An insight into the sialome of the blood-sucking bug Triatoma infestans, a vector of Chagas' disease

Abstract

Triatoma infestans is a hemiptera, vector of Chagas’ disease, that feeds exclusively on vertebrate blood in all life stages. Hematophagous insects’ salivary glands (SG) produce potent pharmacological compounds that counteract host hemostasis, including anti-clotting, anti-platelet, and vasodilatory molecules. To obtain a further insight into the salivary biochemical and pharmacological complexity of this insect, a cDNA library from its salivary glands was randomly sequenced. Also, salivary proteins were submitted to two dimentional gel (2D-gel) electrophoresis followed by MS analysis. We present the analysis of a set of 1,534 (SG) cDNA sequences, 645 of which coded for proteins of a putative secretory nature. Most salivary proteins described as lipocalins matched peptide sequences obtained from proteomic results.

1. Introduction

Triatoma infestans (Hemiptera: Reduviidae) is an important vector of Trypanosoma cruzi, a protozoan parasite and etiological agent of Chagas’ disease (American trypanosomiasis) in Latin America (Dias, 1987). All instar nymphs and adults are hematophagic and need a blood meal to molt and for oviposition. The insect obtains the blood meal by injecting its maxilla into vertebrate’s skin searching for a vessel (Lavoipierre, 1965).

The (SG)s of blood-feeding arthropods show a variety of anti-hemostatic compounds that help the bug to obtain its blood meal. Like other blood-sucking arthropods that have been studied (Ribeiro and Francischetti, 2003), T. infestans is capable of counteracting host hemostatic responses triggered to prevent blood loss following tissue injury, such as vasoconstriction, blood coagulation and platelet aggregation (Ribeiro, 1995). The molecular diversity of hematophagous insect saliva represents a rich field for the discovery of novel pharmacologically active compounds and for understanding the evolutionary mechanisms leading to the insect’s adaptation to this feeding habit. Previous studies describing the sialome (set of RNA message + set of proteins found in (SG)s) of hematophagous insects and ticks (Francischetti et al., 2002; Valenzuela et al., 2002a, b) have revealed that the sialomes of these disease vectors are more complex than expected and contain many proteins to which we cannot yet ascribe a function.

Lipocalins are a large and heterogenous group of proteins that play various roles, mainly as carriers of small ligands in vertebrates and invertebrates (Flower et al., 2000). A great array of (SG) proteins belonging to the lipocalin family has generated a large number of different molecules having anti-hemostatic functions while maintaining the fundamental structure of the protein fold (Montfort et al., 2000). Lipocalins were found in the saliva of other blood-sucking insects such as Rhodnius prolixus (Ribeiro et al., 2004a) and Triatoma brasiliensis (Santos et al., 2007), and also in tick saliva (Paesen et al., 2000). In R. prolixus, three types of salivary lipocalins have been characterised: the nitrophorins consisting in a group of lipocalins working as NO carrier, and also as anti-clotting; the ADP-binding protein RPAI1, which inhibits platelet activation and aggregation (Francischetti et al., 2000); and a group of lipocalins related to T. pallidipenis thrombin inhibitor triabin (Fuentes-Prior et al., 1997). Another lipocalin from the saliva of T. pallidipenis denominated pallidipin has been ascribed as a specific inhibitor of collagen-induced platelet aggregation (Noeske-Jungblut, et al., 1994).

In this work we present the analysis of a set of 1,534 (SG) cDNA sequences, 645 of which code for proteins of a putative secretory nature. Most salivary proteins described as lipocalins – 55% of the transcripts coding for putative secreted proteins – matched peptide sequences obtained from proteomic results. We expect this work will contribute new salivary transcripts that could help the understanding of the role of salivary molecules in host/vector interactions and the discovery of novel pharmacologic agents.

2. Materials and methods

2.1. Triatomines and salivary glands cDNA library construction

Triatoma infestans were reared in an insectary room kept at 27 °C ± 1.0 °C, with a relative humidity ranging from 70 to 75% and a 16 h:8 h light:dark photoperiod. Salivary glands (SG) were dissected from Vth instar nymphs 2 days after a blood meal and transferred to RNA-Later (Ambion) solution in 1.5 mL polypropylene vials. SG were kept at −20°C for isolating polyA+ RNA.

