BACKGROUND: Phlebotomus tobbi is a vector of Leishmania infantum, and P. sergenti is a vector of Leishmania tropica. Le. infantum and Le. tropica typically cause visceral or cutaneous leishmaniasis, respectively, but Le. infantum strains transmitted by P. tobbi can cause cutaneous disease. To better understand the components and possible implications of sand fly saliva in leishmaniasis, the transcriptomes of the salivary glands (SGs) of these two sand fly species were sequenced, characterized and compared. METHODOLOGY/PRINCIPAL FINDINGS: cDNA libraries of P. tobbi and P. sergenti female SGs were constructed, sequenced, and analyzed. Clones (1,152) were randomly picked from each library, producing 1,142 high-quality sequences from P. tobbi and 1,090 from P. sergenti. The most abundant, secreted putative proteins were categorized as antigen 5-related proteins, apyrases, hyaluronidases, D7-related and PpSP15-like proteins, ParSP25-like proteins, PpSP32-like proteins, yellow-related proteins, the 33-kDa salivary proteins, and the 41.9-kDa superfamily of proteins. Phylogenetic analyses and multiple sequence alignments of putative proteins were used to elucidate molecular evolution and describe conserved domains, active sites, and catalytic residues. Proteomic analyses of P. tobbi and P. sergenti SGs were used to confirm the identification of 35 full-length sequences (18 in P. tobbi and 17 in P. sergenti). To bridge transcriptomics with biology P. tobbi antigens, glycoproteins, and hyaluronidase activity was characterized. CONCLUSIONS: This analysis of P. sergenti is the first description of the subgenus Paraphlebotomus salivary components. The investigation of the subgenus Larroussius sand fly P. tobbi expands the repertoire of salivary proteins in vectors of Le. infantum. Although P. tobbi transmits a cutaneous form of leishmaniasis, its salivary proteins are most similar to other Larroussius subgenus species transmitting visceral leishmaniasis. These transcriptomic and proteomic analyses provide a better understanding of sand fly salivary proteins across species and subgenera that will be vital in vector-pathogen and vector-host research.

Department of Parasitology Faculty of Science Charles University Prague Prague Czech Republic

Erratum v

PLoS Negl Trop Dis. 2012 Jun;6(6). doi:10.1371/annotation/fb586825-2ad7-41a5-b7d6-aef0e65c5887 PubMed

Zobrazit více v PubMed

Titus RG, Ribeiro JMC. Salivary-Gland Lysates from the Sand Fly Lutzomyia-Longipalpis Enhance Leishmania Infectivity. Science. 1988;239:1306–1308. PubMed

Rohousova I, Volf P. Sand fly saliva: effects on host immune response and Leishmania transmission. Folia Parasitol. 2006;53:161–171. PubMed

Belkaid Y, Kamhawi S, Modi G, Valenzuela J, Noben-Trauth N, et al. Development of a natural model of cutaneous leishmaniasis: Powerful effects of vector saliva and saliva preexposure on the long-term outcome of Leishmania major infection in the mouse ear dermis. J Exp Med. 1998;188:1941–1953. PubMed PMC

Kamhawi S, Belkaid Y, Modi G, Rowton E, Sacks D. Protection against cutaneous Leishmaniasis resulting from bites of uninfected sand flies. Science. 2000;290:1351–1354. PubMed

Morris RV, Shoemaker CB, David JR, Lanzaro GC, Titus RG. Sandfly maxadilan exacerbates infection with Leishmania major and vaccinating against it protects against L-major infection. J Immunol. 2001;167:5226–5230. PubMed

Valenzuela JG, Belkaid Y, Garfield MK, Mendez S, Kamhawi S, et al. Toward a defined anti-Leishmania vaccine targeting vector antigens: Characterization of a protective salivary protein. J Exp Med. 2001;194:331–342. PubMed PMC

Gomes R, Teixeira C, Teixeira MJ, Oliveira F, Menezes MJ, et al. Immunity to a salivary protein of a sand fly vector protects against the fatal outcome of visceral leishmaniasis in a hamster model. Proc Natl Acad Sci U S A. 2008;105:7845–7850. PubMed PMC

