Identification of novel tick salivary gland proteins for vaccine development

Methods currently used to control Ixodes scapularis ticks rely principally on acaricidal applications which suffer from a number of limitations. Recently, host vaccination against ticks has been shown to be a promising alternative tick control method. In tick salivary glands, numerous genes are induced during the feeding process. Many of these newly expressed proteins are secreted in tick saliva and may play a role in modulating host immune responses and pathogen transmission. We have performed suppression subtraction hybridization to identify unique I. scapularis salary gland proteins specifically expressed during engorgement. We have cloned and sequenced ten unique salivary gland-associated cDNAs that are up-regulated during feeding. The protein products of these genes represent potential vaccine candidates for use in the control of ticks and to prevent transmission of tick-borne diseases.     

Saliva,Salivary Gland,and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) ^1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick ^3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick''s enzootic cycle ^14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva ^9,16-19.As a tick feeds the host typically responds with a strong hemostatic and innate immune response ^11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding ^{3,11,20,21,23}. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens ^3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.     

Vector-host interactions in disease transmission

Tick-borne spirochetes include borreliae that cause Lyme disease and relapsing fever in humans. They survive in a triangle of parasitic interactions between the spirochete and its vertebrate host, the spirochete and its tick vector, and the host and the tick. Until recently, the significance of vector-host interactions in the transmission of arthropod-borne disease agents has been overlooked. However, there is now compelling evidence that the pharmacological activity of tick saliva can have a profound effect on pathogen transmission both from infected tick to uninfected host, and from infected host to uninfected tick. The salivary glands of ticks provide a pharmacopoeia of anti-inflammatory, anti-haemostatic and anti-immune molecules. These include bioactive proteins that control histamine, bind immunoglobulins, and inhibit the alternative complement cascade. The effect of these molecules is to provide a privileged site at the tick-host interface in which borreliae and other tick-borne pathogens are sheltered from the normal innate and acquired host immune mechanisms that combat infections. Understanding the key events at the tick vector-host interface, that promote spirochete infection and transmission, will provide a better understanding of the epidemiology and ecology of these important human pathogens.     

Tick modulation of host immunity: an important factor in pathogen transmission.

Immunological interactions at the tick host interface involve innate and specific acquired host immune defenses and immunomodulatory countermeasures by the tick. Tick feeding stimulates host immune response pathways involving antigen-presenting cells, cytokines, B-cells, T-cells, circulating and homocytotropic antibodies, granulocytes, and an array of biologically active molecules. In response to host immune defenses, tick-mediated host immunosuppressive countermeasures inhibit: host antibody responses; complement activation; T-cell proliferation; and cytokine elaboration by macrophages and Th1-lymphocytes. Immunosuppressive proteins identified in tick salivary glands and saliva have been partially characterised. Tick-induced host immunosuppression facilitates blood meal acquisition and is an important factor in the transmission/establishment of the tick-borne disease-causing agent, Borrelia burgdorferi. A novel strategy for control of tick-borne pathogens is proposed.     

Tick saliva in anti-tick immunity and pathogen transmission

When feeding on vertebrate host ticks (ectoparasitic arthropods and potential vectors of bacterial, rickettsial, protozoal, and viral diseases) induce both innate and specific acquired host-immune reactions as part of anti-tick defenses. In a resistant host immune defense can lead to reduced tick viability, sometimes resulting in tick death. Tick responds to the host immune attack by secreting saliva containing pharmacologically active molecules and modulating host immune response. Tick saliva-effected immunomodulation at the attachment site facilitates both tick feeding and enhances the success of transmission of pathogens from tick into the host. On the other hand, host immunization with antigens from tick saliva can induce anti-tick resistance and is seen to be able to induce immunity against pathogens transmitted by ticks. Many pharmacological properties of saliva described in ticks are shared widely among other blood-feeding arthropods.     

