GRP structural characteristics

The content of Gly in the composition of GRPs varies between proteins, and Gly-rich motifs can be arranged in specific patterns or not. Glycine-rich regions can form loops, giving the region a flexible characteristic (Steinert et al., Reference Steinert, Mack, Korge, Gan, Haynes and Steven1991; Högel et al., Reference Högel, Götz, Kuhne, Ebert, Stelzer, Rand, Scharnagl and Langosch2018). Gly residues themselves can also be displaced randomly or in patterns. Among ticks, the most common patterns described are tripeptides, such as GXG, GXX and GGX, but Gly-rich repeats can also be found in longer arrangements and in varying patterns, as shown in Fig. 1 (Bishop et al., Reference Bishop, Lambson, Wells, Pandit, Osaso, Nkonge, Morzaria, Musoke and Nene2002; Francischetti et al., Reference Francischetti, Sa-Nunes, Mans, Santos and Ribeiro2009; Maruyama et al., Reference Maruyama, Anatriello, Anderson, Ribeiro, Brandão, Valenzuela, Ferreira, Garcia, Szabó, Patel, Bishop and De Miranda-Santos2010; Leal et al., Reference Leal, Alzugaray, Seixas, Vaz and Ferreira2018). For instance, the glycine-rich region of P29 of Haemaphysalis longicornis is present in the middle of the sequence and has been described as a collagen-like domain, as the GXX repeat that predominates resembles those present in vertebrate collagen proteins (Mulenga et al., Reference Mulenga, Sugimoto, Sako, Ohashi, Musoke, Shubash and Onuma1999). The classification of GRPs within ticks has been primarily based on motif similarity, although it also been categorized by function, such as the cement GRPs, or by orthology, such as some Rhipicephalus GRPs, which are further categorized into distinct subclasses (GYG, expansion of large GYG, and superlarge GYG) (Francischetti et al., Reference Francischetti, Sa-Nunes, Mans, Santos and Ribeiro2009; Ribeiro et al., Reference Ribeiro, Anderson, Manoukis, Meng and Francischetti2011; Ribeiro and Mans, Reference Ribeiro and Mans2020).

Figure 1. Gly-repeat patterns in tick GRP sequences. Each repeat is represented with a different color, and pattern variations within sequences are highlighted with the respective color. Sequences presented are: 64P of Haemaphysalis longicornis (AAM09648.1), RmGRP of Rhipicephalus microplus (AQX36208.1), Sal 1 of Rhipicephalus annulatus (AGR45924.1), NPL-2 of Ixodes scapularis (EEC15723.1), Ctenidin-1-like of Ixodes scapularis (XP_029830867.1), and Aam-3099 of Amblyomma americanum (JAG92486.1).

As previously described, Gly residues are involved in the helical conformation of proteins, influencing the helix-helix packing, and, for instance, conferring greater stability to tertiary structures in transmembrane domains (Dong et al., Reference Dong, Sharma, Zhou and Cross2012). Furthermore, the flexibility of proteins may be influenced by the position of Gly residues. Gly can lead to more flexible termini or rigid centres, which in turn alters the hydration and H-bonding of the helix (Högel et al., Reference Högel, Götz, Kuhne, Ebert, Stelzer, Rand, Scharnagl and Langosch2018).

At least three Metastriate ticks present GRPs containing GGY or GGY-related motifs, which, interestingly, present similarities with silk sequences of spiders (Mulenga et al., Reference Mulenga, Blandon and Khumthong2007; Maruyama et al., Reference Maruyama, Anatriello, Anderson, Ribeiro, Brandão, Valenzuela, Ferreira, Garcia, Szabó, Patel, Bishop and De Miranda-Santos2010). In dragline spider silks, these motifs are associated with mechanical characteristics, such as fibre strength, despite the glycine-rich region usually corresponds to a non-elastic domain (Brooks et al., Reference Brooks, Stricker, Joshi, Kamerzell, Middaugh and Lewis2008; Malay et al., Reference Malay, Sato, Yazawa, Watanabe, Ifuku, Masunaga, Hikima, Guan, Mandal, Damrongsakkul and Numata2016 and, Reference Malay, Arakawa and Numata2017). In the insects Bombyx mori and Antheraea pernyi, the GGY sequence appears to be associated with cuticle hardening and plays a fundamental role in humidity-induced behaviour of silks (Suzuki et al., Reference Suzuki, Matsuoka, Iimura and Fujiwara2002; Futahashi et al., Reference Futahashi, Okamoto, Kawasaki, Zhong, Iwanaga, Mita and Fujiwara2008; Wang et al., Reference Wang, Lin, Ren, Pei, Chen, Qi, Shao and Ling2020).