Collin N, Gomes R, Teixeira C, Cheng L, Laughinghouse A, et al. Sand Fly Salivary Proteins Induce Strong Cellular Immunity in a Natural Reservoir of Visceral Leishmaniasis with Adverse Consequences for Leishmania. Plos Pathog. 2009;5:e1000441. PubMed PMC

Xu X, Oliveira F, Chang BW, Collin N, Gomes R, et al. Structure and function of a “yellow” protein from saliva of the sand fly Lutzomyia longipalpis that confers protective immunity against Leishmania major infection. J Biol Chem. 2011;286:32383–32393. PubMed PMC

Barral A, Honda E, Caldas A, Costa J, Vinhas V, et al. Human immune response to sand fly salivary gland antigens: A useful epidemiological marker? Am J Trop Med Hyg. 2000;62:740–745. PubMed

Gomes RB, Brodskyn U, de Oliveira CI, Costa J, Miranda JC, et al. Seroconversion against Lutzomyia longipalpis saliva concurrent with the development of anti-Leishmania chagasi delayed-type hypersensitivity. J Infect Dis. 2002;186:1530–1534. PubMed

Rohousova I, Ozensoy S, Ozbel Y, Volf P. Detection of species-specific antibody response of humans and mice bitten by sand flies. Parasitology. 2005;130:493–499. PubMed

de Moura TR, Oliveira F, Novais FO, Miranda JC, Clarencio J, et al. Enhanced Leishmania braziliensis infection following pre-exposure to sandfly saliva. PLoS Negl Trop Dis. 2007;1:e84. PubMed PMC

Aquino DMC, Caldas AJM, Miranda JC, Silva AAM, Barral-Netto M, et al. Short report: Epidemiological study of the association between anti-Lutzomyia longipalpis saliva antibodies and development of delayed-type hypersensitivity to Leishmania antigen. Am J Trop Med Hyg. 2010;83:825–827. PubMed PMC

Clements MF, Gidwani K, Kumar R, Hostomska J, Dinesh DS, et al. Measurement of recent exposure to Phlebotomus argentipes, the vector of Indian visceral leishmaniasis, by using human antibody responses to sand fly saliva. Am J Trop Med Hyg. 2010;82:801–807. PubMed PMC

Teixeira C, Gomes R, Collin N, Reynoso D, Jochim R, et al. Discovery of Markers of Exposure Specific to Bites of Lutzomyia longipalpis, the Vector of Leishmania infantum chagasi in Latin America. PLoS Negl Trop Dis. 2010;4:e638. PubMed PMC

Gidwani K, Picado A, Rijal S, Singh SP, Roy L, et al. Serological markers of sand fly exposure to evaluate insecticidal nets against visceral leishmaniasis in India and Nepal: a cluster-randomized trial. PLoS Negl Trop Dis. 2011;5:e1296. PubMed PMC

Marzouki S, Ben Ahmed M, Boussoffara T, Abdeladhim M, Ben Aleya-Bouafif N, et al. Characterization of the Antibody Response to the Saliva of Phlebotomus papatasi in People Living in Endemic Areas of Cutaneous Leishmaniasis. Am J Trop Med Hyg. 2011;84:653–661. PubMed PMC

Hostomska J, Rohousova I, Volfova V, Stanneck D, Mencke N, et al. Kinetics of canine antibody response to saliva of the sand fly Lutzomyia longipalpis. Vector Borne Zoonotic Dis. 2008;8:443–450. PubMed

Souza AP, Andrade BB, Aquino D, Entringer P, Miranda JC, et al. Using Recombinant Proteins from Lutzomyia longipalpis Saliva to Estimate Human Vector Exposure in Visceral Leishmaniasis Endemic Areas. PLoS Negl Trop Dis. 2010;4:e649. PubMed PMC

Vlkova M, Rohousova I, Drahota J, Stanneck D, Kruedewagen EM, et al. Canine antibody response to Phlebotomus perniciosus bites negatively correlates with the risk of Leishmania infantum transmission. PLoS Negl Trop Dis. 2011;5:e1344. PubMed PMC