T. infestans SG mRNA was isolated from 15 SG pairs from Vth instar nymphs, using the Micro-FastTrack mRNA isolation kit (Invitrogen). The PCR-based cDNA library was made following the instructions for the SMART (switching mechanism at 5′ end of RNA transcript) cDNA library construction kit (Clontech). This kit provides a method for producing high-quality, full-length cDNA libraries from nanogram quantities of polyA+ or total RNA. It utilises a specially designed oligonucleotide named SMART IV™ in the first-strand synthesis to generate high yields of full-length, double-stranded cDNA. T. infestans SG polyA+ RNA was used for reverse transcription to cDNA using PowerScript reverse transcriptase (Clontech), the SMART IV oligonucleotide, and the CDS III/3′ primer (Clontech). The reaction was carried out at 42 °C for 1 h. Second-strand synthesis was performed by a long-distance PCR-based protocol using the 5′ PCR primer and the CDS III/3′ primer as sense and antisense primers, respectively. These two primers also create Sfi1A and B restriction enzyme sites at the end of the cDNA. Advantage™ Taq polymerase mix (Clontech) was used to carry out the long-distance PCR reaction on a Perkin Elmer GeneAmp® PCR system 9700 (Perkin Elmer Corp.). The PCR conditions were: 95 °C for 1 min; 14 cycles of 95 °C for 10 s, 68 °C for 6 min. A small portion of the cDNA was analysed on a 1.1% agarose/EtBr (0.1 µg/mL) gel to check for the quality and range of the synthesised cDNA. Double-stranded cDNA was immediately treated with proteinase K (0.8 µg/mL) at 45 °C for 20 min and washed three times with water using Amicon filters with a 100 kDa cutoff (Millipore). The clean, double-stranded cDNA was then digested with SfiI restriction enzyme at 50 °C for 2 h followed by size fractionation on a ChromaSpin–400 drip column (Clontech). The profiles of the fractions were checked on a 1.1% agarose/EtBr (0.1 µg/mL), and fractions containing cDNA were pooled in three different groups according to their size: large, medium or small sequences. Each group was concentrated and washed three times with water using an Amicon filter with a 100 kDa cutoff. The concentrated cDNA was then ligated into a λ TriplEx2 vector (Clontech), and the resulting ligation mixture was packaged using GigaPack® Gold III Plus packaging extract (Stratagene), according to the manufacturer's instructions. The packaged library was plated by infecting log-phase XL1-Blue Escherichia coli cells (Clontech). The percentage of recombinant clones was determined by performing a blue-white selection screening on LB/MgSO₄ plates containing X-gal/IPTG.

2.2. Sequencing of the T. infestans cDNA library

The T. infestans SG cDNA library was plated on LB/MgSO₄ plates containing X-gal/IPTG to an average of 250 plaques per 150 mm Petri plate. Recombinant (white) plaques were randomly picked up and transferred to 96-well MICROTEST™ U-bottom plates (BD BioSciences) containing 75 µL of H₂O per well. The phage suspension was either immediately used for PCR or stored at 4 °C until use.

To amplify the cDNA using a PCR reaction, 4 µL of the phage sample were used as a template. The primers were sequences from the λ TriplEx2 vector and named PT2F1 (5′-AAGTACTCTAGCAATTGTGAGC-3’) and PT2R1 (5′-CTCTTCGCTATTACGCCAGCTG-3′), positioned at the 5′ and 3′ end of the cDNA insert, respectively. The reaction was carried out in MicroAmp 96-well PCR plates (Applied Biosystems) using Platinum PCR® SuperMix (Invitrogen), on a Perkin Elmer GeneAmp® PCR system 9700 (Perkin Elmer Corp.). The PCR conditions were: 1 hold of 75 °C for 3 min, 1 hold of 94 °C for 4 min, 33 cycles of 94 °C for 1 min, 49 °C for 1 min, and 72 °C for 1 min and 20 s. The amplified products were analysed on a 1.2% agarose/EtBr gel. cDNA library clones (1800 clones) were PCR amplified, and those showing a single band were selected for sequencing. The PCR products were used as a template for a cycle-sequencing reaction using DTCS labeling kit from Beckman Coulter). The primer used for sequencing (PT2F3) is upstream from the inserted cDNA and downstream from the PT2F1 primer. The sequencing reaction was performed on a Perkin Elmer 9700 thermocycler. Conditions were 1 hold of 75 °C for 2 min, 1 hold of 94 °C for 4 min, and 30 cycles of 96 °C for 20 s, 50 °C for 20 s, and 60 °C for 4 min. After cycle-sequencing the samples, a cleaning step was performed using the multiscreen 96-well plate cleaning system (Millipore). The 96-well multiscreening plate was prepared by adding a fixed amount (manufacturer’s specification) of Sephadex-50 (Amersham Pharmacia) and 300 µL of deionised water. After partially drying the Sephadex in the multiscreen plate, the whole cycle-sequencing reaction was added to the center of each well, centrifuged at 2,500 rpm for 5 min, and the clean sample was collected on a sequencing microtiter plate (Beckman Coulter). The plate was then dried on a Speed-Vac SC110 model with a microtiter plate holder (Savant Instruments). The dried samples were immediately resuspended with 25 µL of formamide, and one drop of mineral oil was added to the top of each sample. Samples were either sequenced immediately on a CEQ 2000 DNA sequencing instrument (Beckman Coulter) or stored at −30 °C. A total of 1,534 cDNA library clones were sequenced.

2.3. Reverse phase liquid chromatography (RPLC)

Approximately 20 µL of saliva from adult insects were diluted in 200 µL buffer (50mM Tris-HCl, 120 mM NaCl pH 7.4). Proteins were loaded in a Microcon 10 (YM-10, Millipore) and centrifuged at 10,000 × g for 30 min. The flow through with proteins of low molecular weight (< 10 kDa) was kept. Microbore reversed-phase liquid chromatography (RPLC) was performed using a 0.3 mm C18 column from Phenomenex (Torrence, CA). After sample injection, the column was washed for 10 min with 95% mobile phase A (0.1% formic acid in water) at 5 µL/min and peptides were eluted using a linear gradient to 90% mobile B (100% acetonitrile and 0.1% formic acid) for 40 min. The column eluate was monitored at 220 nm. A Probot fraction collector (Dionex, Sunnylvale, CA) was used to deliver the fractions to 96-well plates containing 25 µL of water. Fractions of interest were submitted to tryptic digestion, followed by mass spectrometry as indicated below.