The intrinsically disordered nature of many GRPs seems to facilitate liquid-liquid phase separation (LLPS) in the process of forming adhesive structures. In a salivary GRP from Ixodes ricinus, the N-terminal portion is more negatively charged, while the C-terminal portion has more aromatic residues regularly spaced by glycine-rich regions, which allows for extensive cation-π interactions and therefore the accumulation of particles around the GRP, leading to coacervation. Furthermore, it was demonstrated that the mature GRP (without the signal peptide), referred to as Tick-GRP77, undergoes LLPS and forms viscoelastic solid structures, as a transition to adhesive forms, what corroborates with a cement formation function (Ganar et al., Reference Ganar, Turbina, Nandy, Chen, Suylen, van der Beelen, Louise Pascoe, Koenraadt, Dijkgraaf and Deshpande2023).

Tissue and ontogenetic distribution in ticks

GRPs have been frequently identified in the tick cement, and their tissue localization has been studied in several tick species (Kemp et al., Reference Kemp, Stone, Binnington, Frederick and Rachel1982). The components of cement are secreted in saliva, and the host species, feeding, and mouthparts morphology influence its composition (Tabor et al., Reference Tabor, Ali, Rehman, Garcia, Zangirolamo, Malardo and Jonsson2017). Furthermore, GRPs presence is also modulated by biotic and abiotic factors (see Table 1). The following subsections present, compare, and discuss data on when and where these proteins are found and expressed in ticks, as well as the known conditions that may affect them.

Table 1. Distribution of glycine rich proteins in different tissues and developmental stages of ticks, and their response to feeding

Possible roles of GRPs in ticks

As described in the previous sections, tick GRPs may present multiple functions. In a similar manner, four classes of GRPs and more than 12 functions for GRPs in plants have already been described (Mangeon et al., Reference Mangeon, Junqueira and Sachetto-Martins2010). Tick GRPs are often associated with cement formation, especially those expressed in the SG. However, in other tissues they seem to perform alternative and/or unknown functions (Maruyama et al., Reference Maruyama, Anatriello, Anderson, Ribeiro, Brandão, Valenzuela, Ferreira, Garcia, Szabó, Patel, Bishop and De Miranda-Santos2010). The differences described for GRP variability and abundance between adult males and females also suggest additional roles, as males are expected to attach and detach more times than females during feeding and reproduction. In fact, R. appendiculatus and R. microplus showed more total GRP transcripts in males, suggesting potential involvement in other physiological processes (de Castro et al., Reference De Castro, de Klerk, Pienaar, Latif, Rees and Mans2016; Bensaoud et al., Reference Bensaoud, Aounallah, Sciani, Faria, Chudzinski-Tavassi, Bouattour and M'ghirbi2019; Garcia et al., Reference Garcia, Chaves Ribeiro, Maruyama, Gardinassi, Nelson, Ferreira, Andrade and de Miranda Santos2020).

The cement cone of R. microplus was previously described as presenting a high content of glycine (Kemp et al., Reference Kemp, Stone, Binnington, Frederick and Rachel1982). Thereafter, GRPs in the cement were suggested to promote the hardening of the adhesion structure (Bullard et al., Reference Bullard, Allen, Chao, Douglas, Das, Morgan, Ching and Karim2016b). As shown above, this is strongly corroborated by the property of certain GRPs to form a gel-solid structure after LLPS (Ganar et al., Reference Ganar, Turbina, Nandy, Chen, Suylen, van der Beelen, Louise Pascoe, Koenraadt, Dijkgraaf and Deshpande2023).

