The potential of type 2 cystatin in blood feeding and innate immunity of the tick Haemaphysalis doenitzi (Acari: Ixodidae)

doi:10.21203/rs.3.rs-3404101/v1

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The potential of type 2 cystatin in blood feeding and innate immunity of the tick Haemaphysalis doenitzi (Acari: Ixodidae)

https://doi.org/10.21203/rs.3.rs-3404101/v1

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Ticks, which are obligate blood-sucking parasites, transmit a wide range of pathogens during their blood-feeding process. Enzymes and macromolecules that inhibit enzymes play a crucial role in tick physiology. In the present study, genes encoding type 2 cystatin were cloned and characterized from Haemaphysalis doenitzi, and their functions in innate immunity and tick blood feeding were further assessed. Four cystatin genes, namely HDcyst-1, HDcyst-2, HDcyst-3, and HDcyst-4, were successfully cloned from the tick. Their open reading frames vary in length, with sizes of 390, 426, 426, and 426 base pairs (bp), respectively. Proteinase inhibition assays indicated that 0.2 µM of cystatin displayed a minimum of 50% inhibitory activity against cathepsin B and S. The qPCR results imply that HDcyst-2 could play a pivotal role in tick hemophagia, whereas HDcyst-1, HDcyst-3, and HDcyst-4 may be integral to the regulation of blood digestion. Our investigation unveiled that adult ticks subjected to LPS injection or exposed to low-temperature stress exhibited elevated expression levels of type 2 cystatin compared to the control group. RNA interference experiments demonstrated that the expression of type 2 cystatins had a significant impact on engorged weight, the number of eggs laid, hatching rate, and mortality of H. doenitzi, along with an extension of blood-feeding duration by 2 days. These discoveries suggest the involvement of type 2 cystatin in tick innate immunity and blood-feeding processes. Consequently, type 2 cystatin emerges as a promising candidate for tick control and the development of novel immunosuppressive drugs in the future.

Haemaphysalis doenitzi

Cystatin

Innate immunity

RNAi

Blood-feeding

Enzyme activity

Ticks are obligate hemophagous ectoparasites of several mammals, birds, reptiles, and amphibians (Chen and Liu 2022). Ticks can transmit bacterial, parasitic, and viral pathogens, often carrying multiple pathogens simultaneously, examples include tick-borne, babesiosis, emerging disease, Borrelia, Borrelia burgdorferi, Borrelia mayonii, Ixodes, Amblyomma, etc (Madison-Antenucci et al. 2020; Zhao et al. 2021). During blood feeding, ticks transmit pathogens without harming themselves is intimately related to the ability of defense (Fogaça et al. 2021). Besides, tick saliva contains a complex mixture of peptides and non-peptides that may act as a tool to disarm host immunity and assist in the positive effects of blood draw (Ali et al. 2022). Tick blood meal is stored and digested in the midgut, and there is a role for tick-derived proteins in the midgut content, such as defensin, cysteine protease, and serine protease inhibitor (Wu et al. 2022; Song et al. 2022; Chmelař et al. 2017).

Haemaphysalis doenitzi is a three-host tick and is a tick species endemic to southwestern China (Liu et al. 2016; Hoogstraal and Wassef 1973). Southeast Asia and Australia and many other countries are also widely distributed (Fukuoka et al. 2019). H. doenitzi is smaller than Haemaphysalis longicornis, and the adult tick has a yellowish-brown body surface, Mostly parasitic on the body surface of small mammals of hares and birds (Lu et al. 2022; Zhang et al. 2019). Little is known about H. doenitzi, but this research remains important for animal husbandry and medical development.

In the parasite-host relationship, parasites of animals with good immune responses have evolved many strategies to adapt them to survive and reproduce. Parasites are completers of evasion of host immunity, driving diversity within gene families and immune gene polymorphism within populations (Maizels 2009). With the increasing research on functional genes, specifically in the immune responses, among which cystatin is a natural tight-binding reversible inhibitor of cysteine proteases, which are widely present in all organisms (mammals, nematodes, arthropods, etc.) and are involved in the evasion of the host immune system, as well as in the regulation of normal protein hydrolysis and also in disease pathology (Zavasnik-Bergant 2008). They are involved in various biological physiological processes and inhibit the invasion and metastasis of tumor cells in vitro and vivo, adjustment of cysteine proteases, immune system modulation, reconstruction of bone, embryo development, blood digestion, and the inflammatory response, it plays an important role in processes (Xie et al. 2008; Zhou et al. 2010; Wang et al. 2015; Kotál et al. 2021; Zavašnik-Bergant et al. 2017).

According to the structure of cystatin, the cystatin superfamily is divided into 4 types of families: type 1 stefin, type 2 cystatin, type 3 kininogen, and type 4 cystatin-like proteins (fetuins, histidine-rich proteins) (Rawlings and Barrett 1990). Type 2 cystatin was first discovered in 1983, Anastas et al. detected, improved, and purified it from egg white, and it is the same kind of substance as human cystatin C, named Cys-C (Anastasi et al. 1983). Cystatins were first identified from Amblyomma americanum in 2005, and the physiological processes and functions involved in different tick species have been deeply studied (Karim et al. 2005).