Probing the functional role of tick metalloproteases

Tick saliva assists feeding through a complex mixture of compounds that disarm the host homeostasis processes, such as platelet aggregation, vasoconstriction and blood clotting, as well as innate and acquired immune responses. Although the various properties of tick salivary glands have sparked great interest as candidate sources for anti‐tick vaccines to prevent tick and tick‐borne diseases, antigens that can be useful to induce an immune response against tick bites or the pathogens transmitted by ticks have not yet been developed. Metalloproteases, which have been found in tick saliva, salivary gland, ovary and midgut, play an important role in inflammation, immunomodulation, fibrinolysis, blood protein digestion, nociception, vitellogenesis, remodelling of extracellular matrix and pathogen transmission. A large proportion of tick metalloproteases belong to the metzincin group, whose members characteristically have a highly conserved zinc‐binding motif integrated into the central α helix at the active site, and a methionine‐containing triad called Met‐turn followed by a cysteine‐rich domain at the C‐terminal site. This review discusses specifically the biological aspects of metalloproteases in tick physiology that have been published to date.     

Ixodes scapularis salivary gland protein P11 facilitates migration of Anaplasma phagocytophilum from the tick gut to salivary glands

Ixodes ticks harbour several human pathogens belonging to the order Rickettsiales, including Anaplasma phagocytophilum, the agent of human anaplasmosis. When ticks feed on A. phagocytophilum-infected mice, the pathogen enters the ticks' gut. The bacteria then migrate from the gut to infect the salivary glands of the ticks and are transmitted to the next host via the saliva. The molecular mechanisms that enable the migration of A. phagocytophilum from the gut to the salivary glands are poorly understood. Here we show that a secreted tick protein, P11, is important in this process. We show that P11 enables A. phagocytophilum to infect tick haemocytes, which are required for the migration of A. phagocytophilum from the gut to the salivary glands. Silencing of p11 impaired the A. phagocytophilum infection of tick haemocytes in vivo and consequently decreased pathogen infection of the salivary glands. In vitro experiments showed that P11 could bind to A. phagocytophilum and thus facilitate its infection of tick cells. This report provides new insights into A. phagocytophilum infection of ticks and reveals new avenues to interrupt the life cycle of Anaplasma and related Rickettsial pathogens.     

New member of the protein disulfide isomerase (PDI) family identified in Amblyomma variegatum tick

Ticks belonging to arthropoda are blood feeding, geographically widespread ectoparasites of mammals, reptiles and birds. Their saliva contains active substances that protect them from host immune attack and allow for transmission of various pathogens during the feeding process. Characterization of tick saliva components can therefore contribute to the development of effective methods for the control of tick-borne diseases.

Here we describe the identification and basic characterization of a gene encoding a 55 kDa protein found in the salivary glands (SG) of Amblyomma variegatum tick. Based on the primary structure and homology to the family of protein disulfide isomerases (PDI; EC 5.3.4.1) the gene was named AvPDI. The 1461 nt long AvPDI open reading frame codes for a 487 amino acid protein. In vitro expressed AvPDI was exclusively localized in the endoplasmic reticulum. RT-PCR and Western blot analysis revealed that AvPDI expression is not restricted to the SG of the tick. More detailed analysis on tissue slides from SG detected an AvPDI specific signal in granular cells of the acini type II and III. Finally, reductase activity of AvPDI was confirmed in an insulin assay. The structural and functional characteristics suggest that AvPDI is another member of the PDI protein family and represents the first more closely characterized PDI in the ticks.     

Tick-host interaction: a synthesis of current concepts

Ixodid ticks are recognized world-wide as major vectors of arboviruses, rickettsiae, spirochaetes and parasitic protozoa of man and domestic animals. Some ticks also inject a debilitating, sometimes fatal, paralytic toxin in their saliva. All these factors are transmitted via the salivary glands and mouthparts of the feeding tick. Tick feeding is a prolonged and complex process. Major developmental events occurring within the tick during feeding, as well as extensive tick-host interactions, all influence the likelihood of pathogen transmission. In this review, Reuben Kaufman discusses the sequence and complexity of these interactions.     

