Similar mechanisms regulate protein exocytosis from the salivary glands of ixodid and argasid ticks.

Numerous bioactive compounds are secreted from large dense core granules in tick salivary glands during feeding in response to an external stimulus. Investigations into the signalling pathways regulating secretion indicated that they are similar for Argasidae (fast-feeding ticks) and Ixodidae (slow-feeding ticks), but differ in their sensitivity to prostaglandin E(2). In both cases, dopamine is the external signal for inducing exocytosis. Dopamine-induced exocytosis was shown to be strongly calcium dependant. Firstly, it requires extracellular calcium via a L-type voltage-gated calcium channel located on the plasma membrane and, secondly, intracellular calcium which is released presumably in response to inositol 1,4,5-triphosphate (IP(3)). Pathways such as the activation of phospholipase C, inositol-phosphate kinases, G-proteins, GTPases and Na(+)-K(+)-ATPases have been shown to be essential.

Biochemical perspectives on paralysis and other forms of toxicoses caused by ticks.

Tick toxicoses, of which paralysis is the most widespread and dominant form, are important elements of pathogenesis induced by ticks. Tick paralysis is the most widespread and dominant form of tick toxicoses. Non-paralytic forms of tick toxicoses do occur and evidence suggests that these forms of toxicoses are not evolutionary related. While functional significance has been suggested for tick toxins, the advantages for tick survival in general are not clear. This review considers the molecular nature of tick toxins, the possibility that tick toxins have originated more than once independently and whether these toxins could have unrecognized benign functions.

The influence of tick behavior, biotope and host specificity on concerted evolution of the platelet aggregation inhibitor savignygrin, from the soft tick Ornithodoros savignyi.

Ticks are obligate blood-feeding parasites that secrete anti-hemostatic components during feeding to enable control of the hemostatic system of the host. Complex interactions at the tick-host interface are an indication of the important role that the host played during tick evolution. The question is to what extent interaction with the host and the environment influences tick evolution. Previously, two isoforms (97% sequence identity) of savignygrin, an alphaIIbbeta3 antagonist, have been described. The presence of both isoforms within 20 random individuals confirmed that these isoforms must be recent gene duplicates. Analysis of the sequence differences between the isoforms shows a Kn/Ks ratio of 1, which indicates neutral selection for the isoforms. However, the biased localization of differences within the 3' end of the genes suggests that concerted evolution acts on the isoforms. Calculation of the divergence date between the isoforms (1.6-5.2 MYA) also indicates purifying selection, as ample time had passed after duplication, for inactivation of one gene copy. We conclude that concerted evolution has functioned to maintain a high copy number of the savignygrins in order for Ornithodoros savignyi to parasitize a wide host range. This contrasts with O. moubata that expresses the savignygrin homolog, disagregin, as a single copy at lower concentration levels and correlates with the confined habitat and consequently narrow host range of O. moubata. Recent "domestication" of O. savignyi due to animal husbandry practices could however, have reduced the selection constraints acting to maintain the gene copies as evidenced by the structural instability of one of the isoforms. Our results suggest that environmental factors and host associations do play an important role in the evolution of anti-hemostatic components in ticks.

Comparative properties of a three-dimensional model of Plasmodium falciparum ornithine decarboxylase.

The ornithine decarboxylase (ODC) component of the bifunctional S-adenosylmethionine decarboxylase/ornithine decarboxylase enzyme (PfAdoMetDC-ODC) of Plasmodium falciparum was modeled on the crystal structure of the Trypanosoma brucei enzyme. The homology model predicts a doughnut-shaped active homodimer that associates in a head-to-tail manner. The monomers contain two distinct domains, an N-terminal alpha/beta-barrel and a C-terminal modified Greek-key domain. These domains are structurally conserved between eukaryotic ODC enzymes and are preserved in distant analogs such as alanine racemase and triosephosphate isomerase-like proteins. Superimposition of the PfODC model on the crystal structure of the human enzyme indicates a significant degree of deviation in the carbon alpha-backbone of the solvent accessible loops. The surface locality of the ab initio modeled 38 amino acid parasite-specific insert suggests a role in the stabilization of the large bifunctional protein complex. The active site pockets of PfODC at the interface between the monomers appear to be conserved regarding the binding sites of the cofactor and substrate, but each contains five additional malaria-specific residues. The predicted PfODC homology model is consistent with mutagenesis results and biochemical studies concerning the active site residues and areas involved in stabilizing the dimeric form of the protein. Two competitive inhibitors of PfODC could be shown to interact with several parasite-specific residues in comparison with their interaction with the human ODC. The PfODC homology model contributes toward a structure-based approach for the design of novel malaria-specific inhibitors.