In ticks, cold stress enhances their cold tolerance and triggers an appropriate innate immune response (Yu et al. 2014). Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria, and trace amounts of LPS released from infecting pathogens can initiate an effective innate immune response, allowing the immune system to defend against further infection (Park and Lee 2013). Insects have developed an additional innate immune system that cooperates with the RNAi response against invading pathogens (Berkhout 2018). The application of RNAi in ticks has led to the effect of tick function and tick-host-pathogen interactions, as well as tick survival and development (Setten et al. 2019). In this study, the sequence and structure of type 2 cystatin were determined, and the expression level of the type 2 cystatin gene was measured. We evaluated in vitro the cathepsin B and S activity of prokaryotic recombinant cystatins and evaluated for physiological processes of blood feeding using RNAi, using low-temperature stress and LPS injection to deepen our understanding of the molecular mechanisms of the innate immune response, to achieve a new way of thinking about controlling ticks.

Collection and feeding of ticks

Adult ticks were collected from sheep in Cangxi County, Sichuan Province, and incubated in a laboratory incubator with constant light (temperature 25.5°C ± 1°C; relative humidity 75% ± 4%; light L: D of 16h: 8h). All experiments in this study were performed with permission from the Animal Ethics Committee of Hebei Normal University (protocol number: IACUC-156027).

Cloning and sequence analysis of the full-length cDNA of cystatin

A cDNA library of the whole tick of H. doenitzi was constructed using the TransZol™ Up Plus RNA Kit (TransGen Biotech, China) method described previously (Lu et al. 2022). Based on H. doenitzi transcriptome library (accession number: MZ981733). Design specific primers with primer Premier 5.0 (Premier Biosoft International, United States), specific primers for cystatin (HDcyst-1: forward primer 5′-ATGACTGGCTGCTTGGCTCT-3′ and reverse primer 5′-TTACACGCAGTCAAATGAAGTTAG-3′; HDcyst-2: forward primer 5′-ATGGCTCTATCGAATGCTGCTG-3′ and reverse primer 5′-CTACGTAGACGAAGATCCCGTGTT-3′; HDcyst-3: forward primer 5′-ATGGCTCTACGTACTTCGGCG-3′ and reverse primer 5′-CTACGAAGATGAGGAGCCTCTTCC-3′; HDcyst-4: forward primer 5′-ATGGTATCTTCGAGTACAACTGCTT-3′ and reverse primer 5′-CTACGCAGAAGAAGAGGGGGA-3′), perform reverse transcription, the PCR program included high-temperature incubation at 94°C for 10 min, 30 cycles of denaturation at 94°C for 30 s, annealing at 60°C or 61°C for 30 s and extension at 70°C for 1 min, and a final extension at 72°C for 10 min. The PCR products were detected by electrophoresis on 1%-2% agarose gels (including Gold View type 1), and then the target bands were cut off, and the PCR products were recovered using the Easy Pure Quick Gel Extraction Kit. The cDNA was then stored at − 80°C until use. The purified PCR products were ligated with the pEASY-T1 vector, and the sequencing results were compared with the transcriptome library. The confirmed cDNAs of HDcyst-1, HDcyst-2, HDcyst-3, and HDcyst-4 were blasted (National Center for Biotechnology Information (NCBI) (https://blast.ncbi.nlm.nih.gov/Blast.cgi)) and the complete sequences of HDcyst-1, HDcyst-2, HDcyst-3, and HDcyst-4ited in the NCBI Nucleotide database under the separate accession numbers WGH08197.1, WGH08198.1, WGH08199.1, and WGH08200.1. The open reading frames (ORF) of HDcyst-1, HDcyst-2, HDcyst-3, and HDcyst-4 were identified by NCBI ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/). Sequence comparisons of all data used in this experiment were performed using NCBI using DNAMAN (Lynnon Biosoft Bioinformatic Solutions – Home for multiple sequence comparisons. Phylogenetic analyses based on the protein sequence across species were performed using MEGA 7.0, including Dermacentor andersoni (XP_050037667.1), Dermacentor silvarum (AJS11559.1), R. microplus (AIY34385.1), Rhipicephalus appendiculatus (AGB35873.1), Rhipicephalus haemaphysaloides (AIZ78006.1), Ixodes scapularis (AQN67695.1), I. persulcatus (AQN67693.1), H. longicornis (ABZ89554.1), H. flava (QBR53060.1), Ancylostoma caninum (RCN47450.1), Ancylostoma duodenale (KIH58790.1), Toxocara canis (KHN76703.1), Cynoglossus semilaevis (XP_008320464.1), Xiphophorus hellerii (XP_032409752.1), Xiphophorus couchianus (XP_027863228.1), Rhynchospora pubera (KAJ4762774.1), Brachypodium distachyon (XP_003562767.1), Phoenix dactylifera (XP_008811323.2), Argiope bruennichi (KAF8796835.1), Pamphobeteus verdolaga (CAH7646902.1), Cupiennius salei (QDC23069.1). Finally, molecular weight and theoretical isoelectric point were calculated using PI/Mw (https://www.expasy.org/) to predict the signal peptide of this gene using SignalP-5.0 (https://services.healthtech.dtu.dk/services/SignalP-5.0/).

Spatiotemporal expression analysis of cystatin

A total of 50 adult female H. doenitzi engorged with blood for 12 h were selected and four tissues, the salivary gland, ovary, midgut, and marsupial canal, were collected; gene expression was measured in different tissues at different life stages of the ticks: egg (incubated for 10 days), larvae (10 days after hatching), nymphs (10 days after molting), and adult (10 days after molting). Total RNA was extracted according to the above method with the same specific primers as above, and β-actin (accession number AY254898) was used as the internal reference gene (β-actin forward primer 5′ -CGTTCCTGGGTATGGAATCG-3′and β-actin reverse primer 5′-TCCACGTCGCACTTCATG-3′). Then, one-way ANOVA was performed on the qPCR results using GraphPad Prism 8.0.2 software (Prism 8.0.2 Release Notes (graphpad.com)).