Another important function that may be performed by tick GRPs is the modulation of the host immune response. The similarity of some GXX/GGX repeats in tick GRPs compared to the Gly-rich motifs of vertebrate GRPs indicates a probable function on evasion of host defence/haemostasis by mimicking host skin components (Mulenga et al., Reference Mulenga, Sugimoto, Sako, Ohashi, Musoke, Shubash and Onuma1999; Bishop et al., Reference Bishop, Lambson, Wells, Pandit, Osaso, Nkonge, Morzaria, Musoke and Nene2002). This hypothesis is endorsed by the increased inflammation at tick attachment sites in GRP-silenced ticks, which also shows increased haemorrhaging at the tick bite site (Hollmann et al., Reference Hollmann, Kim, Tirloni, Radulović, Pinto, Diedrich, Yates, da Silva Vaz and Mulenga2018). Additionally, Gly can be recognized by receptors and when Gly binds to anion channel receptors, the chloride conductance increases, leading to chloride influx, which causes the hyperpolarization of platelets, thereby inhibiting platelet aggregation (Schemmer et al., Reference Schemmer, Zhong, Galli, Wheeler, Xiangli, Bradford, Conzelmann, Forman, Boyer and Thurman2013). Indeed, Ribeiro et al. (Reference Ribeiro, Alarcon-Chaidez, Ivo, Mans, Mather, Valenzuela and Wikel2006) identified one GRP as a probable inhibitor of platelet aggregation.

GRPs are also described to bind nucleic acids and RNA-binding GRPs are associated with the development of embryos in a range of organisms, as well as in the ecdysis of insects (Zhong et al., Reference Zhong, Mita, Shimada and Kawasaki2006; Ciuzan et al., Reference Ciuzan, Hancock, Pamfil, Wilson and Ladomery2015). In zebrafish, GRPs are also involved in spinal cord regeneration (Ma et al., Reference Ma, Cheng, Li, Deng, Zhang and Zhu2021). In ticks, the presence of GRPs in eggs was reported in H. qinghaiensis, H. longicornis, and R. microplus. Furthermore, silencing GRP genes has been shown to reduce hatchability and weight of larvae (Jiang et al., Reference Jiang, Gao, Wang, Xu, Li, Qi, Yang, Ji, Zhang, Luo and Yin2014; Leal et al., Reference Leal, Alzugaray, Seixas, Vaz and Ferreira2018; Luo et al., Reference Luo, Shen, Ren, Guan, Zhao, Yin, Chen, Zhao, Luo, Li and Liu2020). These data indicate that GRPs are important players in tick development and that further studies can elucidate the specific roles that GRPs play in these processes.

In response to stress, insect GRPs in the cuticle protect against temperature changes, allowing them to adapt to new environmental conditions (Zhang et al., Reference Zhang, Goyer and Pelletier2008). Response to environmental stress mediated by A. americanum GRPs has also been suggested, as temperature changes (cold and heat), oxidative stress, and injury positively or negatively modulate the expression of GRP genes. Interestingly, gene expression of the GRP Aam-40766 decreased in the SG but increased when the entire tick was analysed. On the other hand, the expression of the GRP Aam-36909 gene was downregulated in the whole tick when exposed to cold or injury stresses, but upregulated in the SG, which was suggested as a physiological adaptation (Bullard et al., Reference Bullard, Sharma, Das, Morgan and Karim2019). In eggs of H. longicornis, one GRP is also upregulated in response to temperature stress (25°C) (Luo et al., Reference Luo, Shen, Ren, Guan, Zhao, Yin, Chen, Zhao, Luo, Li and Liu2020).

