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.
tick; salivary gland; hemostasis; coagulation; platelet aggregation; serine proteases; thrombin; kunitz-domain protein; disintegrin; serpin
Cysteine proteases have been discovered in various bloodfeeding ectoparasites. Here, we assemble the available information about the function of these peptidases and reveal their role in hematophagy and parasite development. While most of the data shed light on key proteolytic events that play a role in arthropod physiology, we also report on the association of cysteine proteases with arthropod vectorial capacity. With emphasis on ticks, specifically Ixodes ricinus, we finally propose a model about the contribution of cysteine peptidases to blood digestion, and how their concerted action with other tick midgut proteases leads to the absorbance of nutrients by the midgut epithelial cells.
activity-based probes; arthropod; aspartic aminopeptidase; Babesia; blood feeding; caspase; cathepsin-B; cathepsin-C; cathepsin-D; cathepsin-L; cysteine proteases; ecdysone; ectoparasite; embryogenesis; fat body; hematophagy; hemoglobin; legumain RNAi; leucine aminopeptidase; lysosome; malaria; metamorphosis; midgut; mosquito; oocyte; Plasmodium; tick; vitellin; yolk protein
Saliva of blood-sucking arthropods contains a complex mixture of peptides that affect their host’s hemostasis, inflammation, and immunity. These activities can also modify the site of pathogen delivery and increase disease transmission. Saliva also induces hosts to mount an antisaliva immune response that can lead to skin allergies or even anaphylaxis. Accordingly, knowledge of the salivary repertoire, or sialome, of a mosquito is useful to provide a knowledge platform to mine for novel pharmacological activities, to develop novel vaccine targets for vector-borne diseases, and to develop epidemiological markers of vector exposure and candidate desensitization vaccines. The mosquito Ochlerotatus triseriatus is a vector of La Crosse virus and produces allergy in humans. In this work, a total of 1,575 clones randomly selected from an adult female O. triseriatus salivary gland cDNA library was sequenced and used to assemble a database that yielded 731 clusters of related sequences, 560 of which were singletons. Primer extension experiments were performed in selected clones to further extend sequence coverage, allowing for the identification of 159 protein sequences, 66 of which code for putative secreted proteins. Supplemental spreadsheets containing these data are available at http://exon.niaid.nih.gov/transcriptome/Ochlerotatus_triseriatus/S1/Ot-S1.xls and http://exon.niaid.nih.gov/transcriptome/Ochlerotatus_triseriatus/S2/Ot-S2.xls.
mosquito; salivary gland; sialome; transcriptome; La Crosse virus
Little is known about the composition and function of the saliva in black flies such as Simulium guianense, the main vector of river blindness disease in Brazil. The complex salivary potion of hematophagous arthropods counteracts their host's hemostasis, inflammation, and immunity.
Transcriptome analysis revealed ubiquitous salivary protein families--such as the Antigen-5, Yellow, Kunitz domain, and serine proteases--in the S. guianense sialotranscriptome. Insect-specific families were also found. About 63.4% of all secreted products revealed protein families found only in Simulium. Additionally, we found a novel peptide similar to kunitoxin with a structure distantly related to serine protease inhibitors. This study revealed a relative increase of transcripts of the SVEP protein family when compared with Simulium vittatum and S. nigrimanum sialotranscriptomes. We were able to extract coding sequences from 164 proteins associated with blood and sugar feeding, the majority of which were confirmed by proteome analysis.
Our results contribute to understanding the role of Simulium saliva in transmission of Onchocerca volvulus and evolution of salivary proteins in black flies. It also consists of a platform for mining novel anti-hemostatic compounds, vaccine candidates against filariasis, and immuno-epidemiologic markers of vector exposure.