LPS injection induces the expression of cystatin in adult ticks

Unfed female ticks were selected and injected with PBS in the control group and LPS in the experimental group. Approximately 0.5 µl of LPS (0.2 µg/ml LPS) was injected into the fourth basal segment of the abdomen of each female tick under a microscope using a beveled Hamilton’s needle (10 µl). After the ticks were left at 26°C for 12 h, the total RNA was extracted and subjected to real-time fluorescence quantitative PCR analysis.

Low-temperature stress induces the expression of cystatin

Six groups of H. doenitzi were removed, each with 30 heads. 3 groups were treated at room temperature (26°C) (3 days, 6 days, 9 days) and 3 groups were treated at low temperature (4°C) (3 days, 6 days, 9 days), and RNA was extracted from different tissues and different periods, diluted to the same concentration, and reverse transcribed for qPCR detection.

Expression and purification of cystatin recombinant protein

Redesigned primers to remove signal peptides (HDcyst-1: forward primer 5′-GGATCCATCCCAGGAGGCTGGAGAGA-3′ and reverse primer 5′-CTCGAGCACGCAGTCAAATGAAGTTAGC-3′; HDcyst-2: forward primer 5′-GGATCCGAGCATCGACTGGTTGGAG-3′ and reverse primer 5′-GCGGCCGCCGTAGACGAAGATCCCGTG-3′ ; HDcyst-3: forward primer 5′-GGATCCGACGAGCCCCACCTGGTGGGA-3′ and reverse primer 5′-GCGGCCGCCGAAGATGAGGAGCCTCTTC-3′ ; HDcyst-4: forward primer 5′-GGATCCCAGTTGGGAGTCCGCGGC-3′ and reverse primer 5′-CTCGAGCGCAGAAGAAGAGGGGGATGAG-3′).

HDcyst-1 and HDcyst-4 genes selected Bam I and Xho I as restriction sites to design primers, HDcyst-2 and HDcyst-3 genes selected Bam I and Not I as restriction sites to design primers, reverse transcription was performed, the purified product was ligated into pET-32(a+) vector, transformed into Escherichia coli, and the obtained bacterial broth was subjected to 37°C 200 r/min overnight, and some of the bacterial broth was taken for re-expansion, is the OD value of bacterial broth was between 0.4–0.6, and the screening of different IPTG, temperature and time was carried out, and the expression of cystatin was higher and the least amount of hetero protein above 37°C. Finally, 37°C, induction of 0.5 mM IPTG, and 8h of bacterial broth precipitation were selected as the best expression conditions. The recombinant proteins were purified using the ProteinIso™ Ni-NTA Resin kit (TransGen Biotech, China), firstly, the bacterial precipitate was denatured and dissolved with 8M urea and passed over the membrane; the samples were added to a Ni-NTA-filled column and affinity chromatographed on ice for 30min. then, the samples were eluted with different concentrations of imidazole by gravimetric affinity chromatography to obtain 3ml of elution buffer (0.025 M NaCl, 0.05 M NaHPO4, and 0.02 M imidazole, pH = 7.4) for the complexed recombinant protein; After elution for dialysis, about 15 cm dialysis bags were boiled in 2% sodium bicarbonate and 1 mmol/L EDTA for 10 min, distilled water was used to clean the dialysis bags, and then the bags were boiled in 1 mmol/L EDTA for 10 min, cooled and put at 4°C. Before use, the cleaned bags were filled with water excluded, and placed in dialysis solution for gradient dialysis (dialysis solution composition: 25 mM Tris-Hcl pH = 7.5, 100 mM NaCl, 0.2 mM EDTA, 10% glycerol, 0.2 M of gradient urea). The dialysis-concentrated samples were subjected to the BCA protein concentration assay kit (Transgenic, China), then diluted to the desired concentration, 40 µl aliquots of recombinant protein (0.2 µM) were boiled for 5 min, then verified by SDS-PAGE (12.5%), 10 µl rHDcyst-1 (0.2 µM) was reduced desalted and analyzed by mass spectrometry LC-MS/MS (Waters, USA).

Enzyme activity test

The kits used were the Cathepsin B and S Inhibitor Enzyme Activation Kit (ab185438 Cathepsin B Inhibitor Screening Kit (Fluorometric), ab185437 Cathepsin S Inhibitor Screening Kit (Fluorometric)). First, mix 49µL of CTSB(S) Reaction Buffer, 1 µL of Cathepsin B (S), 50µL/well and mix enough reagents for the assay (sample, inhibitor control, and blank control); 1×, 2×, 4×, 8×, 16×, 32×dilution of recombinant protein with CTSB(S) Reaction Buffer. Add 10µL of diluted Test Inhibitor or CTSB(S) Reaction Buffer to Cathepsin S (Enzyme Control); take 10µL of recombinant protein diluent (CTSB(S) Reaction Buffer for control), add to each well containing Cathepsin B(S) solution and incubate 3 times per sample for 10-15min at room temperature. Add 38µL of CTSB(S) Reaction Buffer and 2µL of CTSB(S) Substrate to each well, mix well, 40µL/well, and react for 30-60min at room temperature. Relative Inhibition (%) = (Slope of EC - Slope of the sample) / Slope of EC×100%.