Volf P, Rohousova I. Species-specific antigens in salivary glands of phlebotomine sandflies. Parasitology. 2001;122:37–41. PubMed

Charlab R, Valenzuela JG, Rowton ED, Ribeiro JMC. 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;96:15155–15160. PubMed PMC

Valenzuela JG, Garfield M, Rowton ED, Pham VM. Identification of the most abundant secreted proteins from the salivary glands of the sand fly Lutzomyia longipalpis, vector of Leishmania chagasi. J Exp Biol. 2004;207:3717–3729. PubMed

Anderson JM, Oliveira F, Kamhawi S, Mans BJ, Reynoso D, et al. Comparative salivary gland transcriptomics of sandfly vectors of visceral leishmaniasis. BMC Genomics. 2006;7:52. PubMed PMC

Kato H, Anderson JM, Kamhawi S, Oliveira F, Lawyer PG, et al. High degree of conservancy among secreted salivary gland proteins from two geographically distant Phlebotomus duboscqi sandflies populations (Mali and Kenya). BMC Genomics. 2006;7:226. PubMed PMC

Oliveira F, Kamhawi S, Seitz AE, Pham VM, Guigal PM, et al. From transcriptome to immunome: Identification of DTH inducing proteins from a Phlebotomus ariasi salivary gland cDNA library. Vaccine. 2006;24:374–390. PubMed

Hostomska J, Volfova V, Mu JB, Garfield M, Rohousova I, et al. Analysis of salivary transcripts and antigens of the sand fly Phlebotomus arabicus. BMC Genomics. 2009;10:282. PubMed PMC

Killick-Kendrick R. The biology and control of phlebotomine sand flies. Clin Dermatol. 1999;17:279–289. PubMed

Svobodova M, Votypka J. Experimental transmission of Leishmania tropica to hamsters and mice by the bite of Phlebotomus sergenti. Microbes Infect. 2003;5:471–4. PubMed

Svobodova M, Votypka J, Peckova J, Dvorak V, Nasereddin A, et al. Distinct transmission cycles of Leishmania tropica in 2 adjacent foci, Northern Israel. Emerg Infect Dis. 2006;12:1860–8. PubMed PMC

Svobodova M, Alten B, Zidkova L, Dvorak V, Hlavackova J, et al. Cutaneous leishmaniasis caused by Leishmania infantum transmitted by Phlebotomus tobbi. Int J Parasitol. 2009;39:251–6. PubMed

Aransay AM, Scoulica E, Tselentis Y, Ready PD. Phylogenetic relationships of phlebotomine sandflies inferred from small subunit nuclear ribosomal DNA. Insect Mol Bio. 2000;9:157–168. PubMed

Maia C, Afonso MO, Neto L, Dionisio L, Campino L. Molecular detection of Leishmania infantum in naturally infected Phlebotomus perniciosus from Algarve region, Portugal. J Vector Borne Dis. 2009;46:268–272. PubMed

Volf P, Volfova V. Establishment and maintenance of sand fly colonies. J Vector Ecol. 2011;36:S1–S9. PubMed

Chmelar J, Anderson JM, Mu J, Jochim RC, Valenzuela JG, et al. Insight into the sialome of the castor bean tick, Ixodes ricinus. BMC Genomics. 2008;9:233. PubMed PMC

Drastichova Z, Bourova L, Hejnova L, Jedelsky P, Svoboda P, et al. Protein alterations induced by long-term agonist treatment of HEK293 cells expressing thyrotropin-releasing hormone receptor and G(11)alpha protein. J Cell Biochem. 2010;109:255–264. PubMed

Guo YJ, Ribeiro JMC, Anderson JM, Bour S. dCAS: a desktop application for cDNA sequence annotation. Bioinformatics. 2009;25:1195–1196. PubMed PMC

Huang XQ, Madan A. CAP3: A DNA sequence assembly program. Genome Res. 1999;9:868–877. PubMed PMC

Ewing B, Hillier L, Wendl MC, Green P. Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res. 1998;8:175–185. PubMed

Ewing B, Green P. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 1998;8:186–194. PubMed

Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25:3389–3402. PubMed PMC

Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, et al. Gene Ontology: tool for the unification of biology. Nat Genet. 2000;25:25–29. PubMed PMC

