Salivary Gland Transcriptomes and Proteomes of Phlebotomus tobbi and Phlebotomus sergenti, Vectors of Leishmaniasis

Introduction Sand flies are bloodsucking nematoceran Diptera that transmit the protozoan parasites of the genus Leishmania. Similar to that of other bloodsucking arthropods, sand fly saliva comprises antihemostatic, immunomodulatory, and antigenic components. The saliva is deposited into the host skin every time the sand fly ingests a blood meal to facilitate feeding. Also during the bite by an infected sand fly, Leishmania parasites are egested into the wound with the saliva. Sand fly saliva can enhance Leishmania infection in naive mice [1], [2]. Conversely, pre-exposure of mice to sand fly saliva conferred a protective effect against Leishmania infection [3], [4]. Even single salivary proteins have been characterized as potential Leishmania vaccine candidates in mouse, hamster, and dog models of cutaneous or visceral leishmaniasis [5]–[9]. The potent effects of sand fly saliva stimulate a protective host cellular immune response [3]–[9], and the antigenic nature of saliva also provides a humoral immunity measurement of host exposure to sand fly bites already used in several human epidemiological studies [10]–[18]. Identifying markers of vector exposure based on anti-saliva antibodies are essential in epidemiologic and vector control surveillance [15], [16], [18], [19]–[21]. However, anti-saliva antibodies are highly specific [12], [16], [22] and with over 80 species of sand flies implicated in Leishmania transmission, it is vital to continue describing the salivary proteins in the search for markers of exposure as well as vaccine candidates. Sand fly salivary gland proteins have been well studied in Lutzomyia longipalpis [23], [24] and Phlebotomus papatasi [6]. Recently, transcriptomic and proteomic data have been published for several other sand fly species, vectors of visceral (P. ariasi, P. argentipes, and P. perniciosus) and cutaneous (P. arabicus, P. duboscqi) forms of leishmaniasis [25]–[28]. To broaden the repertoire of subgenus Larroussius salivary proteins and provide the first report from a subgenus Paraphlebotomus sand fly, we prepared and analyzed the transcriptomes and proteomes of P. tobbi and P. sergenti, both proven vectors in the Old World. Phlebotomus sergenti, subgenus Paraphlebotomus, is the main vector of Le. tropica, principally an agent of cutaneous leishmaniasis [29]–[31]. Phlebotomus tobbi, on the other hand, is an important vector of Le. infantum [32] together with the taxonomically related P. ariasi and P. perniciosus, sand flies of the subgenus Larroussius [29], [33], [34]. In contrast to other members of the subgenus, P. tobbi transmits the cutaneous form of the Le. infantum [32]. Additionaly, we characterized P. tobbi antigens, glycoproteins, and hyaluronidase activity; the later one compared with 6 sand fly species belonging to vectors of cutaneous or visceral leishmaniases. Methods Sand fly salivary glands Colonies of P. tobbi (originating from Turkey), P. papatasi (Turkey), P. sergenti (Israel), P. argentipes (India), P. arabicus (Israel), P. perniciosus (Spain), and L. longipalpis (Brazil) were kept in the insectary of Charles University in Prague as described in [35]. The P. sergenti colony, originating from Turkey, was reared in similar conditions at the Laboratory of Malaria and Vector Research, National Institutes of Health (Rockville, MD, USA). For mRNA extraction, salivary glands (SGs) from non-bloodfed 1- to 2-day-old female sand flies were dissected and stored in RNA Later (Ambion, Inc., Austin, TX, USA). For other assays and analysis, SGs of non-bloodfed 5- to 7-day-old females were stored at −70°C; SGs were stored in NuPAGE LDS sample buffer (Invitrogen, Carlsbad, CA, USA) for proteome analysis and in Tris buffer (20 mM Tris, 150 mM NaCl, pH 7.8) for hyaluronidase assays, affinity blot, and immunoblot. Before use, samples were homogenized by three freeze-thaw cycles in liquid nitrogen. Protein concentration in resulting SG homogenate (SGH) was measured on Qubit Fluorometer (Invitrogen) following manufacturer's guidelines. Salivary gland cDNA library construction and sequencing An SG cDNA library was constructed from P. sergenti (Turkey) and P. tobbi. MicroFastTrack mRNA isolation kit (Invitrogen) was used to isolate SG mRNA from 40 SG pairs dissected into 20 µl of RNA Later (Ambion). A cDNA library was constructed using SMART™ cDNA Library Construction Kit (BD Clontech, Palo Alto, CA, USA) following the manufacturer's protocol, with some modifications as described in [36]. For each species, three cDNA libraries were constructed according to PCR product size – large, medium, and small. PCR amplicons were washed and concentrated to 4–7 µl on Microcon YM-100 columns (Millipore, Billerica, MA, USA). Concentrated samples (3 µl) were ligated into the λTripleEx2 vector and packed into the phage particles with Gigapack III Gold Packaging Extract (Stratagene, La Jolla, CA, USA). Phage libraries were used to infect the log-phase XL-1 Blue Escherichia coli (Clontech) plated onto four LB agar plates per each library size. Transfected plaques were randomly selected and transferred into 96-well V-shape plates with 75 µl of ultrapure water per well. Four 96-well plates of phage were picked per each library size, resulting in 12 plates (1,152 clones) per sand fly species. Phages (3 µl) were subjected to PCR using FastStart PCR Master Mix (Roche, Molecular Biochemicals, Indianopolis, IN, USA) and vector-specific primers (PT2F1 5′-AAGTACTCTAGCAATTGTGAGC-3′ and PT2R1 5′-CTCTTCGCTATTACGCCAGCTG-3′). Amplification conditions were as follows: 1 hold of 75°C for 3 min, 1 hold of 94°C for 2 min, 34 cycles of 94°C for 1 min, 49°C for 1 min, and 72°C for 2 min. The final elongation step lasted for 10 min at 72°C. Products were cleaned using ExcelaPure 96-well UF PCR Purification Plates (Edge Biosystems, Gaithersburg, MD, USA) and cleaned PCR products were used as a template for cycle-sequencing reaction using BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Fullerton, CA, USA) and a vector-specific forward primer (PT2F3 5′-CTCGGGAAGCGCGCCATTGT-3′). Products of cycle-sequencing reaction were cleaned using Sephadex and MultiScreen HV Plates (Millipore), dried, resuspended in formamide, and stored at −20°C until sequenced on an ABI 3730XL 96-Capillary DNA Sequencer (Applied Biosystems). Proteome analysis For mass spectrometric (MS) analysis, SGH samples of P. sergenti (Turkey) and P. tobbi were dissolved in Laemmli sample buffer in parallel with or without 2-mercaptoethanol and electrophoretically separated on 12% polyacrylamide SDS minigel with initial voltage 80 V and 120 V upon entry of sample to the gel. Gels were stained for total proteins with Coomassie Brilliant Blue R-250. Individual bands were cut, destained and digested as was described in [37]. Samples (0.5 µl) were transferred to a 384 spot stainless steel MALDI target (AB Sciex, Framingham, MA, USA) and let to dry. Dried droplets were covered with a 0.5 µl drop of alpha-cyano-hydroxycinnamic acid (Fluka, Switzerland) solution (2 mg/ml in 80% acetonitrile) and allowed to dry. Spectra were acquired with 4800 Plus MALDI TOF/TOF analyzer (AB Sciex) equipped with a Nd∶YAG laser (355 nm; firing rate 200 Hz). Voltages were set as follows: source1 20 kV, grid1 16 kV, source1 lens 10 kV, lens1 5 kV, mirror1 14.085 kV, mirror2 20.3 kV and reflector detector 1.905 kV. Digitizer bin size was set to 0.5 ns, vertical scale 0.5 V, vertical offset 0.0, input bandwith 500 MHz. Spectra were externally calibrated using ProteoMass peptide MALDI calibration kit (Sigma-Aldrich). Spectra were recorded in the range 700 to 4000 Da, focus mass 2100 Da. Spectra were summed from 40 positions per 50 shots, 2000 shots in total. Spectra were processed by 4000 Series Explorer version 3.5.3 (AB Sciex) without smoothing; baseline subtraction was performed with peak width set to 50. Spectra were deisotoped and peaks with a local signal-to-noise ratio greater than 5 were picked and searched by local Mascot v. 2.1 (Matrix Science, Boston, MA, USA) against a database of protein sequences derived from the cDNA library. Database search criteria were as follows: enzyme: trypsin; taxonomy: none; fixed modification: carbamidomethylation; variable modification: methionine oxidation; peptide mass tolerance: 80 ppm; one missed cleavage allowed. Only hits that were scored as significant (P<0.05) were included. The data associated with this manuscript may be downloaded from ProteomeCommons.org Tranche using the following hash: mCZfFsOLaBtSfR+Jh6o8OwEgjrqDp4m3VntpJdAPqPGFNzNpTPry8IhEuGeLw9 TmpHcTRMSiiuiNNRL/6xP65TLvyNwAAAAAAAADGQ =   = . The hash may be used to prove exactly what files were published as part of this manuscript's data set, and the hash may also be used to check that the data has not changed since publication. Bioinformatic and phylogenetic analysis Expression sequence tags (ESTs) were analyzed using the dCAS software (Desktop cDNA Annotation System, version 1.4.3) [38] with all third-party components recommended: CAP3 assembler program [39], Phred [40], [41], and BLAST programs [42]. Sequences with Phred quality scores lower than 25 were removed, as well as primers and vector sequences. Resulting sequences were grouped based on nucleotide homology of 90% identity over 100 residues and aligned into consensus transcript sequences (contigs) using the CAP3 sequence assembly program. BLAST programs were used to compare contigs and singletons (contigs with a single sequence) to the non-redundant protein database of the NCBI, the Gene Ontology database (GO) [43], to COG conserved domains database [44], Protein