Tick Salivary Gland Proteins Research Articles

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+ ( Qian et al., 1998; Yuan et al., 2000). 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 α/β 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.

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.

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 2α 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 μM) stimulated secretion of anticoagulant protein in a dose-dependent manner but the voltage-gated Ca 2+-channel blocker verapamil (1 to 1000 μM) and the receptor-mediated Ca 2+-entry antagonist, SK&F 96365 (1 and 10 μM), and 5 mM ethylene glycol bis(β-aminoethyl ether)-N,N N′, N′-tetraacetic acid (EGTA) had no appreciable effect on inhibiting PGE 2-stimulated secretion of anticoagulant protein. PGE 2 (0.1 μM) and the non-hydrolyzable analog of guanosine triphosphate (GTP), GTPγS (10 μM), 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 μM) 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+.

Kinetics and cross-species comparisons of host antibody responses to lone star ticks and American dog ticks (Acari: Ixodidae).

Understanding of the animal antibody response to tick salivary gland proteins is necessary to identify candidate antibodies that may have use as species- and feeding-duration-specific biomarkers of tick exposure in humans. The kinetics of the humoral immune response of rabbits to challenge feeding by 2 tick species [Amblyomma americanum (L.) and Dermacentor variabilis (Say)] was characterized by both enzyme-linked immunosorbent assay and immunoblot. Western blot analysis revealed that rabbits produced antibodies against both A. americanum and D. variabilis tick salivary gland antigens, with molecular weights ranging from 12.2 to 125 kDa; the antibody response against the saliva of both tick species possessed both unique and common aspects. The presence of antibody against low-molecular-mass (< 20 kDa) salivary gland antigens of A. americanum may be specific for A. americanum exposure. Antibodies directed against D. variabilis salivary gland antigens of 111, 86.3, and 85 kDa may be specific for D. variabilis exposure. The data suggest that host antibodies directed against specific tick salivary gland proteins might have use as species-specific biologic markers of tick exposure.

Tick Avoidance Behaviors Associated with a Decreased Risk of Anti-Tick Salivary Gland Protein Antibody Seropositivity in Military Personnel Exposed to Amblyomma Americanum in Arkansas

During April through September 1990, 399 military personnel who originated from either Fort Chaffee, Arkansas (n = 236) or Fort Wainwright, Alaska (n = 163) were studied during maneuvers in tick-infested areas at Fort Chaffee. Study subjects completed a questionnaire and had pre- and post-maneuvers serum specimens analyzed for antibodies to several rickettsial and ehrlichial agents and to Amblyomma americanum (lone star tick) salivary gland proteins (anti-tick saliva antibodies [ATSA], a biologic marker of tick exposure). Military rank/grade and home station were associated with pre-maneuvers ATSA seropositivity by enzyme-linked immunosorbent assay (ELISA). Fort Wainwright personnel were more likely to show at least a 50% increase in ATSA levels, compared with subjects from Fort Chaffee, from the pre- to the post-maneuvers specimen (adjusted odds ratio [AOR] = 2.7, 95% confidence interval [CI] = 1.2-6.1). Subjects from Fort Wainwright who did not report use of bed netting were at an increased risk of post-maneuvers ATSA seropositivity (AOR = 4.1, 95% CI = 1.5-11.5). In contrast, subjects from Fort Chaffee who did not report tucking pants into socks were at increased risk of post-maneuvers ATSA seropositivity (AOR = 2.8, 95% CI = 1.1-7.1). Subjects from Fort Chaffee who reported an attached tick bite during maneuvers were more likely to be ATSA-seropositive in the post-maneuvers specimen (AOR = 2.5, 95% CI = 1.2-5.2). Western blot assays revealed large differences in tick salivary gland proteins that were recognized on the post-maneuvers specimen among three randomly selected individuals, and small differences within a single individual who reported a tick bite during maneuvers, comparing pre- and post-maneuvers specimens. The ATSA ELISA seropositivity was not associated with seroconversion to the tick-borne infectious agents.