Antioxidant analysis

Weigh 4.8 mg of DPPH-dissolved in 2 ml of anhydrous methanol and dilute 100 times when used (ready-to-use); dilute the protein samples to be tested into 1×, 2×, 4×, 8×, 16×, 32× concentrations with methanol as the sample group; add DPPH-, detect the absorbance value at 520 nm with an enzyme marker, repeat three times, and count the data; formula for free radical scavenging rate: ESC% = [1- (Apositive -Ablank) /Acontrol]×100%; Apositive: sample group; Acontrol: control group; Ablank; blank group.

RNAi experiment

The multiple functions of cystatin in rearing and reproduction were investigated using RNAi technology. Specific primers were designed for selected fragments of 100–200 bp size. (dsHDcyst-1: forward primer 5′ ACTTAGGGCTGCTCTGGGG-3′ and reverse primer 5′-CTGGCTGCTTGGCTCTTTC-3′; dsHDcyst-2: forward primer 5′-GAGAGGTGTCGTTCCGCC-3′ and reverse primer 5′-AATGCTGCTGCCTTCACTCTT-3′; dsHDcyst-3: forward primer 5′-AGTGTCAAAGAACTCCCGCCC-3′ and reverse primer 5′-ATGTTGTTGTGCTGCTACGCC-3′; dsHDcyst-4: forward primer 5′-GCGTGTGGTAATGTTCGTCG-3′ and reverse primer 5′-GTAGTGGCTGCCTGCTTGTG-3′), T7 promoter, and protective bases were added to the 5' end of the upstream and downstream primers, referring to Lu J.¹¹ The control GFP sequence (GFP: forward primer 5′-GCTTCTCGTTGGGGTCTTT-3′ and reverse primer 5′-GACGTAAACGGCCACAAGT-3′) and the interfering group were partial fragments of cystatin, which were used to synthesize dsRNA. 60 unfed female ticks were selected, and the interference group was injected with cystatin dsRNA and the control group was injected with GFP dsRNA. Approximately 0.5 µl of dsRNA (2000 ng) was injected and the injected ticks were placed in the incubator for 24 h for observation. Dead or weak ticks were removed after 24 h. Ticks from the disturbance and control groups were placed in the ear, and a fraction of the surviving ticks were subjected to qPCR to detect the relative expression of the gene. The experimental data were collected using the 2^−△△Ct algorithm and the RQ value of each sample was calculated according to the formula the biostatistical analysis was performed using SPSS 16.0 software (SPSS Inc, USA); the tick satiation process was collected and the blood-sucking time, engorged weight and engorgement rate were recorded. The engorged ticks were placed in an incubator and the egg-laying rate was recorded. Student’s t-test (P < 0.05) was used to determine significant differences between the efficiency of the RNAi for each group, and one-way ANOVA (P < 0.05)

Cloning and sequence analysis of the full-length cDNA of cystatin

The open reading frame (ORF) of HDcyst-1 spans 390 base pairs and encodes 129 amino acids, while the ORF of HDcyst-2, HDcyst-3, and HDcyst-4 consists of 426 base pairs, encoding 141 amino acids (as depicted in Fig. 1). These cystatins feature a predicted N-terminal region with a signal peptide, two disulfide bonds in the middle, a conserved "QXVXG" sequence, and a "PW" module, except for HDcyst-4, where the "LG" structure replaces "PW" (as illustrated in Fig. 2). The physicochemical properties of HDcyst-1, HDcyst-2, HDcyst-3, and HDcyst-4 were also predicted and are presented (Table 1). All four proteins possess signal peptides and exhibit isoelectric points of 4.75, 5.69, 5.32, and 6.39, respectively. Furthermore, a multiple sequence alignment unveiled that HDcyst-1 shared 99% similarity with Hlcyst-3 from H. longicornis, and HDcyst-3 exhibited 99% similarity with the cystatin from H. flava. This suggests a close genetic relationship between these proteins. Additionally, phylogenetic analysis demonstrated that type 2 cystatin proteins from ticks formed a distinct clade, separate from those of other species (as shown in Fig. 3). The type 2 cystatin protein identified in this study displays a particularly strong genetic affinity with cystatin proteins from the Haemaphysalis genus.

Table 1

The physicochemical parameters of cystatin.
Name	ORF(bp)	Amino acids	Molecular weight(kDa)	Isoelectric point	Signal peptide
HDcyst-1	390	129	13.91681	4.75	1–21
HDcyst-2	426	141	15.74268	5.69	1–24
HDcyst-3	426	141	15.60974	5.32	1–23
HDcyst-4	426	141	15.00291	6.39	1–24

Expression levels of the cystatin gene

The qPCR results unequivocally demonstrated the expression of cystatin across all tissues (midgut, salivary gland, Malpighian tubule, and ovary) and developmental stages (eggs, larvae, nymphs, and adults) of H. doenitzi. Notably, through one-way ANOVA analysis, it was evident that HDcyst-1, HDcyst-2, and HDcyst-4 exhibited their highest expression levels in the unfed larval stage, while HDcyst-3 reached its peak expression in the unfed nymph stage (P < 0.05) (see Fig. 4). Further, one-way ANOVA analysis also revealed distinct tissue-specific expression patterns: HDcyst-1 exhibited significantly elevated expression in the midgut compared to other tissues, HDcyst-2 displayed significantly higher expression in the salivary gland than in other tissues, and both HDcyst-3 and HDcyst-4 showed significantly increased expression in the Malpighian tubule relative to other tissues (P < 0.05) (see Fig. 5).

