Transcriptome analysis of the salivary glands of the female tick Amblyomma americanum (Acari: Ixodidae).

Ticks infest a wide range of hosts while bypassing their immune, inflammatory and haemostatic responses during their extended feeding, which may last for more than two weeks. Here, we present a transcriptome analysis of 3868 expressed sequence tags (ESTs) from three cDNA libraries generated from the salivary glands of adult female Ambyomma americanum ticks at different stages of feeding. We applied a normalization step for one library, significantly decreasing the abundance of mitochondrial sequences amongst the 2292 sequences from the normalized library. Our ESTs include homologues that may modulate haemostatic, immune and inflammatory responses of the hosts. Other ESTs probably represent important components of the highly efficient secretory pathways for salivary proteins and concomitantly transmitted pathogens.

RNA interference in ticks: a study using histamine binding protein dsRNA in the female tick Amblyomma americanum.

RNA interference (RNAi), a gene silencing process, 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 putative Amblyomma americanum histamine binding protein (HBP) to test the significance of using this methodology in the assessment of the function and importance of gene products in ectoparasitic ticks. The female salivary glands incubated in vitro with HBP dsRNA had a significantly lower histamine binding ability. In addition, the injection of HBP dsRNA into the unfed females led both to a reduced histamine binding ability in the isolated salivary glands and to an aberrant tick feeding pattern or host response. Molecular data demonstrated less expression of the HBP mRNA in the RNAi group. Taken together, these results suggest that RNAi might be an important tool for assessing the significance of tick salivary gland secreted proteins modulating responses at the tick-host interface.

Identification of SNARE and cell trafficking regulatory proteins in the salivary glands of the lone star tick, Amblyomma americanum (L.).

Prostaglandin E(2) (PGE(2)) stimulates secretion of tick salivary gland proteins via a phosphoinositide signaling pathway and mobilization of intracellular Ca(2+) (). Highly conserved intracellular SNARE (soluble NSF attachment protein receptors) complex proteins are associated with the mechanism of protein secretion in vertebrate and invertebrate neuronal and non-neuronal cells. Proteins in the salivary glands of partially fed female lone star ticks cross-react individually with antibodies to synaptobrevin-2 (vesicle (v)-SNARE), syntaxin-1A, syntaxin-2 and SNAP-25 (target (t)-SNAREs), cytosolic alpha/beta SNAP and NSF (N-ethylmaleimide-sensitive fusion protein), Ca(2+) sensitive synaptotagmin, vesicle associated synaptophysin, and regulatory cell trafficking GTPases Rab3A and nSec1. V-SNARE and t-SNARE proteins form an SDS-resistant, boiling sensitive core complex in the salivary glands. Antibodies to SNARE complex proteins inhibit PGE(2)-stimulated secretion of anticoagulant protein in permeabilized tick salivary glands. We conclude that SNARE and cell trafficking regulatory proteins are present and functioning in the process of PGE(2)-stimulated Ca(2+) regulated protein secretion in tick salivary glands.

Identity and synthesis of prostaglandins in the lone star tick, Amblyomma americanum (L.), as assessed by radio-immunoassay and gas chromatography/mass spectrometry.

High concentrations of PGE(2) and PGF(2alpha) were identified by radio-immunoassay (RIA) and/or gas chromatography/mass spectrometry (GC/MS) in the hemolymph, salivary glands and saliva of the lone star tick Amblyomma americanum (L.). Binding studies indicated that PGE(2) was free and not bound to any proteins in the hemolymph. A small amount of 6-keto-PGF(1alpha) (breakdown product of PGI(2); prostacyclin) was also found in the salivary glands but not in the hemolymph or saliva. Neither PGD(2) nor PGA(2)/B(2) was detected in any tick material investigated. Although PGE(2) was found in the gut contents, only small amounts of label crossed the gut into the hemolymph during artificial feeding with labeled PGE(2), indicating that the high amounts of PGE(2) in hemolymph and salivary glands are not sequestered from the host blood meal. Isolated salivary glands and salivary gland homogenates demonstrated robust synthesis of PGE(2) at high concentrations of exogenous arachidonic acid. Synthesis by the salivary glands was monitored by measuring increasing PGE(2) with increasing arachidonic acid by RIA, GC/MS and labeled PGE(2) in the presence of labeled arachidonic acid. Synthesis was inhibited in a dose-dependent manner by indomethacin indicating that the cyclooxygenase synthesizing prostaglandins in ticks shares similarities to the enzyme found in mammals.