Identification of extrinsic blood coagulation pathway inhibitors from the tick Ornithodoros savignyi (Acari: Argasidae).

The salt BaSO(4) selectively adsorbs two proteins from crude Ornithodoros savignyi salivary gland extract. They co-purify during reversed-phase HPLC, but can be separated by hydrophobic-interaction chromatography. Their molecular masses are 9333 and 9173Da. The 9.3kDa protein was designated BSAP1 and the 9.1kDa protein BSAP2. Their amino acid compositions show significant differences, in particular the presence of seven and eight cysteine residues in BSAP1 and BSAP2, respectively. The proteins do not contain gamma-carboxyglutamic acid, hydroxyproline, or hydroxylysine. The proteins do not inhibit the intrinsic coagulation cascade, but inhibit the extrinsic pathway. The observed inhibition is not due to inhibition of factor VII. Both proteins bind to membranes. BSAP1 binds neutral and negatively charged membranes more strongly than BSAP2. Its affinity for negative membranes is, however, much lower than for neutral membranes. In contrast, BSAP2 binds both membranes equally strongly. The binding of the proteins to the membranes was significantly lowered upon pre-incubation with Ca(2+).

Amino acid sequence and structure modeling of savignin, a thrombin inhibitor from the tick, Ornithodoros savignyi.

The full-length gene of savignin, a potent thrombin (E.C. 3.4.21.5) inhibitor from the tick Ornithodoros savignyi has been cloned and sequenced. Both 5' and 3' UTR's, a signal peptide from the translated amino acid sequence and an unusual poly-adenylation signal (AATACA) has been identified. The translated protein sequence shows high identity (63%) with ornithodorin, the thrombin inhibitor from the tick, Ornithodoros moubata. Molecular modeling using the structure of ornithodorin as reference gave a structure with an RMSD of 0.25 A for the full-length protein, 0.11 A for the N-terminal BPTI-like domain and 0.11 A for the C-terminal BPTI-like domain, indicating that maximum deviation occurs in the mobile bridge (0.18 A) between the two domains. Docking of savignin to thrombin shows that the interaction is similar to the ornithodorin-thrombin complex. The N-terminal amino acid residues of savignin bind inside the active site cleft, while the C-terminal domain of savignin has a net negative electrostatic potential and interacts with the basic fibrinogen recognition exosite of thrombin through hydrogen bonds and hydrophobic interactions. These results correlate with kinetic data obtained, which showed that savignin is a competitive, slow, tight-binding inhibitor that requires thrombin's fibrinogen-binding exo-site for optimal inhibition.

Structure-based inhibitor screening: a family of sulfonated dye inhibitors for malaria parasite triosephosphate isomerase.

The crystal structure of malaria triosephosphate isomerase (TIM) was screened against the National Cancer Institute database of three-dimensional molecular structures. Ten top-scoring commercially available compounds were analyzed for inhibition of recombinant TIM. Two anionic dyes showed inhibition of TIM at concentrations of <100 mM. Four related sulfonated dyes were identified from the literature, docked, and screened in vitro. All showed inhibition of malaria TIM. Models indicate that these compounds bind in two suggested conformations to the active site region of the TIM enzyme. These compounds may be used in rational modification procedures for the synthesis of lead anti-TIM drugs.

Identification of putative proteins involved in granule biogenesis of tick salivary glands.

Ticks secrete bioactive components during feeding that assist them in gaining a blood meal. Compounds secreted are stored in granules until a stimulus induces secretion during feeding. Biogenesis of tick secretory granules has not been investigated before. An adequate understanding of granule biogenesis could advance our understanding of tick salivary gland biology and could aid in the rational design of tick control methods. Putative tick salivary gland proteins 1-4 (TSGP1-4) involved in granule biogenesis were identified in this study based on their abundance in salivary gland extracts and granule preparations and their ability to aggregate under conditions of slight acidity and high calcium concentration. TSGP2 and TSGP3 have been identified as previously described toxic and nontoxic homologues, respectively, while toxicity was also associated with TSGP4.

