Soft Tick Ornithodoros Moubata Research Articles

Molecular cloning and comparative analysis of fibrinogen-related proteins from the soft tick Ornithodoros moubata and the hard tick Ixodes ricinus

Among disease-vectors, the evolution of the tick innate immune system is still lagging when compared to insects. Such an investigation, which was initiated, by first cloning and sequencing lectins associated in the innate immunity of invertebrates and having fibrinogen related domains, helped in the sequencing of cDNA encoding for OMFREP from the soft tick, Ornithodoros moubata. Also obtained were Ixoderin A and Ixoderin B cDNA sequences from the hard tick Ixodes ricinus. Tissue-specific expression of OMFREP showed that it was present primarily in the hemocytes and salivary glands. Ixoderin A besides sharing a similar expression profile was also expressed in the midgut. Both showed significantly high homology to the lectin Dorin M, from O. moubata. Further, phylogenetic comparisons between these molecules of the soft and hard ticks showed their relatedness to Tachylectins 5A and 5B, involved in the innate immunity of Tachypleus tridentatus and ficolins from both vertebrates and invertebrates. Ixoderin B showing tissue-specific expression only in the salivary glands and the sequence displaying certain motif differences in homology point towards a possible function different from the other two molecules. This is the first report of lectin-like sequences, with a fibrinogen-domain, from the hard tick I. ricinus and a preliminary phylogenetic study of these tick sequences with related fibrinogen-domain containing sequences highlights a possible role for them in the innate immunity of the ticks.

Complement Inhibitor of C5 Activation from the Soft Tick Ornithodoros moubata

Blood-feeding ticks must control C activation or be damaged by the host inflammatory response. We report the characterization and expression of a novel, relatively small, broad-acting C inhibitory protein (termed OmCI) from the soft tick Ornithodoros moubata. The native 17-kDa nonglycosylated protein inhibits both human and guinea pig classical and alternative C activation pathways. The IC50 values for each pathway were 12 and 27 nM, respectively, in hemolytic assays using human serum diluted 40-fold. The cDNA encodes a protein of 168 aa, including an 18-aa secretion signal sequence that is absent in the mature form. The inhibitor has 46% amino acid identity with moubatin, a platelet aggregation inhibitor also from O. moubata that is an outlying member of the lipocalin family. Native OmCI had no inhibitory effect on the addition of C8 and C9 to preformed C5b-C7 and C5b-C8 to form the membrane attack complex and no effect on the rate of C3a production by the C3 convertase enzymes C4bC2a, C3(H2O)Bb, or C3bBb. Both recombinant and native OmCI abolish production of C5a by human classical (C4bC3bC2a) and alternative (C3bC3bBb) C5 convertases. Addition of excess C5 but not C3 competes away the inhibitory activity of OmCI, indicating that OmCI targets C5 itself rather than inhibiting the C5 convertase C4bC3bC2a itself. Direct binding of OmCI to C5 was demonstrated by Western blotting and gel filtration chromatography using 125I-labeled proteins. OmCI is the first lipocalin family member shown to inhibit C and also the first natural inhibitor that specifically targets the C5 activation step.

Immune-responsive lysozymes from hemocytes of the American dog tick, Dermacentor variabilis and an embryonic cell line of the Rocky Mountain wood tick, D. andersoni

Immune-responsive lysozyme encoding cDNAs were identified from two medically important tick species by an expressed sequence tag approach of D. variabilis hemocytes (Dv Lys) and a D. andersoni embryonic derived cell line, DAE100. Comparative sequence analyses indicated the Dermacentor molecules to be products of orthologous genes and to be most similar to arthropod c-type lysozymes. Northern blotting analyses demonstrated that Dv Lys expression levels were most abundant in tick hemocytes and to a much lesser degree in the midgut while barely detectable in ovary, salivary gland, and Malpighian tubule tissues. Involvement of the Dermacentor c-type lysozymes in innate immunity was demonstrated by Escherichia coli challenges of D. variabilis ticks by injection resulting in a temporal profile of significantly elevated transcript abundances above those of naive controls that was similarly observed of the D. andersoni cells co-cultured with E. coli. In contrast to that reported of the digestive gut lysozyme of the soft tick Ornithodoros moubata, Dv Lys levels were not statistically differentially regulated by blood meal digestion. Additionally, given the differences in tissue distribution, sequence characteristics and phylogenetic placements between the Dermacentor and Ornithodoros lysozymes demonstrates that ticks possess differently adapted c-type lysozymes that are spatially and temporally differentially expressed.