Tick salivary secretion as a source of antihemostatics

Ticks are mostly obligatory blood feeding ectoparasites that have an impact on human and animal health. In addition to direct damage due to feeding, some tick species serve as the vectors for the causative agents of several diseases, such as the spirochetes of the genus Borrelia causing Lyme disease, the virus of tick-borne encephalitis, various Rickettsial pathogens or even protozoan parasites like Babesia spp. Hard ticks are unique among bloodfeeders because of their prolonged feeding period that may last up to two weeks. During such a long period of blood uptake, the host develops a wide range of mechanisms to prevent blood loss. The arthropod ectoparasite, in turn, secretes saliva in the sites of bite that assists blood feeding. Indeed, tick saliva represents a rich source of proteins with potent pharmacologic action that target different mechanisms of coagulation, platelet aggregation and vasoconstriction. Tick adaptation to their vertebrate hosts led to the inclusion of a powerful protein armamentarium in their salivary secretion that has been investigated by high-throughput methods. The resulting knowledge can be exploited for the isolation of novel antihemostatic agents. Here we review the tick salivary antihemostatics and their characterized functions at the molecular and cellular levels.     

Saliva-activated transmission (SAT) of Thogoto virus: dynamics of SAT factor activity in the salivary glands ofRhipicephalus appendiculatus,Amblyomma variegatum,andBoophilus microplus ticks

Thogoto (THO) virus is transmitted from infected to uninfected ticks when co-feeding on uninfected guinea-pigs, even though the guinea-pigs do not develop a detectable viraemia. This form of non-viraemic transmission is potentiated by a factor (s) secreted by the saliva of ticks and hence has been termed saliva-activated transmission (SAT). The synthesis of the SAT factor by the salivary glands of three ixodid tick species was determined by placing uninfected nymphal ticks on guineapigs that were subsequently inoculated with a mixture of THO virus and salivary gland extract (SGE) derived from one of the tick species. SAT factor activity was measured by determining the number of nymphs that acquired THO virus. For the three-host ixodid species,Rhipicephalus appendiculatus andAmblyomma variegatum, maximum enhancement of THO virus transmission was observed when salivary glands were derived from uninfected, female ticks that had fed for a period of 6 or 8 days, respectively. In contrast, when salivary glands were derived form uninfected femaleBoophilus microplus, a one-host ixodid tick species, enhancement of THO virus transmission was observed throughout the tick feeding period. Thus, the natural feeding behaviour of ticks appears to be an important factor in determining the relative importance of these vectors in mediating SAT.     

Salivary glands in ixodid ticks: control and mechanism of secretion

The salivary glands are vital to the biological success of ixodid ticks and the major route for pathogen transmission. Important functions include the absorption of water vapor from unsaturated air by free-living ticks, excretion of excess fluid for blood meal concentration, and the secretion of bioactive protein and lipid compounds during tick feeding. Fluid secretion is controlled by nerves. Dopamine is the neurotransmitter at the neuroeffector junction regulating secretion via adenylate cyclase and an increase in cellular cAMP. Dopamine also affects the release of arachidonic acid which is subsequently converted to prostaglandins. Prostaglandin E(2) (PGE(2)) is secreted at extremely high levels into tick saliva for export to the host where it impacts the host physiology. Additionally, PGE(2) has an autocrine or paracrine role within the salivary gland itself where it interacts with a PGE(2) receptor to induce secretion (exocytosis) of bioactive saliva proteins via a phosphoinositide signalling pathway and an increase in cellular Ca(2+). Regulation of fluid secretion has been extensively studied, but little is known about the mechanism of fluid secretion. Continuing advances in tick salivary gland physiology will be made as key regulatory and secretory gland proteins are purified and/or their genes cloned and sequenced.     

Mx1-Based Resistance to Thogoto Virus in A2G Mice Is Bypassed in Tick-Mediated Virus Delivery

The interferon-induced mouse Mx1 protein has intrinsic antiviral activity against orthomyxoviruses, including Thogoto virus. Thus, Mx1⁺ A2G mice are apparently resistant to infection following needle- or tick-borne virus challenge. However, tick-borne challenge and, to a lesser degree, injection of virus mixed with tick salivary gland extract resulted in virus transmission to uninfected ticks feeding on the A2G mice. The data indicate that immunomodulatory components in tick saliva can overcome a natural antiviral mechanism.