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.
Table 1.
Salivary protein transcripts of Phlebotomus tobbi.
Table 2.
Salivary protein transcripts of Phlebotomus sergenti.
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 (*).
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.
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 (•).
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.
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).
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.
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 (*).
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.
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.
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.
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.
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 (*).
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.
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.
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.
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.
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.
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.
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.
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.
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.
Table 3.
N-glycosylation sites of Phlebotomus tobbi salivary proteins.