Toward an Understanding of the Molecular Mechanism for Successful Blood Feeding by Coupling Proteomics Analysis with Pharmacological Testing of Horsefly Salivary Glands

Horseflies are economically important blood-feeding arthropods and also a nuisance for humans and vectors for filariasis. They rely heavily on the pharmacological properties of their saliva to get a blood meal and suppress immune reactions of hosts. Little information is available on antihemostatic substances in horsefly salivary glands; especially no horsefly immune suppressants have been reported. By proteomics or peptidomics and coupling transcriptome analysis with pharmacological testing, several families of proteins or peptides, which act mainly on the hemostatic system or immune system of the host, were identified and characterized from 30,000 pairs salivary glands of the horsefly Tabanus yao (Diptera, Tabanidae). They are: (i) a novel family of inhibitors of platelet aggregation including two members, which possibly inhibit platelet aggregation by a novel mechanism and act on platelet membrane, (ii) a novel family of immunosuppressant peptides including 12 members, which can inhibit interferon-γ production and increase interleukin-10 secretion, (iii) a serine protease inhibitor with 56 amino acid residues containing anticoagulant activity, (iv) a serine protease with anticoagulant activity, (v) a protease with fibrinogenolytic activity, (vi) three families of antimicrobial peptides including six members, (vii) a hyaluronidase, (viii) a vasodilator peptide, which is an isoform of vasotab identified from Hybomitra bimaculata, and interestingly (ix) two metallothioneins, which are the first metallothioneins reported from invertebrate salivary glands. The current work will facilitate the understanding of the molecular mechanisms of the ectoparasite-host relationship and help in identifying novel vaccine targets and novel leading pharmacological compounds. Horseflies are economically important blood-feeding arthropods and also a nuisance for humans and vectors for filariasis. They rely heavily on the pharmacological properties of their saliva to get a blood meal and suppress immune reactions of hosts. Little information is available on antihemostatic substances in horsefly salivary glands; especially no horsefly immune suppressants have been reported. By proteomics or peptidomics and coupling transcriptome analysis with pharmacological testing, several families of proteins or peptides, which act mainly on the hemostatic system or immune system of the host, were identified and characterized from 30,000 pairs salivary glands of the horsefly Tabanus yao (Diptera, Tabanidae). They are: (i) a novel family of inhibitors of platelet aggregation including two members, which possibly inhibit platelet aggregation by a novel mechanism and act on platelet membrane, (ii) a novel family of immunosuppressant peptides including 12 members, which can inhibit interferon-γ production and increase interleukin-10 secretion, (iii) a serine protease inhibitor with 56 amino acid residues containing anticoagulant activity, (iv) a serine protease with anticoagulant activity, (v) a protease with fibrinogenolytic activity, (vi) three families of antimicrobial peptides including six members, (vii) a hyaluronidase, (viii) a vasodilator peptide, which is an isoform of vasotab identified from Hybomitra bimaculata, and interestingly (ix) two metallothioneins, which are the first metallothioneins reported from invertebrate salivary glands. The current work will facilitate the understanding of the molecular mechanisms of the ectoparasite-host relationship and help in identifying novel vaccine targets and novel leading pharmacological compounds. Hematophagous arthropods have developed effective mechanisms to get a blood meal and overcome their host's immune responses. Such arthropods produce a wide array of antihemostatic and immune suppressant compounds in their salivary glands (1Ribeiro J.M. Blood-feeding arthropods: live syringes or invertebrate pharmacologists?.Infect. Agents Dis. 1995; 4: 143-152PubMed Google Scholar, 2Charlab R. Valenzuela J.G. Rowton E.D. Ribeiro J.M. Toward an understanding of the biochemical and pharmacological complexity of the saliva of a hematophagous sand fly Lutzomyia longipalpis.Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 15155-15160Crossref PubMed Scopus (214) Google Scholar, 3Takác P. Nunn M.A. Mészáros J. Pechánová O. Vrbjar N. Vlasáková P. Kozánek M. Kazimírová M. Hart G. Nuttall P.A. Labuda M. Vasotab, a vasoactive peptide from horse fly Hybomitra bimaculata (Diptera, Tabanidae) salivary glands.J. Exp. Biol. 2006; 209: 343-352Crossref PubMed Scopus (46) Google Scholar, 4Ribeiro J.M.C. Characterization of a vasodilator from the salivary glands of the yellow fever mosquito Aedes aegypti.J. Exp. Biol. 1992; 165: 61-71Crossref PubMed Google Scholar, 5Arca B. Lombardo F. de Lara Capurro M. della Torre A. Dimopoulos G. James A.A. Coluzzi M. Trapping cDNAs encoding secreted proteins from the salivary glands of the malaria vector Anopheles gambiae.Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1516-1521Crossref PubMed Scopus (130) Google Scholar). Antihemostatic compounds of blood-sucking arthropods have been distinguished into several groups such as inhibitors of coagulation factors (Factors VII, V, and Xa and thrombin), platelet functions, and fibrinolytic enzymes. Immunosuppressive factors from ticks have been extensively studied (6Arocha-Piñango C.L. Marchi R. Carvajal Z. Guerrero B Invertebrate compounds acting on the hemostatic mechanism.Blood Coagul. Fibrinolysis. 1999; 10: 43-68Crossref PubMed Scopus (45) Google Scholar, 7Markwardt F. Coagulation inhibitors from animals feeding on blood.Rev. Iberoam. Thromb. Hemost. 1994; 7: 225-231Google Scholar, 8Kazimírová M. Sulanová M. Kozánek M. Takác P. Labuda M. Nuttall P.A. Identification of anticoagulant activities in salivary gland extracts of four horsefly species (Diptera, tabanidae).Haemostasis. 2001; 31: 294-305PubMed Google Scholar). Tick salivary gland extract (SGE) 1The abbreviations used are: SGE, salivary gland extract; LPS, lipopolysaccharide; RP, reverse phase; pNA, p-nitroanilide; IL-10, interleukin-10; IFN-γ, interferon-γ; aa, amino acid(s); GP, glycoprotein; BDS, base-deactivated silica. can suppress innate and adaptive immunity. It has been reported that tick SGE inhibits host complement activation and depresses macrophage function by inhibiting lipopolysaccharide (LPS)-induced nitric-oxide synthesis and proinflammatory cytokine production (9Wikel S.K. The Immunology of Host-Ectoparasitic Arthropod Relationships. CAB International, Wallingford, UK1996Google Scholar, 10Ribeiro J.M.C. Ixodes dammini: salivary anti-complement activity.Exp. Parasitol. 1987; 64: 347-353Crossref PubMed Scopus (123) Google Scholar, 11Ribeiro J.M.C. Spielman A. Ixodes dammini: salivary anaphylatoxin inactivating activity.Exp. Parasitol. 1986; 62: 292-297Crossref PubMed Scopus (78) Google Scholar, 12Ribeiro J.M.C. Weiss J.J. Telford III, S.R. Saliva of the tick Ixodes dammini inhibits neutrophil function.Exp. Parasitol. 1990; 70: 382-388Crossref PubMed Scopus (147) Google Scholar, 13Urioste S. Hall L.E. Telford >III, S.R. Titus R. Saliva of the Lyme disease vector, Ixodes dammini, blocks cell activation by a nonprostaglandin E2-dependent mechanism.J. Exp. Med. 1994; 180: 1077-1085Crossref PubMed Scopus (127) Google Scholar, 14Kopecky J. Kuthejlová M. Suppressive effect of Ixodes ricinus salivary gland extract on mechanisms of natural immunity in vitro.Parasite Immunol. (Oxf.). 1998; 20: 169-174PubMed Google Scholar, 15Ferreira B.R. Silva J.S. Successive tick infestations selectively promote a T-helper 2 cytokine profile in mice.Immunology. 1999; 96: 434-439Crossref PubMed Scopus (115) Google Scholar, 16Kopecky J. Kuthejlová M. Pechová J. Salivary gland extract from Ixodes ricinus ticks inhibits production of interferon-γ by the upregulation of interleukin-10.Parasite Immunol. (Oxf.). 