Family database (Pfam) [45], Simple Modular Architecture Tool database (SMART) [46], and to rRNA Nucleotide Sequences, and Mitochondrial and Plasmid Sequence (MIT-PLA) databases available from NCBI. The three frame translations of each dataset were submitted to the SignalP server [47] to detect signal peptides. The grouped and assembled sequences, BLAST results, and SignalP results combined by dCAS software in an Excel spreadsheet were manually verified and annotated. Additionally, glycosylation sites were determined in selected sequences using NetNGlyc prediction server [48]. For phylogenetic analysis, protein sequences without signal peptide were aligned using ClustalX (version 2.0) [49] with related sequences obtained from GenBank and manually refined in BioEdit 7.0 editing software. For each alignment, best substitution matrix was determined by ProtTest software, version 2.0 [50]. This matrix was then used by TREE-PUZZLE 5.2 [51] to reconstruct maximum likelihood phylogenetic trees from the protein alignments using quartet puzzling with 1000 puzzling steps. Resulting trees were visualized in MEGA 4 [52]. All protein and nucleotide accession numbers mentioned in the text, tables and figures are listed in Text S1. Hyaluronidase activity Hyaluronidase activity was compared between seven sand fly species: P. tobbi, P. sergenti (Israel), P. papatasi, P. argentipes, P. arabicus, P. perniciosus, and L. longipalpis. Hyaluronidase activity in SGs was quantified using a sensitive assay on microtitration plates coupled with biotinylated HA (bHA). bHA, prepared as described in [53], was immobilized onto Covalink NH microtiter plates (Nunc, Placerville, NJ, USA) using the method in [54] at a final concentration of 1 µg bHA per well. The plates were incubated overnight at 4°C and washed three times in PBS (containing 2 M NaCl and 50 mM MgSO4, pH 7.2). Plates with immobilized bHA were blocked with 1% BSA in PBS for 45 min, washed and equilibrated to pH 5.0 (0.1 M acetate, 0.1 M NaCl, 0.1% Triton X-100, pH 5.0), the pH optimum for sand fly salivary hyaluronidase [53]. SGHs were incubated for 45 min at 37°C in triplicate at a final concentration of 0.5 gland per well. As a standard, bovine hyaluronidase (Sigma) at a concentration of 0.01 Turbidity Reducing Units (TRU)/µl was serially diluted in 0.1 M acetate buffer (0.1 M NaCl, 0.1% Triton X-100, pH 4.5). Wells without bHA or enzyme were used as controls. The reaction was terminated by 6 M guanidine 200 µl/well. Plates were washed in PBS (containing 2 M NaCl, 50 mM MgSO4, 0.05% Tween 20, pH 7.2) and then equilibrated with PBS, 0.1% Tween 20, pH 7.2. Avidin-peroxidase (Sigma) was added at a final concentration of 0.2 µg/well and incubated for 30 min at room temperature. Color reaction was developed with o-phenylenediamine substrate in 0.1 M citrate-phosphate buffer (pH 5.5). After 10 min in dark, plates were read at 492 nm (Tecan-Infinite M 200 Fluorometer; Schoeller Instruments, Prague, Czech Republic). The obtained results were expressed as relative TRU (rTRU). Three independent experiments were performed with a different set of SGH samples in each experiment. For hyaluronidase zymography, 8% polyacrylamide gels (0.75 mm thick) were copolymerized with 0.002% hyaluronic acid (HA). As the hyaluronidase activities and band patterns varied among sand fly species, different loads were used per lane to obtain bands of equal intensity. The equivalent of 1/2 gland (L. longipalpis and P. sergenti) or 1/20 gland (other tested species) was loaded for zymography under non-reducing conditions, and the equivalent of 2.5 glands (L. longipalpis and P. sergenti) or 1/4 gland (other tested species) was loaded for zymography under reducing conditions. The total protein content per lane was as follows (non-reducing/reducing conditions): L. longipalpis = 110/550 ng; P. papatasi = 12.5/62.5 ng; P. sergenti = 140/700 ng; P. argentipes = 14/70 ng; P. arabicus = 10.5/52.5 ng; P. tobbi = 10/50 ng; P. perniciosus = 10.5/52.5 ng. For reducing conditions, samples were treated with 3% 2-mercaptoethanol for 40 min at 45°C. SDS-PAGE electrophoresis was carried out using Mini-Protean II apparatus (Bio-Rad, Hercules, CA, USA) and constant voltage at 150 V. After electrophoresis, gels were rinsed 2×20 min in 0.1 M Tris, pH 7.8, and 20 min in 0.1 M acetate buffer, pH 5.5 (both with 1% Triton X-100 to wash out SDS) and then incubated in 0.1 M acetate buffer (without detergent) for 120 min at 37°C. The gels were then washed in water, soaked in 50% formamide for 30 min and stained in Stains-all (Sigma, St. Louis, MO, USA) solution (100 mg/ml in 50% formamide) for 24 h in the dark. Hyaluronidase activity was visible as a pink band on a dark blue background. Immunoblotting Immunoblot was performed using P. tobbi SGH separated by SDS-PAGE on 10% polyacrylamide gel under non-reducing conditions using the Mini-Protean III apparatus (Bio-Rad). Separated proteins were electrotransferred onto nitrocellulose (NC) membrane by iBlot Dry Blotting System (Invitrogen). After transfer, the NC membrane was cut into strips with the equivalent of four glands/strip and free binding sites were blocked by 5% low-fat dried milk in 20 mM Tris buffer with 0.05% Tween (Tris-Tw) overnight at 4°C. The strips were then incubated with serum obtained from rabbit repeatedly exposed to P. tobbi females. Serum was diluted 1∶250 in Tris-Tw and incubated with P. tobbi proteins for 1 h, followed by 1 h incubation with peroxidase-conjugated swine anti-rabbit IgG (Sevapharma, Prague, Czech Republic) diluted 1∶1,000 in Tris-Tw. Substrate solution contained Tris buffer, diaminobenzidine, and H2O2. Affinity blotting Affinity blot was performed using P. tobbi SGH separated and electrotransferred as described for Immunoblot. After transfer, free binding sites on NC membrane were blocked by 5% BSA in 20 mM Tris-Tw overnight at 4°C. The strip was then incubated for 1 h at room temperature with biotinylated lectin from Canavalia ensiformis (Concanavalin A, Sigma) diluted 0.2 µg/ml in Tris-Tw. To control the reaction specificity, another strip was incubated with lectin preincubated for 30 min with the ligand, 0.5 M methyl-α-D-mannopyranoside. Avidin-peroxidase (Sigma) was added at a final concentration of 2.5 µg/ml and incubated for 1 h at room temperature. Substrate solution contained Tris buffer, diaminobenzidine, and H2O2. Ethics statement All animals used in this study were maintained and handled strictly in accordance with institutional guidelines and legislation for the care and use of animals for research purpose Czech Act No. 246/1992 coll. on Protection Animals against Cruelty in present statues at large that complies with all relevant European Union and international guidelines for experimental animals. The experiments were approved by the Committee on the Ethics of Animal Experiments of the Charles University in Prague (Permit Number: 24773/2008-10001) and were performed under the Certificate of Competency (Registration Number: CZU 934/05) in accordance with the Examination Order approved by Central Commission for Animal Welfare of the Czech Republic. All efforts were made to minimize suffering of experimental animals within the study. Results and Discussion Salivary gland transcripts analysis Phlebotomus tobbi and P. sergenti cDNA libraries were constructed from SGs of female sand flies dissected one day after emergence. From each cDNA library, 1,152 randomly selected clones were sequenced. Obtained ESTs were deposited in the NCBI dbEST database under accession numbers GW814275–GW815416 (1,142 sequences) for P. tobbi and GW813185–GW814274 (1,090 sequences) for P. sergenti. High-quality sequences were grouped together based on sequence homology, and resulting assembled sequences were analyzed using the dCAS cDNA annotation software [38] and verified by manual annotation. In the P. tobbi cDNA library, 997 high-quality sequences were grouped into 68 contigs and 125 singletons (one sequence in cluster); in P. sergenti, 853 high-quality sequences were grouped into 56 contigs and 196 singletons. Similar to other sand flies studied so far, the most abundant transcripts in both libraries were those coding for putative secretory proteins. BLAST comparison of translated nucleotide sequences with the non-redundant (NR) protein database showed high similarity with other sand fly secreted salivary proteins. In P. tobbi, 81 clusters containing 863 sequences (average 10.7 sequences per cluster) matched to sand fly salivary proteins. Of them, we found 62 clusters (796 sequences) with predicted signal peptide sequence. In P. sergenti, 50 clusters containing 553 sequences (average 11.1 sequences per cluster) matched to sand fly salivary proteins. Of them, 32 clusters (482 sequences) with predicted signal protein sequence were found. Tables 1 and 2 list representative secreted salivary proteins from P. tobbi and P. sergenti, respectively, deposited into NCBI GenBank database. The tables show GenBank accession numbers, putative mature protein features, best match to NR protein database, and presence in the proteome analysis as confirmed by MS (Figure 1). Additionally, Figure 1A and 1B show detailed analysis of MS results for P. tobbi and P. sergenti, respectively, including cluster name, Gen Bank accession number, and molecular weight of mature proteins under reducing and non-reducing conditions. 10.1371/journal.pntd.0001660.g001 Figure 1 Proteome analysis of sand fly salivary proteins. (A) Phlebotomus tobbi and (B) P. sergenti salivary gland homogenate were separated under reducing and non-reducing conditions. Resulting protein bands were cut from Coomassie-stained gel and analyzed by mass spectrometry. Obtained data were compared to relevant cDNA library. Identified proteins are listed with their GenBank accession number, cluster name, and molecular weight of the protein band (kDa). ND means not determined due to insignificant results. 