Neuropathogenic properties of Argas (Persicargas) walkerae larval homogenates.

Several tick species have been demonstrated, described, or suspected to cause paralysis in their host during the repletion process, presumably by impairing neurotransmission. The resulting polyneuropathy gradually spreads to the upper limbs causing incoordination and ends in respiratory failure. This form of paralysis is commonly confused with Guillain-Barrè syndrome, botulism and myasthenia gravis and although the clinical symptoms of these diseases are similar, it is not clear whether the pathogenesis is also the same. During this study we investigated the mechanism of paralysis by the tick Argas (Persicargas) walkerae by determining the effect of larval homogenates on both potassium-stimulated (calcium-dependent) and veratridine-stimulated (external calcium-independent) release of [3H]glycine from crude rat brain synaptosomes. The results indicated that larval homogenates inhibited both processes. These findings are reconcilable with the results obtained for two other paralysis-causing tick species, Ixodes holocyclus and Dermacentor andersoni, which were indicated to cause paralysis by decreasing the synthesis or release of acetylcholine at the neuromuscular junction.

Disaggregation of aggregated platelets by apyrase from the tick, Ornithodoros savignyi (Acari: Argasidae).

Apyrase, secreted by ticks during feeding, is a platelet aggregation inhibitor that functions as a regulator of the host's hemostatic system. This present study concerns the disaggregation effect of salivary gland apyrase from the tick Ornithodoros savignyi. Secondarily aggregated platelets, disaggregated by apyrase, exhibited a reversal of shape from a spherical (aggregated) form to a discoid form, reminiscent of reversible aggregation at low ADP concentrations in citrated platelet-rich plasma. However, they showed a dilatory open canaliculary system and an absence of granules indicating disaggregation after degranulation had taken place. In contrast, disaggregation by the fibrin(ogen)olytic enzyme, plasmin, showed that platelets degranulated, but retained a spherical form with numerous extended pseudopods. While thrombin had no effect on aggregation or clotting of platelets disaggregated with plasmin, it did activate those platelets disaggregated with apyrase and clotted the plasma. This is the first study to describe the disaggregating effects of tick derived apyrase on aggregated platelets. It also shows that apyrase can disaggregate platelets even after secondary aggregation and degranulation of platelets has taken place. Platelet aggregation is one of the main barriers encountered by ticks during feeding and counteraction of this process by ticks is an important factor for successful feeding.

Detection and micro-scale isolation of a low molecular mass paralysis toxin from the tick, Argas (Persicargas) walkerae.

This study describes the isolation of a 11 kDa paralysis toxin from crude larval extracts of Argas (Persicargas) walkerae by exploiting the cross-reactivity of a monoclonal antibody (4B12), directed against the paralysis toxin of Rhipicephalus evertsi evertsi. This low molecular mass is in contrast to previous findings of a 60-70 kDa toxin for A. (P.) walkerae, but is similar to neurotoxins isolated from venomous forms of the class Arachnida, which comprise the orders Araneae (spiders). Scorpionida (Scorpions) and Acari (ticks and mites). Since numerous antigenic bands, ranging between 11 and 115 kDa, were detected by the monoclonal antibody 4B12, the possibility of toxin-complex formation and the effect of pH on the latter were investigated by means of HPLC and ammonium sulphate precipitation. The results suggest that physiological conditions, with respect to pH and ionic strength, promote the formation of heterogeneous toxin-complexes while an acidic pH favours the formation of a more homogeneous toxin-containing complex. Furthermore, the effect of partially purified toxin on neurotransmitter release from crude rat brain synaptosomes was investigated, since tick paralysis toxins are hypothesised to inhibit neurotransmitter release from the presynaptic terminal. Both calcium-dependent, as well as calcium-independent release was inhibited by the toxin-containing sample.

Savignin, a potent thrombin inhibitor isolated from the salivary glands of the tick Ornithodoros savignyi (Acari: Argasidae).