1999; 21: 351-356Crossref PubMed Scopus (64) Google Scholar, 17Koník P. Slavíková V. Salát J. Reznícková J. Dvoroznáková E. Kopecký J. Anti-tumour necrosis factor-α activity in Ixodes ricinus saliva.Parasite Immunol. (Oxf.). 2006; 28: 649-656Crossref PubMed Scopus (19) Google Scholar). As an important hematophagous arthropod, there have been comparatively few studies on antihemostatic substances in horsefly salivary glands, although anticoagulant activity has been identified. Only a vasoactive peptide (vasotab) was reported from Hybomitra bimaculata (Diptera, Tabanidae) salivary glands (3Takác P. Nunn M.A. Mészáros J. Pechánová O. Vrbjar N. Vlasáková P. Kozánek M. Kazimírová M. Hart G. Nuttall P.A. Labuda M. Vasotab, a vasoactive peptide from horse fly Hybomitra bimaculata (Diptera, Tabanidae) salivary glands.J. Exp. Biol. 2006; 209: 343-352Crossref PubMed Scopus (46) Google Scholar). No immunosuppressive substances have been described before in horsefly salivary glands. Female horseflies require substantial amounts of blood (up to 0.5 ml) for egg production. They can ingest up to 200 mg of blood within only 1–3 min, suggesting that they must possess very potent antihemostatic mechanisms (3Takác P. Nunn M.A. Mészáros J. Pechánová O. Vrbjar N. Vlasáková P. Kozánek M. Kazimírová M. Hart G. Nuttall P.A. Labuda M. Vasotab, a vasoactive peptide from horse fly Hybomitra bimaculata (Diptera, Tabanidae) salivary glands.J. Exp. Biol. 2006; 209: 343-352Crossref PubMed Scopus (46) Google Scholar, 18Hollander A.L. Wright R.E. Impact of tabanids on cattle: blood meal size and preferred feeding sites.J. Econ. Entomol. 1980; 73: 431-433Crossref PubMed Scopus (26) Google Scholar). More than one feeding episode is needed for horseflies, and approximately 10 landings on a host are necessary to complete one blood meal, suggesting that they must possess very potent immunosuppressive mechanisms. To identify and characterize interesting salivary compounds for understanding the molecular mechanisms of the ectoparasite-host relationship and to help in identifying novel vaccine targets, we used proteomics or peptidomics and transcriptome analysis coupled with pharmacological testing of the activities to investigate pharmacological molecules in the salivary glands of the horsefly T. yao Macquart. T. yao Macquart horseflies (about 30,000; average weight, 0.17 g) were collected in Shanxi Province of China in July 2004. Collections were performed between 17:00 and 20:00 during optimal weather (sunny, 30–35 °C, and no wind). All the flies were transported to the laboratory alive and kept at −80 °C. Horseflies were glued to the bottom of a Petri dish and placed on ice. They were then dissected under a microscope. The salivary gland was excised and transferred into 0.1 m phosphate buffer solution, pH 6.0, and kept in the same solution at −80 °C. 30,000 pairs of horsefly salivary glands were homogenized in 0.1 m phosphate buffer solution, pH 6.0, and centrifuged at 5000 × g for 10 min. The supernatant was termed SGE and lyophilized. The lyophilized SGE sample (2.1 g, total A280 nm of 600) was dissolved in 10 ml of 0.1 m phosphate buffer solution, pH 6.0, and then was applied to a Sephadex G-75 (Superfine, Amersham Biosciences, 2.6 × 100-cm) gel filtration column equilibrated with 0.1 m phosphate buffer, pH 6.0. Elution was performed with the same buffer, of The of the was at nm was to pharmacological testing including of platelet of serine protease activity on on blood of cytokine secretion, fibrinogenolytic activity, activity, and activity as under The containing pharmacological activities were and by or reverse or × column as in The amino acid of the and amino acid from the were by on an The were placed on a were a in and The were as The was the of a was and as The of was within was performed under were a were a The was by a with as a was from pairs of horsefly salivary glands of T. yao Macquart. was by by a synthesis The first was by or or and The was by was with a the of the a of × for of was used for and the with The in the as in to the peptide by and as under and in the were used in The was from The 2 at by of 10 at °C, at °C, and at °C. was performed on an were performed to the described by R. Valenzuela J.G. Rowton E.D. Ribeiro J.M. Toward an understanding of the biochemical and pharmacological complexity of the saliva of a hematophagous sand fly Lutzomyia longipalpis.Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 15155-15160Crossref PubMed Scopus (214) Google Scholar). protease and fibrinogenolytic activity were to the described by A. novel from and molecular Biol. 1995; PubMed Scopus Google Scholar). and were to J. R. J. peptidomics of PubMed Scopus Google Scholar). The and were from activities of platelet aggregation were to the described J. Salivary gland extracts from the a potent inhibitor of platelet Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). aggregation was by in an with at °C. The on the of were to the reported by Takác (3Takác P. Nunn M.A. Mészáros J. Pechánová O. Vrbjar N. Vlasáková P. Kozánek M. Kazimírová M. Hart G. Nuttall P.A. Labuda M. Vasotab, a vasoactive peptide from horse fly Hybomitra bimaculata (Diptera, Tabanidae) salivary glands.J. Exp. Biol. 2006; 209: 343-352Crossref PubMed Scopus (46) Google Scholar). were as reported in 16Kopecky J. Kuthejlová M. Pechová J. Salivary gland extract from Ixodes ricinus ticks inhibits production of interferon-γ by the upregulation of interleukin-10.Parasite Immunol. (Oxf.). 1999; 21: 351-356Crossref PubMed Scopus (64) Google and 17Koník P. Slavíková V. Salát J. Reznícková J. Dvoroznáková E. Kopecký J. Anti-tumour necrosis factor-α activity in Ixodes ricinus saliva.Parasite Immunol. (Oxf.). 2006; 28: 649-656Crossref PubMed Scopus (19) Google All the were by of of The for pharmacological testing can in the All of the peptides used for the in were by a peptide at and by and to that the was than All peptides were dissolved in As in the supernatant of the horsefly salivary gland extract was into six Sephadex G-75 gel inhibit platelet aggregation and and for serine also inhibit platelet and inhibit inhibit and the activity of on interferon-γ secretion, and the of and activity, and we J. J. and R. from Sephadex G-75 gel filtration was to Amersham as in The at as in inhibit platelet in was by reverse column as in and the at as in the to inhibit platelet The inhibitor was at in and was to a with a molecular of by at in for serine and and was by column as in the at as in the to the and The serine protease was in was applied to a column as in and and platelet aggregation inhibitor activity, and were to as in and The as in was platelet inhibitor as in and an antimicrobial as in were from the of in was by as in More than were molecules were They are as in and 12 as and in as in as in and vasotab as in and are antimicrobial and 12 increase interleukin-10 production and interferon-γ is a serine protease is a vasodilator platelet aggregation and were from T. yao Macquart amino acid of the and amino acid from the are in on the amino acid of the were to the encoding and The complete encoding and 2 are and are and They two of amino acid residues including peptides and containing the family in and 2 10 that possibly and 2 with Aedes containing 12 is an at the of 2 and an at the of the of with platelet aggregation inhibitors such as novel of and platelet aggregation inhibitor from the tick Biol. 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PubMed Scopus Google Scholar). and have an the on the They can with for to and inhibit platelet aggregation by such as and and 2 have an and possibly act as an of the in to inhibit platelet aggregation as platelet aggregation inhibitors As a blood-feeding arthropod, is effective and that the horsefly a to inhibit the for platelet is on is to is also interesting to investigate the of the in the as as the of the of the to platelet or immunosuppressant peptides, and were from T. yao Macquart amino acid are and They are to the secreted peptide from bimaculata were from the of T. yao Macquart salivary glands as in are of amino acid