10.1371/journal.pntd.0001660.t001 Table 1 Salivary protein transcripts of Phlebotomus tobbi. Putative mature protein Best match to NR protein database Cluster name GenBank Accn Comment In proteome pI Mw AA GenBank Accn Species E Value PtSP49 HM173648 41.9-kDa 8.3 45.5 410 ABA43063 P. perniciosus 0 PtSP38 HM140619 yellow-related Yes 8.5 42.6 375 ABA43050 P. perniciosus 0 PtSP37 HM140618 yellow-related Yes 6.0 41.5 370 ABA43049 P. perniciosus 0 PtSP73 HM173639 ParSP25-like Yes 4.4 38.8 336 ABA43056 P. perniciosus 4E-87 PtSP10 HM135952 apyrase Yes 9.1 35.7 311 ABB00906 P. perniciosus 1E-180 PtSP4 HM135951 apyrase Yes 9.1 35.2 311 ABB00907 P. perniciosus 1E-174 PtSP66 HM173645 33-kDa Yes 9.0 33.9 288 ABA43054 P. perniciosus 4E-155 PtSP77 HM140620 antigen 5-related Yes 9.1 31.2 272 ABA43055 P. perniciosus 3E-151 PtSP78 HM140621 antigen 5-related 9.2 30.2 263 ABA43055 P. perniciosus 1E-149 PtSP79 HM140622 antigen 5-related Yes 9.2 28.8 252 ABA43055 P. perniciosus 2E-151 PtSP76 HM173641 ParSP25-like 4.5 28.0 244 ABA43056 P. perniciosus 7E-95 PtSP75 HM173640 ParSP25-like 4.6 27.8 243 ABA43056 P. perniciosus 3E-77 PtSP56 HM164147 D7-related Yes 8.1 27.1 233 ABA43051 P. perniciosus 1E-122 PtSP60 HM164150 D7-related Yes 8.3 27.0 234 ABA43052 P. perniciosus 8E-119 PtSP54 HM164151 D7-related 8.3 27.0 233 ABA43051 P. perniciosus 3E-125 PtSP58 HM164149 D7-related Yes 9.4 26.8 230 ABA43052 P. perniciosus 4E-117 PtSP44 HM164146 D7-related Yes 8.9 26.7 233 ABA43058 P. perniciosus 5E-124 PtSP57 HM164148 D7-related Yes 8.7 25.9 225 ABA43052 P. perniciosus 8E-116 PtSP42 HM164145 D7-related 9.5 25.3 216 ABA43058 P. perniciosus 1E-119 PtSP28 HM173643 PpSP32-like Yes 10.0 24.5 227 ABA43053 P. perniciosus 1E-100 PtSP29 HM173644 PpSP32-like Yes 10.1 24.5 227 ABA43053 P. perniciosus 4E-99 PtSP27 HM173642 PpSP32-like 10.1 24.3 225 ABA43053 P. perniciosus 7E-92 PtSP9 HM164139 PpSP15-like Yes 8.6 14.9 122 ABA43048 P. perniciosus 2E-65 PtSP17 HM164140 PpSP15-like 8.0 14.7 123 AAX55748 P. ariasi 8E-40 PtSP32 HM164144 PpSP15-like Yes 8.7 14.6 121 ABA43057 P. perniciosus 1E-69 PtSP31 HM164143 PpSP15-like 8.7 14.4 119 ABA43057 P. perniciosus 4E-51 PtSP18 HM164141 PpSP15-like 8.6 13.8 118 ABA43059 P. perniciosus 5E-54 PtSP23 HM164142 PpSP15-like Yes 9.1 13.2 112 ABA43059 P. perniciosus 3E-63 PtSP8 HM173646 unknown 10.2 5.0 43 ABB00905 P. perniciosus 1.5 PtSP71 HM173638 unknown 10.6 4.5 42 ABA43060 P. perniciosus 5E-12 PtSP81 HM173647 unknown 9.5 3.7 34 ABB00905 P. perniciosus 5E-10 PtSP125 JN192442 hyaluronidase ACS93505 P. arabicus 2E-61 Putatively secreted salivary proteins from Phlebotomus tobbi with cluster name, GenBank accession number (GenBank Accn), presence in the proteome analysis as confirmed by mass spectrometry (Figure 1A), putative mature protein features (pI, predicted isoelectric point; Mw, predicted molecular weight; AA, number of amino acid residues), and best match to non-redundant protein database. 10.1371/journal.pntd.0001660.t002 Table 2 Salivary protein transcripts of Phlebotomus sergenti. Putative mature protein Best match to NR protein database Cluster name GenBank Accn Comment In proteome pI Mw AA GenBank Accn Species E Value PsSP82 HM569371 41.9-kDa 4.74 56.6 508 ABI20189 P. duboscqi 1E-78 PsSP26 HM569362 yellow-related Yes 8.06 43.9 382 ABI15938 P. duboscqi 0 PsSP19 HM560865 yellow-related Yes 8.86 42.5 377 AAL11051 P. papatasi 1E-176 PsSP20 HM560866 yellow-related Yes 9.80 42.4 377 AAL11051 P. papatasi 1E-178 PsSP22 HM560867 yellow-related Yes 5.70 42.3 377 ABI20172 P. duboscqi 1E-164 PsSP18 HM569361 yellow-related Yes 9.02 42.2 377 AAL11051 P. papatasi 1E-171 PsSP42 HM560861 apyrase Yes 8.91 35.9 317 AAG17637 P. papatasi 1E-135 PsSP40 HM560860 apyrase Yes 8.87 35.6 315 AAG17637 P. papatasi 1E-123 PsSP41 HM560862 apyrase Yes 8.31 33.7 295 AAG17637 P. papatasi 1E-134 PsSP49 HM569369 33-kDa Yes 9.00 32.9 279 ABI20155 P. duboscqi 1E-131 PsSP52 HM537134 antigen 5-related Yes 8.75 29.0 254 ABA54266 P. papatasi 1E-121 PsSP4 HM560863 D7-related Yes 8.93 26.8 233 AAL11048 P. papatasi 1E-101 PsSP5 HM569360 D7-related Yes 8.93 26.8 233 AAL11048 P. papatasi 1E-102 PsSP7 HM560864 D7-related Yes 8.41 26.7 233 AAL11048 P. papatasi 1E-102 PsSP44 HM569368 PpSP32-like 9.3 22.5 204 AAL11050 P. papatasi 1E-67 PsSP14 HM560870 PpSP15-like 8.76 17.1 142 AAL11047 P. papatasi 1E-40 PsSP15 HM560868 PpSP15-like Yes 9.07 14.7 122 AAL11047 P. papatasi 3E-45 PsSP54 HM569365 PpSP15-like 8.61 14.6 121 AAL11046 P. papatasi 2E-52 PsSP55 HM569363 PpSP15-like 8.61 14.6 121 AAL11046 P. papatasi 3E-52 PsSP98 HM569366 unknown 4.73 14.3 127 ABA12153 P. argentipes 3E-16 PsSP9 HM569364 PpSP15-like Yes 9.06 14.0 120 AAL11045 P. papatasi 4E-51 PsSP10 HM560869 PpSP15-like Yes 8.92 14.0 120 AAL11045 P. papatasi 2E-52 PsSP11 HM560871 PpSP15-like Yes 8.05 13.9 120 AAL11045 P. papatasi 7E-53 PsSP73 HM569367 unknown 4.51 12.2 118 AAX55657 P. ariasi 2E-20 PsSP28 HM569370 unknown 10.68 3.0 27 ABI20185 P. duboscqi 4E-6 Putatively secreted salivary proteins from Phlebotomus sergenti with cluster name, GenBank accession number (GenBank Accn), presence in the proteome analysis as confirmed by mass spectrometry (Figure 1B), putative mature protein features (pI, predicted isoelectric point; Mw, predicted molecular weight; AA, number of amino acid residues), and best match to non-redundant protein database. The putative secreted salivary proteins of P. tobbi and P. sergenti could be divided into ten main protein families (Figure S1): antigen 5-related protein, apyrase, hyaluronidase, D7-related and PpSP15-like protein (odorant-binding proteins superfamily), ParSP25-like protein, PpSP32-like protein, yellow-related protein, the 33-kDa salivary proteins, and the 41.9-kDa superfamily. The following paragraphs describe these families in detail, focusing on protein family characteristics, possible function, biochemical, immunomodulatory, and antigenic properties, and phylogenetic analysis in context with related proteins from other sand flies. Antigen-5 related protein Antigen 5-related proteins (Ag5r) are present in saliva of all sand fly species studied so far [55], [56], including P. tobbi (PtSP77/HM140620, PtSP78/HM140621, PtSP79/HM140622) and P. sergenti (PsSP52/HM537134). Sand fly Ag5r proteins are members of CAP superfamily consisting of mammalian Cysteine-rich secretory proteins (CRISPs), Antigen 5 (Ag5) originally described from wasp venom, and plant Pathogenesis-related 1 proteins (PR-1) [55]. Proteins with CAP domain occur across all living organisms, including prokaryotes [57], and are mostly extracellular/secreted. All sand fly Ag5r proteins have similar predicted molecular mass (ranging from 28.8 to 31.2 kDa) and are alkaline (Table S1). In P. tobbi and P. sergenti, the predicted molecular mass corresponded well with the one measured in proteomic analysis (Figure 1, Tables 1 and 2) suggesting single-domain protein and negligible post-translational modifications. We identified 14 highly conserved cysteine residues proportionally distributed through the whole sequence length (Figure 2), possibly involved in disulfide bonding. 10.1371/journal.pntd.0001660.g002 Figure 2 Multiple sequence alignment of the antigen 5-related family of salivary proteins. Multiple sequence alignment of sand fly antigen 5-related proteins from Phlebotomus arabicus (Ara), P. argentipes (Arg), P. ariasi (Ari), P. duboscqi (Dub), P. papatasi (Pap), P. perniciosus (Per), P. sergenti (Ser), P. tobbi (Tob), and Lutzomyia longipalpis (Lon). Sequences without signal peptide were aligned using ClustalX and manually refined using BioEdit sequence-editing software. Accession numbers are indicated in the sequence name. Identical amino acid residues are highlighted black and similar residues grey. Conserved cysteine residues are indicated above the alignment by letter C and T cell epitopes predicted for P. duboscqi by Kato et al. [26] are indicated by asterisk (*). Although the members of this family were described in sialotranscriptomes of all bloodsucking arthropods characterized [55], [56], their role is mostly unknown with a few exceptions. In Stomoxys calcitrans, Ag5r protein possesses immunoglobulin Fc binding activity [58]. In Tabanus yao, members of the Ag5r protein family can probably serve as an inhibitors of angiogenesis (RTS disintegrin motif) [59] or a potent platelet inhibitor (RGD motif) [60]. The Ag5r proteins are not specific for salivary glands thus they may possess other functions not associated with feeding [23], [56]. Several studies showed antigenic properties associated with Ag5r proteins. Plasmid coding for Ag5r protein from P. ariasi (ParSP05/AAX44092) induced a cell-mediated immune response in Swiss Webster mice [27], showing that sand fly Ag5r proteins might modulate cell-mediated host immune response. This presumption is also supported by several T cell epitopes predicted for P. duboscqi Ag5r proteins [26] that include regions highly conserved among sand flies (Figure 2). Antibody response to sand fly Ag5r proteins was demonstrated in P. perniciosus; Ag5r protein (PpeSP07/ABA43055) reacted with IgG antibodies from sera of P. perniciosus bitten dogs [21]. In other bloodsucking diptera, Ag5r proteins are mostly associated with IgE antibody response. Ag5r protein of Simulium vittatum seems to be the major allergen for insect bite hypersensitivity sharing common IgE-binding epitopes with Ag5r protein from Culicoides nubeculosus [61], [62]. Specific anti-Ag5r IgE antibodies were also observed in Ugandan individuals bitten by Glossina morsitans [63]. Phylogenetic analysis of Ag5r proteins from sand flies and other insects showed a strongly supported distinct clade of sand fly Ag5r proteins (Figure 3) similar to a previous analysis by [28]. The relationship within the sand fly clade reflected phylogenetic relationship within phlebotomine sand flies [33], showing three distinct branches: clade I with species belonging to subgenera Euphlebotomus, Larroussius, and Adlerius; clade II with Phlebotomus and Paraphlebotomus species (P. papatasi, P. duboscqi, and P. sergenti); and Lutzomyia in clade III (Figure 3). 