Prostaglandin E(2)-stimulated secretion of protein in the salivary glands of the lone star tick via a phosphoinositide signaling pathway.

Previous studies identified a prostaglandin E(2) (PGE(2)) receptor in the salivary glands of partially fed female lone star ticks, Amblyomma americanum (L.). In the present studies, protein secretion from dispersed salivary gland acini was shown to be specific for PGE(2), as compared with PGF(2alpha) or the thromboxane analog U-46619, in accordance with their respective binding affinities for the PGE(2) receptor. Furthermore, the selective PGE(2) EP1 receptor agonist, 17-phenyl trinor PGE(2), was as effective as PGE(2) in stimulating secretion of anticoagulant protein. Calcium ionophore A-23187 (1 to 100 microM) stimulated secretion of anticoagulant protein in a dose-dependent manner but the voltage-gated Ca(2+)-channel blocker verapamil (1 to 1000 microM) and the receptor-mediated Ca(2+)-entry antagonist, SK&F 96365 (1 and 10 microM), and 5mM ethylene glycol bis(beta-aminoethyl ether)-N,NN', N'-tetraacetic acid (EGTA) had no appreciable effect on inhibiting PGE(2)-stimulated secretion of anticoagulant protein. PGE(2) (0.1 microM) and the non-hydrolyzable analog of guanosine triphosphate (GTP), GTPgammaS (10 microM), directly activated phospholipase C (PLC) in a membrane-enriched fraction of the salivary glands after PLC was first incubated with the PGE(2) EP1 receptor antagonist AH-6809, which presumably antagonized endogenous PGE(2) (0.3 microM) in the broken-cell-membrane-enriched fraction. TMB-8, an antagonist of intracellular inositol trisphosphate (IP(3)) receptors, inhibited PGE(2)-stimulated secretion. The results support the hypothesis that PGE(2) stimulates secretion of tick salivary gland protein via a phosphoinositide signaling pathway and mobilization of intracellular Ca(2+).

Molecular cloning of cAMP-dependent protein kinase catalytic subunit isoforms from the lone star tick, Amblyomma americanum (L.).

The salivary glands of ixodid ticks are central to tick feeding and to survival during off-host periods. They produce and secrete a number of molecules critical to maintaining the complex host-vector interface and to maintaining osmotic balance. We have previously shown that a cyclic AMP-dependent protein kinase (cAPK) is involved in the mechanism of salivary gland secretion. We have now cloned cDNAs encoding three isoforms of the catalytic subunit (cAPK-C) of the cAPK from Amblyomma americanum, which are probably produced from alternative RNA processing of a single cAPK-C gene. The cDNAs contain unique N-termini of variable lengths that are linked to a common region containing the alpha A helix, catalytic core, and a C-terminal tail. The common region is highly similar to both insect and vertebrate cAPK-Cs. We have examined mRNA profiles in whole ticks and in isolated salivary glands throughout feeding and find that a single cAPK-C isoform is expressed in the salivary glands of both unfed and feeding females.

Prostaglandin E2 in the salivary glands of the female tick, Amblyomma americanum (L.): calcium mobilization and exocytosis.