The evolution of insects to a blood diet leads to the development of a saliva that antagonizes their hosts' hemostasis and inflammation. Hemostasis and inflammation are redundant processes, and thus a complex salivary potion comprised of dozens or near one hundred different polypeptides is commonly found by transcriptome or proteome analysis of these organisms. Several insect orders or families evolved independently to hematophagy creating unique salivary potions in the form of novel pharmacological use of endogenous substances, and in the form of unique proteins not matching other known proteins, these probably arriving by fast evolution of salivary proteins as they evade their hosts' immune response. In this work we present a preliminary description of the sialome (from the Greek Sialo = saliva) of the common bed bug Cimex lectularius, the first such work from a member of the Cimicidae family. This manuscript is a guide for the supplemental database files http://exon.niaid.nih.gov/transcriptome/C_lectularius/S1/Cimex-S1.zip and http://exon.niaid.nih.gov/transcriptome/C_lectularius/S2/Cimex-S2.xls
Bedbug; saliva; salivary transcriptome; salivary proteome
Aegyptin is a 30-kDa mosquito salivary gland protein that binds to collagen and inhibits platelet aggregation. We have studied biophysical properties of aegyptin and its mechanism of action. Light scattering plot shows that aegyptin displays a monomeric elongated form which explains the apparent molecular mass of 110 kDa estimated by gel-filtration chromatography. Surface plasmon resonance identified the sequence RGQOGVMGF (O is hydroxyproline) that mediates collagen interaction with von Willebrand Factor (vWF) as high-affinity (KD ≈ 10 nM) binding site for aegyptin. Additionally, aegyptin interacts with linear RGQPGVMGF peptide and heat-denatured collagen, implying that the triple-helix and hydroxyproline are not a prerequisite for binding. In contrast, aegyptin does not interact with scrambled RGQPGVMGF peptide. Aegyptin also recognizes with low affinity (µM range) peptides (GPO)10 and GFOGER which respectively represent glycoprotein VI and integrin α2β1 binding sites in collagen, and prevents platelet adhesion and aggregation. Truncated forms of aegyptin were engineered, and the C-terminus fragment was shown to interact with collagen and to attenuate platelet aggregation. In addition, aegyptin prevents laser-induced carotid thrombus formation in the presence of Rose Bengal in vivo, without observable bleeding in rats. In conclusion, aegyptin interacts with distinct binding sites in collagen, and is useful tool to inhibit platelet-collagen interaction in vitro and in vivo.
aegyptin; blood-sucking; hematophagy; mosquitoes; yellow fever; GPVI
A salivary protein from a malaria-transmitting mosquito uses a single domain to bind to thromboxane A2 and cysteinyl leukotrienes and prevent blood clotting and inflammation in the host on which it feeds.
The highly expressed D7 protein family of mosquito saliva has previously been shown to act as an anti-inflammatory mediator by binding host biogenic amines and cysteinyl leukotrienes (CysLTs). In this study we demonstrate that AnSt-D7L1, a two-domain member of this group from Anopheles stephensi, retains the CysLT binding function seen in the homolog AeD7 from Aedes aegypti but has lost the ability to bind biogenic amines. Unlike any previously characterized members of the D7 family, AnSt-D7L1 has acquired the important function of binding thromboxane A2 (TXA2) and its analogs with high affinity. When administered to tissue preparations, AnSt-D7L1 abrogated Leukotriene C4 (LTC4)-induced contraction of guinea pig ileum and contraction of rat aorta by the TXA2 analog U46619. The protein also inhibited platelet aggregation induced by both collagen and U46619 when administered to stirred platelets. The crystal structure of AnSt-D7L1 contains two OBP-like domains and has a structure similar to AeD7. In AnSt-D7L1, the binding pocket of the C-terminal domain has been rearranged relative to AeD7, making the protein unable to bind biogenic amines. Structures of the ligand complexes show that CysLTs and TXA2 analogs both bind in the same hydrophobic pocket of the N-terminal domain. The TXA2 analog U46619 is stabilized by hydrogen bonding interactions of the ω-5 hydroxyl group with the phenolic hydroxyl group of Tyr 52. LTC4 and occupies a very similar position to LTE4 in the previously determined structure of its complex with AeD7. As yet, it is not known what, if any, new function has been acquired by the rearranged C-terminal domain. This article presents, to our knowledge, the first structural characterization of a protein from mosquito saliva that inhibits collagen mediated platelet activation.
When feeding, a female mosquito must inhibit the blood clotting and inflammatory responses of the host. To do this, the insect produces salivary proteins that neutralize key host molecules participating in clotting and inflammation. Here, we describe a salivary protein AnSt-D7L1 that scavenges both thomboxane A2 and cysteinyl leukotrienes, two substances involved in blood vessel constriction, platelet aggregation, and inflammatory responses to an insect bite. We produced this protein in bacteria and showed that it tightly binds both these molecules, inhibiting the processes in which they are involved. We then determined its structure using X-ray crystallography and showed that there is a single binding site in one domain of the protein, accommodating both thromboxane A2 and cysteinyl leukotrienes, and that this site is responsible for the scavenging effect of the protein. These studies reveal the structural features of proteins needed to bind to key molecules of potential pharmacological importance and add to our understanding of the process of mosquito blood feeding, which is essential for transmission of the malaria parasite.