10.1371/journal.pntd.0001660.g003 Figure 3 Phylogenetic analysis of the antigen 5-related family of sand fly salivary proteins. Phylogenetic analysis of antigen 5-related proteins from Phlebotomus arabicus (Pab), P. argentipes (Pag), P. ariasi (Par), P. duboscqi (Pdu), P. papatasi (Pp), P. perniciosus (Ppe), P. sergenti (Ps), P. tobbi (Pt), Lutzomyia longipalpis (LJL), and antigen 5 sequences from Simulium vittatum, Culicoides nubeculosus, and Drosophila willistoni. Phylogenetic analysis was conducted on amino acid sequences without signal peptide using Tree Puzzle (version 5.2) by maximum likelihood (WAG model), quartet puzzling, and automatically estimated internal branch node support (10,000 replications). Sequence names, accession numbers, and branch node values are indicated. Apyrase Apyrase (EC 3.6.1.5) appears to be a universal enzyme used to prevent blood coagulation by diverse hematophagous animals such as bloodsucking leeches, ticks, triatomine bugs, fleas, and mosquitoes. This enzyme hydrolyses both ATP and ADP to AMP, thus destroying an important physiologic stimulus of platelet aggregation released from damaged tissues and blood cells. Apyrases of bloodsucking insects are divided into three families: CD-39 (the actin/heat shock 70/sugar kinase superfamily); 5′-nucleotidase; and Cimex-type [55], [56]. Sand flies are not an exception; transcripts coding for apyrases have been found in the saliva of all tested species [6], [23]–[28], including P. tobbi (PtSP4/HM135951, PtSP10/HM135952) and P. sergenti (PsSP40/HM560860, PsSP41/HM560862, PsSP42/HM560861) (Tables 1 and 2). The predicted molecular mass of the translated molecules is uniform for all sand fly species, varying between 35 and 36 kDa (Table S1). All sand fly apyrases deposited in GenBank have also been found in the proteomic analysis (Table S1). In P. tobbi and P. sergenti, the predicted molecular mass corresponds well with the molecular weight measured under non-reducing conditions (33.0–37.6 kDa) (Figure 1; Tables 1 and 2). Sand fly apyrases belong to the Cimex-type apyrase family. They hydrolyze ADP at a faster rate than ATP [64] and, similar to Cimex lectularius, the activity strictly depends on Ca2+ but not Mg2+ ions [6], [23], [64]–[66]. Apyrase activity has been demonstrated in the saliva of L. longipalpis [23], [64], P. argentipes [65], P. colabaensis [65], P. duboscqi [66] P. papatasi [65], [67], P. perniciosus [65], and as well as in recombinant apyrases of P. papatasi (PpApy/AF261768) [67] and P. duboscqi (PduApy2/DQ834331) [66]. Bacterially expressed P. duboscqi apyrase inhibited ADP- as well as collagen-induced platelet aggregation [66], indicating that post-translational modifications such as glycosylation are not necessary for apyrase activity. Orthologs of the Cimex apyrase family have also been identified in vertebrates and termed calcium-activated nucleotidases (CANs) [68]. In contrast to sand flies, human soluble CAN-1 (SCAN-1) preferentially hydrolyses UDP and GDP; however, the engineered SCAN-1 mutant Glu92Tyr shows five times and seven times higher hydrolysis activity for ADP and ATP, respectively [69]. This mutated tyrosine is conserved among species of the genus Phlebotomus (Figure 4), supporting its key role in substrate specificity for phlebotomine apyrases [69]. In human SCAN-1, other amino acid residues essential for binding nucleotide and Ca2+ were identified [70], some of them being absolutely conserved among the analyzed apyrase proteins (Asp44, Ser100, Asp114, Glu216, Arg232, Ser277), while others were uniformly mutated within sand fly apyrases (Asp101Glu, Gly160Ser, Ile214Trp) (Figure 4). 10.1371/journal.pntd.0001660.g004 Figure 4 Multiple sequence alignment of the apyrase family of salivary proteins. Multiple sequence alignment of sand fly apyrases from Phlebotomus arabicus (Ara), P. argentipes (Arg), P. ariasi (Ari), P. duboscqi (Dub), P. papatasi (Pap), P. perniciosus (Per), P. sergenti (Ser), P. tobbi (Tob), Lutzomyia longipalpis (Lon), and related sequences from Cimex lectularius and Homo sapiens. Sequences without signal peptide were aligned using ClustalX and manually refined using BioEdit sequence-editing software. Accession numbers are indicated in the sequence name. Identical amino acid residues are highlighted black and similar residues grey. Nucleotide binding sites (*) and Ca2+ binding sites (+), as predicted for human apyrase by Dai et al. [70], are indicated. The position of E92Y point mutation of human apyrase described by Yang and Kirley [69] is indicated by (•). Besides hydrolyzing activity, sand fly apyrases also possess antigenic properties. Antibodies from dogs experimentally or naturally exposed to P. perniciosus strongly recognized PpeSP01 (ABB00906) and PpeSP01B (ABB00907) apyrases [21]. In humans naturally exposed to sand flies, anti-sand fly saliva IgG antibodies recognized a protein band corresponding, in molecular weight, to apyrase [11], [12]. Moreover, antibodies elicited by P. duboscqi saliva also recognized bacterially expressed P. duboscqi apyrase [66], indicating that not all antibodies are specific for possible glycan modifications of sand fly apyrases. Phylogenetic analysis of sand fly apyrases reflects the same taxonomic relationship as Ag5r proteins. Figure 5 shows three distinct clades separating species in clade I (P. arabicus, P. argentipes, P. ariasi, P. perniciosus, P. tobbi) from Phlebotomus and Paraphlebotomus subgenera in clade II (P. papatasi, P. duboscqi, and P. sergenti), and genus Lutzomyia in clade III. This analysis showed a very close relationship within the Larroussius species, P. tobbi and P. perniciosus (Figure 5). 10.1371/journal.pntd.0001660.g005 Figure 5 Phylogenetic analysis of the apyrase family of sand fly salivary proteins. Phylogenetic analysis of apyrases from Phlebotomus arabicus (Pab), P. argentipes (Pag), P. ariasi (Par), P. duboscqi (Pdu), P. papatasi (Pp), P. perniciosus (Ppe), P. sergenti (Ps), P. tobbi (Pt), Lutzomyia longipalpis (Lulo), and related apyrase sequences from Cimex lectularius and Homo sapiens. Phylogenetic analysis was conducted on amino acid sequences without signal peptide using Tree Puzzle (version 5.2) by maximum likelihood (WAG model), quartet puzzling, and automatically estimated internal branch node support (10,000 replications). Sequence names, accession numbers, and branch node values are indicated. Hyaluronidase Hyaluronidase is an enzyme that catalyzes the hydrolysis of hyaluronic acid, a major component of the extracellular matrix in vertebrates. It is an ubiquitous enzyme found in mammals, bacteria and in the venom of bees, wasps, spiders, and snakes [71]. In bloodsucking Diptera, hyaluronidase activity has been found primarily in the saliva of telmophagic insects: horse flies, black flies, biting midges, and sand flies [72]. Thus, hyaluronidase is believed to decreases host skin tissue viscosity, assisting other salivary components to diffuse and create a pool of blood [60], [72], [73]. Sand fly hyaluronidase belongs to the same family as mammalian and Hymenopteran hyaluronidases (endo-β-N-acetyl-hexosaminidases, E.C. 3.2.1.35) and is different from that of bloodsucking leeches and nematodes (endo-β-glucuronidases, E.C. 3.2.1.36) [71], [74]. Hyaluronidase activity has been detected in all eight sand fly species studied to date ([23], [28], [53], [73], Figure 6). Our zymographic analyses of P. tobbi (Figure 6) and P. sergenti originating from Israel (Figure 6) and Turkey [53] showed the potent activity of sand fly hyaluronidase. Based on the microplate method, P. tobbi hyaluronidase activity is one of the highest measured (Figure 6A). In contrast, hyaluronidase of P. sergenti had the lowest activity among the species of the genus Phlebotomus (Figure 6A). Under non-reducing conditions, P. tobbi and P. sergenti hyaluronidase revealed diffuse bands with the molecular weight of around 110 and 135 kDa, respectively (Figure 6B). Hyaluronidase of P. sergenti is probably a homodimer, because under reducing conditions, the activity was observed at about half of the molecular weight, both in the Israeli (Figure 6B) and Turkish strains [53], while hyaluronidase of P. tobbi was monomeric with similar molecular weight under non-reducing and reducing conditions and the activity reduced to minimum when denaturated and treated with β-mercaptoethanol (Figure 6B). Similar features were observed for the hyaluronidase of P. perniciosus, the other Larroussius species ([53], Figure 6), which suggests common biochemical characteristics of this enzyme between closely related species. In general, the remarkably high activity of salivary hyaluronidase may aid the spread of other salivary components as well as transmitted pathogens. Indeed, hyaluronidase coinjected with Le. major promotes infection in BALB/c mice [72]; however, no association was found between hyaluronidase activity and the sand fly capacity to vector either cutaneous or visceral leishmaniasis (Figure 6A). 