Antimicrobial activity is a known function of GRPs and glycine-rich antimicrobial peptides (GR-AMPs), including silk GRPs from spiders, scorpions, and insects. GR-AMPs are molecules of the innate immune response that act against a wide range of pathogens. In general, GR-AMP sequences contain a signal peptide, a pro-peptide and a glycine-rich region, which usually present domains with repeats containing Gly residues (Yi et al., Reference Yi, Chowdhury, Huang and Yu2014; Wang and Wang, Reference Wang and Wang2016), similar to what can be found in tick GRPs. Attacins, ctenidins, gloverins, diptericins, serrulins, acanthoscurrins, hyastatin, and prolixins are some GR-AMPs that have already been described and characterized (Yi et al., Reference Yi, Chowdhury, Huang and Yu2014; Wang and Wang, Reference Wang and Wang2016; Meraj et al., Reference Meraj, Dhari, Mohr, Lowenberger and Gries2024). Most of assays employing GR-AMPs indicated activity against gram-negative bacteria, although effects against gram-positive bacteria, fungi, protozoa (Meraj et al., Reference Meraj, Dhari, Mohr, Lowenberger and Gries2024) and virus are also reported (Yi et al., Reference Yi, Chowdhury, Huang and Yu2014; Wang and Wang, Reference Wang and Wang2016). For example, serrulin from the scorpion Tityus serrulatus inhibits the growth of E. coli, Pseudomonas aeruginosa (gram-negative bacteria), Micrococcus luteus (gram-positive bacteria), Aspergillus niger (filamentous fungus) and Candida albicans (yeast) at low concentrations and its sequence presented similarity to acanthoscurrins (Acantho 1 and 2) from the spider Acanthoscurria gomesiana, which present a reported antimicrobial activity (Lorenzini et al., Reference Lorenzini, da Silva, Fogaça, Bulet and Daffre2003; de Jesus Oliveira et al., Reference de Jesus Oliveira, Oliveira and da Silva Junior2019). Interestingly, serrulin is also similar to a secreted GRP from the tick I. scapularis (de Jesus Oliveira et al., Reference de Jesus Oliveira, Oliveira and da Silva Junior2019), suggesting a possible antimicrobial role for this tick GRP.

GRPs/Gly-rich motifs may also present different activities not directly responsible for cellular killing. One such example is the GRP hyastatin of Hyas Araneus, which presents three domains: an N-terminal region containing Gly residues, a Pro/Arg-rich region and a C-terminal region containing six cysteine residues. Native hyastatin exhibits antimicrobial activity against bacteria (E. coli and Corynebacterium glutamicum) and yeast (S. cerevisiae and C. albicans), whereas the recombinant N-terminal region does not present the same effect, although both proteins are able to bind chitin. Therefore, it is suggested that the Gly-rich region may be primarily involved in pathogen attachment rather than in cell disruption (Sperstad et al., Reference Sperstad, Haug, Vasskog and Stensvåg2009). Insect attacins, the most extensively studied GR-AMPs, were first identified in the giant silk moth Hyalophora cecropia and are probably the most promising antimicrobials among the GRPs (Yi et al., Reference Yi, Chowdhury, Huang and Yu2014; Wang and Wang, Reference Wang and Wang2016). Attacins may increase outer membrane permeability, bind LPS, and inhibit the synthesis of specific proteins, such as the OMPs, without reaching the internal membrane or cytoplasm (Carlsson et al., Reference Carlsson, Nyström, de Cock and Bennich1998). In terms of mechanism of action, attacins can be compared to polymyxin. The polymyxin-resistant strain of P. mirabilis EH193 was found to be sensitive to an attacin at a concentration 100-fold lower than polymyxin (Carlsson et al., Reference Carlsson, Nyström, de Cock and Bennich1998; Yi et al., Reference Yi, Chowdhury, Huang and Yu2014). Additionally, a peptide with 50% identity to Bactrocera dorsalis attacin B showed antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) (Shin and Park, Reference Shin and Park2019). Although the antimicrobial activity of tick GRPs was not directly tested for tick GRPs, it has been previously suggested that members of the GGY GRP family may possess this activity (Ribeiro et al., Reference Ribeiro, Alarcon-Chaidez, Ivo, Mans, Mather, Valenzuela and Wikel2006; Mulenga et al., Reference Mulenga, Blandon and Khumthong2007; Francischetti et al., Reference Francischetti, Sa-Nunes, Mans, Santos and Ribeiro2009). If tick GRPs do have antimicrobial properties, then modulation of GRP levels by pathogens may represent another component of the arms race in the host-parasite-vector relationship.

Vaccine potential

Tick infestations and tick-borne diseases are emerging global health concerns for both livestock and humans. Conventionally, acaricides are employed to control these ectoparasites, but resistant ticks and the worrisome effects of chemicals emphasizes the need for the development of alternative strategies, such as vaccines (Schetters et al., Reference Schetters, Bishop, Crampton, Kopáček, Lew-Tabor, Maritz-Olivier, Miller, Mosqueda, Patarroyo, Rodriguez-Valle, Scoles and De La Fuente2016). In this context, tick GRPs have already been identified as protective antigens, and the outcomes of immunization protocols can be seen summarized in Table 2 (Trimnell et al., Reference Trimnell, Davies, Lissina, Hails and Nuttall2005; Shahein et al., Reference Shahein, Abouelella, Hussein, Hamed, El-Hakim, Abdel-Shafy and Tork2013; Antunes et al., Reference Antunes, Couto, Ferrolho, Rodrigues, Nobre, Santos, Margarida Santos-Silva, de la Fuente and Domingos2018; Couto et al., Reference Couto, Seixas, Stutzer, Olivier, Maritz-Olivier, Antunes and Domingos2021).

