Tick Haemaphysalis Longicornis Research Articles

Tick troponin I-like molecule is a potent inhibitor for angiogenesis

This is the first report identifying the anti-angiogenesis saliva molecule of the ixodid tick. In our previous study, we identified a troponin I-like molecule (HLTnI) of the ixodid hard tick Haemaphysalis longicornis, a vector for various pathogens. To investigate its potential inhibitory effects for angiogenesis, we expressed and purified recombinant HLTnI in Escherichia coli. In a vascular endothelial growth factor (VEGF) competitive angiogenesis assay, the recombinant HLTnI significantly inhibited the capillary formation of human vascular endothelial cells (HUVEC) in vitro. The inhibition was dose-dependent with an IC50 of 18.95 nM. These results indicated that HLTnI is a potent angiogenesis inhibitor.

A secreted cystatin from the tick Haemaphysalis longicornis and its distinct expression patterns in relation to innate immunity

Proteins capable of selective and specific inhibition of cysteine protease have been identified as cystatins and are isolated from a variety of microbes and tissues of animals and plants. The physiological function of these proteins has been proposed to be the regulation of protein turnover and defense against pathogens as well as the balance of the host–parasite immune relationship. Genes encoding cystatins have been found in several species of ticks, but the function of cystatin in ticks is not understood. We cloned a gene encoding cystatin from tick H. longicornis and designated it as Hlcyst-2 ( H. longicornis cystatin-2). Its full-length cDNA is 569 bp, and it encodes a putative 133 amino acid protein with an obvious signal peptide. Sequence analysis demonstrated that it has significant homology with the known cystatin. The recombinant protein was expressed in a GST-fused soluble form in Escherichia coli and purified by affinity chromatography. The inhibitory activity of the recombinant protein against papain, cathepsin L, and cathepsin B was identified by fluorogenic substrate analysis. Cystatin was mostly expressed in the tick midgut and hemocyte. Blood feeding induced significantly increased expression in the midgut. Real-time PCR confirmed that LPS-injected adult ticks expressed Hlcyst-2 1.6 more times than the PBS-injected control; Babesia gibsoni-infected larvae ticks expressed Hlcyst-2 1.8 more times than normal larvae ticks. The recombinant protein also showed a significant growth-inhibitory effect on Babesia bovis cultured in vitro. These results indicated this cystatin Hlcyst-2 is involved in tick innate immunity.

Identification of a follistatin-related protein from the tick Haemaphysalis longicornis and its effect on tick oviposition

The identification of ovary-associated molecules will lead to a better understanding of the physiology of tick reproduction and vector–pathogen interactions. A gene encoding a follistatin-related protein (FRP) was obtained by random sequencing from the ovary cDNA library of the tick Haemaphysalis longicornis. The full-length cDNA is 1157 bp, including an intact ORF encoding an expected protein with 289 amino acids. Three distinct domains were present in the deduced amino acids, namely, the follistatin-like domain, KAZAL, and two calcium-binding motifs, EFh. The sequence shows homology with the follistatin-related protein (FRP), which was thought to play some roles in the negative regulation of cellular growth. RT–PCR showed that the gene was expressed throughout the developing stages and mainly in the ovary as well as in fat body, hemocytes, salivary glands, and midgut. This gene was expressed in GST-fused recombinant protein with an expected size. The mouse antiserum against the recombinant protein recognized a 56-kDa native protein in both tick ovary and hemolymph. The recombinant proteins were found to have binding activity for both activin A and bone morphogenetic protein-2 (BMP-2). Silencing of FRP by RNAi showed a decrease in tick oviposition, which is consistent with the effect of a recombinant protein vaccine on the adult tick. These results showed that the tick FRP might be involved in tick oviposition. This is the first report of a member of follistatin family proteins in Chelicerata, which include ticks, spiders, and scorpions.