2.4. 2D-gel electrophoresis

2D-gel electrophoresis was performed using ZOOM IPGRunner System (Invitrogen) under manufacturer's recommended running conditions. Briefly, approximately 500 µg of sample proteins of T. infestans saliva were solubilised with 155 µL rehydration buffer (7 M urea, 2 M thiourea, 2% CHAPS, 20 mM DTT, 0.5% carrier ampholytes, pH 3–10). The samples were absorbed by rehydration ZOOM strips (7 cm; pH 3–10 NL) overnight at room temperature and then focused under manufacturer's recommended conditions. The focused IPG strips were reduced/alkylated/equilibrated with reducing and alkylating reagents dissolved in the sample buffer. The strips were then applied onto NuPAGE 4–12% Bis-Tris ZOOM gels (Invitrogen). The gels were run under MOPS buffer and stained with SeeBlue staining solution (Bio-Rad).

2.5. Protein identification by mass spectrometry

Protein identification of either RPLC or 2D gel-separated proteins was performed on reduced and alkylated trypsin-digested samples prepared by standard mass spectrometry protocols. Tryptic digests were analysed by coupling the Nanomate (Advion BioSciences)—an automated chip-based nano-electrospray interface source—to a quadrupole time-of-flight mass spectrometer, QStarXL MS/MS System (Applied Biosystems/Sciex). Computer-controlled, data-dependent automated switching to MS/MS provided peptide sequence information. AnalystQS software (Applied Biosystems/Sciex) was used for data acquisition. Data processing and databank searching were performed with Mascot software (Matrix Science). The NR protein database from the NCBI, National Library of Medicine, NIH, was used for the search analysis, as was a protein database generated during the course of this work.

2.6. Bioinformatic tools and procedures

Expressed sequence tags (ESTs) were trimmed of primer and vector sequences, clusterised, and compared with other databases as previously described. (Valenzuela et al., 2003). The BLAST tool (Altschul et al., 1996), CAP3 assembler (Huang et al., 1999), ClustalW (Thompson et al., 1994), and TREEview software (Page, 1996) were used to compare, assemble and align sequences, and to visualise alignments. For functional annotation of the transcripts we used the tool BlastX (Altschul et al., 1997) to compare the nucleotide sequences with the nonredundant (NR) protein database of the NCBI and to the Gene Ontology (GO) database (Ashburner et al., 2000). The tool rpsBlast (Schaffer et al., 2001) was used to search for conserved protein domains in the Pfam (Bateman et al., 2000), SMART (Letunic et al., 2002), Kog (Tatusov et al., 2003), and conserved domains (CDD) databases (Marchler-Bauer et al., 2002). We have also compared the transcripts with other subsets of mitochondrial and rRNA nucleotide sequences downloaded from NCBI and to several organism proteomes downloaded from NCBI (yeast), Flybase (D. melanogaster), or ENSEMBL (An. gambiæ). Segments of the three-frame translations of the EST (since the libraries were unidirectional, we did not use six-frame translations) starting with a methionine in the first 100 predicted amino acids (aa)—or the predicted protein translation, in the case of complete coding sequences—were submitted to the SignalP server (Nielsen, 1997) to help identify translation products that could be secreted. O-glycosylation sites on the proteins were predicted with the program NetOGlyc (http://www.cbs.dtu.dk/services/NetOGlyc/) (Hansen et al., 1998). Functional annotation of the transcripts was based on all the comparisons above. Following inspection of all results, transcripts were classified as either Secretory (S), Housekeeping (H), or of unknown (U) function, with further subdivisions based on function and/or protein families. Sequence alignments were done with the ClustalX software package (Thompson et al., 1997). Phylogenetic analysis and statistical neighbor-joining bootstrap tests of the phylogenies were done with the Mega package (Kumar et al., 2004). Hyperlinked Excel spreadsheets of the assembled ESTs and of the salivary protein database are supplied as Supplemental Tables 1 and 2 at the journal site.

3. Results

General Description of the Salivary Transcriptome Database

3.1. Description of the clusterised data set / cDNA library characteristics

1,534 sequences were used to assemble a clusterised database, yielding 657 clusters of related sequences, 500 of which contained only one EST. The consensus sequence of each cluster is named either a contig (deriving from two or more sequences) or a singleton (deriving from a single sequence). In this paper, we will use the denomination contig to address sequences deriving from both consensus sequences and from singletons. The 657 contigs were compared by the program BlastX, BlastN, or rpsBlast (Altschul et al., 1997) to the nonredundant protein database of the National Center of Biological Information (NCBI), to the gene ontology database (Ashburner et al., 2000), to the conserved domains database of the NCBI (Marchler Bauer et al., 2002), and to a custom-prepared subset of the NCBI nucleotide database containing either mitochondrial or rRNA sequences. Because the libraries are unidirectional, the three frame translations of the dataset were also derived, and open reading frames (ORF) starting with methionine and longer than 40 aa residues were submitted to SignalP server (Nielsen et al., 1997) to help identify putative secreted proteins. The EST assembly, BLAST, and signal peptide results were transferred into an Excel spreadsheet for manual annotation. Five categories of expressed genes derived from the manual annotation of the contigs (Table 1). The putatively secreted (S) category contained 18% of the clusters and 42% of the sequences, with an average number of 5.5 sequences per cluster. The housekeeping (H) category had 36.4% and 35.9% of the cluster and sequences, respectively, and an average of 2.3 sequences per cluster. Forty-four percent of the clusters, containing 21% of all sequences, were classified as unknown (U) because no assignment for their function could be made; most of these consisted of singletons. Possible transposable elements originated 7 clusters, mostly singletons. We have also identified viral transcripts in our dataset. These data can be downloaded as Supplemental Table 1 for the EST data, and Supplemental File 2 for the proteome set.

Table 1.

Types of transcripts found in Triatoma infestans salivary glands.

