Hard Tick Haemaphysalis Longicornis Research Articles

Structural Characterization and Cytolytic Activity of a Potent Antimicrobial Motif in Longicin, a Defensin-Like Peptide in the Tick Haemaphysalis longicornis

Longicin, a defensin-like peptide, was recently identified in the hard tick Haemaphysalis longicornis. Longicin and one of its synthetic partial analogs (P4) displayed antimicrobial/fungicidal/parasiticidal activity. In the present study, we compared longicin-derived synthetic analogs in order to characterize the antimicrobial motif (P4) by analyzing some structural features using various bioinformatic tools and/or CD spectroscopy. According to the chemicophysical characteristics, P4 is suggested to be a cationic peptide with hydrophobic and amphipathic character. The predicted secondary structure indicated the existence of a beta-sheet, which was also observed in the modeled tertiary structure. CD spectroscopic results also showed the existence of a beta-sheet and transition to a helical conformation in the presence of membrane-mimicking conditions. These structural observations on P4 suggested that the antimicrobial activity could be due to the beta-sheet as well as the alpha-helix. In addition, a sequence homology search showed that molecules identified in other ticks and organisms also have the P4 analogous domain at their C-terminal, which indicates P4 as a conserved domain. The peptide P4 also showed low cytolytic activity. Based on the present result and previously reported studies, the peptide P4 could be suggested as a novel antimicrobial domain indicating future therapeutic agent against bacteria.

Hemoglobinase activity of a cysteine protease from the ixodid tick Haemaphysalis longicornis

We report here the molecular characterization and possible function of a cysteine protease (termed HlCPL-A) identified in the midgut of the hard tick Haemaphysalis longicornis. HlCPL-A is a 333 amino acid protein belonging to the papain family of the cysteine protease. A construct encoding proHlCPL-A was expressed in Escherichia coli and purified as both procathepsin L and active processed cathepsin L forms. The HlCPL-A gene expression was up-regulated by blood-feeding process. HlCPL-A exhibited substrate specificity against synthetic peptidyl substrates (Z-Phe-Arg-MCA and Z-Arg-Arg-MCA; k cat / K m = 0.19 and 0.0023 M − 1 S − 1 , respectively). The proteolytic activity of HlCPL-A was inhibited by leupeptin, antipain and E-64 but was unaffected by pepstatin. HlCPL-A was capable of degrading bovine hemoglobin at pH 3.2 to 5.6. These results suggest that HlCPL-A may play important roles in the digestion of host hemoglobin in ticks.

Hemalin, a thrombin inhibitor isolated from a midgut cDNA library from the hard tick Haemaphysalis longicornis

A full-length sequence of a thrombin inhibitor (designated as hemalin) from the midgut of parthenogenetic Haemaphysalis longicornis has been identified. Sequence analysis shows that this gene belongs to the Kunitz-type family, containing two Kunitz domains with high homology to boophilin, the thrombin inhibitor from Rhipicephalus ( Boophilus) microplus. The recombinant protein expressed in insect cells delayed bovine plasma clotting time and inhibited both thrombin-induced fibrinogen clotting and platelet aggregation. A 20-kDa protein was detected from the midgut lysate with antiserum against recombinant hemalin. The gene is expressed at all stages of the tick except for the egg stage, and hemalin mRNA mainly in the midgut of the female adult tick. Real-time PCR analysis shows that this gene has a distinctly high expression level in the rapid bloodsucking period of the larvae, nymphs, and adults. Disruption of the hemalin gene by RNA interference led to a 2-day extension of the tick blood feeding period, and 27.7% of the RNA-treated ticks did not successfully complete the blood feeding. These findings indicate that the newly identified thrombin inhibitor from the midgut of H. longicornis might play an important role in tick blood feeding.

A set of serine proteinase paralogs are required for blood-digestion in the ixodid tick Haemaphysalis longicornis

We present evidence demonstrating that genes encoding enzymes essential for successful blood-feeding are differentially induced in the midgut of the hard tick Haemaphysalis longicornis. Three serine proteinase genes ( HlSP, HlSP2 and HlSP3) isolated from H. longicornis midgut exhibit protein sequence similarity with other trypsin-like serine proteinases reported from arthropods and vertebrate animal species. The endogenous enzymes were mainly detected in the midgut epithelial cells and in the lumen of an adult tick. The recombinant enzymes expressed in Escherichia coli efficiently hydrolyzed synthetic substrates specific for serine proteinases over a broad range of pH and temperature values. Notably, the transcript levels of HlSP2 and HlSP3 were detected to significantly increase at 96 h post infestation, while the transcript of HlSP was induced in the earlier stage of blood-feeding. Further, silencing of HlSP, HlSP2 and HlSP3 genes by RNA interference led to a significant reductions in the engorged tick body weight, suggesting synergetic roles of these serine proteinases in blood-feeding and digestion.