LPS injection tick and low-temperature stress induce cystatin expression

The injection of LPS effectively triggered the transcription of cystatin genes. In adult ticks, the relative expression of cystatin was notably elevated in the LPS-treated group at the 12-hour mark following injection. Specifically, HDcyst-1 and HDcyst-2 exhibited an approximately 1.8-fold increase compared to the PBS control group, while HDcyst-3 showed an approximately 1.6-fold increase, and HDcyst-4 displayed a roughly 1.9-fold increase (P < 0.05) (see Fig. 6). Furthermore, low-temperature stress at 4°C had a discernible impact on cystatin gene transcription, as indicated by qPCR results. The relative expression in the entire tick population initially exhibited a gradual decline over the first 3 days of low-temperature treatment, followed by an increase at the 6-day mark, and ultimately another decrease (P < 0.05) (see Fig. 7).

Purification of rHDcyst-1, rHDcyst-2, rHDcyst-3, and rHDcyst-4

The cystatin gene was successfully incorporated into the bacterial expression vector pET32(a+). Subsequently, rHDcyst-1, rHDcyst-2, rHDcyst-3, and rHDcyst-4 were expressed as recombinant proteins, exhibiting the expected size of 32 kDa (13 kDa for immature proteins). The expression of recombinant cystatin led to the formation of inclusion bodies, which were subsequently purified through affinity chromatography. The optimal elution concentration for the initial purification step was determined to be 200 mM imidazole. Following this initial purification, further refinement was achieved through dialysis and ultrafiltration. Subsequent analysis via SDS-PAGE confirmed the purity of the isolated cystatin (as evidenced in Fig. 8). To validate the identity of the purified cystatin, mass spectrometric data were subjected to a search in the UniProt protein database containing the recombinant protein sequence, utilizing Proteome Discoverer 2.2 by Thermo Fisher Scientific. The results revealed that the recombinant protein peptide, as identified by LC-MS/MS, accurately corresponded to the cystatin protein (Table 2). This outcome attests to the correctness of the E. coli prokaryotic expression system we established for the expression of cystatin in H. doenitzi.

Table 2

Mass spectrometric identifcation of recombinational cystatn.
Recombinant proteins	Number of specific peptides amino acid sequence	MH+ [Da]	XCor
rHDcyst-1	VEGLDNYDTVLDLTKVETQVVAGMNYR	3042.50884	2.55
	NLAHYAVAQRVEGLDNYDTVLDLTK	2818.437	3.29
	LTFTIAGSSCK	1184.59799	2.15
rHDcyst-2	RNDTSHPR	982.48132	2.95
	NDTSHPR	826.3802	1.84
	TAESTCPVTETYTRELCRPK	2399.13297	3.21
rHDcyst-3	LAHFALAK	870.5196	1.74
	DICTAQIYEVPWLNTTSLTSFTCGGR	2990.40227	4.02
	EFFDTLLEITAVETQLVAGTNYR	2630.33482	5.07
rHDcyst-4	ITFNTAESTCPISATYSK	1990.94263	3.51
	KTNTDDEHYHTLAHLAISQQVQGR	2762.36048	5.04
	DTCTAVINEGLGGHETLVR	2041.99713	3.61

Inhibitory activity of recombinant cystatin on cysteine proteases

An assessment of the inhibitory potency of purified cystatin against cysteine proteinases cathepsin B and S was conducted. This was measured through the hydrolysis of Ac-RR-AFC and Z-VVR-AFC substrates. Remarkably, a concentration of 0.2 µM cystatin resulted in a significant 50% relative inhibition of cathepsin B and S at 0.05 mM. Furthermore, complete inhibition, reaching 100%, was observed for cathepsin B and S with the application of just 0.006 µM of purified cystatin (refer to Fig. 9). The data highlights the potent inhibitory action of cystatin on these enzymes.

Analysis of the antioxidant activity of recombinant cystatin

The findings from the purification of cystatin and its impact on DPPH free radical scavenging ability indicate a progressive enhancement in the scavenging rate of the recombinant protein cystatin with increasing concentrations ranging from 0 to 80 µM, as illustrated in Fig. 10. This suggests its potential involvement in regulating oxidative processes within the midgut.

Effect of RNAi on biological characteristics of H. doenitzi

After the interference with HDcyst-1, HDcyst-2, HDcyst-3, and HDcyst-4, their relative expression levels decreased significantly by 79.4%, 88.2%, 77%, and 66.9%, respectively (see Fig. 11). Consequently, the satiation rate of female ticks was notably reduced (refer to Fig. 12). Moreover, the engorged weights in the HDcyst-1, HDcyst-2, HDcyst-3, and HDcyst-4 inoculated groups were 10%, 13%, 17%, and 10% lower compared to the control group, respectively. Similarly, the egg-laying weights in these groups exhibited reductions of 10%, 11%, 22%, and 11%, respectively, when compared to the control group. Additionally, the egg hatching rate in the HDcyst-1, HDcyst-2, HDcyst-3, and HDcyst-4 inoculated groups was lower by 6%, 6%, 11%, and 5%, respectively, in comparison to the control group. Significantly, there was a noticeable difference in the blood-feeding duration between the experimental and control groups. Furthermore, a marked discrepancy was observed in the number of eggs laid and egg hatchability between the HDcyst-3 interference group and the other cystatin interference groups (P < 0.05) (Table 3). It's noteworthy that the group subjected to interference with type 2 cystatin exhibited a more pronounced upward trend in mortality. These results imply that cystatin plays a pivotal role in the blood-feeding site and may be involved in maintaining blood meals within the intestine.