Types of transcripts	Clusters	Sequences	Sequences/Cluster
Secreted (S)	118	645	5.5
Housekeeping (H)	239	550	2.3
Unknown (U)	292	324	1.1
Transposable element (TE)	7	11	1.6
Viral product	1	4	4.0
Total	657	1534

3.2. Housekeeping (H) genes

The 239 gene clusters (comprising 550 EST) attributed to H genes expressed in the (SG)s of T. infestans were further characterised into 20 groups, according to their possible function (Table 2). According to an organ specialised in secreting polypeptides and as observed in previous sialotranscriptomes (Francischetti et al., 2002; Ribeiro et al., 2004a, b; Calvo et al., 2007), the two larger sets were associated with protein synthesis machinery (298 EST in 51 clusters) and with energy metabolism (17 clusters containing 57 EST). We have also included in this category a group of 66 EST that grouped into 52 clusters and represent conserved proteins of unknown function, presumably associated with cellular metabolism. Other sequences with homology to housekeeping protein include those coding for ribosomal, cytochrome, ATP synthase subunit, and NADH-ubiquinone oxidoreductase, among other molecules.

Table 2.

Functional classification of the housekeeping genes expressed in Triatoma infestans salivary glands.

Types of transcripts	Clusters	%	Sequences	%
Conserved, unknown function	52	21.8	66	11.3
Protein synthesis machinery	51	21.3	298	54.2
Signal transduction	18	7.5	18	3.3
Metabolism, energy	17	7.1	57	10.4
Transport/Storage	14	5.9	15	2.7
Cytoskeletal	12	5.0	13	2.4
Nuclear regulation	12	5.0	13	2.4
Protein export machinery	10	4.2	12	2.2
Protein modification	10	4.2	16	2.9
Transcription machinery	9	3.8	10	1.8
Proteasome machinery	8	3.3	10	1.8
Metabolism, carbohydrate	6	2.5	6	1.1
Metabolism, amino acid	4	1.7	4	0.7
Transcription factor	3	1.3	3	0.5
Metabolism, intermediate and detoxication	3	1.3	3	0.5
Metabolism, nucleotide	3	1.3	3	0.5
Lysosomal products	2	0.8	2	0.4
Extracellular matrix	2	0.8	2	0.4
Metabolism, oxidant	2	0.8	2	0.4
Metabolism, lipid	1	0.4	1	0.2
Total	239		550

3.3. Viral product

Contig 110 produced a best BlastX match with gi|20451030|, a capsid protein precursor from a Triatoma virus (TrV), scoring with other reported capsid proteins. TrV infects triatomines and belongs to a new family of RNA viruses known as Dicistroviridae (Czibener, et al., 2000).

3.4. Transposable elements

Seven contigs on our database possibly derive from transposable elements (TE). Their translation products are similar to those of Drosophila melanogaster proteins annotated as endonuclease and reverse transcriptase. These transcripts may indicate active ongoing transposition activity in T. infestans, or with regulatory transcripts inhibiting TE genomic mobilisation.

3.5. Transcripts coding for putative secreted proteins in Triatoma infestans salivary glands

3.5.1. Lipocalins

The most abundant group of putative secreted proteins in T. infestans (SG)s is the lipocalins, corresponding to 55% of the transcripts in the S class. Lipocalins are widely distributed and heterogeneous proteins occurring in animals, plants and bacteria. Even though they have various molecular masses, the domain, determining the specific properties, is about 18–20 kDa (Flower et al., 1993). The lipocalin primary sequence shows a low percentage of similarity when comparing randomly selected members of the family. By contrast, an interesting feature of the lipocalins is their well conserved three-dimensional structure (Flower, 1995). They are typically small, extracellular proteins sharing several common molecular properties: the binding of small, principally hydrophobic molecules, binding to specific cell-surface receptors; the formation of covalent and non-covalent complexes with other soluble macromolecules. Although they have been classified mainly as transport proteins, it is now clear that members of the lipocalin family fulfill a wide variety of different functions (Flower, 2000). The aim to discover the role of salivary lipocalins in blood-sucking insects through functional genomic and proteomic studies has been able to identify anticoagulants, antiplatelets, and vasodilatory molecules (Andersen et al., 2005).

We found lipocalins similar to salivary proteins of other species of the genus Triatoma, such as pallidipin, an inhibitor of collagen-induced platelet aggregation (Noeske-Jungblut et al., 1994); and triabin, a potent and selective thrombin inhibitor, both from the bug Triatoma pallidipennis (Noeske-Jungblut et al., 1995); and procalin, a salivary allergen from T. protacta (Paddock et al., 2001). The South American T. brasiliensis was also shown to contain salivary cDNA sequences similar to these three previously described Triatoma lipocalins (Sant’Anna et al., 2002). More recently, the sialotranscriptome of T. brasiliensis revealed a high content of lipocalins in its (SG)s, comprising 93.8% of the transcripts coding for putative secreted protein (Santos et al., 2007). Lipocalins have also been found in tick saliva and in Rhodnius performing similar functions, such as histamine and serotonin binding (Ribeiro et al., 2004a; Paesen et al., 2000; Sangamnatdej et al., 2002). This scenario contrasts with mosquitoes and sandflies, where no salivary lipocalins have been described to date.