A cholera toxin-sensitive, prostaglandin E2 (PGE2) specific receptor has been identified in the plasma membrane fraction of tick salivary glands. In the present study, we report that stimulation of dispersed salivary glands of the lone star tick Amblyomma americanum (L.) with 1 nM to 10 microM PGE2 increased the intracellular concentration of inositol trisphosphate (IP3) in a dose-dependent manner. Incubation of dispersed tissue with 1 nM to 10 microM PGE2 also stimulated release of 45Ca2+ from preloaded tissue. PGE2 (10 microM) did not stimulate an influx of 45Ca2+. Therefore, the PGE2 receptor in the salivary glands appears to activate a phosphoinositide phospholipase C signalling pathway to increase formation of intracellular IP3 and, thus, mobilize Ca2+ from intracellular stores. Incubation of dispersed salivary glands with 1 nM to 1 microM PGE2 stimulated secretion of anticoagulant protein, but not at < 1 nM or > 1 microM PGE2. In addition, the mammalian PGE2 EP1 receptor antagonist AH-6809 affected secretion of anticoagulant by dispersed salivary gland tissue at a low concentration supporting the hypothesis that the PGE2 receptor in tick salivary glands is EP1-like. We propose that a major function for PGE2 in tick salivary glands is to mobilize Ca2+ and stimulate secretion (exocytosis) of bioactive proteins into the tick's saliva during feeding.

A novel phospholipase A2 activity in saliva of the lone star tick, Amblyomma americanum (L.).

Saliva from female lone star ticks, Amblyomma americanum, contained a novel phospholipase A2 (PLA2) activity that hydrolyzed 14C-arachidonate from 14C-arachidonyl phosphatidylcholine. The tick saliva PLA2 (ts-PLA2) was active over a broad pH range (4.5-11.5) with two distinct pH optima of pH 5.5 and 9.5. Though extracellular PLA2s are reported to be activated by millimolar Ca2+, ts-PLA2 was sensitive to submicromolar Ca2+ and was half-maximally activated by 3.5 microM Ca2+. Tick saliva contains > 500 microM Ca2+ and the feeding lesion in the host is expected to contain millimolar Ca2+. Saliva exhibited a single peak of PLA2 activity corresponding to a molecular weight of 55.7 +/- 1.3 kDa by size exclusion chromatography. The ts-PLA2 was unaffected by a variety of compounds known to inhibit either secreted or cytosolic PLA2s from other sources. However, ts-PLA2 was inhibited by the substrate analog, oleyloxyethyl phosphorylcholine (IC50 = 1.4 microM), and the end product, arachidonic acid (IC50 = 38 microM). Low concentrations of dithiothreitol did not greatly affect ts-PLA2, but activity was reduced at higher concentrations. The PLA2 activity found in A. americanum salivary glands showed many similarities to ts-PLA2, but also some distinct differences. Secreted at the tick-host interface, ts-PLA2 is thought to play an important, but unknown, role during the prolonged tick feeding.

Isolation and characterization of americanin, a specific inhibitor of thrombin, from the salivary glands of the lone star tick Amblyomma americanum (L.).

A thrombin (EC 3.4.21.5) inhibitor (americanin) was isolated from the salivary glands of the lone star tick Amblyomma americanum (L.) using reversed-phase chromatography and anion-exchange chromatography. Americanin did not inhibit any other protease tested, including factor Xa, plasmin, trypsin, chymotrypsin, elastase, papain, pepsin, and carboxypeptidase. The inhibition of thrombin by americanin decreased dramatically with dilution of the reaction mixture including thrombin, its substrate, and americanin. When thrombin assays were performed in the presence of americanin, the reaction curve showed a time-dependent inhibition. Significant inhibition was observed when americanin concentration was approximately equal to that of thrombin, with a Ki of 0.073 nM. The results suggest that americanin is a specific, reversible, competitive, slow, tight-binding inhibitor of thrombin.

Cloning and sequence of a gene for the homologue of the stearoyl CoA desaturase from salivary glands of the tick Amblyomma americanum.