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

Baculoviruses are specific insect pathogens used as selective biological insecticides on lepidopteran insects. We have tested a recombinant baculovirus expressing a chitinase gene for its efficacy as a tick bioacaricide. The recombinant Autographa californica multiple nuclear polyhedrosis virus expressing a chitinase enzyme (AcMNPV-CHT1) from the hard tick, Haemaphysalis longicornis, was constructed and found to have a novel bioacaricidal effect against ticks. The recombinant baculovirus was used to express the chitinase enzyme in Spodoptera frugiperda (Sf9) insect cells. Topical application of the supernatant harvested from the insect cell culture was found to cause mortality in nymphal ticks of H. longicornis. High temperature (>30 degrees C) and infrared radiation affected the chitinase enzyme activity and recombinant baculovirus infectivity by reducing the speed of tick killing by 60%. A mixture of recombinant virus and chitinase was found to kill ticks faster (p < 0.01) than pure chitinase and recombinant virus alone. Thus, the recombinant virus showed a synergistic effect with the foreign chitinase gene. In order to reduce the excessive use and cost of acaricides, it was found that a mixture of recombinant virus and flumethrin could halve the dose of the chemical acaricide used. These findings are important for the safe use of the recombinant virus expressing chitinase as a bioacaricide against ticks.

Molecular cloning and functional characterization of an aspartic protease from the hard tick Haemaphysalis longicornis

Haemaphysalis longicornis cDNA encoding an aspartic protease (longepsin) was identified from a midgut cDNA library. The longepsin cDNA contains 1176 bp that code for 392 amino acid residues with a predictable molecular weight of 39.3 kDa. The cDNA has a signal peptide sequence associated with the N-terminal domains and domain structure analysis revealed that the deduced protein has two aspartic acid residues that are characteristic of a single active site for aspartic proteases. This novel longepsin cDNA exhibits 57% identity to the lysosomal aspartic protease of Aedes aegypti, 52% to Bombyx mori cathepsin D, 38% to Ancylostoma caninum, 44% to Schistosoma mansoni and 28% to Boophilus microplus aspartic proteases. The DNA fragment coding for longepsin was cloned into a pGEX-4T-3 vector and expressed in Escherichia coli. The recombinant longepsin, once activated was able to hydrolyze casein substrate as well as hemoglobin (Hb) under acidic conditions (pH 3.5). RT-PCR analysis showed that the longepsin mRNA transcripts were expressed in salivary glands and midgut and not in the ovary. Northern blot analysis revealed that longepsin (1.5 kb) was expressed in unfed and partially fed ticks and expression levels increased during feeding. The finding that longepsin is expressed in the midgut and salivary glands, proteolytic activity occurs under acidic conditions and longepsin can be gene silenced of longepsin provides compelling support for the hypothesis that longepsin plays an integral role in the proteolysis of erythrocyte Hb obtained from a host blood meal.

Contribution of the N-Terminal and C-Terminal Domains of Haemaphysalin to Inhibition of Activation of Plasma Kallikrein-Kinin System

Haemaphysalin is a kallikrein-kinin system inhibitor from hard tick Haemaphysalis longicornis, and consists of two Kunitz type protease inhibitor domains. Each domain as well as haemaphysalin inhibited intrinsic coagulation by inhibiting activation of the kallikrein-kinin system without affecting the amidolytic activities of intrinsic coagulation factors, indicating that both domains were involved in the inhibition through a similar mechanism to that for haemaphysalin. Reconstitution experiments showed that the C-terminal domain contributed more predominantly to this inhibition. Direct binding assaying showed that the C-terminal domain could bind to the cell-binding region of high molecular weight kininogen (HK), suggesting that it also binds to the cell-binding region of factor XII. Judging from these findings, the C-terminal domain may more effectively inhibit the association of factor XII and HK with the cell surface by binding to cell-binding regions, and hence would predominantly contribute to the inhibition of activation of the kallikrein-kinin system.