Identification of two forms of cyclophilin from the hard tick Haemaphysalis longicornis

We have shown here the identification and characterization of two cyclophilin, cyclophilin A (CyPA) and B (CyPB), from the ixodid tick, Haemaphysalis longicornis. Both CyPA and CyPB contain the conserved peptidyl-prolyl isomerase (PPIase) domain, with CyPA consisting of 188 amino acids and CyPB of 216 amino acids. CyPA and CyPB share 50–67% amino acid sequence identity with the cyclophilins of other organisms, and are found in multiple organs throughout the developmental stages of ixodid ticks. In addition, recombinant CyPA and CyPB exhibited PPIase activity that could be inhibited by the cyclic peptides cyclosporin A (CsA). Silencing of CyPA through RNA interference has led to a significant reduction in the body weight of engorged ticks and their failure to lay eggs, in contrast to CyPB whose silencing did not result in any detectable phenotypic changes. Our results indicate that CyPA represents the major cyclophilin protein in H. longicornis involved in blood ingestion, viability, and oocyte development. This is the first report of cyclophilin proteins from ixodid and argasid ticks.

Molecular characterization of a blood‐induced serine carboxypeptidase from the ixodid tick Haemaphysalis longicornis

Ticks feed exclusively on blood to obtain their nutrients, but the gene products that mediate digestion processes in ticks remain unknown. We report the molecular characterization and possible function of a serine carboxypeptidase (HlSCP1) identified in the midgut of the hard tick Haemaphysalis longicornis. HlSCP1 consists of 473 amino acids with a peptidase S10 family domain and shows structural similarity with serine carboxypeptidases reported from other arthropods, yeasts, plants and mammals. Endogenous HlSCP1 is strongly expressed in the midgut and is supposed to localize at lysosomal vacuoles and on the surface of epithelial cells. Endogenous HlSCP1, identified as a 53 kDa protein with pI value of 7.5, was detected in the membrane/organelle fraction isolated from the midgut, and its expression was upregulated during the course of blood-feeding. Enzymatic functional assays revealed that a recombinant HlSCP1 (rHlSCP1) expressed in yeast efficiently hydrolyzed the synthetic substrates specific for cathepsin A and thiol protease over a broad range of pH and temperature values. Furthermore, rHlSCP1 was shown to cleave hemoglobin, a major component of the blood-meal. Our results suggest that HlSCP1 may play a vital role in the digestion of the host's blood-meal.

Identification and characterisation of a leucine aminopeptidase from the hard tick Haemaphysalis longicornis

Aminopeptidases responsible for blood digestion have yet to be identified in haematophagous ticks. We report here the cloning and molecular characterisation of a cDNA encoding leucine aminopeptidase, a member of the M17 cytosolic aminopeptidase family, from the hard tick Haemaphysalis longicornis (HlLAP). Endogenous HlLAP was detected in the soluble fraction of adult tick extracts by immunoblotting. Immunohistochemical studies demonstrated that endogenous HlLAP expression mainly took place in the cytosol of midgut epithelial cells. Furthermore, expression of HlLAP was induced by a blood-feeding process. A functional recombinant HlLAP expressed in Escherichia coli efficiently hydrolyses synthetic substrates for aminopeptidase, a leucyl (with the K m value 0.19 ± 0.011 mM and V max value 157.2 ± 3.17 nmol/min/mg protein) and a methionyl substrate (with the K m value 0.12 ± 0.0052 mM and V max value 171.9 ± 2.31 nmol/min/mg protein). Enzyme activity was found to be optimum at pH 8 and 35 °C. The recombinant HlLAP enzyme activity was strongly dependent on metal divalent cations, Mn 2+, and was inhibited by bestatin. These results indicate that HlLAP play an important role for host’s blood digestion process.

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.

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.

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.

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.

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.