Tatusov RL, Fedorova ND, Jackson JD, Jacobs AR, Kiryutin B, et al. The COG database: an updated version includes eukaryotes. BMC Bioinformatics. 2003;4:4. PubMed PMC

Bateman A, Birney E, Durbin R, Eddy SR, Howe KL, et al. The Pfam protein families database. Nucleic Acids Res. 2000;28:263–266. PubMed PMC

Schultz J, Copley RR, Doerks T, Ponting CP, Bork P. SMART: a web-based tool for the study of genetically mobile domains. Nucleic Acids Res. 2000;28:231–234. PubMed PMC

Bendtsen JD, Nielsen H, von Heijne G, Brunak S. Improved prediction of signal peptides: SignalP 3.0. J Mol Biol. 2004;340:783–795. PubMed

NetNGlyc 1.0 Server. Available: http://www.cbs.dtu.dk/services/NetNGlyc/

Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, et al. Clustal W and clustal X version 2.0. Bioinformatics. 2007;23:2947–2948. PubMed

Abascal F, Zardoya R, Posada D. ProtTest: selection of best-fit models of protein evolution. Bioinformatics. 2005;21:2104–2105. PubMed

Schmidt HA, Strimmer K, Vingron M, von Haeseler A. TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics. 2002;18:502–504. PubMed

Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol. 2007;24:1596–1599. PubMed

Cerna P, Mikes L, Volf P. Salivary gland hyaluronidase in various species of phlebotomine sand flies (Diptera : Psychodidae). Insect Biochem Mol Biol. 2002;32:1691–1697. PubMed

Frost GI, Stern R. A microtiter-based assay for hyaluronidase activity not requiring specialized reagents. Anal Biochem. 1997;251:263–269. PubMed

Ribeiro JMC, Arca B. From Sialomes to the Sialoverse: An Insight into Salivary Potion of Blood-Feeding Insects. Adv In Insect Phys. 2009;37:59–118.

Ribeiro JMC, Mans BJ, Arca B. An insight into the sialome of blood-feeding Nematocera. Insect Biochem Mol Biol. 2010;40:767–784. PubMed PMC

Yeats C, Bentley S, Bateman A. New knowledge from old: In silico discovery of novel protein domains in Streptomyces coelicolor. BMC Microbiol. 2003;3:3. PubMed PMC

Ameri M, Wang X, Wilkerson MJ, Kanost MR, Broce AB. An immunoglobulin binding protein (Antigen 5) of the stable fly (Diptera : Muscidae) salivary gland stimulates bovine immune responses. J Med Entomol. 2008;45:94–101. PubMed PMC

Ma DY, Gao L, An S, Song YZ, Wu J, et al. A horsefly saliva antigen 5-like protein containing RTS motif is an angiogenesis inhibitor. Toxicon. 2010;55:45–51. PubMed

Xu XQ, Yang HL, Ma DY, Wu J, Wang YP, et al. Toward an understanding of the molecular mechanism for successful blood feeding by coupling proteomics analysis with pharmacological testing of horsefly salivary glands. Mol Cell Proteomics. 2008;7:582–590. PubMed

Schaffartzik A, Weichel M, Crameri R, Bjornsdottir PS, Prisi C, et al. Cloning of IgE-binding proteins from Simulium vittatum and their potential significance as allergens for equine insect bite hypersensitivity. Vet Immunol Immunopathol. 2009;132:68–77. PubMed

Schaffartzik A, Marti E, Crameri R, Rhyner C. Cloning, production and characterization of antigen 5 like proteins from Simulium vittatum and Culicoides nubeculosus, the first cross-reactive allergen associated with equine insect bite hypersensitivity. Vet Immunol Immunopathol. 2010;137:76–83. PubMed

Caljon G, Broos K, De Goeyse I, De Ridder K, Sternberg JM, et al. Identification of a functional Antigen5-related allergen in the saliva of a blood feeding insect, the tsetse fly. Insect Biochem Mol Biol. 2009;39:332–41. PubMed

Ribeiro JMC, Rossignol PA, Spielman A. Blood-Finding Strategy of A Capillary-Feeding Sandfly, Lutzomyia-Longipalpis. Comp Biochem Physiol A Comp Physiol. 1986;83:683–686. PubMed

Ribeiro JMC, Modi GB, Tesh RB. Salivary apyrase activity of some Old World Phlebotomine sand flies. Insect Biochem. 1989;19:409–412.