Immunization of mice with recombinant P27/30 protein confers protection against hard tick Haemaphysalis longicornis (Acari: Ixodidae) infestation

The success of immunological control methods is dependent upon the use of potential key antigens as tick vaccine candidates. Previously, we cloned a gene encoding 27 kDa and 30 kDa proteins (P27/30) of Haemaphysalis longicornis, and identified the P27/30 is a troponin I-like protein. In this study, the recombinant P27/30 (rP27/30) expressed in Escherichia coli was used to immunize mice and the mice were challenge-infested with ticks at different developmental stages of the same species. The rP27/30 protein stimulated a specific protective anti-tick immune response in mice, evidenced by the statistically significant longer pre-feeding periods in adult ticks. Furthermore, significantly longer feeding periods were noted in both larval and adult ticks. On the other hand, only larval ticks exhibited low attachment rates (31.1%). Immunization of mice with rP27/30 protein confers protection against hard tick Haemaphysalis longicornis infestation. These results demonstrated that the rP27/30 protein might be a useful vaccine candidate antigen for biological control of ticks.

Cloning and molecular characterization of a cubilin-related serine proteinase from the hard tick Haemaphysalis longicornis

Serine proteinases are one of the largest proteolytic families of enzymes, and have diverse cellular activities in mammalian tissues. We report here the cloning and molecular characterization of a cDNA encoding the serine proteinase of the hard tick Haemaphysalis longicornis (HlSP). The HlSP cDNA is 1570 bp long and the deduced precursor protein consists of 464 amino acids with a predicted molecular mass of 50.4 kDa and a pI of 8.2. The preprotein, consisting of 443 amino acids, was predicted to include a complement C1r/C1s, Uegf, and bone morphogenic protein-1 domain, a low-density lipoprotein receptor class A domain, and a catalytic domain. HlSP sequence analysis showed high similarity to serine proteinases reported from arthropods and vertebrate animal species. Two-dimensional immunoblot analysis revealed endogenous HlSP in adult tick extracts at 50 kDa. Endogenous HlSP was also expressed in all lifecycle stages of H. longicornis. Immunohistochemical studies detected the endogenous enzyme in the midgut epithelial cells of an adult tick. The Escherichia coli-expressed recombinant HlSP was demonstrated to degrade bovine serum albumin and hydrolyze the substrate Bz- l-Arg-pNA at the rate of 30.2 μmol/min/mg protein. Further, HlSP expression was up-regulated during a blood-feeding process, indicating its involvement in the digestion of host blood components.

Identification of the Coxiella sp. detected from Haemaphysalis longicornis ticks in Korea.

Two Haemaphysalis longicornis ticks were found positive in PCR assay of com-1 gene to detect Coxiella burnetii DNA from 100 ticks. The nucleotide sequences of com-1 and 16S rRNA gene were determined from 2 ticks and compared with those of other C. burnetii strains. The results suggest that H. longicornis harbor Coxiella sp. bacteria in Korea. Furthermore, icd, cbhE', and cbbE' genes are C. burnetii specific genes whereas com-1 gene is Coxiella genus specific gene. This study gives the first documentation to prove the existence of Coxiella sp. in tick collected in Korea.

CLONING AND SEQUENCING OF PUTATIVE CALRETICULIN COMPLEMENTARY DNAs FROM FOUR HARD TICK SPECIES

Calreticulin (CRT) is a calcium-binding protein and has many functions in eukaryotic cells. CRT is possibly involved in parasite host immune system evasion. To better understand the molecular basis of CRT in ticks, we cloned and sequenced 4 full-length complementary DNAs (cDNAs) from the hard tick species, Dermacentor variabilis, Haemaphysalis longicornis, Ixodes scapularis, and Rhipicephalus sanguineus, using the technique of rapid amplification of cDNA ends. The deduced amino acid sequences share high identities (between 77 and 98%) with 3 known tick CRT sequences. The major characteristics of known CRTs are observed in all 4 of our deduced tick CRTs. These include 3 major domains, a signal peptide sequence at the beginning of the coding region, 2 triplets of conserved regions, cysteine sites providing disulfide bridges for N-terminal folding, and a nuclear localization signal. Remarkably, the replacement of the endoplasmic reticulum retention signal KDEL by HEEL, which is believed to be associated with secretion of CRT into the host during feeding and was previously recorded only in 2 ticks and a hookworm, is also present in all 4 of our tick putative CRTs. In addition, the CRT gene is potentially useful for tick phylogenetic reconstruction.