Table 2. Glycine rich proteins from ticks used in immunization protocols, the respective outcomes and organ/tissue distribution

P29, a GRP of H. longicornis, was used for immunization of rabbits, reducing the feeding time and increasing the mortality of larvae and nymphs (Mulenga et al., Reference Mulenga, Sugimoto, Sako, Ohashi, Musoke, Shubash and Onuma1999). A recent study used HLMLP, a cysteine-glycine-rich protein, to immunize rabbits, affecting engorgement (Luo et al., Reference Luo, Shen, Ren, Guan, Zhao, Yin, Chen, Zhao, Luo, Li and Liu2020). Two other H. longicornis GRPs were used to immunize mice. Hlim2 reduced the weight of engorged females, while Hlim3 reduced tick attachment and duration of feeding time (Harnnoi et al., Reference Harnnoi, Watchabunsook, Sakaguchi, Xuan and Fujisaki2006).

Another potential antigen candidate is RH50 from Rhipicephalus haemaphysaloides. In ticks fed to RH50-immunized rabbits, the attachment of nymphs and adults decreased, and nymphal mortality increased (Zhou et al., Reference Zhou, Gong, Zhou, Xuan and Fujisaki2006). Vaccination of Holstein calves with Rm39, a GRP of R. microplus, resulted in weak immune recognition, and significant anti-Rm39 IgG levels were only obtained at the end of the challenge. However, egg weight and hatch rate were reduced in vaccinated calves, while fed adult ticks were capable of full engorgement, albeit with a pale colour (Maruyama et al., Reference Maruyama, Garcia, Teixeira, Brandão, Anderson, Ribeiro, Valenzuela, Horackova, Veríssimo, Katiki, Banin, Zangirolamo, Gardinassi, Ferreira and De Miranda-Santos2017).

Mice immunized with the R. appendiculatus salivary protein 64TRP reduced the transmission of tick-borne encephalitis virus (TBEV) when transmission occurred from infected mice to nymphs and infected ticks to mice. Also, secreted cement proteins demonstrated an anamnestic response after 64TRP vaccination (Labuda et al., Reference Labuda, Trimnell, Ličková, Kazimírová, Davies, Lissina, Hails and Nuttall2006). In guinea pigs, the immunization with 64TRP reduced the attachment rate of R. sanguineus, and the vaccination with three different recombinant protein versions of truncated 64TRP fragments (64TRP2, 64TRP3 and 64TRP5) reduced the attachment of R. appendiculatus. I. ricinus fed in rabbits immunized with 64TRP2 reduced engorged weight and egg mass weight, and increased mortality, whereas immunization with 64TRP2/6 increased mortality of nymphs fed on guinea pigs. The feasibility of a broad-spectrum 64TRP-based vaccine against four ticks (R. appendiculatus, I. ricinus, A. variegatum and R. microplus) has been suggested based on the presence of cross-reactive conserved protective epitopes (Trimnell et al., Reference Trimnell, Hails and Nuttall2002, Reference Trimnell, Davies, Lissina, Hails and Nuttall2005).

Other GRPs have been identified as potential vaccine candidates, but further studies are needed to assess their antigenic and immunogenic effects in the host and tick (de la Fuente and Contreras, Reference de la Fuente and Contreras2022). Proteins present in tick saliva are interesting targets, as they must interact directly with the host immune system. Moreover, an increase in tick-borne disease transmission and tick territorial dispersal, both of which are influenced by climatic changes, highlights the potential health risks that ticks may pose to the animal food chain and public health (Madison-Antenucci et al., Reference Madison-Antenucci, Kramer, Gebhardt and Kauffman2020; Tardy et al., Reference Tardy, Vincenot, Bouchard, Ogden and Leighton2022). This reinforces the necessity for the development of anti-tick vaccines, particularly those of broad-spectrum.