Hamasaki R, Kato H, Terayama Y, Iwata H, Valenzuela JG. Functional characterization of a salivary apyrase from the sand fly, Phlebotomus duboscqi, a vector of Leishmania major. J Insect Physiol. 2009;55:1044–1049. PubMed PMC

Valenzuela JG, Belkaid Y, Rowton E, Ribeiro JMC. The salivary apyrase of the blood-sucking sand fly Phlebotomus papatasi belongs to the novel Cimex family of apyrases. J Exp Biol. 2001;204:229–237. PubMed

Smith TM, Kirley TL. The calcium activated nucleotidases: A diverse family of soluble and membrane associated nucleotide hydrolyzing enzymes. Purinergic Signal. 2006;2:327–333. PubMed PMC

Yang MY, Kirley TL. Site-directed mutagenesis of human soluble calcium-activated nucleotidase 1 (hSCAN-1): Identification of residues essential for enzyme activity and the Ca2+-induced conformational change. Biochemistry. 2004;43:9185–9194. PubMed

Dai JY, Liu J, Deng YQ, Smith TM, Lu M. Structure and protein design of a human platelet function inhibitor. Cell. 2004;116:649–659. PubMed

Stern R. Devising a pathway for hyaluronan catabolism: are we there yet? Glycobiology. 2003;13:105R–115R. PubMed

Volfova V, Hostomska J, Cerny M, Votypka J, Volf P. Hyaluronidase of Bloodsucking Insects and Its Enhancing Effect on Leishmania Infection in Mice. PLoS Negl Trop Dis. 2008;2:e294. PubMed PMC

Ribeiro JMC, Charlab R, Rowton ED, Cupp EW. Simulium vittatum (Diptera : Simuliidae) and Lutzomyia longipalpis (Diptera : Psychodidae) salivary gland hyaluronidase activity. J Med Entomol. 2000;37:743–747. PubMed

Stern R, Jedrzejas MJ. Hyaluronidases: Their genomics, structures, and mechanisms of action. Chem Rev. 2006;106:818–839. PubMed PMC

Markovic-Housley Z, Miglierini G, Soldatova L, Rizkallah PJ, Muller U, et al. Crystal structure of hyaluronidase, a major allergen of bee venom. Structure. 2000;8:1025–1035. PubMed

Wilson AD, Heesom KJ, Mawby WJ, Mellor PS, Russell CL. Identification of abundant proteins and potential allergens in Culicoides nubeculosus salivary glands. Vet Immunol Immunopathol. 2008;122:94–103. PubMed

Vinhas V, Andrade BB, Paes F, Bomura A, Clarencio J, et al. Human anti-saliva immune response following experimental exposure to the visceral leishmaniasis vector, Lutzomyia longipalpis. Eur J Immunol. 2007;37:3111–3121. PubMed

Valenzuela JG, Charlab R, Gonzalez EC, de Miranda-Santos IKF, Marinotti O, et al. The D7 family of salivary proteins in blood sucking diptera. Insect Mol Bio. 2002;11:149–155. PubMed

James AA, Blackmer K, Marinotti O, Ghosn CR, Racioppi JV. Isolation and Characterization of the Gene Expressing the Major Salivary-Gland Protein of the Female Mosquito, Aedes-Aegypti. Molecul Biochem Parasitol. 1991;44:245–254. PubMed

Geng YJ, Gao ST, Huang DN, Zhao YR, Liu JP, et al. Differentially expressed genes between female and male adult Anopheles anthropophagus. Parasitol Res. 2009;105:843–851. PubMed

Calvo E, Mans BJ, Andersen JF, Ribeiro JMC. Function and evolution of a mosquito salivary protein family. J Biol Chem. 2006;281:1935–1942. PubMed