Molecular cloning, structure and bait region splice variants of α 2-macroglobulin from the soft tick Ornithodoros moubata

The sequence of a α 2-macroglobulin (α 2M) from the soft tick Ornithodoros moubata (TAM) was determined by cloning and sequencing of overlapping polymerase chain reaction (PCR) and rapid amplification of cDNA ends PCR products. The TAM cDNA sequence is 4944 bp long and contains one open reading frame coding for a protein precursor composed of 1494 amino-acid residues, including a 24-residue signal sequence. The mature protein is cleaved into two subunits similarly to the C3 and C4 components of complement and fish α 2Ms. Phylogeny analysis revealed that TAM is closely related to Limulus α 2M and displays the highest similarity to the partial sequence of α 2M from hard tick Ixodes scapularis. The comparison of conserved cysteine residues between TAM and human and Limulus α 2Ms made it possible to predict the pattern of disulfide bridges and explain the atypical molecular arrangement of TAM. Four variants of the TAM bait region differing only in a short central segment were found; our data indicate that TAM exists as a single-copy gene in the tick genome and its bait region variants likely arise by alternative splicing. TAM is produced by tick hemocytes and it is also significantly expressed in salivary glands. TAM mRNA levels were shown to be up-regulated upon blood meal.

Lysozyme from the gut of the soft tick Ornithodoros moubata: the sequence, phylogeny and post-feeding regulation

Sequence of a tick gut lysozyme (TGL) from the soft tick Ornithodoros moubata was determined by cloning and sequencing of overlapping polymerase chain reaction (PCR) and RACE PCR products. It is the first lysozyme sequence representing the subphylum Chelicerata. The resulting open reading frame codes for a putative signal peptide of 22 amino-acid residues and a mature protein composed of 124 amino-acids. Calculated mass of the protein is 14037.75 Da and a theoretical isoelectric point is 8.16. The phylogenetic analysis revealed that the TGL belongs to the c-type lysozymes. It forms a distinct monophyletic group together with multiple lysozyme-like sequences found in the gene products agCP6542 from Anopheles gambiae strain PEST and CG8492-PA from Drosophila melanogaster. This group is referred to as an H-branch due to a unique histidine residue at position 52 which replaces the highly conserved tyrosine present in the vast majority of c-type lysozymes. TGL seems to be an interesting case in which the features of lysozymes with anti-bacterial and digestive function are combined. Semi-quantitative RT-PCR and Northern blotting analysis demonstrated that TGL is strongly up-regulated at the transcriptional level after a bloodmeal. The maximum lysozyme mRNA level was detected 16 h post bloodmeal and the message remained stable for 5 days and then it slowly dropped down to the level of non-fed ticks within 2 weeks.

Molecular cloning, expression and isolation of ferritins from two tick species— Ornithodoros moubata and Ixodes ricinus

Genes encoding ferritins were isolated and cloned from cDNA libraries of hard tick Ixodes ricinus and soft tick Ornithodoros moubata. Both tick ferritins are composed of 172 amino-acid residues and their calculated mass is 19,667.2 Da and 19,974.5 Da for I. ricinus and O. moubata, respectively . The sequences of both proteins are closely related to each other as well as to the ferritin from another tick species Dermacentor variabilis (>84% similarity) . The proteins contain the conserved motifs for ferroxidase center typical for heavy chains of vertebrate ferritins. The stem-loop structure of a putative iron responsive element was found in the 5′ untranslated region of ferritin mRNA of both ticks. Antibodies against fusion ferritin from O. moubata were raised in a rabbit and used to monitor the purification of a small amount of ferritins from both tick species. The authenticity of ferritin purified from O. moubata was confirmed by mass-fingerprinting analysis. In the native state, the tick ferritins are apparently larger (~500 kDa) than horse spleen ferritin (440 kDa). On SDS-PAGE tick ferritins migrate as a single band of about 21 kDa. These results suggest that tick ferritins are homo-oligomers of 24 identical subunits of heavy-chain type. The Northern blot analysis revealed that O. moubata ferritin mRNA level is likely not up-regulated after ingestion of a blood meal.