A thrombin (E.C. 3.4.21.5) inhibitor, savignin, was isolated from the salivary glands of Ornithodoros savignyi by a combination of size exclusion, anion-exchange, and reversed-phase chromatography. The inhibitor has a molecular mass of 12,430.4 Da as determined by electrospray mass spectrometry. The behavior of savignin during anion-exchange chromatography indicated that it has an acidic pI. The available N-terminal sequence (residues 1-11) differed from that of ornithodorin with only one residue. Savignin inhibits thrombin-induced platelet aggregation, but has no effect on ADP- or collagen-induced aggregation. Kinetic studies indicated that savignin is a competitive, slow-, tight-binding inhibitor of alpha-thrombin (K(i) = 4.89 +/- 1.39 pM). Tight-binding kinetics showed that the inhibitor has a lower affinity for gamma-thrombin (K(i) = 22.3 +/- 5.9 nM). Plasmin, factor Xa, and trypsin are not inhibited by savignin.

Apyrase activity and platelet aggregation inhibitors in the tick Ornithodoros savignyi (Acari: Argasidae).

Ticks are ectoparasites that cause considerable damage to their hosts while feeding. The feeding process is facilitated by anti-haemostatic factors present in the tick saliva. Apyrase (ATP diphosphohydrolase, EC 3.6.1.5) is a platelet aggregation inhibitor found in most haematophagous organisms studied. The present study describes the identification and characterization of such an activity in the tick Ornithodoros savignyi. The enzyme conformed to many properties common to apyrases. These included a low substrate specificity, dependence on bivalent metal ions for activity and insensitivity to the classical ATPase inhibitors. Heat denaturation studies, pH optima and similar effects of inhibitors on the enzyme's ATP and ADP hydrolysing activitives supported its classification as an apyrase. Salivary gland extracts inhibited the platelet aggregation induced by ADP, collagen and thrombin and disaggregated aggregated platelets. The results suggest the presence of two or more anti-platelet factors present in the salivary glands of this tick species.

Cloning, nucleotide sequence and expression of the gene encoding factor Xa inhibitor from the salivary glands of the tick, Ornithodoros savignyi.

The N-terminal sequence of the competitive and slow tight-binding factor Xa inhibitor (fXaI; Ki = 0.83 +/- 0.10 nM) isolated from the salivary glands of Ornithodoros savignyi ticks (Acari: Argasidae) was employed to design a degenerate gene-specific primer (GSP) for 3'-rapid amplification of cDNA ends (3'-RACE). The primer consisted of a sequence encoding for amino acid residues 5-11. A full-length gene was next constructed from the 3'-RACE product in a two-step PCR procedure and successfully expressed by the BAC-TO-BAC baculovirus expression system. The deduced amino acid sequence of the gene showed 46% identity and 78% homology to an fXaI (TAP) from Ornithodoros moubata. Recombinant fXaI (rfXaI) consists of 60 amino acid residues, has a molecular mass of approximately 7 kDa and inhibited fXa by approximately 91%. The availability of the rfXaI will aid further investigations of its potential for therapeutic applications and as vaccine against tick infestation. The authentic nucleotide sequence of the gene encoding tick fXaI furthermore enables studies at the genetic level and probing of other tick species for similar and related genes.

Isolation and characterization of an anticoagulant from the salivary glands of the tick, Ornithodoros savignyi (Acari: Argasidae).

An inhibitor of activated coagulation factor X (fXa) was isolated from salivary gland extracts prepared from Ornithodoros savignyi using a two-step procedure, involving reversed-phase high-performance liquid chromatography (RP-HPLC) and diethylaminoethyl (DEAE) ion-exchange chromatography. From its behaviour during DEAE chromatography it could be deduced that it possesses an acidic pI (approximately 4.6). Capillary zone electrophoresis (CZE) of the purified inhibitor showed it to be homogeneous. The molecular mass was determined as 12 kDa using capillary gel electrophoresis (CGE) and as 7183.4 using laser desorption mass spectrometry (LDMS). The N-terminal amino acid sequence (residues 1-12) was determined and found to share a 66% identity with tick anticoagulant peptide (TAP). The O. savignyi peptide is a slow, tight-binding inhibitor of fXa (Ki = 0.83 +/- 0.10 nM). The interaction of the fXa--inhibitor was found to be competitive and dependent on ionic strength. Preliminary investigations show that the inhibitor may be specific for fXa.

