remains a challenge in the development of suitable anti-tick vaccine antigens.

Introduction

Cystatins are reversible tightly binding peptidase inhibitors that are found inside cells and in body fluids (Abrahamson et al., 2003). The cystatin superfamily is divided in three families (types 1, 2 and 3) sharing a common cystatin domain, a cleft-shape structure responsible for binding to the active site of target peptidases. This domain is typically formed by three motifs: (i) the G amino acid residue in the N-terminal region; (ii) the QXVXG and (iii) PW amino acid residues in the C-terminal region (Bode et al., 1988). Cystatins of type 1 are intracellular inhibitors found inside lysosomes; they have one cystatin domain, no disulfide bonds, and neither a signal peptide or glycosylation site. Cystatins of type 2 are non-glycosylated extracellular inhibitors composed of two loops, five stranded β-sheets and one alpha helix in perpendicular position (Bode et al., 1988). The type 3 cystatins, also known as kininogens, are glycosylated proteins with multiple cystatin domains in their structure, being found in mammal intravascular tissue (Sharma, 2006, Schwarz et al., 2012).

Cystatins interact with cysteine proteases through a well-established mechanism, where the cysteine peptidase active site cleft is completely covered by the wedge-shaped edge formed by the three conserved motifs of cystatins (Bode et al., 1988, Stubbs et al., 1990). For example, the cystatin-cathepsin binding mode is based on the configuration in which all cystatins insert the N-terminus and loop L1 into the active site of cathepsin, while loop L2 provides additional interactions on the external surface of the active site (Bode et al., 1988, Alvarez-Fernandez et al., 2005, Ljunggren et al., 2007, Nandy and Seal, 2016).

In ticks, only type 1 and 2 cystatins have been identified, and currently, the specific target enzymes for tick cystatins are not fully known. One recognized role of cystatins in tick physiology is the modulation of blood digestion through inhibition of cathepsins B, L, and C, which are involved in hemoglobin proteolysis (Franta et al., 2010). These inhibitors also participate in the modulation of peptidases involved in host immune response (Hewitson et al., 2009), such as cathepsins S and L, affecting antigen processing and presentation, MHC maturation, and angiogenesis (Hsing and Rudensky, 2005, Obermajer et al., 2008). Cathepsin L, specifically, has a well-known role in the development of adaptive immune response in mammals (Lombardi et al., 2005, Hsing and Rudensky, 2005). In addition, it has been reported that tick cystatins can interact with host cathepsin L with high affinity (Kotsyfakis et al., 2006, Kotsyfakis et al., 2007, Wang et al., 2015a, Wang et al., 2015b, Parizi et al., 2015), suggesting the role of tick cystatins in host-parasite interactions.

The taiga tick, Ixodes persulcatus, is distributed mainly in Russia and southern Eurasia (Korenberg, 2000, Eremeeva et al., 2007) and parasitizes a number of vertebrate hosts, including humans (Estrada-Peña and Jongejan, 1999). It transmits Lyme borreliosis through infection with the spirochetes Borrelia adzelii and Borrelia garinii (Steere et al., 2004). Presently, Lyme borreliosis is the most important vector-borne disease in the temperate northern hemisphere (Moyer, 2015) and a better understanding of the physiology and ecology of the tick vectors, including I. persulcatus, is necessary to devise improved disease control strategies.

Anti-tick vaccines are being promoted as critical to integrated tick control, which seeks to reduce the amount and frequency of acaricides use (Schetters et al., 2016), counteract acaricide resistance in ticks (Willadsen, 2006), and control tick borne-diseases (Sprong et al., 2014, Moyer, 2015). A number of tick vaccine antigens have been evaluated in both homologous and heterologous challenge systems (Parizi et al., 2012a, de la Fuente and Contreras, 2015). However, little data is available on such trials with the taiga tick I. persulcatus (Konnai et al., 2011, Gomes et al., 2015).

Cystatins have been evaluated as vaccine antigen candidates against infestations by hard and soft ticks, resulting in partial protection against Ixodes scapularis (Kotsyfakis et al., 2008) and Ornithodoros moubata (Salát et al., 2010), respectively. Interestingly, cross-protection trials demonstrated the potential use of conserved tick proteins to control different tick species (Parizi et al., 2012a). Antibodies against BrBmcys2c, a cystatin from Rhipicephalus microplus salivary glands and gut (Parizi et al., 2015), present cross-reaction with other R. microplus cystatins (Parizi et al., 2013). Moreover, the similarity in primary and secondary structures among various tick cystatins suggests that antibodies against recombinant BrBmcys2c (rBrBmcys2c) could recognize I. persulcatus cystatins.

The objective of the present investigation was to identify and characterize novel cystatins from I. persulcatus using molecular and structural approaches. In addition, a vaccine trial was conducted to evaluate the potential of rBrBmcys2c cystatin from R. microplus to protect against I. persulcatus infestation.

Section snippets

Ticks and hamsters

Adult I. persulcatus ticks were collected by flagging from the lower vegetation in forests of Hokkaido, Japan. The ticks were maintained at 28 °C and 85% of humidity in a climate controlled room. Hamsters (Mesocricetus auratus) were obtained from laboratory colonies of Syrian hamsters (Japan SLC, Shizuoka, Japan). The animals were kept in a P3 animal facility at Graduate School of Veterinary Medicine, Hokkaido University. The experimental protocol followed the Institutional Animal Care and Use

Sequence analysis

The deduced amino acid sequences of the three cystatins complete ORFs (JpIpcys2a, JpIpcys2b, and JpIpcys2c) were 133, 140, and 132 amino acids long, respectively (Fig. 1). The predicted amino acid sequences of these cystatins have theoretical molecular weights of 14.3, 15.6, 14.6 kDa, and predicted isoelectric points of 5.53, 8.80 and 7.63, respectively. Moreover, all three novel cystatins, as well as BrBmcys2c, have signal peptide and four cysteine residues responsible for two disulfide bridges

Discussion

The I. persulcatus cystatins described in this work have molecular characteristics of secretory type 2 cystatins. The importance of cystatin motifs for the interaction between the inhibitor and the enzyme active site cleft has been previously described (Abrahamson et al., 1987, Bode et al., 1988, Mihelic et al., 2008). Among the three cystatins identified in the present study, only JpIpcys2c showed amino acid substitutions on all three conserved cystatin motifs. The N-terminal glycine (the only

Acknowledgments

This work was supported by grants from CNPq-Instituto Nacional de Ciência e Tecnologia de Entomologia Molecular, CAPES, CNPq, FAPERJ and FAPERGS (Brazil), and MEXT (Japan).

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    1

    Equal contributors.

    2

    Current affiliation: Tick Unit, International Livestock Research Institute, Nairobi, Kenya.

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