Salivary gland changes and host antibody responses associated with feeding of male lone star ticks (Acari:Ixodidae).

The goal of this study was to demonstrate that male lone star ticks, Amblyomma americanum (L.), actively feed on rabbits during attachment and that the host is capable of mounting an immune response against male salivary gland proteins. During attachment, it was shown that male ticks salivary glands hypertrophy. An enzyme-linked immunosorbent assay was used to detect rabbit serum proteins in the midgut of previously attached male ticks. SDS-polyacrylamide gel electrophoresis showed that the proteins expressed in the male tick salivary gland changed during feeding, with several new proteins in the 15- to 50-kDa range synthesized during attachment. Rabbits mounted a detectable antibody response against male tick salivary gland proteins after 2 sequential feedings of male ticks. The antibodies were directed against a spectrum of male salivary gland proteins ranging from 18 to 160 kDa. Several of these proteins were not recognized by antibodies directed against female tick saliva proteins, and thus may be specific male salivary gland components. This evidence indicates that male A. americanum ticks actively feed during attachment, that their salivary gland proteins change during feeding, and that male salivary gland proteins are immunogenic.

A secreted calreticulin protein in ixodid tick ( Amblyomma americanum) saliva

A complementary DNA clone from salivary glands of feeding female Amblyomma americanum ticks has been characterized as encoding calreticulin. Calreticulin, a major endoplasmic reticulum (ER) calcium-binding protein, appears to be secreted in Amblyomma and Dermacentor saliva. Evidence is accummulating that calreticulin performs roles unrelated to calcium storage. Unlike most known calreticulins, tick-secreted calreticulin lacks the ER retention signal, KDEL. This is the first molecular cloning of a specific tick salivary gland protein. The finding of a secreted calreticulin in tick saliva suggests a role for calreticulin in blood feeding through host immunosuppression or antihemostasis.

Spleen accessory cell antigen processing and in vitro induction of specific lymphocyte proliferation in BALB/c mice infested with nymphal Ixodes ricinus ticks.

Ticks are hematophagous organisms that transmit numerous human and veterinary diseases. They also induce an anti tick immune response during their blood meal. Thus, rabbits repeatedly infested with I. ricinus nymphs and adults produce IgG antibodies reacting against some salivary gland and integumental proteins from female ticks1. In contrast, experimental infestations of BALB/c mice with nymphal I. ricinus do not generate anti tick antibodies (IgG) or only occasionally. However mice immunized with salivary gland extract (SGE) develop antibodies that recognize tick integumental and salivary gland proteins (Fig 1).KeywordsSalivary GlandBlood MealAntigen ProcessingTritiated ThymidineFemale TickThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Antibodies to Borrelia burgdorferi and tick salivary gland proteins in New Jersey outdoor workers.

In 1990, a second cross-sectional study of outdoor workers (n = 758) at high risk for Lyme disease was conducted. A questionnaire was administered, and antibodies to Borrelia burgdorferi and tick salivary gland proteins (antitick saliva antibody, a biologic marker of tick exposure) were assayed by enzyme-linked immunosorbent assay. The statewide Lyme disease seroprevalence increased from 8.1% in 1988 to 18.7% in 1990. Antitick saliva antibody seropositivity varied by county and was associated with measures of self-reported tick exposure. The data suggested that the prevalence of B. burgdorferi infection increased in New Jersey outdoor workers from 1988 to 1990.

Protein phosphatase activity is controlled by an inhibitor phosphoprotein in tick salivary glands

Protein phosphatase activity in tick salivary glands was inhibited by heat-stable protein(s) from tick salivary glands as well as by an inhibitor protein from rabbit skeletal muscle. Inhibitor activity was increased after phosphorylation of inhibitor proteins with the catalytic subunit (C) of cyclic AMP-dependent protein kinase and ATP. C inhibited protein phosphatase activity of the partially purified enzyme, while purified cyclic AMP-dependent protein kinase inhibitor protein prevented inhibition of tick salivary gland protein phosphatase by C suggesting that the inhibitor phosphoprotein coelutes with the partially purified enzyme. A soluble heat-stable protein with a molecular weight of approx. 26 kDa was phosphorylated by C, suggesting that a protein phosphatase inhibitor protein similar to inhibitor-1 in mammalian tissue, is present in tick salivary glands.