Isolation and characterization of Dorin M, a lectin from plasma of the soft tick Ornithodoros moubata

A lectin with high hemagglutinating activity, which we have named Dorin M, was identified in the plasma of the soft tick Ornithodoros moubata. The activity of the plasma lectin could be efficiently inhibited by sialic acid, N-acetyl- d-hexosamines and sialoglycoproteins. Dorin M was purified to homogeneity using two different isolation systems: affinity chromatography on a column of bovine submaxillary mucin conjugated to Sepharose 4B with specific elution by N-acetyl- d-glucosamine and chromatography on Blue-Sepharose followed by anion exchange FPLC on a MonoQ column. The purified lectin is a glycoprotein which, in the native state, forms aggregates with molecular mass of about 640 kDa. Non-reducing SDS PAGE revealed that the lectin consists of two noncovalently bound subunits migrating closely around 37 kDa. Dorin M is a glycoprotein, probably modified by N-type glycosylation. After chemical deglycosylation, only one band of about 32 kDa was detected. Dorin M is the first lectin purified from ticks.

Characterization of an alpha-macroglobulin-like glycoprotein isolated from the plasma of the soft tick Ornithodoros moubata.

We report the identification of the first representative of the alpha-2-macroglobulin family identified in terrestrial invertebrates. An abundant acidic glycoprotein was isolated from the plasma of the soft tick Ornithodoros moubata. Its molecular mass is about 420 kDa in the native state, whereas in SDS/PAGE it migrates as one band of 190 kDa under nonreducing conditions and a band of 92 kDa when reduced. Chemical deglycosylation reveals that it is composed of two different subunits, designated A and B. The N-terminal amino-acid sequence of subunit A is similar to the N-terminus of invertebrate alpha-2-macroglobulin. Sequence analysis of several internal peptides confirms that the tick protein belongs to the alpha-2-macroglobulin family, and the protein is therefore referred to as tick alpha-macroglobulin (TAM). Functional analyses strengthen this assignment. TAM inhibits trypsin and thermolysin cleavage of the high-molecular-weight substrate azocoll in a manner similar to that of bovine alpha-2-macroglobulin. This effect is abolished by pre-treatment of TAM with methylamine. In the presence of TAM, trypsin is protected against active-site inhibition by soybean trypsin inhibitor. We cloned and sequenced a PCR product containing sequences from both subunits and spanning the N-terminus of subunit B and the putative 'bait region' (a segment of alpha-2-macroglobulin which serves as target for various proteases). This indicates that the two subunits are generated from a precursor polypeptide by post-translational processing.

Endosymbionts of ticks and their relationship to Wolbachia spp. and tick-borne pathogens of humans and animals.

The presence, internal distribution, and phylogenetic position of endosymbiotic bacteria from four species of specific-pathogen-free ticks were studied. These included the hard ticks Ixodes scapularis (the black-legged tick), Rhipicephalus sanguineus (the brown dog tick), and Haemaphysalis longicornis and the African soft tick Ornithodoros moubata. PCR assays for bacteria, using two sets of general primers for eubacterial 16S and 23S rRNA genes (rDNAs) and seven sets of specific primers for wolbachial, rickettsial, or Francisella genes, indicated that I. scapularis possessed symbiotic rickettsiae in the ovaries and that the other species harbored eubacteria in both the ovaries and Malpighian tubules. Phylogenetic analysis based on the sequence of 16S rDNA indicated that the symbiont of I. scapularis belonged to the alpha subgroup of proteobacteria and was closely related to the members of the genus Rickettsia. The other species had similar microorganisms in the ovaries and Malpighian tubules, which belonged to the gamma subgroup of proteobacteria, and formed a monophyletic group with the Q-fever pathogen, Coxiella burnetii. O. moubata harbored another symbiont, which formed a monophyletic group with Francisella tularensis and Wolbachia persica, the latter a symbiont previously isolated from Malpighian tubules of the soft tick Argas (Persicargas) arboreus. Thus, the symbionts of these four tick species were not related to the Wolbachia species found in insects. The two symbionts that live in the Malpighian tubules, one closely related to C. burnetii and the other closely related to F. tularensis, appear to be of ancient origin and be widely distributed in ticks.