Triatomine lipocalin sequences were aligned and a neighbor-joining phylogenetic-tree constructed (Fig. 1). The bootstrap values indicated that these sequences have evolved beyond recognition of a common ancestor (not shown). These results may indicate a long evolutionary history for these proteins, or alternatively, a fast rate of evolution. It is interesting to note here that lipocalins were not found in the sialotranscriptome of Oncopeltus fasciatus, a closely related seed feeding bug, indicating that the expansion of this gene family expressed in the salivary glands of triatomines occurred as an adaptation to blood feeding (Francischetti et al., 2007).

Figure 1.

Dendrogram of the T. infestans salivary lipocalins with other insect salivary lipocalins. The sequences derived from the nonreduntant (NR) protein database of the National Center for Biotechnology Information (NCBI) are represented by five letters followed by the NCBI gi| accession number. The five letters derive from the first three letters of the genus and the first two letters from the species name. The protein sequences were aligned by the Clustal program (Thompson et al., 1997), and the dendrogram was done with the Mega package (Kumar et al., 2004) after 1,000 bootstraps with the neighbor-joining (NJ) algorithm. The bar at the bottom represents 20% amino acid substitution. The colorful squares indicate each insect species whose sequences were used: blue, T. infestans sequences from cDNA library; dark green, T. infestans proteins described previously; red, T. brasiliensis; purple, T. pallidipennis; brown, T. protacta; magenta, Rhodnius prolixus; gray, Tribolium castaneum; light green, Galleria mellonella.

3.5.1.2. Enzymes

Some transcripts coding for enzymes are identifiable. A serine protease could be involved with specific host proteolytic events that could affect clotting or the complement cascade. It could also be involved in invertebrate immunity, since prophenoloxidase-activating enzymes are serine proteinases (Söderhäll and Cerenius, 1998). A serine protease with trypsin-like activity was described in T. infestans saliva. This salivary proteolytic activity was denominated triapsin and shown to be released with ejected saliva in active form, suggesting a role in blood-feeding (Amino et al., 2001). Since some Hemiptera species utilise cysteine and aspartic proteases for proteolytic digestion (Terra et al., 1996), the presence of a serine protease could be more related to blood acquisition rather than its digestion. Or it could play a role in the innate immunity of the insect, considering the serine protease as a part of the prophenoloxidase system. This EST has similarities in the alignment with serine proteases from other insects, and phylogenetic analysis shows a strong bootstrap support for the triatomine clade (Fig. 2).

Figure 2.

(A) ClustalW alignment of members of the serine protease family deriving from salivary glands of T. infestans and from other insects. (B) Neighbor-joining phylogram. The numbers in the phylogram nodes indicate percent bootstrap support for the phylogeny. The bar at the bottom indicates 10% amino acid divergence in the sequences.

3.5.1.3 Apyrase

Partial coding sequences (truncated in the 5′ region) for the enzyme apyrase, a member of the 5′ nucleotidase family, were found. This enzyme was described before in T. infestans (SG)s (Faudry et al., 2004), where it helps the acquisition of blood meals by the degradation of adenosine diphosphate (ADP), a mediator of platelet aggregation and inflammation (Ribeiro and Francischetti, 2003).

3.5.1.4 Inositol phosphatase family

Inositol phosphates are involved in many cellular processes related to signal transduction, secretion and cytoskeletal structure. Numerous enzymes are involved in the metabolism of inositol phosphates and phosphoinositides, including kinases, phosphatases, and phospholipases (Erneux et al., 1998; Guo et al., 1999). Inositol polyphosphate 5-phosphatases (IPPs) share a 5-phosphatase domain and consist of a large family of enzymes acting on different substrates. They regulate the pool of 5-phosphorylated inositol phosphates and phosphoinositides, thereby influencing cellular processes, for which second messenger functions have been reported: Ins(1,4,5)P₃, Ins(1,3,4,5)P₄, and the lipids PtdIns(4,5)P₂ and PtdIns(3,4,5)P₃ (Majerus, et al., 1999); (Toker et al., 1997).

Alignment of two contigs from the cDNA library with other IPP sequences from vertebrates and insects showed they share similarities and conserved (aa) sequence (Fig. 3A). The phylogram shows a clade with Triatoma and Rhodnius sequences but apart from other insects, and even the Strongylocentrotus purpuratus sequence is more conserved with vertebrates than with insects (Fig. 3B).

Figure 3.

(A) ClustalW alignment of the T. infestans inositol polyphosphate 5-phosphatases (IPP) with IPP from other organisms. (B) Neighbor-joining phylogram. The numbers in the phylogram nodes indicate percent bootstrap support for the phylogeny. The bar at the bottom indicates 10% amino acid divergence in the sequences.

Rhodnius prolixus also secretes into its saliva an inositol polyphosphate 5-phosphatase during blood feeding (Ribeiro et al., 2004a). It seems to reduce the concentration of some phospholipids in the plasma membrane of cells and platelets; this could have effects such as elimination of substrates for phospholipase C and PI3-kinase, changes in cytoskeletal architecture, and changes in membrane trafficking and vesicle secretion (Andersen et al., 2006). The presence of this phosphatase in Triatoma saliva can also be related to antithemostatic response, since changes in platelets membrane could interfere with their aggregation and facilitate the acquisition of blood meal. This protein was also identified in the 2D gel (Fig. 12).

Figure 12.

Two-dimensional (2D)-gel electrophoresis of Triatoma infestans salivary proteins. Numbers on the left indicate molecular weight marker positions in the gel. The + and − signs indicate the anode or cathode side of the isoelectrophocusing dimension, which ranged from pH 3–10. Gel bands that were identified to a protein (following tryptic digestion and mass spectrometry) are shown in the gel. In some cases, more than one band accounted for the same protein, possibly due to trailing or multiple isoforms. For experimental details, see Materials and methods.