Table 3

Effects of the RNAi of cystatin on biological characters of *H. doenitzi*.
Parameters	RNA interference
	GFP	HDcyst-1	HDcyst-2	HDcyst-3	HDcyst-4
No. test	60	60	60	60	60
Average death rate(%)	41.67%	51.67%	46.67%	60%	63.33%
Engorgement time (d)	7.31^b	9.10^a	9.13^a	9.25^a	9.14^a
Engorgement weight (mg)	215.6 ± 5.1^a	194.9.21 ± 2.6^b	188.3 ± 4.2^bc	179.6 ± 6.9^c	195.1 ± 4.4^b
Egg laying amount(mg)	106.5 ± 6.0^a	95.7 ± 3.0^b	94.4 ± 6.9^b	83.1 ± 5.3^c	94.9 ± 4.5^b
Egg hatchability (%)	85.3 ± 1.7^a	80.3 ± 0.5^b	80.3 ± 1.5^b	75.7 ± 0.8^c	80.8 ± 3.6^b
Note: one-way ANOVA was conducted to analyze the data; Different letters indicate statistical differences (P < 0.05).

This study introduces four novel cystatins from H. doenitzi, namely HDcyst-1, HDcyst-2, HDcyst-3, and HDcyst-4. In egg serum, these cystatins, similar to human cystatin C, and exhibit distinct structural characteristics as secreted proteins. The amino acid sequences of putative HDcyst-1, HDcyst-2, HDcyst-3, and HDcyst-4 suggest their membership in the type 2 cystatin group, characterized by two disulfide bonds, an N-terminal glycine, a signal peptide, and a PW module, with the exception of HDcyst-4. Interestingly, HDcyst-4 differs from cystatin C due to a 'LG' substitution in the conserved 'PW' module within hairpin 2 (Fig. 2) (Delbridge and Kelly 1990; Yu et al. 2011). This distinction underscores the high conservation of cystatin genes across various animal species. Most tick cystatins are known for their ability to inhibit cysteine protease activity. In our study, recombinant cystatins efficiently inhibited the activity of cathepsin B and S, consistent with findings in other tick cystatins. Notably, HDcyst-4 exhibited reduced enzyme activity effectiveness due to two amino acid substitutions in the amino acid sequence, such as the absence of the conserved SND motif present in all cystatins that inhibit bean proteins, thus explaining their lack of inhibition on bean proteins (Kotsyfakis et al. 2006). Experimental data on the relative activity of cystatins in inhibiting cysteine proteases suggest that tick cystatins are equally proficient in inhibiting protein function as those found in the salivary glands of snakes and schistosomes (Richards et al. 2011; Dvořáková et al. 2020). Human cystatin C's influence on cathepsin S and B inhibitors in T cell migration, as well as the reciprocal regulation of cathepsins and cystatins, underscores their significant roles in immune regulation. Moreover, the similarity in potency between recombinant cystatins in H. doenitzi and known cystatins suggests that HDcyst-1, HDcyst-2, HDcyst-3, and HDcyst-4 may also be involved in T cell physiological processes (Chauhan and Tomar 2016; Cipollini et al. 2008). Cysteine cathepsins emerge as pivotal regulators of both the innate and adaptive arms of the immune system. They modulate lysosomal biogenesis and autophagic flux, impacting the survival and polarization of innate immune cells (Jakoš et al. 2019). Cystatin F, known for its role in regulating immune cell cytotoxicity, contributes to the control of innate and adaptive immune responses, serving as a crucial mediator employed by bystander cells to attenuate NK and T-cell cytotoxicity (Kos et al. 2018). This underscores the potential significance of cystatin as a key enzyme in innate immunity and suggests its vital role in the survival of parasites.