10.1371/journal.pntd.0001660.g006 Figure 6 Comparison of hyaluronidase activity in seven sand fly species. (A) Hyaluronidase activity was compared in the same species using salivary gland homogenate equivalent to 0.5 gland using the microtitration plate method. The results are expressed in relative Turbidity Reducing Units ± standard error, using bovine testicular hyaluronidase as a standard: L. longipalpis = 0.04±0.001 rTRU, P. papatasi = 0.20±0.01 rTRU, P. sergenti (Israel) = 0.07±0.001 rTRU, P. argentipes = 0.18±0.02 rTRU, P. arabicus = 0.16±0.01 rTRU, P. tobbi = 0.31±0.04 rTRU, P. perniciosus = 0.24±0.03 rTRU. Three independent experiments were done. (B) SDS-PAGE zymography assay under reducing and non-reducing conditions on 8% polyacrylamide gel with incorporated hyaluronan for detection of hyaluronidase activity in salivary gland homogenate of seven sand fly species: Lutzomyia longipalpis (Lon), Phlebotomus papatasi (Pap), P. sergenti (Ser), P. argentipes (Arg), P. arabicus (Ara), P. tobbi (Tob), and P. perniciosus (Per). Although sand fly hyaluronidase is a very potent enzyme, it is scarcely found in transcriptomic and proteomic approaches probably due to the low abundance of transcripts combined with the large size of the protein. Hyaluronidase transcripts have been reported in only two of seven salivary cDNA libraries, namely in L. longipalpis and P. arabicus [23], [24], [28]. In P. sergenti, no transcript was found, and in P. tobbi, only one 3′-truncated transcript was identified (PtSP125/JN192442). Amino acid residues that constitute the catalytic site (Asp111, Glu113, and Glu247) and form disulfide bridges (Cys22–Cys313 and Cys189–Cys201) in bee hyaluronidase [75] are conserved among the sand fly hyaluronidase sequences (Figure 7). Based on the NetNGlyc prediction server [48], several putative glycosylation sites were predicted in sand fly hyaluronidases, including one highly conserved among aligned sequences (Figure 7). 10.1371/journal.pntd.0001660.g007 Figure 7 Multiple sequence alignment of the hyaluronidase family of salivary proteins. Multiple sequence alignment of hyaluronidases from Phlebotomus arabicus (Ara), P. tobbi (Tob), Lutzomyia longipalpis (Lon), and related sequences from Apis mellifera, Culicoides nubeculosus (CUL), Tabanus yao (TAB), Anoplius samariensis (ANO), and Homo sapiens. Sequences without signal peptide were aligned using ClustalX and manually refined using BioEdit sequence-editing software. Accession numbers are indicated in the sequence name. Identical amino acid residues are highlighted black and similar residues grey. Active site residues (*) and cysteine residues forming disulfide bridges (C) as predicted for Apis hyaluronidase by Markovic-Housley et al. [75] are indicated. Red residues (N) denote predicted N-glycosylation sites, including one (+) highly conserved among aligned sequences. Allergenic properties of sand fly hyaluronidase are not known, although it has been identified or suspected as the main allergen in the saliva of other bloodsucking Diptera, namely biting midges and horseflies [59], [76]. However, there is no record of typical IgE-mediated allergic reaction to sand fly saliva; only negligible amount of anti-saliva IgE was measured in hosts repeatedly bitten by sand flies [11], [19], [77]. Odorant binding-related proteins Two sand fly salivary protein families, D7-related proteins and PpSP15-like proteins, are related to the arthropod pheromone/odorant binding protein superfamily (OBP, protein domain PBP-GOBP, pfam01395) [56], [78]. D7-related (D7r) proteins are named after the D7 protein, originally described in Aedes aegypti as a major salivary protein exclusively synthesized in bloodsucking females [79], [80]. Salivary proteins related to D7 have also been found in black flies and biting midges [56], [78] and all sand fly species studied to date. In the P. tobbi SG cDNA library we found seven clusters homologous to D7r sequences (HM164145–HM164151) and three clusters in the P. sergenti cDNA library (PsSP4/HM560863, PsSP5/HM569360, PsSP7/HM560864) (Tables 1 and 2). Within all sand flies, D7r proteins have similar predicted molecular mass (25.3–28.1 kDa) and wide range of pI (4.82–9.5) (Table S1). Based on the results from NetNGlyc prediction server [48], we found a mixture of putative glycosylated and non-glycosylated D7r sequences in most of the sand fly species studied with the exception of L. longipalpis, P. sergenti, and P. papatasi, where no N-glycosylation sites were found (Figure 8). All sand fly D7r predicted proteins contain nine highly conserved cysteine residues (Figure 8), implying there is a single non-disulphide-bond-forming cysteine. 10.1371/journal.pntd.0001660.g008 Figure 8 Multiple sequence alignment of the D7-related family of salivary proteins. Multiple sequence alignment of sand fly D7-related proteins from Phlebotomus arabicus (Ara), P. argentipes (Arg), P. ariasi (Ari), P. duboscqi (Dub), P. papatasi (Pap), P. perniciosus (Per), P. sergenti (Ser), P. tobbi (Tob), Lutzomyia longipalpis (Lon), and related sequence from Anopheles stephensi (Ans). Sequences without signal peptide were aligned using ClustalX and manually refined using BioEdit sequence-editing software. Accession numbers are indicated in the sequence name. Identical amino acid residues are highlighted black and similar residues grey. The cysteinyl leukotriene binding motif [56] is indicated by (*). The other family related to OBPs, PpSP15-like proteins, is closely related to larger D7-related proteins [25], [56] and are named after 15-kDa salivary protein of P. papatasi (PpSP15/AF335487) [6]. They have not been identified in any Diptera other than sand flies [25], [56]. It is the most abundant family among sand fly salivary proteins, and P. tobbi and P. sergenti are not exceptions; six and seven members of this family were found in each cDNA library, respectively (Tables 1 and Table 2). Several members were also detected by proteomic analysis, having similar molecular mass as predicted based on the amino acid sequences (Tables 1 and Table 2; Figure 1). Within the sand flies, PpSP15-like proteins have a similar predicted molecular mass (12.2–17.1 kDa) and surprisingly wide range of pI (6.33–9.44) (Table S1). In accordance with previous reports [25], [28], all sand fly PpSP15-like proteins show high degree of variability of around six conserved cystine residues (Figure S2). In mosquitoes, some salivary D7 strongly bind biogenic amines and leukotrienes as well as components of the coagulation cascade, thus promptly antagonizing the host defense system [81]–[83]. D7r and PpSP15-like sand fly salivary proteins have not yet been characterized functionally; however, the motif [ED]-[EQ]-x(7)-C-x(12,17)-W-x(2)-W-x(7,9)-[TS]-x-C-[YF]-x-[KR]-C-x(8,22)-Q-x(22,32)-C-x(2)-[VLI], found in mosquito D7 salivary proteins that bind cysteinyl leukotrienes [83], is also found in the sand fly D7r proteins (Figure 8). Sand fly PpSP15-like proteins and D7r proteins possess antigenic properties. PpSP15-like proteins were reported as promising anti-Leishmania vaccine candidates [6], [27], [84]. Phlebotomus papatasi SP15 protein is able to protect mice against Le. major challenge, and a DNA vaccine containing the PpSP15 cDNA provided the same protection [6]. ParSP03 (AAX56359), a PpSP15-like protein from P. ariasi, elicited similar delayed-type hypersensitivity and humoral immune responses upon DNA vaccination [27]. D7r could serve as a marker of exposure to sand fly bites. In humans, all tested serum samples from individuals naturally exposed to P. papatasi strongly bound to a P. papatasi protein band with a molecular mass corresponding to PpSP30 D7r protein (AAL11049) [12], [18]. As an ideal marker of exposure, this protein was recognized by both IgE and IgG antibodies, including all tested IgG subclasses [18]. D7r proteins seem to be applicable also for measurement of dog exposure, the main reservoir host for visceral leishmaniasis, since IgG antibodies from animals bitten by P. perniciosus [21] or L. longipalpis [16], [85] recognized D7r proteins of the respective species (PpeSP4/DQ150623, PpeSP04B/DQ150624, PpeSP10/DQ153104, LJL13/AF420274). Moreover, L. longipalpis-bitten dogs bind also to the LJL13 D7r recombinant form [16]. Phylogenetic analysis of D7r proteins showed several major clades (Figure 9). Phlebotomus sergenti sequences clustered together forming a distinct subclade within clade III that contains P. papatasi and P. duboscqi. In contrast, P. tobbi D7r protein sequences are divided among clades I and II, which contain sequences from P. arabicus, P. ariasi, P. argentipes, P. perniciosus, and L. longipalpis. Interestingly, clade II only contained sequences with predicted N-glycosylation sites, which may suggest a unique functional characteristic of D7 molecules within this clade that have arisen after gene duplication. Similarly, phylogenetic analysis of PpSP15-like proteins (Figure 10) revealed several separated groups, consistently clustering P. sergenti sequences with P. duboscqi and P. papatasi, and P. tobbi sequences with those from P. perniciosus and other sand flies studied to date, including a single member from L. longipalpis. PpSP15 could be a multicopy gene, as more than two alleles were found in several P. papatasi individuals, some of them unique to the population origin [86]. 10.1371/journal.pntd.0001660.g009 Figure 9 Phylogenetic analysis of the D7-related family of sand fly salivary proteins. Phylogenetic analysis of D7-related proteins from Phlebotomus arabicus (Pab), P. argentipes (Pag), P. ariasi (Par), P. duboscqi (Pdu), P. papatasi (Pp), P. perniciosus (Ppe), P. sergenti (Ps), P. tobbi (Pt), Lutzomyia longipalpis (LJL), and related sequences from Anopheles gambiae (D7r4) and Simulium vittatum. Phylogenetic analysis was conducted on amino acid sequences without signal peptide using Tree Puzzle (version 5.2) by maximum likelihood (WAG model), quartet puzzling, and automatically estimated internal branch node support (10,000 replications). Sequence names, accession numbers, and branch node values are indicated. Underlined sequences possess predicted N-glycosylation sites. 