Effect of methoprene and 20-hydroxyecdysone on salivary gland development of the lone star tick, Amblyomma americanum (L.)

Topically applied juvenile hormone III analogues 7-S-methoprene and 7-S-hydroprene (50 μg/tick) modestly stimulated attached virgin Amblyomma americanum females to feed and attain average body weights of 27.1 mg (178% of controls, just 4% of mated controls), however, tick salivary gland protein levels were stimulated 133%, into the range of mated, early-rapid feeding female ticks (> 100 mg body weight). Na K - ATPase activity was also increased to levels seen in salivary glands of mated, early-rapid feeding ticks (212% of controls) by methoprene. Electron microscopy of salivary glands from treated ticks revealed euchromatic nuclei, partially swollen alveolar lumina, proliferation of basolateral labyrinth and increased rough endoplasmic reticulum, morphological changes normally seen in later feeding stages of mated females, but not in virgins. Treatment of attached, feeding females with 100 μg 20-hydroxyecdysone/tick also stimulated virgins to gain additional weight (195%) and total salivary gland protein (144%), but not Na K - ATPase activity. Methoprene stimulated protein level was not potentiated by additional treatments with 20-hydroxyecdysone. Tick salivary glands are thus partially, but differentially activated by methoprene and 20-hydroxyecdysone treatment while pre-mating weight arrest (feeding) is released only slightly.

Acquired resistance to Rhipicephalus appendiculatus (Acari: Ixodidae): identification of an antigen eliciting resistance in rabbits.

Immune resistance to infestation by Rhipicephalus appendiculatus , an ixodid tick, was induced in rabbits by repeated tick infestation. Host resistance, which interferes with tick feeding, was expressed as a reduction in tick engorgement weight. Antibodies from resistant hosts were used to isolate several tick salivary gland antigens. These antigens were tested for their ability to produce tick feeding resistance upon injection into susceptible hosts. The results of these experiments suggest that a 90-kilodalton tick salivary gland protein may be useful to induce host resistance to the feeding of ixodid ticks.

Kinetics of the phosphotransferase reaction of the catalytic subunit of the tick salivary gland cAMP-dependent protein kinase

The catalytic subunit of the cAMP dependent protein kinase was purified 100-fold from tick salivary glands. The enzyme mechanism of the phosphotransferase reaction catalyzed by this subunit was investigated. Highly purified enzyme did not show ATP-ase activity in the absence of protein substrates. Initial velocities were measured using histone H1 or a synthetic heptapeptide, Kemptide, as P i acceptors and [γ- 32P]ATP as a phosphodonor. At varying levels of ATP and fixed concentrations of protein cosubstrates and vice versa, double reciprocal plots gave a family of lines converging to the left of the 1 v axis. Secondary plots of intercepts and slopes versus fixed cosubstrate concentrations were linear. At high concentration (2 Km), histone showed substrate inhibition which was noncompetitive versus ATP. Product inhibition by Mg·ADP was competitive versus ATP and noncompetitive with respect to H1. Phosphohistone on the other hand was noncompetitive with respect to H1, but gave parabolic competitive inhibition against ATP. Dead end inhibition by AMP-PNP, an analogue of ATP, was competitive and noncompetitive against ATP and H1, respectively. Dead end inhibition by the protein kinase inhibitor was noncompetitive and competitive against ATP and H1, respectively. These studies strongly suggest that the tick salivary gland protein kinase has a sequential mechanism with predominately ordered addition of ATP followed by protein substrate and ordered release of phosphoprotein and ADP.