Isawa H, Orito Y, Iwanaga S, Jingushi N, Morita A, et al. Identification and characterization of a new kallikrein-kinin system inhibitor from the salivary glands of the malaria vector mosquito Anopheles stephensi. Insect Biochem Mol Biol. 2007;37:466–477. PubMed

Alvarenga PH, Francischetti IMB, Calvo E, Sa-Nunes A, Ribeiro JMC, et al. The Function and Three-Dimensional Structure of a Thromboxane A(2)/Cysteinyl Leukotriene-Binding Protein from the Saliva of a Mosquito Vector of the Malaria Parasite. Plos Biol. 2010;8:e1000547. PubMed PMC

Oliveira F, Lawyer PG, Kamhawi S, Valenzuela JG. Immunity to Distinct Sand Fly Salivary Proteins Primes the Anti-Leishmania Immune Response towards Protection or Exacerbation of Disease. PLoS Negl Trop Dis. 2008;2:e226. PubMed PMC

Bahia D, Gontijo NF, Leon IR, Perales J, Pereira MH, et al. Antibodies from dogs with canine visceral leishmaniasis recognise two proteins from the saliva of Lutzomyia longipalpis. Parasitol Res. 2007;100:449–454. PubMed

Elnaiem DEA, Meneses C, Slotman M, Lanzaro GC. Genetic variation in the sand fly salivary protein, SP-15, a potential vaccine candidate against Leishmania major. Insect Mol Bio. 2005;14:145–150. PubMed

Coutinho-Abreu IV, Wadsworth M, Stayback G, Ramalho-Ortigao M, McDowell MA. Differential Expression of Salivary Gland Genes in the Female Sand Fly Phlebotomus papatasi (Diptera: Psychodidae). J Med Entomol. 2010;47:1146–1155. PubMed

Schmitzova J, Klaudiny J, Albert S, Schroder W, Schreckengost W, et al. A family of major royal jelly proteins of the honeybee Apis mellifera L. Cell Mol Life Sci. 1998;54:1020–1030. PubMed PMC

Geyer PK, Spana C, Corces VG. On the Molecular Mechanism of Gypsy-Induced Mutations at the Yellow Locus of Drosophila-Melanogaster. EMBO J. 1986;5:2657–2662. PubMed PMC

Albert S, Bhattacharya D, Klaudiny J, Schmitzova J, Simuth J. The family of major royal jelly proteins and its evolution. J Mol Evol. 1999;49:290–297. PubMed

Alves-Silva J, Ribeiro JMC, Van Den Abbeele J, Attardo G, Hao ZR, et al. An insight into the sialome of Glossina morsitans morsitans. BMC Genomics. 2010;11:213. PubMed PMC

Johnson JK, Li J, Christensen BM. Cloning and characterization of a dopachrome conversion enzyme from the yellow fever mosquito, Aedes aegypti. Insect Biochem Mol Biol. 2001;31:1125–1135. PubMed

Volf P, Skarupova S, Man P. Characterization of the lectin from females of Phlebotomus duboscqi sand flies. Eur J Biochem. 2002;269:6294–6301. PubMed

Sergentomyia schwetzi: Salivary gland transcriptome, proteome and enzymatic activities in two lineages adapted to different blood sources

Human antibody reaction against recombinant salivary proteins of Phlebotomus orientalis in Eastern Africa

Insights into the sand fly saliva: Blood-feeding and immune interactions between sand flies, hosts, and Leishmania

The Diversity of Yellow-Related Proteins in Sand Flies (Diptera: Psychodidae)

Hyaluronidase Activity in Saliva of European Culicoides (Diptera: Ceratopogonidae)

De novo assembly and sex-specific transcriptome profiling in the sand fly Phlebotomus perniciosus (Diptera, Phlebotominae), a major Old World vector of Leishmania infantum

Comparative analysis of salivary gland transcriptomes of Phlebotomus orientalis sand flies from endemic and non-endemic foci of visceral leishmaniasis

Recombinant antigens from Phlebotomus perniciosus saliva as markers of canine exposure to visceral leishmaniases vector

Leishmania development in sand flies: parasite-vector interactions overview

Zobrazit více v PubMed

GENBANK
JN192442