10.1371/journal.pntd.0001660.g010 Figure 10 Phylogenetic analysis of the PpSP15-like family of sand fly salivary proteins. Phylogenetic analysis of the PpSP15-like proteins from Phlebotomus arabicus (Pab), P. argentipes (Pag), P. ariasi (Par), P. duboscqi (Pdu), P. papatasi (Pp), P. perniciosus (Ppe), P. sergenti (Ps), P. tobbi (Pt), Lutzomyia longipalpis (Lulo), and related sequence from Anopheles gambiae (XP_551869). Phylogenetic analysis was conducted on amino acid sequences without signal peptide using Tree Puzzle (version 5.2) by maximum likelihood (WAG model), quartet puzzling, and automatically estimated internal branch node support (10,000 replications). Sequence names, accession numbers, and branch node values are indicated. PpSP32-like proteins This family is named PpSP32 from the original identification in P. papatasi (AAL11050) [24] and due to the lack of homology to a conserved protein domain. PpSP32-like proteins have been described solely in sand flies and are found in all species studied so far; we identified homologous sequences also in P. tobbi (PtSP27/HM173642, PtSP28/HM173643, PtSP29/HM173644) and P. sergenti (PsSP44/HM569368). The predicted molecular mass of P. tobbi PpSP32-like proteins (24.5 kDa) is slightly lower than what was measured in proteomic analysis (Figure 1, Tables 1 and 2). All sequences have a wide range of predicted molecular mass (ranging from 22.5 to 34.9 kDa), no protein domain match, and are alkalic (pI ranging from 9.3 to 10.6) (Table S1). An interesting common feature of this protein family is that it possesses highly conserved N- and C- terminal regions with extremely variable internal sequence (Figure 11). Within the genus Phlebotomus there are predicted N-glycosylation sites in the variable and C-terminal regions (Figure 11). 10.1371/journal.pntd.0001660.g011 Figure 11 Multiple sequence alignment of the PpSP32-like family of salivary proteins. Multiple sequence alignment of sand fly PpSP32-like proteins from Phlebotomus arabicus (Ara), P. argentipes (Arg), P. ariasi (Ari), P. duboscqi (Dub), P. papatasi (Pap), P. perniciosus (Per), P. sergenti (Ser), P. tobbi (Tob), and Lutzomyia longipalpis (Lon). Sequences without signal peptide were aligned using ClustalX and manually refined using BioEdit sequence-editing software. Accession numbers are indicated in the sequence name. Identical amino acid residues are highlighted black and similar residues grey. Red residues (N) denote predicted N-glycosylation sites. To date, no function has been associated with sand fly PpSP32-like proteins, although L. longipalpis and P. perniciosus proteins have been hypothesized to possess collagen binding activity [24], [25] and in P. papatasi, PpSP32 transcripts are expressed independently of either diet or age [87], indicating a vital role for these molecules in feeding. Phylogenetic analysis of PpSP32-like proteins reflects again the taxonomic relationship within Phlebotomine sand flies [33]. True to form, phylogenetic position of P. tobbi PpSP32-like proteins are within a subclade I with P. perniciosus and the P. sergenti PpSP32-like protein is within the Phlebotomus and Paraphlebotomus clade II (Figure 12). 10.1371/journal.pntd.0001660.g012 Figure 12 Phylogenetic analysis of the PpSP32-like family of sand fly salivary proteins. Phylogenetic analysis of PpSP32-like proteins from Phlebotomus arabicus (Pab), P. argentipes (Pag), P. ariasi (Par), P. duboscqi (Pdu), P. papatasi (Pp), P. perniciosus (Ppe), P. sergenti (Ps), P. tobbi (Pt), and Lutzomyia longipalpis (LJL). Phylogenetic analysis was conducted on amino acid sequences without signal peptide using Tree Puzzle (version 5.2) by maximum likelihood (WAG model), quartet puzzling, and automatically estimated internal branch node support (10,000 replications). Sequence names, accession numbers, and branch node values are indicated. Yellow-related proteins Phlebotomine yellow-related proteins are characterized by the presence of major royal jelly protein domain (MRJP; pfam03022). Originally, MRJP proteins were described from honeybee larval jelly, making up to 90% of the protein content [88]. Sequences related to MRJP proteins were described in Drosophila, where it is related to cuticle pigmentation and, when mutated, it produced a yellow phenotype and thus named Yellow proteins [89], [90]. In bloodsucking Diptera, salivary yellow-related proteins have only been described in sand flies [55], [56] and black flies [91]. Yellow-related proteins are found in all sand fly species studied to date. In the P. sergenti cDNA library, five different clusters were found (PsSP18/HM569361, PsSP19/HM560865, PsSP20/HM560866, PsSP22/HM560867, PsSP26/HM569362) compared with P. tobbi, where only two clusters were found (PtSP37/HM140618 and PtSP38/HM140619) (Tables 1 and 2). Sand fly yellow-related proteins have a similar predicted molecular mass (41.5–45.2 kDa), wide range of pI (4.75–9.8), and contain four conserved cysteine residues shown to form two disulfide bonds in LJM11 (AAS05318) [9] (Table S1, Figure 13). Yellow-related proteins are modulated on a transcriptional level [87] and are likely post-translationally modified, as variants with different mobility have been detected on SDS-PAGE [6], [25] (Figure 13). 10.1371/journal.pntd.0001660.g013 Figure 13 Multiple sequence alignment of the yellow-related family of salivary proteins. Multiple sequence alignment of yellow-related proteins from Phlebotomus arabicus (Ara), P. argentipes (Arg), P. ariasi (Ari), P. duboscqi (Dub), P. papatasi (Pap), P. perniciosus (Per), P. sergenti (Ser), P. tobbi (Tob), Lutzomyia longipalpis (Lon), and related sequence from Drosophila simulans (XP_002103634). Sequences without signal peptide were aligned using ClustalX and manually refined using BioEdit sequence-editing software. Accession numbers are indicated in the sequence name. Identical amino acid residues are highlighted black and similar residues grey. Red residues (N) denote predicted N-glycosylation sites. The span of the MRJP protein domain (pfam03022) is marked by arrows. Based on the crystal structure of Lon AAS05318 [9], the cystine residues forming disulfide bonds are indicated by letter C and conserved amino acids contained in the ligand binding pocket by an asterisk (*). Ribeiro and Arca [55] proposed that in Phlebotomines, salivary yellow-related proteins work as kratagonists, the binders of biogenic amines. Indeed, Xu et al. [9] proved that the bacterially expressed L. longipalpis yellow-related proteins (LJM11, LJM17/AAD32198, and LJM111/ABB00904) bind biogenic amines, namely serotonin, catecholamines, and histamine. The proteins differed in affinity to the particular ligand, suggesting functional divergence within the family [9]. The midgut yellow protein in Aedes aegypti is involved in the melanization pathway as a dopachrome conversion enzyme [92]; however, in sand flies the yellow-related proteins found in the midgut lumen probably originating from swallowed saliva [93] and researchers failed to detect dopachrome convertase activity in salivary yellow-related proteins [28], [56]. In Glossina morsitans, the ubiquitous tissue expression of the protein suggests also a housekeeping role for yellow-related proteins [91]. Sand fly salivary yellow proteins possess antigenic properties as they are recognized by serum antibodies of experimentally bitten mice [12] and dogs [19], [21], as well as naturally exposed dogs, humans, and foxes [11], [16], [18], [21], [77], [85]. Additionally, a combination of recombinant LJM17 and LJM11 successfully substituted L. longipalpis whole SG sonicate in probing sera of individuals for vector exposure [16], [20]. Yellow proteins are also under consideration for anti-Leishmania vector-based vaccines. LJM17 from L. longipalpis elicited leishmanicidal Th1 cytokines in immunized dogs [8], and LJM11 protected laboratory animals against both Le. major and Le. infantum [7], [9]. In contrast, mice immunized with P. papatasi yellow-related proteins PpSP42 or PpSP44 (AAL11052 and AAL11051, respectively) elicited Th2 cytokines and exacerbated Le. major infection [84]. It remains to be elucidated whether the protection induced by yellow-related proteins is related to particular protein immunogenicity, to sand fly species, or to the vector-Leishmania-host combination, as all of these factors can contribute to vaccine efficacy. Recently, Xu et al. [9] showed that L. longipalpis LJM11 but not LJM111 produces a DTH response in mice challenged by SGH. The authors related this immunogenicity to electrostatic potential on the protein surface, which is positive in LJM11; thus the protein is probably more attractive to antigen-presenting cells [9]. Yellow-related proteins are highly conserved among sand flies. Phylogenetic analysis produced three major clades combining Larroussius, Adlerius and Euphlebotomus (clade I); Phlebotomus and Paraphlebotomus (clade II); and Lutzomyia (clade III), while subclades discerned each subgenus (Figure 14). Interestingly, P. sergenti illustrates a gene duplication event that preceded speciation and was followed by a clear gene duplication expansion that is seen in one of the subclades. Gene duplication in bloodsucking arthropod salivary molecules is fundamental for the functional diversification of proteins, as can be seen with the range of substrates bound by the L. longipalpis yellow-related proteins [9]. Within clade II, two subclades can be seen distinguishable by the presence of putative N-glycosylation sites. Moreover, sequences in clade IIa have a slightly higher predicted isoelectric point than the glycosylated sequences in clade IIb (Figure 14, Table S1), indicating another feature that might be responsible for functional diversification. 