residues including a peptide by an peptide and the peptides containing or amino acid residues at the is a for between peptide and for the have peptide the of and 12 on and interferon-γ by in As in with or 12 in increase the of the of was in a or only a effect on the two of in with the All the that in horsefly salivary glands act as an immunosuppressant and inhibit the host's by of proinflammatory is a cytokine of and cytokine responses. can inhibit the of proinflammatory for J. Kuthejlová M. Pechová J. Salivary gland extract from Ixodes ricinus ticks inhibits production of interferon-γ by the upregulation of interleukin-10.Parasite Immunol. (Oxf.). 1999; 21: 351-356Crossref PubMed Scopus (64) Google Scholar, 17Koník P. Slavíková V. Salát J. Reznícková J. Dvoroznáková E. Kopecký J. Anti-tumour necrosis factor-α activity in Ixodes ricinus saliva.Parasite Immunol. (Oxf.). 2006; 28: 649-656Crossref PubMed Scopus (19) Google Scholar, S. J. J.M. Ixodes ricinus tick salivary gland extract inhibits and by and in B Immunol. (Oxf.). PubMed Scopus Google Scholar). possibly the production to inhibit in the current It has been reported that horseflies needed approximately 10 landings on a host to a complete meal M. Sulanová M. Kozánek M. Takác P. Labuda M. Nuttall P.A. Identification of anticoagulant activities in salivary gland extracts of four horsefly species (Diptera, tabanidae).Haemostasis. 2001; 31: 294-305PubMed Google Scholar, 18Hollander A.L. Wright R.E. Impact of tabanids on cattle: blood meal size and preferred feeding sites.J. Econ. Entomol. 1980; 73: 431-433Crossref PubMed Scopus (26) Google Scholar). The landings on a host an for innate and adaptive immune to the horseflies have developed a of to overcome the host immune responses. As in peptides in horsefly salivary glands can suppress the proinflammatory cytokine by facilitate the blood feeding and the of in the blood-feeding arthropods such as ticks a to overcome the host's immune responses. As described in the first of and three antimicrobial peptides or proteins were from the horsefly They are and The cDNAs encoding three two and and a were from the of T. yao Macquart salivary glands as in also that has a of which is from antimicrobial antimicrobial peptides are and antimicrobial activities the as in They are the first antimicrobial factors from horsefly salivary glands. animals and antimicrobial peptides to host by Horseflies have to of their feeding The antimicrobial peptides in horsefly salivary glands can the blood meal and inhibit of horseflies have developed antimicrobial factors to their from during blood serine protease inhibitor was from the horsefly The encoding was also from the salivary gland of T. yao Macquart. is amino acid residues including a peptide of amino acid residues and of 56 amino acid residues The has a molecular of and six and a serine protease inhibitors T. E. P. S.R. gland peptide with protease activity in Biol. PubMed Google Scholar, S. M. peptides with a activity the inactivating Biol. 1998; PubMed Scopus Google Scholar, and of a peptide from the 1987; Scopus Google Scholar, R. J. inhibitor from the PubMed Scopus (64) Google Scholar, J. V. R. M. A. N. J. G. M. and of inhibitors from PubMed Scopus Google Scholar, M.A. The of a serine protease inhibitor from in the of fly and Parasitol. PubMed Scopus Google Scholar, Valenzuela J.G. Ribeiro J.M. a novel inhibitor from the salivary gland of the Ixodes of and Xa as for the of PubMed Scopus Google Scholar, A. E. R. M. M. A. protease inhibitor is in salivary glands and into the during Biol. PubMed Scopus Google Scholar). The is to serine protease inhibitors and from the S. M. peptides with a activity the inactivating Biol. 1998; PubMed Scopus Google than serine protease It also with serine protease inhibitors such as tick R. J. inhibitor from the PubMed Scopus (64) Google and Valenzuela J.G. 