Evolution of ecdysteroids and of their apolar conjugates during the post-embryonic development of the tickOrnithodoros moubata

The ecdysteroid (ES) content of the soft tick Ornithodoros moubata was investigated during the five successive nymphal molting cycles by means of an enzyme immunoassay (EIA). Samples were submitted to esterase hydrolysis in order to release free ecdysteroids from the acyl-ester conjugates (AP = apolar products). Crude and hydrolysed extracts were then analyzed by EIA using two different antibodies, a monoclonal raised against 20-hydroxyecdysone (20E) and a polychlonal raised against ecdysone (E). With the crude extracts, each molting cycle was associated with an ES peak, occurring in the middle of the instar. 20E was preponderant during the first 2 nymph cycles, but the proportion of E and other ES increased during the following instars. The shape of the peak also seemed to be different. Hydrolysis with esterase revealed that the total immunoreactivity was increased by 10- to 20-fold as compared to crude extracts in N1, N2, and N3, then by approximately 50- and 80-fold in N4 and N5 (last instar), respectively. The use of both antibodies and of HPLC fractionation showed that not only 20E was conjugated as AP, but also two other ES tentatively identified as ecdysone and 2-deoxyecdysone. It is suspected that a conversion of conjugated 20E to less immunoreactive ES occurs. Arch. Insect Biochem. Physiol. 35:159–168, 1997. © 1997 Wiley-Liss, Inc.

The ornithodorin-thrombin crystal structure, a key to the TAP enigma?

Ornithodorin, isolated from the blood sucking soft tick Ornithodoros moubata, is a potent (Ki = 10(-12) M) and highly selective thrombin inhibitor. Internal sequence homology indicates a two domain protein. Each domain resembles the Kunitz inhibitor basic pancreatic trypsin inhibitor (BPTI) and also the tick anticoagulant peptide (TAP) isolated from the same organism. The 3.1 A crystal structure of the ornithodorin-thrombin complex confirms that both domains of ornithodorin exhibit a distorted BPTI-like fold. The N-terminal portion and the C-terminal helix of each domain are structurally very similar to BPTI, whereas the regions corresponding to the binding loop of BPTI adopt different conformations. Neither of the two 'reactive site loops' of ornithodorin contacts the protease in the ornithodorin-thrombin complex. Instead, the N-terminal residues of ornithodorin bind to the active site of thrombin, reminiscent of the thrombin-hirudin interaction. The C-terminal domain binds at the fibrinogen recognition exosite. Molecular recognition of its target protease by this double-headed Kunitz-type inhibitor diverges considerably from other members of this intensely studied superfamily. The complex structure provides a model to explain the perplexing results of mutagenesis studies on the TAP-factor Xa interaction.

Effects of ingested phytoecdysteroids in the female soft tick Ornithodoros moubata.

The effects of the ingestion of some phytoecdysteroids were studied in the soft tick Ornithodoros moubata. Supernumerary moulting and malformations of first leg pairs were obtained with 22-oxo-20-hydroxy-ecdysone, 20-hydroxyecdysone-22-acetate, and 20-hydroxyecdysone-22-benzoate. Egg-yield was reduced with 20-hydroxyecdysone-22-acetate and carthamosterone. Finally, drying-out of eggs was observed with carthamosterone and 22-deoxy-20,26-dihydroxyecdysone. In addition, we demonstrated that there is a correlation between the number of completed gonotrophic cycles and the impossibility of inducing supernumerary moulting.