10.1371/journal.pntd.0001660.g014 Figure 14 Phylogenetic analysis of the yellow-related family of sand fly salivary proteins. Phylogenetic analysis of yellow-related proteins from Phlebotomus arabicus (Pab), P. argentipes (Pag), P. ariasi (Par), P. duboscqi (Pdu), P. papatasi (Pp), P. perniciosus (Ppe), P. sergenti (Ps), P. tobbi (Pt), Lutzomyia longipalpis (Lulo or LJM), and related sequence from Drosophila simulans (XP_002103634). Phylogenetic analysis was conducted on amino acid sequences without signal peptide using Tree Puzzle (version 5.2 by) maximum likelihood (WAG model), quartet puzzling, and automatically estimated internal branch node support (10,000 replications). Sequence names, accession numbers, and branch node values are indicated. Underlined sequences possess predicted N-glycosylation sites. ParSP25-like proteins ParSP25-like transcripts were found in P. tobbi but not in P. sergenti SG library. Phlebotomus tobbi ParSP25-like molecules (PtSP73/HM173639, PtSP75/HM173640, and PtSP76/HM173641) have predicted molecular mass ranging from 27.8 to 38.8 kDa and contain a large proportion of acidic residues resulting in a pI of 4.5±0.1. The sequences share similarity with eight other sand fly salivary proteins from three sand fly species [25], [27], [28] (Figure 15), all of them with predicted pI between 4.4 and 5.0 (Table S1). Analysis of the putative protein sequences revealed highly conserved regions rich in amino acid residues such as Asp, Tyr, Glu, and Ser and no predicted N-glycosylation sites (Figure 15). Though the function is not known, some members of this family were shown to be highly antigenic. Mice immunized with a plasmid coding for ParSP25 (AAX55664) elicited high levels of anti-P. ariasi IgG1 and a strong DTH reaction when challenged with P. ariasi saliva [27]. Moreover, dogs exposed to P. perniciosus bites strongly bind to protein band characterized as PpeSP08 (ABA43056) [21]. Sand fly ParSP25-like proteins are most likely genus-specific because, so far, the sequences have been found only in Adlerius (P. arabicus) and Larroussius species (P. ariasi, P. perniciosus, P. tobbi) and not in representatives of the other subgenera (Figure 15). 10.1371/journal.pntd.0001660.g015 Figure 15 Multiple sequence alignment of the ParSP25-like family of sand fly salivary proteins. Multiple sequence alignment of Phlebotomus tobbi PtSP73 (HM173639), PtSP75 (HM173640), and PtSP76 (HM173641) with related sequences from P. arabicus (Ara), P. ariasi (Ari), and P. perniciosus (Per). Sequences without signal peptide were aligned using ClustalX and manually refined using BioEdit sequence-editing software. Accession numbers are indicated in the sequence name. Identical amino acid residues are highlighted black and similar residues grey. The 33-kDa family These proteins, named by Anderson et al. [25] as members of the 33-kDa family, have not yet been found in any Diptera other than sand flies. PsSP49 (HM569369) and PtSP66 (HM173645) share sequence similarity with seven other sand fly salivary proteins from six sand fly species both from both New and Old World sand flies [24]–[28] (Figure 16). All sand fly 33-kDa family proteins have similar predicted molecular weight (32.3–34.5 kDa) and alkalic pI (8.2–9.1) (Table S1). PsSP49 and PtSP66 were both identified in the proteomic analysis (Figure 1). Two highly conserved N-glycosylation sites were predicted among all sand fly sequences (Figure 16) and both PsSP49 and PtSP66 were found above the predicted molecular weight in the proteomic analysis (Figure 1, Tables 1 and 2), indicating a post-translational modification. Indeed, the two proteins from P. arabicus (PabSP32/ACS93510 and PabSP34/ACS93511) showed glycosylation by ProQ Emerald staining [28]. The function is unknown; however, P. perniciosus PpeSP06 (ABA43054) and the L. longipalpis LJL143 (AAS05319) were identified as antigens for dogs living in endemic areas of Le. infantum [16], [21], the later one also shown to be a candidate for vaccine against canine leishmaniasis [8]. 10.1371/journal.pntd.0001660.g016 Figure 16 Multiple sequence alignment of the 33-kDa salivary protein family. Multiple sequence alignment of Phlebotomus sergenti PsSP49 (HM569369) and P. tobbi PtSP66 (HM173645) proteins with related sequences from P. arabicus (Ara), P. argentipes (Arg), P. ariasi (Ari), P. duboscqi (Dub), P. perniciosus (Per), and Lutzomyia longipalpis (Lon). Sequences without signal peptide were aligned using ClustalX and manually refined using BioEdit sequence-editing software. Accession numbers are indicated in the sequence name. Identical amino acid residues are highlighted black and similar residues grey. Red residues (N) denote predicted N-glycosylation sites. 41.9-kDa superfamily 41.9-kDa protein superfamily is specific to bloodsucking Nematocera encompassing members of mosquitoes, biting midges, black flies, and sand flies [56]. The P. sergenti and P. tobbi members of this superfamily, PsSP82 (HM569371) and PtSP49 (HM173648), share sequence similarity with five other sand fly salivary proteins from five sand fly species (Figure 17). These sand fly proteins have a wide range of predicted molecular weight (27.5–56.6 kDa) and pI (4.3–8.5) (Table S1) but only one of them, P. perniciosus PpeSP19 (ABA43063), has been found by proteomic analysis [25]. All sequences are rich in putative N-glycosylation sites (Figure 17) and the function is not known. 10.1371/journal.pntd.0001660.g017 Figure 17 Multiple sequence alignment of the sand fly members of 41.9-kDa salivary protein superfamily. Multiple sequence alignment of Phlebotomus sergenti PsSP82 (HM569371) and P. tobbi PtSP49 (HM173648) proteins with related sequences from P. arabicus (Ara), P. ariasi (Ari), P. duboscqi (Dub), P. perniciosus (Per), and Lutzomyia longipalpis (Lon). Sequences without signal peptide were aligned using ClustalX and manually refined using BioEdit sequence-editing software. Accession numbers are indicated in the sequence name. Identical amino acid residues are highlighted black and similar residues grey. Red residues (N) denote predicted N-glycosylation sites. Other putative salivary proteins Several other putative salivary proteins were identified in the transcriptomes of P. tobbi and P. sergenti SGs. They are smaller than 15 kDa, their function is not known, and are, thus far, unique to sand flies. Additionally, none of these small proteins have been found in the proteomic analysis (Figure 1, Tables 1 and 2). PsSP28 (HM569370), PtSP8 (HM173646), and PtSP81 (HM173647) share sequence similarity with P. ariasi ParSP23 (AAX55663) and P. perniciosus PpeSP15 (ABB00905) (Figure 18). The proteins have a low predicted molecular weight (2.4–5.0 kDa) and an alkalic pI (9.2–10.7). 10.1371/journal.pntd.0001660.g018 Figure 18 Multiple sequence alignment of the PsSP28, PtSP8, and PtSP81 salivary proteins. Multiple sequence alignment of Phlebotomus sergenti PsSP28 (HM569370) and P. tobbi PtSP8 (HM173646) and PtSP81 (HM173647) proteins with related sequences from P. ariasi (Ari) and P. perniciosus (Per). Sequences without signal peptide were aligned using ClustalX and manually refined using BioEdit sequence-editing software. Accession numbers are indicated in the sequence name. Identical amino acid residues are highlighted black and similar residues grey. PsSP98 (HM569366) has a predicted molecular weight similar to PpSP15-like proteins (14.3 kDa) but is highly acidic (pI = 4.73). The protein sequence is related to 16-kDa proteins from P. arabicus (PabSP64/ACS93507, PabSP63/ACS93506) and P. argentipes (PagSP73/ABA12153) (Figure 19). 10.1371/journal.pntd.0001660.g019 Figure 19 Multiple sequence alignment of the Phlebotomus sergenti PsSP98 salivary protein. Multiple sequence alignment of Phlebotomus sergenti PsSP98 protein (HM569366) with related sequences from P. arabicus (Ara) and P. argentipes (Arg). Sequences without signal peptide were aligned using ClustalX and manually refined using BioEdit sequence-editing software. Accession numbers are indicated in the sequence name. Identical amino acid residues are highlighted black and similar residues grey. PsSP73 (HM569367) has a predicted molecular weight 12.2 kDa and is highly acidic (pI = 4.51). The predicted protein sequence is related to proteins found in P. arabicus (PabSP75/ACS93508) and P. ariasi (ParSP13/AAX55657) (Figure 20). 10.1371/journal.pntd.0001660.g020 Figure 20 Multiple sequence alignment of the Phlebotomus sergenti PsSP73 salivary protein. Multiple sequence alignment of Phlebotomus sergenti PsSP73 protein (HM569367) with related sequences from P. arabicus (Ara) and P. ariasi (Ari). Sequences without signal peptide were aligned using ClustalX and manually refined using BioEdit sequence-editing software. Accession numbers are indicated in the sequence name. Identical amino acid residues are highlighted black and similar residues grey. PtSP71 (HM173638) has a low predicted molecular weight (4.5 kDa) and an alkalic pI (10.6). The protein sequence is related to molecules identified in P. perniciosus (PpeSP12/ABA43060, PpeSP13/ABA43061) and P. ariasi (ParSP15/AAX55658) (Figure 21), indicating these sequences might be unique to Larroussius species. 