3.5.1.5 Serpins

Serpins constitute a family of proteins, most of which function as serine proteinase inhibitors. They are important regulators of serine proteinases involved in inflammation, blood coagulation, fibrinolysis, and complement activation. Other roles for serpins could be protection against microbial proteinases, regulation of both endogenous proteinases and phenoloxidase activation (Kanost, 1999). Among arthropods, serpins have been described from lepidopteran insects, crayfish, ticks, and others (Jiang, et al., 1996; Liang et al., 1995; Prevot, et al, 2006). One of the contigs from the cDNA library matched for a serpin but the sequence was truncated (Table 3).

Table 3.

Classification of transcripts coding for putative secreted proteins in Triatoma infestans salivary glands.

Types of transcripts	Clusters	Sequences	% S type sequence
Lipocalins	69	355	55.0
Trialysin	6	74	11.5
Peptide with trialysin signal peptide	7	64	9.9
Hemolysin-domain protein	7	32	5.0
Defensin and immunity related peptides	6	32	5.0
Enzymes	7	20	3.1
Kazal domain-containing peptides	3	17	2.6
Antigen 5 protein family	2	13	2.0
Other peptides	5	9	1.4
Odorant binding protein family	2	9	1.4
Triatox	1	8	1.2
Similar to Culicoides salivary protein	1	6	0.9
Serpin	1	4	0.6
Thrombospondin-like	1	2	0.3
Total	118	645

3.5.1.6 Kazal domain-containing peptides

Two contigs with transcripts encoding a polypeptide with a mature molecular mass of 8.9 kDa and containing a single Kazal domain are predicted. Although this domain is associated with serine protease inhibitors, it is also found in other extracellular proteins without this function and containing antimicrobial activity (Fogaça et al., 2005). Kazal-type serine protease inhibitors are single- or more commonly multi-domain proteins, can be classical or non-classical Kazal-type inhibitors, and share a conserved sequence motif and molecular conformation (one central α-helix and three small antiparallel β- sheets) (Stubbs et al., 1997). Several Kazal-type family members have been previously described to be present in vertebrate and invertebrate animals. The role of Kazal-type inhibitors is not always known. Some invertebrate Kazal-type inhibitors, such as rhodniin, bdellin B-3 and infestin 1–2 (Friedrich et al., 1993; Fink, et al., 1986; Sommerhoff et al., 1994; Campos, et al., 2002) are identified as non-classical Kazal-type inhibitors. Thrombin inhibitors of the Kazal-type family have been found in triatomines. Infestin is the longest Kazal-type proteinase inhibitor precursor described in triatomines. It seems to be similar to rhodniin and dipetalogastin precursors but has more Kazal-type domains (Lovato et al., 2006). Rhodniin, isolated from the blood-sucking insect R. prolixus, is such a Kazal-type inhibitor composed of two domains, and strongly inhibits thrombin, the key enzyme of the blood coagulation cascade (van de Locht et al., 1995).

Alignment with other sequences containing a Kazal domain shows the presence of five conserved cysteines (Fig. 4A). These sequences also showed similarity to the vasodilator named vasotab from the horse fly Hybomitra bimaculata. Vasotab is a member of the Kazal-type protease inhibitor family (Takác et al., 2006). The phylogram analysis indicates a strong bootstrap support for the triatomine clade (Fig. 4B). A transcript encoding this polypeptide was also found in the mosquitoes Ae. aegypti (Ribeiro et al., 2007) and Ae. albopictus (Arcà et al., 2007). And recently, the sialotranscriptome of the triatomine Triatoma brasiliensis demonstrated the presence of nine clusters coding for Kazal-type peptides (Santos, et al., 2007).

Figure 4.

The salivary Kazal-domain containing peptides of T. infestans. (A) ClustalW alignment. Six conserved cysteines are shown in black with white background and marked with an asterisk (*). (B) Phylogenetic tree showing the sequence distance relationships between members of the family. The bar shows 10% divergence at the amino acid level. The numbers indicate the bootstrap value from 1000 trials.

3.5.1.7 – Antigen-5 protein family

This is a family of secreted proteins that belong to the CAP family (cysteine-rich secretory proteins; AG5 proteins of insects; pathogenesis-related protein 1 of plants) (Megraw et al., 1998). The CAP family is related to venom allergens in social wasps and ants (Hoffman, 1993; King and Spangfort, 2000) and to antifungal proteins in plants (Stintzi et al., 1993; Szyperski et al., 1998). Members of this protein family are found in the (SG)s of many blood-sucking insects (Francischetti et al., 2002; Li et al., 2001; Valenzuela et al., 2002b; Arcà et al., 2005; Calvo et al., 2007). The function of any AG5 protein in the saliva of any blood-sucking arthropod is still unknown. Other animals have shown some insight into their activity: snake venom proteins of the same family have been shown to contain smooth muscle-relaxing activity (Yamazaki et al., 2002; Yamazaki and Morita, 2004), and the salivary neurotoxin of the venomous lizard Heloderma horridum is also a member of this protein family (Nobile et al., 1996).