When considering the cystatin molecules previously reported in other tick species, Hlcyst-3 (ABZ89554.1) exhibits a high degree of homology with HDcyst-1 in H. doenitzi. However, these ticks also display some distinctive structural features shared with HDcyst-2, HDcyst-3, and HDcyst-4 in H. doenitzi. All four of these cystatins are secreted proteins, and interestingly, in H. longicornis, both Hlcyst-3 and Hlcyst-2 (ABV71390.1) are identified as secreted cystatins with similar characteristics (Zhou et al. 2009). Hlcyst-2 has been found to play a significant role in tick innate immunity and tick midgut physiology (Zhou et al. 2006). On the other hand, HDcyst-1, HDcyst-2, HDcyst-3, and HDcyst-4, while identified as similar cystatins, exhibit some variations in their sequence structures and primary functions. Notably, cystatin has been detected in various developmental stages of H. doenitzi, indicating its crucial physiological roles throughout the tick's life cycle. HDcyst-1, HDcyst-2, and HDcyst-4 are most highly expressed in larvae, while HDcyst-3 dominates during the nymph stage. This observation suggests that cystatin may play roles in growth and development, making it a potential target for future tick control strategies. Examining specific tick tissues, such as the midgut, malpighian tubule, salivary gland, and ovary of engorged female ticks, within the first 12 hours after feeding, reveals interesting patterns of cystatin expression. The highest expression of HDcyst-1 is found in the midgut, HDcyst-2 in the salivary gland, and HDcyst-3 and HDcyst-4 in the malpighian tubule. Hlcyst-2, similar to HDcyst-1, is abundant in the intestine and exhibits antioxidant properties, indicating its involvement in the tick's physiological processes. Additionally, HlSC-1 (AB510962.1), another cystatin from H. longicornis, shares similarities with HDcyst-1 and is associated with the blood-feeding process but shows high expression levels in the midgut. Intriguingly, Hlcyst-2, expressed solely in the midgut, differs from HlSC-1's broader tissue distribution. This suggests that cystatin not only participates in tick innate immunity and blood digestion within the midgut but also plays a role in feeding and reproduction, possibly influencing the host's immune response (Lu et al. 2020). Furthermore, HDcyst-2, HDcyst-3, and HDcyst-4 exhibit a high degree of sequence structural similarity to cystatin in H. flava. An examination of protein profiles in H. flava's hemolymph reveals a complex protein system involved in anticoagulation, blood meal digestion, and innate immunity, with cystatin being a noteworthy component. It's worth mentioning that OmC1 also possesses immunomodulatory properties and is predominantly expressed in tick midguts, much like HDcyst-1 (Liu et al. 2022). These findings shed light on the multifaceted roles of cystatin in ticks, spanning immune response, blood digestion, feeding, and reproduction, and underscore its potential significance in tick-host interactions (Grunclová et al. 2006).

Cystatin plays a pivotal role in the blood-feeding process of hematophagous arthropods, facilitating ticks in their parasitic endeavors by aiding in host parasitization, evading host immune responses, and facilitating successful blood meal acquisition and digestion (Schwarz et al. 2012). At the outset of tick feeding, the host initiates an immune rejection response, prompting ticks to secrete specialized immunomodulatory proteins into their saliva and the wound site, thus thwarting the host's attempts to disrupt their blood-feeding process (Dantas-Torres et al. 2012). HDcyst-2 exhibits predominant expression in the salivary glands, where it orchestrates the regulation of proteolytic activities associated with ingested host factors and immune responses. It demonstrates a remarkable preventive effect against autoimmune diseases. The immunosuppressive properties of the tick salivary gland protein DsCystatin (ADZ23478.1) are noteworthy, as it directly interacts with and inhibits the enzymatic activity of human cathepsin L and B. DsCystatin also suppresses the expression of inflammatory cytokines like IL1β, IFNγ, and TNFα (Denisov and Dijkgraaf 2021; Sun et al. 2018). Conversely, HDcyst-1 is primarily expressed in substantial quantities within the midgut, the digestive organ of ticks. This localization suggests a role in enhancing the metabolic processes of the tick's digestive system, thereby facilitating efficient blood digestion. Notably, HDcyst-3 and HDcyst-4 exhibit significant expression in the malpighian tubules, distinguishing them from HDcyst-1 and HDcyst-2. This observation suggests that HDcyst-3 and HDcyst-4 may play a rapid role in tick metabolism during the period immediately following tick satiation and before ovary development commences.

In the realm of plants, cystatin emerges as a formidable defense protein against phytophagous insects and ticks (Martinez et al. 2016). Furthermore, in the animal kingdom, the snake plasma cystatin superfamily, known as SVMI, stands as the first line of defense against snake venom, endowing them with innate resistance (Bastos et al. 2016). In the context of H. longicornis, the expression of cystatin is notably induced by the injection of endotoxin, specifically LPS. Notably, LPS is a component found on the surface of Gram-negative bacteria and has the remarkable ability to activate the immune systems of humans, as well as mice and ticks (Makjaroen et al. 2023). Cystatin plays multifaceted roles in the regulation of dendritic cell activity. For instance, Sialostatin L2 has been identified as an inhibitor of LPS-induced dendritic cell maturation. On the contrary, cystatin C deficiency has been linked to an increase in LPS-induced sepsis and the activation of NLRP3 inflammatory vesicles in mice (Wang et al. 2016). Through transcriptional studies of cystatin mRNA, we have gained insights into the functions of ticks in both blood digestion and innate immunity. The induction of LPS significantly elevates the expression of four cystatins, strongly implying their pivotal role in the innate immune system of ticks. Moreover, the conspicuous expression of HDcyst-1 and HDcyst-2 genes in the midgut and salivary glands after H. doenitzi feeding suggests the presence of distinct defense responses mediated by these well-defined immune response molecules. Sophoricoside attenuates autoimmune‑mediated liver injury through the regulation of oxidative stress and the NF‑κB signaling pathway, the NF‑κB transcription factor family is a key player that controls both innate and adaptive immunity (Chen 2023) and thus participates in the innate immunity of ticks. The tick's life cycle encompasses a response to cold stress that triggers an autoimmune reaction. For instance, engorged female Rhipicephalus sanguineus exhibit heightened sensitivity to prolonged cold exposure, rendering them inactive during winter. Intrathecine proteins come into play during the tick's response to cold stress, with D. silvarum heat shock proteins 70, 90, and microtubulin playing a pivotal role in the adaptation of ticks to cold temperatures, ultimately contributing to their survival and environmental adaptation (Dantas-Torres and Otranto 2011; Agwunobi et al. 2021). The study's findings illuminate the regulatory trend of cystatin expression during low-temperature treatment, with a rapid upsurge observed after 6 days, enabling ticks to maintain their vital functions and gradually recover, a crucial aspect of cold adaptation. In conclusion, cystatin is an integral component of the innate immunity of ticks, strengthening the evidence supporting this hypothesis. Preliminary experiments involving recombinant cysteine protease inhibition demonstrate a potential avenue for modulating innate immunity by altering oxidative levels in ticks.