Blood-sucking arthropods have evolved a number of inhibitors of platelet aggregation and blood coagulation. In this report we have molecularly and functionally characterized aegyptin, a member of the family of 30-kDa salivary allergens from Aedes aegypti, whose function remained elusive thus far. Aegyptin displays a unique sequence characterized by glycine, glutamic acid, and aspartic acid repeats and was shown to specifically block collagen-induced human platelet aggregation and granule secretion. Plasmon resonance experiments demonstrate that aegyptin binds to collagen types I-V (Kd ≈ 1 nM) but does not interact with vitronectin, fibronectin, laminin, fibrinogen, and von Willebrand factor (vWf). In addition, aegyptin attenuates platelet adhesion to soluble or fibrillar collagen. Furthermore, aegyptin inhibits vWf interaction with collagen type III under static conditions and completely blocks platelet adhesion to collagen under flow conditions at high shear rates. Notably, aegyptin completely prevents collagen but not convulxin binding to recombinant glycoprotein VI. These findings indicate that aegyptin recognizes specific binding sites for glycoprotein VI, integrin α2β1, and vWf, thereby preventing collagen interaction with its three major ligands. Aegyptin is a novel tool to study collagen-platelet interaction and a prototype for development of molecules with antithrombotic properties.
Saliva of adult female mosquitoes help sugar and blood feeding by providing enzymes and polypeptides that help sugar digestion, control microbial growth and counteract their vertebrate host hemostasis and inflammation. Mosquito saliva also potentiates the transmission of vector borne pathogens, including arboviruses. Culex tarsalis is a bird feeding mosquito vector of West Nile Virus closely related to C. quinquefasciatus, a mosquito relatively recently adapted to feed on humans, and the only mosquito of the genus Culex to have its sialotranscriptome so far described.
A total of 1,753 clones randomly selected from an adult female C. tarsalis salivary glands (SG) cDNA library were sequenced and used to assemble a database that yielded 809 clusters of related sequences, 675 of which were singletons. Primer extension experiments were performed in selected clones to further extend sequence coverage, allowing for the identification of 283 protein sequences, 80 of which code for putative secreted proteins.
Comparison of the C. tarsalis sialotranscriptome with that of C. quinquefasciatus reveals accelerated evolution of salivary proteins as compared to housekeeping proteins. The average amino acid identity among salivary proteins is 70.1%, while that for housekeeping proteins is 91.2% (P < 0.05), and the codon volatility of secreted proteins is significantly higher than those of housekeeping proteins. Several protein families previously found exclusive of mosquitoes, including only in the Aedes genus have been identified in C. tarsalis. Interestingly, a protein family so far unique to C. quinquefasciatus, with 30 genes, is also found in C. tarsalis, indicating it was not a specific C. quinquefasciatus acquisition in its evolution to optimize mammal blood feeding.
Ixodes scapularis ticks transmit the Lyme disease agent in the US. Although strong anti-tick immunity mediates tick rejection by certain vertebrates, only a few antigens have been molecularly characterized. We show that guinea pig vaccination against a secreted tick salivary immunomodulator—sialostatin L2—can lead to decreased feeding ability of I. scapularis nymphs. Increased rejection rate, prolonged feeding time and apparent signs of inflammation were observed for nymphs attached to vaccinated animals, indicating a protective host immune response. Interestingly, sialostatin L2 humoral recognition does not take place upon repeated tick exposure in control animals, but only in the vaccinated animals that neutralize sialostatin L2 action. Therefore, we demonstrate an essential sialostatin L2 role upon nymphal infestation that can be blocked by vertebrate immunity and we propose the discovery of similarly ‘silent’ antigens towards the development of a multicomponent vaccine that will protect against tick bites and the pathogens they transmit.
‘silent’ antigens; immunity; protection; vaccine; vector
Mosquito saliva, consisting of a mixture of dozens of proteins affecting vertebrate hemostasis and having sugar digestive and antimicrobial properties, helps both blood and sugar meal feeding. Culicine and anopheline mosquitoes diverged ~150 MYA, and within the anophelines, the New World species diverged from those of the Old World ~95 MYA. While the sialotranscriptome (from the Greek sialo, saliva) of several species of the Cellia subgenus of Anopheles has been described thoroughly, no detailed analysis of any New World anopheline has been done to date. Here we present and analyze data from a comprehensive salivary gland (SG) transcriptome of the neotropical malaria vector Anopheles darlingi (subgenus Nyssorhynchus).