We here report a salivary member of the antigen-5 family found in T. infestans (SG)s. Alignment and phylogenetic analysis of insect members of this family indicates that T. infestans salivary antigen-5 protein clusters with other reported antigen-5 proteins from R. prolixus, vespids and other insects. The hymenoptera proteins, from vespids, have an additional domain after the signal peptide (Fig. 5), indicating that probably their evolution diverged at some point from the others. Both Rhodnius and Triatoma proteins have a basic tail. This content of a poly-Lys tail in salivary proteins may direct these proteins to the surface of activated platelets, where they could bind and/or release biologically active ligands or interact with other proteins on the platelet surface. They could also block the interaction of coagulation factors with the membrane surface or direct the inhibitor to negatively charged membranes critical for productive blood coagulation complex assembly (Broze Jr., 1995). Demonstration that proteins rich in positively charged residues effectively block the coagulation cascade comes from studies performed with a recombinant R. prolixus salivary lipocalin (nitrophorin-7, NP-7). NP-7 contains a cluster of positively charged residues in the N-terminus and specifically binds to anionic phospholipids, preventing thrombin formation by the prothrombinase complex (Andersen et al., 2004).

Figure 5.

(A) ClustalW alignment of T. infestans salivary antigen-5 with members of the same family. (B) Phylogenetic tree of selected members of the antigen-5 family of proteins from Triatominae, Vespoidea and other Insecta, obtained by the NJ method. The numbers in the phylogram nodes indicate percent bootstrap support for the phylogeny. The bar at the bottom indicates 10% amino acid divergence in sequences. For details, see text.

3.5.1.8 Odorant-binding protein family

Many airborne molecules, such as hydrophobic odorants and pheromones, must be recognised by a specialised class of proteins that facilitate their delivery to the olfactory receptors (OR) (Forêt and Maleszka, 2006). In both insects and vertebrates this function is provided by odorant-binding proteins (OBPs) (Pelosi et al., 1996; Krieger and Breer, 1999; Deyu and Leal, 2002). They are generally thought to solubilise hydrophobic odorants and carry them to the respective receptors (Vogt et al., 1991; Pelosi et al., 1994; Prestwich et al., 1995). Besides contributing to the recognition of odorants in insects, they may also function as carriers in other developmental and physiological processes.

Insect OBPs are small, water soluble molecules expressed in both olfactory and gustatory sensilla, as well as in other specialised tissues (Pelosi et al., 2005). OBPs have also been found in non-olfactory tissue, suggesting that their roles may be related to general carrier capabilities with broad specificity for lipophilic compounds (Forêt and Maleszka, 2006). The heme-binding protein of R. prolixus (Paiva-Silva et al., 2002) is one of these OBPs implicated in non-olfactory functions. In fact, there is increasing evidence that OBPs do play an active role in odorant recognition rather than merely serving as passive odorant carriers. One line of evidence is the large number of OBPs present within a variety of insect species. Several proteins of the odorant-binding protein (OBP) family have been described in D. melanogaster, Apis mellifera, and the hemiptera R. prolixus, where a putative OBP with a signal peptide indicative of secretion was identified (Hekmat-Scafe et al., 2000; Forêt and Maleszka, 2006; Ribeiro et al., 2004a). We found transcripts coding for members of the OBP family in T. infestans. They share similarities with OBPs from the mosquitoes An. gambiae and Ae. aegypti. The OBP-like sequences have four conserved cysteines and good bootstrap support for the triatomine clade (Fig. 6). Sequences in Diptera clade do not have a strong bootstrap support, except for some members that probably originate from genetic duplication and/or function conservation. It has been proposed that a moderate number of OBPs could act in a combinatorial manner with a moderate number of OR to greatly increase the discriminating power of an insect’s olfactory system (Hekmat-Scafe, et al., 2002).

Figure 6.

(A) ClustalW alignment of the OBP family of salivary peptides. Four conserved cysteines are shown in black with white background and marked with an asterisk (*). (B) Neighbor-joining phylogram. The numbers in the phylogram nodes indicate percent bootstrap support for the phylogeny. The bar at the bottom indicates 20% amino acid divergence in sequences.

It is possible to speculate whether these salivary OBP’s could be acting as partners in gustatory perception in Triatoma, as they are related to gustatory perception in Drosophila (Galindo and Smith, 2001). If this is the case, saliva would be important not only for its anti-hemostatic function, but also in the chemoreception of phagostimulants (Friend and Smith, 1977). Against this hypothesis is the previous observation that Rhodnius prolixus surgically deprived of salivary glands were able to feed normally on an artificial feeder (Ribeiro and Garcia, 1981).

3.5.1.9 Similar to Culicoides salivary protein

This EST sequence found in T. infestans shows similarity with Culicoides and Fungi sequences (Fig. 7). Culicoides are competent vectors of a wide range of economically important pathogens that affect both domestic and wild animals (Mellor et al., 2000). A broad range of genes encoding salivary proteins were characterised from a (SG) cDNA library of C. sonorensis and revealed many proteins involved in antihemostasis, immunomodulation, and digestion (Campbell et al., 2005). In addition to acting as a vector, Culicoides are the primary cause of an extremely pruritic allergic dermatitis, known colloquially as “summer eczema”, or “insect bite hypersensitivity” in horses (Anderson, et al., 1993; Kurotaki, et al., 1994). The saliva of kissing bugs (Triatominae) contains several allergens responsible for severe allergic reactions such as urticaria, dyspnea, and anaphylaxis in humans (Moffitt et al., 2003). This transcript, similar to one found in Culicoides, could be related to hypersensitivity or allergic responses by the human host.

Figure 7.

(A) ClustalW alignment of a predicted sequence from a contig that showed similarity with a sequence from Culicoides. (B) Neighbor-joining phylogram. The numbers in the phylogram nodes indicate percent bootstrap support for the phylogeny. The bar at the bottom indicates 5% amino acid divergence in sequences.