Gene Silencing of a Cubilin-Related Serine Proteinase from the Hard Tick Haemaphysalis longicornis by RNA Interference

RNA interference (RNAi) has been recently exploited to determine gene function by degrading specific mRNAs in several eukaryotic organisms. We constructed a double stranded RNA (dsRNA) from a previously cloned Haemaphysalis longicornis serine proteinase (HlSP) gene to test the importance of the function of the HlSP gene product during blood-feeding. Growth of unfed ticks treated with HlSP dsRNA was significantly inhibited compared to that of PBS-treated ticks. This inhibition was supported by the level of HlSP mRNA. HlSP may play a crucial role for blood-feeding in these ticks. This is the first report on gene silencing of a functional serine proteinase in hard ticks.

Enzymatic Characterization of a Cubilin-Related Serine Proteinase from the Hard Tick Haemaphysalis longicornis

In the present study, we performed enzymatic characterization of Haemaphysalis longicornis serine proteinase (HlSP) with a view to shed light on the mechanisms of blood digestion in the hard ticks. Escherichia coli-expressed recombinant HlSP (rHlSP) was shown to potently hydrolyze the synthetic substrates Bz-(DL)-Arg-pNA, Z-Ala-Ala-Leu-pNA and Suc-Ala-Ala-Ala-pNA and yielded an activity of 31.5, 88.2 and 18.3 mumol/min/mg protein, respectively at an optimum temperature of 25 degrees C. However, the enzyme showed little activity to hydrolyze the substrates Suc-Arg-Pro-Phe-His-Leu-Leu-Val-Tyr-MCA and Pyr-Phe-Leu-pNA. The optimum pH for the enzyme was shown to be 4.0 to 5.0. Several inhibitors such as antipain, leupeptin and phenylmethylsulfonyl fluoride (PMSF), specific for serine proteinase were shown to inhibit enzyme activity by 20-82%, while E-64 (specific for cysteine proteinases) and pepstatinA (specific for aspartic proteinases) had shown only little inhibitory effects on it. This is the first report on enzymatic characterization of a functional serine proteinase from the hard ticks.

Immunization effect of recombinant P27/30 protein expressed in Escherichia coli against the hard tick Haemaphysalis longicornis (Acari: Ixodidae) in rabbits

We investigated the induction of resistance to Haemaphysalis longicornis infestation in rabbits that had been immunized with recombinant H. longicornis P27/30 protein. The success of immunological control methods is dependent upon the use of potential key antigens as tick vaccine candidates. Previously, we cloned a gene encoding 27 kDa and 30 kDa proteins (P27/30) of H. longicornis, and identified P27/30 as a troponin I-like protein. In this study, rabbits that were immunized with recombinant P27/30 expressed in Escherichia coli showed the statistically significant longer feeding duration for larval and adult ticks (P<0.05), low engorgement rates in larval ticks (64.4%), and an apparent reduction in egg weights, which suggest that H. longicornis P27/30 protein is a potential candidate antigen for a tick vaccine. These results demonstrated that the recombinant P27/30 protein might be a useful vaccine candidate antigen for biological control of H. longicornis.