A total of 2,371 clones randomly selected from an adult female An. darlingi SG cDNA library were sequenced and used to assemble a database that yielded 966 clusters of related sequences, 739 of which were singletons. Primer extension experiments were performed in selected clones to further extend sequence coverage, allowing for the identification of 183 protein sequences, 114 of which code for putative secreted proteins.
Comparative analysis of sialotranscriptomes of An. darlingi and An. gambiae reveals significant divergence of salivary proteins. On average, salivary proteins are only 53% identical, while housekeeping proteins are 86% identical between the two species. Furthermore, An. darlingi proteins were found that match culicine but not anopheline proteins, indicating loss or rapid evolution of these proteins in the old world Cellia subgenus. On the other hand, several well represented salivary protein families in old world anophelines are not expressed in An. darlingi.
Anopheles funestus, together with Anopheles gambiae, is responsible for most malaria transmission in sub-Saharan Africa, but little is known about molecular aspects of its biology. To investigate the salivary repertoire of this mosquito, we randomly sequenced 916 clones from a salivary-gland cDNA library from adult female F1 offspring of field-caught An. funestus. Thirty-three protein sequences, mostly full-length transcripts, are predicted to be secreted salivary proteins. We additionally describe 25 full-length housekeeping-associated transcripts. In accumulating mosquito sialotranscriptome information—which includes An. gambiae, Anopheles stephensi, Anopheles darlingi, Aedes aegypti, Aedes albopictus, Culex pipiens quinquefasciatus, and now An. funestus—a pattern is emerging. First, ubiquitous protein families are recruited for a salivary role, such as members of the antigen-5 family and enzymes of nucleotide and carbohydrate catabolism. Second, a group of protein families exclusive to blood-feeding Nematocera includes the abundantly expressed D7 proteins also found in sand flies and Culicoides. A third group of proteins, only found in Culicidae, includes the 30-kDa allergen family and several mucins. Finally, ten protein and peptide families, five of them multigenic, are exclusive to anophelines. Among these proteins may reside good epidemiological markers to measure human exposure to anopheline species such as An. funestus and An. gambiae.
Malaria; Hematophagy; Salivary glands; Vector; Saliva
Saliva of blood-sucking arthropods contains a cocktail of antihemostatic agents and immunomodulators that help blood feeding. Mosquitoes additionally feed on sugar meals and have specialized regions of their glands containing glycosidases and antimicrobials that might help control bacterial growth in the ingested meals. To expand our knowledge on the salivary cocktail of Ædes ægypti, a vector of dengue and yellow fevers, we analyzed a set of 4,232 expressed sequence tags from cDNA libraries of adult female mosquitoes.
A nonredundant catalogue of 614 transcripts (573 of which are novel) is described, including 136 coding for proteins of a putative secretory nature. Additionally, a two-dimensional gel electrophoresis of salivary gland (SG) homogenates followed by tryptic digestion of selected protein bands and MS/MS analysis revealed the expression of 24 proteins. Analysis of tissue-specific transcription of a subset of these genes revealed at least 31 genes whose expression is specific or enriched in female SG, whereas 24 additional genes were expressed in female SG and in males but not in other female tissues. Most of the 55 proteins coded by these SG transcripts have no known function and represent high-priority candidates for expression and functional analysis as antihemostatic or antimicrobial agents. An unexpected finding is the occurrence of four protein families specific to SG that were probably a product of horizontal transfer from prokaryotic organisms to mosquitoes.
Overall, this paper contributes to the novel identification of 573 new transcripts, or near 3% of the Æ. ægypti proteome assuming a 20,000-protein set, and to the best-described sialome of any blood-feeding insect.
Aedes (Stegomyia) aegypti is an important dengue vector in tropical and subtropical zones throughout the world. A transcriptome of Ae. aegypti vitellogenic fat bodies is described here. The fat body is a dynamic tissue that participates in multiple biochemical functions of intermediate metabolism. A total of 589 randomly selected cDNAs were assembled into 262 clusters based on their primary sequence similarities. The putative translated proteins were classified into categories based on their function in accordance with significant similarity using the BlastX at NCBI FTP site and Pfam (Bateman et al. 2000) and SMART (Schultz et al. 2000) databases. The characterization of transcripts expressed in the fat body of Ae. aegypti at 24 hours post blood meal provides a basic tool for understanding the processes occurring in this organ and could identify putative new genes whose promoters can be used to specifically express transgenes in the fat bodies of Ae. aegypti.