Amino acid and protein depletion in medium of cell cultures infected with Cowdria ruminantium.

Cowdria ruminantium (Rickettsiales) causes heartwater in ruminants of Africa, and some islands off Africa and in the Caribbean Sea. The in vitro culture method for the organism devised in 1985, which provided for the first time a means for production of adequate quantities of live organisms and their products, is erratic and requires improvement. We studied depletion of amino acids (AAs) and major proteins in culture medium taken daily from infected and uninfected ovine and bovine vascular endothelial cell cultures. AAs of these samples were analyzed by Pico Tag reversed phase HPLC precolumn derivatization, and major proteins determined by capillary electrophoresis using a 57 cm x 75 microns fused silica tube at high pH. In both ovine and bovine cell cultures, significant depletion of arginine and glutamine occurred over a 5-day observation period regardless of whether they were infected or uninfected. This indicates that supplementation of nutrient media with these AAs might improve conditions for growth of the organism. Both AAs are essential for survival of cultured cells, and probably for the rickettsia (although the metabolism of C. ruminantium is poorly understood). Concentrations of several AAs increased in infected cultures, implying de novo synthesis and/or proteolysis on the part of the organism. In fact, several protein fractions did decrease in culture medium throughout the course of infection, while increasing or remaining unchanged in uninfected control cultures. Proteolytic activity by C. ruminantium may be essential for nitrogen metabolism by the organism. It is suggested that studies such as these will facilitate the development of a specific medium for optimal in vitro growth of the heartwater organism, and may also lead to an understanding of the metabolic stratagem of C. ruminantium. This knowledge, in turn, could reveal the mechanism for pathogenesis of heartwater, with implications for control.

Identification of anticoagulant activities in the salivary glands of the soft tick, Ornithodoros savignyi.

Salivary gland extracts of the sand tampan, Ornithodoros savignyi, prolonged the activated partial thromboplastin time (APTT) and prothrombin time (PT) significantly in a concentration-dependent manner. There was also a pronounced inhibition of human activated factor Xa (fXa) by salivary gland extracts. The salivary gland extracts inhibited chromogenic assays specific for both fXa and thrombin. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the salivary gland proteins followed by elution of specific areas or bands from a polyvinylidene difluoride (PVDF)-membrane, showed that various anticoagulant factors are present when screened by means of the APTT assay. The most active component was associated with a band of M(r) of 14 kDa. Partial purification of this component was achieved using isoelectric focusing (IEF) and size-exclusion high-performance liquid chromatography (HPLC).

Isolation and characterization of an anticoagulant present in the salivary glands of the bont-legged tick, Hyalomma truncatum.

A low molecular mass anticoagulant (17 kDa) was isolated from the salivary glands of prefed female Hyalomma truncatum ticks by means of reverse phase and anion-exchange HPLC. Trypsin digestion and amino acid analysis confirmed the protein nature of the anticoagulant. The inhibitor appears to be uncompetitive with a Ki of 6.9 x 10(-10)M. The target of the anticoagulant is factor Xa at the junction of the extrinsic and intrinsic pathways. This may be crucial for the survival of the tick, making it feasible to investigate the possibility of vaccination with this antihaemostatic against tick feeding. In addition, tick anticoagulants may possibly have therapeutic application in controlling thrombosis.

A shared epitope found in the major paralysis inducing tick species of Africa.

Cross-reactivity between all the paralysis inducing tick species of veterinary relevance in Africa was demonstrated, by using a monoclonal antibody directed against the paralysis inducing toxin of Rhipicephalus evertsi evertsi. Western blot results, together with amino acid composition studies indicated that this monoclonal antibody recognizes protein bands of similar molecular mass and amino acid composition in R. evertsi evertsi and Ixodes rubicundus. This suggests that the Karoo paralysis toxin of I. rubicundus is possibly also a trimer with a high degree of homology to the spring lamb paralysis toxin of R. evertsi evertsi. The conclusive identity of these protein bands of I. rubicundus could not be shown. Bio-assay studies performed on 1-day-old chickens suggested that the anti-spring lamb paralysis toxin monoclonal antibody also recognizes the paralysis toxin present in Argas (Persicargas) walkerae, by rendering some degree of protection against the effect of this toxin.