A serine proteinase inhibitor (serpin) from ixodid tick Haemaphysalis longicornis; cloning and preliminary assessment of its suitability as a candidate for a tick vaccine

Rapid amplification of the cDNA ends (RACE) and primers designed based on a conserved serpin amino acid motif (NAVYFKG) were used to clone a 1350 bp cDNA which encodes a 378 polypeptide with high sequence similarity to several known serpins. We have named this gene as Haemaphysalis longicornis serpin-1 (HLS1). Northern blotting and reverse transcription (RT)-PCR analysis of total RNA from unfed or partially fed whole ticks as well as dissected tick organs revealed that transcription of HLS1 mRNA was induced by blood meal feeding during the slow feeding phase (24–48 h post-attachment) only in the tick midguts. Vaccination of rabbits with recombinant HLS1 (rHLS1) expressed in Escherichia coli resulted in 43.9 and 11.2% mortality of nymph and adult ticks which were fed on immunized rabbits. Polyclonal rabbit antibodies to tick saliva did not react with rHLS1, suggesting that native HLS1 was not secreted into the host during tick feeding. rHLS1 could be a potential candidate for a cocktail anti-tick vaccine.

Identification and Molecular Characterization of a Chitinase from the Hard Tick Haemaphysalis longicornis

A cDNA encoding tick chitinase was cloned from a cDNA library of mRNA from Haemaphysalis longicornis eggs and designated as CHT1 cDNA. The CHT1 cDNA contains an open reading frame of 2790 bp that codes for 930 amino acid residues with a coding capacity of 104 kDa. The deduced amino acid sequence shows a 31% amino acid homology to Aedes aegypti chitinase and a multidomain structure containing one chitin binding peritrophin A domain and two glycosyl hydrolase family 18 chitin binding domains. The endogenous chitinase of H. longicornis was identified by a two-dimensional immunoblot analysis with mouse anti-rCHT1 serum and shown to have a molecular mass of 108 kDa with a pI of 5.0. A recombinant baculovirus AcMNPV.CHT1-expressed rCHT1 is glycosylated and able to degrade chitin. Chitin degradation was ablated by allosamidin in a dose-dependent manner. The optimal temperature and pH for activity of the purified chitinase were 45 degrees C and pH 5-7. The CHT1 cDNA has an ELR motif for chemokine-mediated angiogenesis and appears to be a chitinase of the chemokine family. Localization analysis using mouse anti-rCHT1 serum revealed that native chitinase is highly expressed in the epidermis and midgut of the tick. AcMNPV.CHT1 topically applied to H. longicornis ticks exhibited replication. This is the first report of insect baculovirus infection of ticks. The importance of AcMNPV.CHT1 as a novel bio-acaricide for tick control is discussed.

Molecular characterization of a troponin I-like protein from the hard tick Haemaphysalis longicornis

A cDNA expression library prepared from mRNA of Haemaphysalis longicornis ( H. longicornis) was screened with a H. longicornis-infested rabbit serum. A cDNA encoding 27/30 kDa proteins was cloned and designated P27/30 gene. The predicted amino acid sequence of the P27/30 gene shows a rather high homology (58% amino acid identities and 11% amino acid similarity) with Drosophila melanogaster troponin I clone E2. H. longicornis P27/30 possesses amino acid sequence of actin-binding domains of troponin I at the amino acid residues 128–148, suggesting that H. longicornis P27/30 is a troponin I-like protein. By immunoblot analysis, mouse anti-recombinant P27/30 serum reacted with major constituent protein bands in extracts of adult ticks, and also immunoreacted with muscle, cuticle, gut, and salivary gland in H. longicornis ticks. Moreover, immunohistochemistry using the anti-P27/30 serum showed a strong reactivity in muscle, suggesting that native P27/30 is expressed abundantly in that tissue.