An Inhibitor from the Argasid Tick Ornithodoros moubata of Cell Adhesion to Collagen

An inhibitor of the adhesion of human platelets to collagen was identified in soluble extracts of the soft tick Ornithodoros Moubata and purified by four chromatographic steps. The isolated inhibitor, TAI (Tick Adhesion Inhibitor), is a ∼15-kDa protein that completely blocks the adhesion of platelets to collagen-coated microtiter plates with an IC50 of 8 nM. In the same concentration range it does not inhibit collagen-induced platelet aggregation or platelet adhesion to fibrinogen and has a partial inhibitory effect on platelet adhesion to fibronectin. TAI also blocks the adhesion of human endothelial cells to collagen, thus its inhibitory effect is not limited to platelets. TAI competes for the binding to platelets of a radiolabeled monoclonal antibody against the platelet glycoprotein Ia-IIa integrin complex. Based on its selective activity and small size, TAI is a promising new molecule for exploring cell-collagen interactions.

Cloning of the cDNA and expression of moubatin, an inhibitor of platelet aggregation.

Moubatin, a new type of specific inhibitor of collagen-induced platelet aggregation, has been isolated from the soft tick Ornithodoros moubata (Waxman, L., and Connolly, T. M. (1993) J. Biol. Chem. 268, 5445-5449). A polymerase chain reaction-generated hybridization probe, produced using primers based on moubatin protein sequence, identified phage containing the entire cDNA sequence of moubatin. Analysis of the predicted amino acid sequence yielded a mature protein of 156 amino acids with a putative prepeptide of 15 amino acids. Comparison of the sequence of moubatin to that of other proteins in the Swiss PROT data base revealed no significant homology. The cDNA sequence was cloned into the yeast expression vector pKH4 alpha 2, producing a biologically active protein which inhibited collagen-stimulated aggregation of washed human platelets with an IC50 of about 100 nM, which is similar to the potency of native tick moubatin. A concentration of recombinant moubatin that fully inhibited collagen-stimulated aggregation did not inhibit aggregation induced by a variety of other platelet agonists, again demonstrating comparable properties of the recombinant and native proteins. Moubatin did not inhibit platelet adhesion to collagen even at a concentration up to 16 times its IC50 for the inhibition of aggregation. This specificity for inhibiting collagen-stimulated aggregation and not adhesion to collagen indicates that moubatin is unique among the natural product inhibitors of collagen stimulation of platelets. Further examination of the mechanism of moubatin-mediated inhibition of collagen-stimulated aggregation revealed that 1-6 microM moubatin diminished the second phase of aggregation induced by ADP, inhibited aggregation in response to submaximal concentrations of the thromboxane A2 mimetic U46619, and competed for the binding of a thromboxane A2 receptor antagonist to platelet membranes. Therefore, at higher concentrations, moubatin may affect more than one aspect of platelet signal transduction including the thromboxane A2 receptor. The availability of recombinant moubatin will allow further investigation of its unique activities in vitro and in vivo.

Isolation of an inhibitor selective for collagen-stimulated platelet aggregation from the soft tick Ornithodoros moubata.

Soluble extracts from the soft tick Ornithodoros moubata were found to inhibit collagen-, ADP-, and thrombin-stimulated platelet aggregation. One inhibitory component was purified to homogeneity by a combination of gel filtration, ion-exchange, and reverse phase high pressure liquid chromatography. The purified activity, named moubatin, is a protein of molecular weight 17,000 and it inhibits the aggregation of washed human platelets stimulated by collagen with an IC50 of approximately 50 nM in the standard assay. At a concentration of moubatin that maximally inhibited collagen-stimulated platelet aggregation, no inhibition of aggregation initiated by other effectors, including arachidonic acid, thrombin, ristocetin, and the calcium ionophore A23187, was observed. Moubatin also inhibits collagen-dependent aggregation in plasma. At a higher concentration of moubatin (> 1 microM) it was also possible to demonstrate an inhibitory effect on the final extent of aggregation induced by a low concentration of ADP. Although moubatin selectively inhibits platelet activation by collagen, it has only a minimal effect on the adhesion of platelets to collagen. The amino acid sequences of peptides derived from proteolytic cleavage of moubatin suggest that moubatin is a unique protein, consistent with its novel functional activity.