In our current investigation, we focused on H. doenitzi. Female ticks subjected to dsRNA injection exhibited significantly reduced engorged weight, egg mass weight, and egg hatching rates. Additionally, they displayed prolonged blood-sucking durations, underscoring the suppressive impact of decreased cystatin expression. The disruption of cystatin and synaptic vesicle protein via RNA interference (RNAi) impairs the ticks' ability to successfully feed (Karim et al. 2005). This mechanism suggests that ticks employ a subtle strategy to evade the host immune system by secreting cystatin, thereby interfering with the normal antigen processing in host antigen-presenting cells. These findings highlight the potential role of cystatin in regulating tick growth, development, and reproductive processes during their blood-feeding activities.

The primary function of cystatin lies in its ability to inhibit cysteine protease activity, which, in turn, plays a pivotal role in a variety of physiological responses. In the case of H. longicornis, it appears to regulate blood digestion by inhibiting cysteine protease activity, thereby altering the blood digestion process within the midgut HICPL-A. This interaction between Om-cystatin 2 and Sialostatin L with cathepsin S leads to the inhibition of dendritic cell maturation, endowing them with potent host immunosuppressive properties (Valenzuela et al. 2022). Additionally, Cystatin Bm-CPI-2, secreted by parasitic filarial nematodes, effectively blocks conventional cysteine proteases like papain, as well as aspartate endopeptidases crucial in the human B-cell class II antigen processing pathway (Maizels et al. 2001). In the realm of the immune system, cystatins mediate tissue protease activity and antigen presentation. They also induce the synthesis of tumor necrosis factor α and interleukin 10 while stimulating nitric oxide production through interferon γ-activated murine macrophages (Vray et al. 2002). These cystatin molecules represent a novel class of immunomodulatory molecules that warrant further exploration in mammalian systems.

Cystatin plays a pivotal role in various aspects of adult tick physiology, including egg production, egg hatchability, blood-feeding, digestion, and innate immunity. However, it's important to acknowledge the limitations of this study. Firstly, the immune regulation induced by the host remains unknown, and the intricate interplay between naturally occurring proteins in this conformation and their effects on other proteins, as well as the precise mechanisms of action in this pathway, have not been thoroughly explored. These areas present exciting opportunities for future research. Furthermore, investigating the target specificity of cystatin variants such as HDcyst-1, HDcyst-2, HDcyst-3, and HDcyst-4, and the development of anti-tick vaccines, represents intriguing avenues for upcoming studies (Wang et al. 2023).

In this study, we investigated the cathepsin B, S, and oxidative activities of recombinant cystatin proteins. The relative expression levels of the four cystatin genes were notably higher in the midgut, salivary gland, and malpighian tubules of engorged ticks, respectively. However, ovarian expression was not significant, suggesting that cystatin may serve unique functions in various tick tissues. Additionally, our findings indicated that cystatin prolongs the time ticks take to ingest blood. Knockdown of HDcyst-1, HDcyst-2, HDcyst-3, and HDcyst-4 through RNAi had a significant impact on H. doenitzi ticks. We also initiated investigations into the innate immune responses of ticks through experiments involving LPS injection stress and low-temperature stress, with further testing planned in our future research. In summary, our results suggest that cystatin may play a role not only in the innate immunity of ticks but also in the physiological processes that regulate tick blood-sucking and development.

Author contributions

Funding, Xiao-Long Yang; Conceptualization, planning and experimental design, Song-Bo Zhang, Zhi-Hua Gao, Xiao-Long Yang; Conducted the experiment and data recording, Song-Bo Zhang, Zhi-Hua Gao; Data analysis, Yi-Kui Wang, Wen-Xia Lv, Ke-Xin Dong; Original manuscript draft, Song-Bo Zhang, Fei-Di Guo, Run-Ying Wang; Manuscript review, editing and approval, Song-Bo Zhang, Xiao-Long Yang.

Acknowledgments

This work was supported by the Natural Science Foundation of Hebei Province (C2023205011; C2022205026), the Foundation of Hebei Normal University (L2023T05), and the National Natural Science Foundation of China (31472050).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Conflict of interest The authors declare no competing fnancial interest.

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The potential of type 2 cystatin in blood feeding and innate immunity of the tick Haemaphysalis doenitzi (Acari: Ixodidae)

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Abstract

Figures

Introduction

Materials and methods

Collection and feeding of ticks

Cloning and sequence analysis of the full-length cDNA of cystatin

Spatiotemporal expression analysis of cystatin

LPS injection induces the expression of cystatin in adult ticks

Low-temperature stress induces the expression of cystatin

Expression and purification of cystatin recombinant protein

Enzyme activity test

Antioxidant analysis

RNAi experiment

Results

Cloning and sequence analysis of the full-length cDNA of cystatin

Expression levels of the cystatin gene

LPS injection tick and low-temperature stress induce cystatin expression

Purification of rHDcyst-1, rHDcyst-2, rHDcyst-3, and rHDcyst-4

Inhibitory activity of recombinant cystatin on cysteine proteases

Analysis of the antioxidant activity of recombinant cystatin

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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