A 1488 base pair cDNA clone has been isolated from a cDNA library made from salivary glands from 3-day feeding adult female ticks. The sequence of this cDNA suggests it is the gene for the tick homologue of the stearoyl CoA desaturase. This gene is expressed in eggs and all feeding stages of the adult examined, but appears to be transcribed to an 8 kb mRNA as well as a 1.5 kb mRNA. Because ticks have the ability to synthesize monounsaturated fatty acids and demonstrate a large increase in salivary monounsaturated fatty acids during tick feeding, we hypothesize that stearoyl CoA desaturase may be a key enzyme in the morphogenesis of tick salivary glands during feeding.

A specific prostaglandin E2 receptor and its role in modulating salivary secretion in the female tick, Amblyomma americanum (L.).

Prostaglandins of the 2-series (e.g. PGE2) are typically synthesized from arachidonic acid (AA) after AA is released from cellular phospholipids after activation of an intracellular phospholipase A2 (PLA2). Treatment of isolated salivary glands with PLA2 inhibitor oleyloxyethyl phosphorylcholine (OPC) or prostaglandin synthetase inhibitors reduced dopamine-induced fluid secretion and cyclic AMP (cAMP) levels in isolated salivary glands. PGE2 and its analog, 17-phenyl trinor PGE2, partly reversed the inhibition of secretion and cAMP level by OPC, suggesting that prostaglandins may have an autocrine effect in modulating tick salivary gland function. A specific PGE2 receptor was identified in the plasma membrane fraction of the salivary glands. The receptor exhibits a single, high affinity PGE2 binding site with a KD approximately 29 nM, is saturable, reversible, and specific for PGE2 and coupled to a cholera toxin-sensitive guanine nucleotide regulatory protein. Assay of adenylate cyclase activity in salivary gland membranes showed that PGE2 neither stimulated nor inhibited adenylate cyclase activity, indicating that the PGE2 effects on cAMP levels and possibly secretion are indirect, and that the PGE2 receptor stimulates an alternate "second messenger" pathway.

Cloning and sequence of a gene for a homologue of the C subunit of the V-ATPase from the salivary gland of the tick Amblyomma americanum (L).

A 1084 base pair partial cDNA showing similarity to the C subunit of the vacuolar ATPase (V-ATPase) was isolated on a clone from a cDNA library made from salivary glands from 3-day-old feeding adult Amblyomma americanum (L.) female ticks. The 5' end was completed using primer extension and the two pieces joined to form a complete cDNA of 1373 bp. This mRNA is expressed in embryos and the salivary glands of unfed adults and adult females at all stages of feeding. Specific inhibitors of the V-ATPase decrease the rate of dopamine-stimulated secretion of isolated salivary glands, but not as much as ouabain, an inhibitor of the Na+, K+ ATPase, indicating that a V-ATPase may participate in the mechanism of salivary fluid secretion in A. americanum, but the volume of saliva secreted is more dependent on an active Na+, K+ ATPase.

Identification and comparison of macrophage-induced proteins and proteins induced under various stress conditions in Brucella abortus.

Brucella abortus is a facultative intracellular pathogen of cattle and humans that is capable of survival inside macrophages. In order to understand how B. abortus copes with the conditions during intracellular growth in macrophages, the protein synthesis pattern of the bacteria grown inside bovine macrophages has been compared with that of bacteria grown in the cell culture medium by two-dimensional polyacrylamide gel electrophoresis. Approximately 24 new proteins that are not detected in the bacteria grown in the cell culture medium have been induced during intracellular growth in macrophages. In contrast, approximately 50 proteins that were expressed during growth in cell culture medium were completely repressed during intracellular growth. The level of expression of 19 proteins increases while that of 54 proteins decreases during intracellular growth. To understand these results, the protein synthesis pattern of B. abortus during intracellular growth was compared with those during other stress conditions. Under each stress condition studied, several new proteins were induced that were not present during regular growth conditions. Comparison of the protein synthesis pattern of B. abortus during intracellular growth with those obtained under various stress conditions has indicated that the response to intracellular growth was not just a simple sum of stress conditions studied so far.

Cloning and characterization of the glucokinase gene of Brucella abortus 19 and identification of three other genes.