Saliva of the soft tick, ornithodoros moubata, contains anti-platelet and apyrase activities

1. 1. Pilocarpine-induced saliva of the soft tick Ornithodoros moubata inhibits platelet aggregation induced by ADP or collagen, even when diluted 2000 times into platelet rich plasma. 2. 2. Saliva contains apyrase (ATP-diphosphohydrolase) activity, which has an optimal pH of 7.0 for ADP and of 8.0 for ATP hydrolysis, respectively. Both Ca 2+ and Mg 2+ activate the reactions. 3. 3. The mean specific activities for ATP and ADP hydrolysis at pH7.5 were 0.97 and 0.74/moles orthophosphate/min/mg protein. 4. 4. These results, which demonstrate for the first time such activities in the saliva of soft ticks, support the hypothesis that the saliva of blood sucking arthropods serves an anti-hemostatic role during feeding and that large amounts of salivary apyrase activity have evolved independently in hematophagous arthropods.

Yolk degradation in tick eggs: I. Occurrence of a cathepsin L‐like acid proteinase in yolk spheres

In crude extracts of eggs of the soft tick Ornithodoros moubata, maximum degradation of vitellin is at pH 3-3.5, whereas no proteolysis is detected at neutral or weakly acidic pHs. Acidic proteolysis is maintained at high level throughout embryonic development, and rapidly decreases in the larva, during the high phase of yolk degradation. Proteinase, acid phosphatase, and N-acetylglucosaminidase are localized within the yolk spheres; these can be considered as lysosomal-like organelles containing both substrate (vitellin) and the degradative machinery. Proteolytic activity has been essentially attributed to a cathepsin L-like enzyme through substrate specificity and inhibitors. The molecular weight is 37,000 to 39,000 as shown using gelatin-containing SDS-PAGE activity gels. At neutral pH the enzyme binds to vitellin, as demonstrated by gel filtration and PAGE under nondenaturing conditions. Acid proteinase activity at pH 5-6 is undetectable both with proteins and synthetic substrates, but is strongly increased after preincubation at pH 3-4. Activation at low pH could be important in the regulation of yolk degradation.

Metabolism of [ 3H]ecdysone by isolated tissues of the female ixodid tick Amblyomma hebraeum (Ixodoidea; Ixodidae)

Malpighian tubules, gut, ovaries and carcasses of the adult female tick Amblyomma hebraeum were incubated in vitro in the presence of 2 μM [ 3H]ecdysone. Organs and media were separately extracted after 6, 24 and 48 h incubations and the patterns of ecdysone metabolites were analyzed by HPLC. Esterase-susceptible apolar metabolites similar to the AP2 already described in the soft tick Ornithodoros moubata and thus presumably corresponding to the same conjugates (C-22 esters with fatty acids) were rapidly produced in all tissues investigated. They were mainly found within the organs but they were also released into the medium to some extent. By contrast, less apolar metabolites corresponding to the AP1 esters were mainly found in the media. Malpighian tubules and gut were the most active organs regarding the conversion of ecdysone into 20-hydroxyecdysone (20E). However, only low quantities of 20E were formed, reaching respectively 12.5% and 11.6% of the total metabolites after 48 h incubations. In the carcass and in the ovary the formation of 20E was only a minor pathway (1.7% and 3.1% of the total metabolites after 48 h). In ovaries we observed a massive conversion of ecdysone into 3-epiecdysone, which (as in insects) presumably proceeded through the intermediate formation of 3-dehydroecdysone. These two compounds were identified among the metabolites by CI/D mass spectrometry. The 3-epimer was released into the media, in contrast with the AP2 which were essentially stored within ovaries. Epimerization was also realized to some extent by carcasses, and again the epimer was released into the culture media. The different pathways are compared with those found in other tick species and in insects, and the significance of the various metabolites is discussed.