10.1371/journal.pntd.0001660.g021 Figure 21 Multiple sequence alignment of the Phlebotomus tobbi PtSP71 salivary protein. Multiple sequence alignment of Phlebotomus tobbi PtSP73 protein (HM173639) with related sequences from P. ariasi (Ari) and P. perniciosus (Per). Sequences without signal peptide were aligned using ClustalX and manually refined using BioEdit sequence-editing software. Accession numbers are indicated in the sequence name. Identical amino acid residues are highlighted black and similar residues grey. Antigens and glycoproteins of Phlebotomus tobbi salivary proteins To identify antigens and glycoproteins in P. tobbi SGH, electrophoretically separated proteins were incubated with anti-P. tobbi rabbit serum and a lectin Concanavalin A (ConA), respectively (Figure 22). When compared with the proteome analysis in Figure 1, the protein bands visible by silver staining are most likely yellow-related proteins (PtSP37 and PtSP38), apyrases (PtSP4 and PtSP10), antigen 5-related proteins (PtSP77 and PtSP79), PpSP32-like proteins (PtSP28 and PtSP29), D7-related proteins (PtSP58 and PtSP60), and PpSP15-like proteins (PtSP9, PtSP23, and PtSP32). Anti-P. tobbi antibodies recognized all identified bands as well as other six high molecular weight proteins not visible by silver staining (Figure 22, lane 2). 10.1371/journal.pntd.0001660.g022 Figure 22 Phlebotomus tobbi salivary gland antigens and glycoproteins. Salivary gland homogenate of Phlebotomus tobbi was separated on 8% polyacrylamide gel under non-reducing conditions. Separated proteins were silver stained (lane 1) or electrotransferred to nitrocellulose membrane and incubated with (2) serum from rabbit repeatedly exposed to bites of P. tobbi females, (3) non-immune rabbit serum, (4) Concanavalin A lectin, or (5) ConA preincubated with methyl-α-D-mannopyranoside to control specificity of the ConA reaction. Probable cluster name as compared to mass spectrometry results in Figure 1 and molecular weight (kDa) are indicated on the left and right side, respectively. Most of the P. tobbi proteins reacted with ConA, indicating they are N-glycosylated. The lectin binding was specific, as the reactivity was totally inhibited when ConA was preincubated with specific monosaccharide methyl-α-D-mannopyranoside. The most intense reaction was observed with the high molecular weight band not visible by silver staining, and with the bands of molecular weight similar to one yellow-related protein (PtSP38) and both apyrases. Among the nine silver-stained bands, three did not react with ConA, namely bands with molecular weight similar to D7-related proteins, PpSP15-like proteins, and one yellow-related protein (PtSP37) (Figure 22, lane 4). The reactivity with ConA is in agreement with N-glycosylation as predicted by NetNGlyc server [48], with the exception of PtSP10 apyrase (Table 3). 10.1371/journal.pntd.0001660.t003 Table 3 N-glycosylation sites of Phlebotomus tobbi salivary proteins. Cluster name GenBank Accn Comment kDa Position Potential score Jury agreement N-Glyc result ConA result PtSP38 HM140619 yellow-related 41.7 29 NISY 0.6242 8/9 + + PtSP37 HM140618 yellow-related 39.2 No sites predicted in this sequence − PtSP10 HM135952 apyrase 37.6 No sites predicted in this sequence + PtSP4 HM135951 apyrase 33.0 163 NISK 0.6696 9/9 ++ + 248 NFSD 0.5678 8/9 + + PtSP79 HM140622 antigen 5-related 29.6 160 NITR 0.6879 9/9 ++ + PtSP77 HM140620 antigen 5-related 29.6 159 NITR 0.6931 9/9 ++ + PtSP29 HM173644 PpSP32-like 29.6 111 NPTG* 0.6866 9/9 ++ + PtSP28 HM173643 PpSP32-like 29.6 111 NPTG* 0.6868 9/9 ++ + PtSP60 HM164150 D7-related 24.5 No sites predicted in this sequence − PtSP58 HM164149 D7-related 24.5 No sites predicted in this sequence − PtSP32 HM164144 PpSP15-like 13.5 No sites predicted in this sequence − PtSP23 HM164142 PpSP15-like 13.5 No sites predicted in this sequence − PtSP9 HM164139 PpSP15-like 13.5 No sites predicted in this sequence − Selected mature proteins from Phlebotomus tobbi were subjected to the NetNGlyc 1.0 Server [48] using the default setting (by default, predictions are done only on the Asn-Xaa-Ser/Thr sequons and the treshold is set up at 0.5). Cluster names and GenBank accession numbers are indicated. Molecular weight (kDa) was calculated based on the Figure 1A. The Position column defines predicted glycosylated sites. The Potential score is the averaged output of nine neural network, and the Jury agreement column indicates how many of the nine networks support the prediction. The N-Glyc Result column shows putative glycosylated sites: + denotes Potential >0.5, ++ Potential >0.5 and Jury agreement (9/9) or Potential >0.75, +++ Potential >0.75 and Jury agreement, ++++ Potential >0.90 and Jury agreement. *: Proline occurs just after the asparagine residue that makes it highly unlikely that the asparagine is glycosylated, presumably due to conformational constraints. ConA column shows reactivity with lectin concanavalin A based on the Figure 22, lane 4. We can speculate that the most glycosylated band with the highest molecular weight might be hyaluronidase. Although producing a minor unstainable band, it is predicted to be highly glycosylated (Figure 7) and its activity is clearly visible around 135 kDa in zymography analyses (Figure 6). Within sand fly yellow-related proteins, it is common that glycosylated and non-glycosylated forms occur in the same species. As proved for P. papatasi [93] and predicted for protein sequences of Phlebotomus (P. papatasi and P. duboscqi) and Paraphlebotomus (P. sergenti) species, at least one form is glycosylated, forming a well supported subclade with glycosylated sequences from other species (Figure 14). Glycosylated and non-glycosylated forms are also present in P. tobbi, as proven by blot analysis (Figure 22), although the closely related P. perniciosus possesses only glycosylated forms. Interestingly, in sand fly species within the clades I and III (Figure 14), all published sequences are glycosylated with an exception of P. tobbi and P. ariasi, which has at least one non-glycosylated form. Further research is needed to investigate whether the presence of sugar side chains may contribute to the antigenicity of the yellow-related proteins. Conclusions With over 80 species of sand flies implicated in Leishmania transmission, it is vital to continue describing their salivary proteins in the search for vaccine candidates and markers of exposure. In this study, we prepared and analyzed the transcriptome and proteome data of P. tobbi and P. sergenti to broaden our knowledge on the repertoire of Larroussius salivary proteins and provide the first report from a Paraphlebotomus sand fly, respectively. P. tobbi has been reported to transmit Le. infantum that causes cutaneous leishmaniasis [32]. Interestingly, the salivary proteins of P. tobbi are highly homologous to those of P. perniciosus, a vector of Le. infantum that causes visceral disease. It is likely that, in this instance, the salivary proteins of P. tobbi are not the determining factor for these different disease manifestations. However, in general, it is possible that the divergence, diversity or amount of sand fly salivary proteins or non proteinaceous components of the saliva correlate with different disease manifestations of the same species of Leishmania. The transcriptome data can be utilized to prepare recombinant proteins that can be used to test their potential as anti-Leishmania vaccines or in epidemiologic studies to develop more specific and efficient methods for measurement of vector exposure. Finally, recombinant salivary proteins may also help us to understand the mechanism of blood sucking or find biological activities of many of these novel sequences. Supporting Information Figure S1 Phlebotomus tobbi and P. sergenti protein families. Analysis of salivary proteins from Phlebotomus tobbi (Pt) and P. sergenti (Ps). Phylogenetic analysis was conducted on amino acid sequences with signal peptide using Tree Puzzle (version 5.2) by maximum likelihood (WAG model), quartet puzzling, and automatically estimated internal branch node support (10,000 replications). Sequence cluster names and branch node values are indicated. Protein families are listed on the right. (TIFF) Click here for additional data file. Figure S2 Multiple sequence alignment of the PpSP15-like family of salivary proteins. Multiple sequence alignment of the PpSP15-like proteins from Phlebotomus arabicus (Ara), P. argentipes (Arg), P. ariasi (Ari), P. duboscqi (Dub), P. papatasi (Pap), P. perniciosus (Per), P. sergenti (Ser), P. tobbi (Tob), and Lutzomyia longipalpis (Lon). Sequences without signal peptide were aligned using ClustalX and manually refined using BioEdit sequence-editing software. Accession numbers are indicated in the sequence name. Identical amino acid residues are highlighted black and similar residues grey. (TIFF) Click here for additional data file. Table S1 List of sand fly salivary proteins with their identifiers and selected protein features. Published sand fly salivary proteins from Phlebotomus arabicus (Ara), P. argentipes (Arg), P. ariasi (Ari), P. duboscqi (Dub), P. papatasi (Pap), P. perniciosus (Per), P. sergenti (Ser), P. tobbi (Tob), and Lutzomyia longipalpis (Lon). The proteins are listed with their house name, GenBank accession numbers (Accn) for both nucleotide and protein sequences, protein name, presence in the proteome analysis as confirmed by mass spectrometry or Edman degradation, protein family, predicted signal peptide (SignalP), putative mature protein features (pI, predicted isoelectric point; Mw, predicted molecular weight; AA, number of amino acid residues), and reference. (XLS) Click here for additional data file. Text S1 Accession numbers for genes and proteins mentioned in the text including tables, figure and supplemental files. (DOC) Click here for additional data file.