A clone from Brucella abortus 19 complemented an Escherichia coli strain deficient in phosphorylation of glucose. Open reading frames similar to E. coli mepA, glk, and genes encoding ATP-coupled exporters were found in the sequence. A fourth affected growth on minimal media of the ptsI glk strain with various carbon sources.

Tick salivary gland physiology.

The multifunctional, morphologically complex salivary glands are essential to the biological success of ticks and are intricately involved in the transmission of pathogens. They are innervated, and there is convincing evidence that dopamine is a neurotransmitter at the neuroeffector junction controlling fluid secretion. As feeding progresses, the rate of salivary fluid secretion increases greatly, enabling the ixodid tick to concentrate the bloodmeal by returning excess water and ions to the host. Saliva in feeding ticks is rich in bioactive components and exhibits a range of pharmacological properties. Factors identified in saliva or salivary glands include cement to help anchor the mouthparts to the host, various enzymes and inhibitors, histamine agonists and antagonists, prostaglandins, antihemostatic factors, and immuno-modulating factors. A secretion from the salivary glands allows ticks to absorb water from the air during the lengthy periods off their hosts. The physiology of this remarkable organ provides a striking example of strategies that have evolved to meet the challenge of a unique parasitic life style.

Distribution of arachidonic acid among phospholipid subclasses of lone star tick salivary glands.

The subclass composition of choline- and ethanolamine-containing phospholipids was determined by analysis of acyl-linked fatty acids released by base hydrolysis of diradylglycerobenzoates formed from lone star tick salivary gland diacyl, alkylacyl and alkenylacyl phospholipids. The diacyl subclass comprises 87% of all choline-containing phospholipids, while th alkylacyl subclass comprises c. 9% and the alkenylacyl subclass c. 4%. The diacyl subclass comprises 72-77% of ethanolamine-containing phospholipids and about 14 and 13% of this subclass of phospholipid are alkylacyl and alkenylacyl lipids, respectively. Arachidonic acid (20:4) is the most abundant fatty acid (28% of all fatty acids) esterified in the alkylacyl form of phosphatidylcholine (PC) and it comprises 17% of the fatty acids in alkenylacyl-PC. The alkylacyl form of phosphatidylethanolamine (PE) is also rich in 20:4 (24%) while the alkenylacyl-PE subclass contains only 9% 20:4. Despite the relatively high amounts of 20:4 within the ether-linked phospholipids, the majority of the salivary gland 20:4 (> 83%) is found in the diacyl phospholipid subclass because of the preponderence of this subclass in tick salivary glands. Isolated salivary glands incorporated [3H]-20:4 primarily (> 98%) into the sn-2 position of diacyl PC > PE, with some incorporation into triglycerides. Continued incubation in the absence of labeled 20:4 demonstrated remodeling of [3H]-20:4 from PC into PE, and from the diacyl subclass to the alkylacyl subclass in the choline containing phospholipids.

Protein phosphatase 1 and 2A in tick salivary glands as assessed by responses to okadaic acid.

Crude protein phosphatase activity was inhibited 80% by nanomolar okadaic acid (OA) in salivary glands of unfed ticks but only 40% in salivary glands of feeding ticks. An additional 40% of protein phosphatase was inhibited by micromolar OA in the salivary glands of feeding ticks but only 10% in salivary glands of unfed ticks. Cyclic AMP and OA alone or together increased the phosphorylation of multiple proteins in a plasma membrane-enriched 900 g supernatant fraction of tick salivary glands. Exogenous cyclic AMP stimulated increased incorporation of phosphate into proteins with approximate molecular weights of 109, 70, 64, 51, 48, 42 and 18.5 kDa. Micromolar OA in the absence of exogenous cyclic AMP stimulated increased incorporation of phosphate into proteins with apparent molecular weights of 109, 93, 74.5, 70, 51, 48, 42 and 18.5 kDa. Cyclic AMP and OA (10(-6) and 10(-9) M) stimulated significantly greater phosphorylation of an 18.5 kDa mol. wt protein above that observed in response to stimulation by OA (10(-6) and 10(-9) M) or exogenous cyclic AMP alone. Micromolar okadaic acid inhibited the amount and number of proteins but not volume of saliva secreted by whole ticks in response to stimulation by DA and theophylline. However, micromolar and nanomolar okadaic acid inhibited the ability of dopamine to stimulate fluid secretion by isolated salivary glands. Overall, the data support the existence of type 1 and 2A protein phosphatases in tick salivary glands and a role for protein phosphatases in modulating tick salivary secretion.