CDNA cloning, characterization and vaccine effect analysis of Haemaphysalis longicornis tick saliva proteins

Immunological control of ticks is currently the only sustainable and practical alternative method to the current use of acaricides which has serious limitations. The success of this method is dependent upon identification and cloning of potential tick vaccine antigens. We used a combination of immuno-screening of an adult tick cDNA library as well as the 3 and 5 rapid amplification of cDNA ends to clone two cDNAs, encoding tick saliva proteins from Haemaphysalis longicornis. The two cDNAs herein named HL 34 and 35 are 1000 bp each and encode polypeptides with 292 and 321 amino acid residues respectively. Northern blotting analysis of total RNA from ticks at different feeding stages revealed that expression of both HL 34 and HL35 mRNAs is induced during the slow feeding phase. We speculate that the functions of both genes are closely associated with blood feeding. Expression analysis by RT-PCR showed that both genes are expressed in other tick organs in addition to salivary glands. Recombinant HL 34 was successfully expressed in Escherichia coli and its suitability as a tick vaccine antigen was analyzed in rabbits. We propose that rHL34 could be a useful component of a cocktail tick vaccine.

Characterization of two cDNAs encoding serine proteinases from the hard tick Haemaphysalis longicornis

Host vaccination against tick infestation is at present the most practical and sustainable alternative tick control method to the current acaricide use which has serious limitations. However the success of this approach to control ticks depends upon the identification of target vaccine antigens. Members of the serine proteinase gene family may represent an interesting group of proteins to target as candidate antigens because of their involvement in regulation of many physiological functions and development processes in a wide range of organisms. We used RT–PCR with the 3′ and 5′ RACE to clone two cDNAs encoding full-length serine proteinases from the hard tick, Haemaphysalis longicornis. RT–PCR degenerate primers were designed from amino acid sequences surrounding active sites, His 57 and Ser 195 conserved among most known serine proteinase. Gene specific primers designed from nucleotide sequences of the RT–PCR products were used to prime the 3′ and 5′ RACE. Southern blotting analysis showed that both HLSG-1 and -2 are single copy. The 2 cDNAs, HLSG-1 and -2 are 1.2 and 1.0 kb long in size with open reading frames encoding polypeptides with 37.7 and 31.2 kDa predicted molecular mass respectively. Northern blotting analysis of total RNA from unfed and partially fed whole ticks showed that the expression of mRNAs for both HLSG-1 and -2 was induced by blood feeding. Expression analysis by RT–PCR showed that both HLSG-1 and -2 are expressed in other tick organs in addition to salivary glands and midguts. The 6 serine proteinase consensus cyteine residues are well conseverd in both HLSG-1 and -2. We have discussed our findings with respect to tick vaccine development research.

Molecular characterization of a peroxiredoxin from the hard tick Haemaphysalis longicornis.

Antioxidant enzymes in eukaryotes play an important role in protection against the oxygen radicals generated during aerobic metabolism. Here we report the cloning and characterization of a cDNA encoding the antioxidant enzyme peroxiredoxin from the hard tick Haemaphysalis longicornis (HlPrx). HlPrx is 939 bp long and contains a 101 bp non-translated sequence at the 5' end and a polyadenylation singnal followed by a poly(A) tail at the 3' end. HlPrx encodes a full-length protein with a predicted molecular mass of 26 kDa that possesses one cysteine residue at amino acid 49 that is conserved among Prx proteins of various species. GenBanktrade mark analysis showed that the deduced amino acid sequence had significant similarity to mammalian and plant Prxs at the amino acid level. A DNA-nicking assay revealed that Escherichia coli-expressed recombinant HlPrx (rHlPrx) inhibited oxidative-nicking of supercoiled plasmid DNA. Two-dimensional immunoblot analysis with mouse antirHlPrx serum showed reaction with a major constituent protein spot in extracts of adult ticks. In addition, immunoblot analysis showed that rHlPrx was immunoreacted with serum from rabbits repeatedly infested with H. longicornis. Localization analysis using mouse antirHlPrx serum revealed that native HlPrx was highly expressed in the salivary gland of the tick. Moreover, Northern blot analysis showed that the level of HlPrx transcripts was increased during blood sucking. The present results indicate that HlPrx may be an important detoxifying enzyme during the normal life span as well as during blood sucking in ticks.

EB8 マダニの抗酸化酵素ベルオキシレドキシンの分子性状

EB8 マダニの抗酸化酵素ベルオキシレドキシンの分子性状