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Vasotab, a vasoactive peptide from horse fly Hybomitra bimaculata (Diptera, Tabanidae) salivary glands.J. Exp. Biol. 2006; 209: 343-352Crossref PubMed Scopus (46) Google Scholar). The peptides have only one amino acid and the peptides have only four amino acid suggesting that family of peptides is in of has been reported to inhibit of by As in vasotab the same function as aggregation and are hemostatic in As by Takác (3Takác P. Nunn M.A. Mészáros J. Pechánová O. Vrbjar N. Vlasáková P. Kozánek M. Kazimírová M. Hart G. Nuttall P.A. Labuda M. Vasotab, a vasoactive peptide from horse fly Hybomitra bimaculata (Diptera, Tabanidae) salivary glands.J. Exp. Biol. 2006; 209: 343-352Crossref PubMed Scopus (46) Google horsefly in the antihemostatic during blood serine protease was from the SGE of T. yao Macquart and molecular is as by The encoding was also from the of T. yao Macquart salivary glands. The is of amino acid residues It of with serine such as the serine and from A. and inhibit blood coagulation in a as in The of the of in a of the was than with the It has been reported that and horsefly have anticoagulant and serine protease activities M. Sulanová M. Kozánek M. Takác P. Labuda M. Nuttall P.A. Identification of anticoagulant activities in salivary gland extracts of four horsefly species (Diptera, tabanidae).Haemostasis. 2001; 31: 294-305PubMed Google Scholar). the we characterized the anticoagulant serine protease from the horsefly of T. It is anticoagulant It is that factors to inhibit blood coagulation as serine from protease with an molecular of and a with an molecular of were from the horsefly SGE as in B and They were to amino acid their were to their amino acid By transcriptome a encoding a with amino acid residues was from the of T. yao Macquart salivary glands. The in a as in are in and arthropods P. P. Salivary gland in species of sand flies Biol. PubMed Scopus Google Scholar). They can facilitate the of compounds by of the the by G. of Sci. 1995; 4: PubMed Scopus Google blood-sucking the activity was also in the tick and amino in the salivary and of the tick J. Google and the fly J.M.C. R. Rowton E.D. and Lutzomyia salivary gland Med. Entomol. PubMed Scopus Google Scholar). The activity is to an important in blood meal by the of host for pharmacological compounds in from T. yao SGE as in for with 2 of in of was by The of suggesting that was by also that on the of an fibrinogenolytic with a molecular of was from the of amino acid is R. a novel from PubMed Scopus Google Scholar). and fibrinogenolytic activity was also with tick salivary in Ixodes Ribeiro J.M. of a salivary gland and of and activities in the saliva of the Lyme disease tick vector Ixodes PubMed Scopus Google Scholar). with effective fibrinogenolytic activity will possibly to inhibit coagulation and facilitate blood meal effect on to metallothioneins with an amino acid of and with an amino acid of were identified from the of T. yao Macquart salivary glands and have been reported to in salivary glands Ribeiro J.M. of a salivary gland and of and activities in the saliva of the Lyme disease tick vector Ixodes PubMed Scopus Google Scholar). and are the first reported metallothioneins identified from invertebrate salivary glands. are proteins that have been as factors for and from and F. of in the salivary Google Scholar). work is needed to the that metallothioneins in the horsefly salivary glands. the pharmacological molecular profile of the horsefly salivary glands is that the current was with the pharmacological molecular profile acting on and responses. the current platelet aggregation inhibitors serine protease fibrinogenolytic and were identified to the molecular array for antihemostatic responses. of were and and their cDNAs were families of antimicrobial peptides which act as innate antimicrobial were and family of peptides of which act as were and metallothioneins were also in the salivary glands of the increase of the salivary gland function in the will a understanding of the molecular between horseflies and their and at the same will to the of novel compounds and immunity. are to Ribeiro for and help for the with