Cell membrane receptors and regulation of cell function in ticks and blood-sucking insects.

Immunoglobulins cross the midgut epithelium and enter the haemolymph of many blood-feeding arthropods without losing their immunological properties. Antigens essential to the survival of the blood-sucking arthropods which may be affected by the small amounts of specific antibody that cross the gut epithelium include membrane receptors or other factors which regulate cell function. Membrane receptors implicated in transmembrane signalling in response to specific neural and endocrine factors fall into three major classes: (1) gated ion channels, (2) agonist-stimulated tyrosine kinases and (3) receptors that interact with GTP-binding (G) proteins. Examples of all three types have been found in insects and ticks. A dopamine receptor interacts with a G-protein essential for controlling fluid secretion by the salivary glands of ixodid ticks. Another receptor in the ixodid tick salivary gland binds a neuropeptide from the tick synganglion and stimulates turnover of plasma membrane phosphoinositides, but its mechanisms of transmembrane signalling and function remain elusive. Another large class of membrane receptors are those concerned with endocytosis. Examples of receptor-mediated endocytosis include incorporation of vitellogenin by developing oocytes in mosquitoes and ticks and uptake of lysed blood-meal components by digest cells of the tick gut. Many cell membrane receptors and possibly hormones could serve as targets for vaccines in blood-feeding insects and ticks. The major challenge is to identify and characterize essential internal receptors and cellular components that are accessible to and affected by specific antibodies that are introduced into the body of blood-feeding arthropods.

Changes in lipids of the salivary glands of the lone star tick, Amblyomma americanum, during feeding.

The lipid composition of salivary glands from male and female lone star ticks, Amblyomma americanum, was investigated at progressive stages of tick feeding. The amounts of fatty acids from both phospholipid and neutral lipid fractions increased dramatically during the initial stage of feeding and peaked in partially fed females weighing 100-250 mg. Percentage compositions of myristic (14:0) and palmitic acid (16:0) decreased, but stearic (18:0), oleic (18:1), linoleic (18:2), and arachidonic acid (20:4) increased during tick feeding. Arachidonic acid, the precursor to eicosanoids including the 2-series of prostaglandins, increased from 1.3% of all fatty acids in salivary glands from unfed female ticks to 8.2% in salivary glands from fully engorged female ticks. Arachidonic acid was found in the triglyceride fraction of unfed and fed virgin females but only in phosphatidylcholine and phosphatidylethanolamine from salivary glands of other fed female ticks. Comparisons between fed and unfed male ticks and fed/virgin, fed/mated, and unfed females demonstrate that feeding is necessary for accumulation of arachidonic acid in salivary gland phospholipids.

Cloning of genes for proline and leucine biosynthesis from Brucella abortus by functional complementation in Escherichia coli.

By selecting for growth of Escherichia coli mutant strains in the absence of the required amino acid, clones were found in a Brucella abortus library carrying genes for glutamyl phosphate reductase (proA) and beta-isopropylmalate dehydrogenase (leuB). These clones hybridized to unique fragments in a genomic digest of B. abortus DNA. The proA-complementing DNA was found in a region of 1.3 kb, which directed the synthesis of a protein of 48,000 Da with a pI of 6.3 in maxicells. The leuB-complementing activity was in a region of 1.4 kb and directed synthesis of a protein of 46,000 Da with a pI of 5.9.