3.5.1.10 Defensin and immunity-related peptides

Antimicrobial peptides (AMPs) are widely distributed throughout the animal and plant kingdoms. Despite sharing some common features, such as a small size (often below 10 kDa) and a cationic character, most AMPs differ in their aa sequence and mode of action (Fogaca et al., 2004). Within this AMP family, defensins constitute one of the major families that have been characterised (Bulet et al., 2004). Defensins are cationic peptides with molecular weights of about 4 kDa, three disulphide bridges, formed by six cysteines residues and three characteristic domains, an amino terminal flexible loop, followed by an α-helix and a carboxy-terminal anti-parallel β-sheet (Bonmantin et al., 1992; Bulet et al., 1999). This peptide can be found in different organisms, such as plants, fungi, mollusks, scorpions, insects, and birds, and also in different cells of various mammals. In humans, insects, and plants, defensins contribute significantly to the host defense against invasion by microorganisms (Raj et al., 2002). In addition to the diversity of structure and mode of action, the site and the regulation of the synthesis of AMPs also differs among arthropod groups. In insects, AMPs are mainly synthesised in the fat body and their gene transcription is strongly induced after an injury and/or infection (Bulet et al., 2002). Defensins have been isolated and characterised as part of the innate immune response from the hemolymph of all insect species so far investigated, like Odonata, Diptera, Coleoptera, Lepidoptera, Acari and Hemiptera (Bartholomay et al., 2004; Bulet et al., 1992; Ceraul et al., 2003; Ishibashi et al., 1999; Johns et al., 2001; Lamberty et al., 1999; Lopez et al., 2003).

Tick defensins have also been found to exist in (SG)s, an important organ for blood feeding and pathogen transmission (Valenzuela et al., 2002). A defensin-like molecule from Ixodes ricinus was recently described and found to be induced following microbial challenge (Rudenko et al., 2005). In triatomines, defensins have been investigated in R. prolixus, being isolated, purified and sequenced (Lopez et al., 2003).

From the alignment of the defensin sequence found in T. infestans (SG)s with other defensins, we notice the presence of the six conserved cysteines, characteristic in this family and important for the disulphide bridges conformation (Fig. 8A). The phylogram reveals a triatomine clade sharing sequences from T. brasiliensis and R. prolixus, and showing a bootstrap support (Fig. 8B).

Figure 8.

(A) ClustalW alignment of the defensin family of salivary peptides. Six conserved cysteines are shown in black with white background and marked with an asterisk (*). (B) Neighbor-joining phylogram. The numbers in the phylogram nodes indicate percent bootstrap support for the phylogeny. The bar at the bottom indicates 10% amino acid divergence in sequences.

3.5.1.11 Similarity to bacterially induced peptide Hdd1 (from Hyphantria cunea)

In a previous work, 11 inducible genes were isolated after bacterial challenge in H. cunea, one of the most serious insect pests in Korea. Hdd1 (Hyphantria differentially displayed clones) was one of this immune-related cDNA but it didn’t produce any significant homology with any known protein (Shin et al., 1998). One contig from the T. infestans cDNA library matched this peptide but the function remains unknown.

3.5.2 Coding for proteins found only in triatomines

3.5.2.1 Trialysin

We identified two contigs that matched the protein trialysin, a pore-forming molecule present in the (SG)s of T. infestans (Amino et al., 2002; Martins et al., 2006) (Fig. 9). It is a 22-kDa protein synthesised as a precursor and processed by limited proteolysis. It exerts its potent cytolytic activity on a large variety of cell types, from bacteria to mammalian cells, and it has been hypothesised that it favors the maintenance of the salivary fluid free of microorganisms and parasites. Trialysin possesses a basic amphipathic lytic motif in the N-terminal region containing 27 aa residues, similar to antimicrobial lytic peptides, and a protein portion that increases the lytic specificity toward eukaryotic cells. Studies in which synthetic peptides containing portions of the N-terminus of trialysin have been tested for their lytic properties against the infective stage of T. cruzi, Escherichia coli and human erythrocytes revealed differences in specificity and activity for the tested targets. The cell membrane is the main target of antimicrobial peptides, thus the diverse lipid composition and distribution or the presence of other components in the cell membrane of the various organisms play an important role in the modulation of peptide/membrane interaction, leading to the differentiation of their action (Amino et al., 2002; Martins et al., 2006).

Figure 9.

(A) ClustalW alignment of the unique family of trialysin salivary peptides. The sequences shown are from the cDNA library (TI-6, TI-8) and trialysin from NCBI database (|gi18920644|). (B) ClustalW alignment of the trialysin sequences with other proteins that showed similarity to trialysin. (C) Neighbor-joining phylogram. The numbers in the phylogram nodes indicate percent bootstrap support for the phylogeny. The bar at the bottom indicates 20% amino acid divergence in sequences.

Further analysis of the near relatives of trialysin found in the nonredundant database indicates related bacterial genes, a proteobacteria clade being strongly associated with the T. infestans sequences (Fig 9b,c).

3.5.2.2 Peptides with trialysin signal peptide

This cDNA library also revealed a group of short proteins sharing the signal peptide from trialysin protein. They do not share similarities with other proteins and their function is unknown.

3.5.2.3. Hemolysin-domain protein

Hemolysins are proteins considered to permeate target cells, forming pores in cytoplasmic membranes of erythrocytes, leukocytes, and other cells, causing the modification of cellular functions and/or lysis of host cells. Hemolysins are more often described in microorganisms, such as Escherichia coli, Staphylococcus aureus, Clostridium septicum, and others (Gouaux, 1998; Melton et al., 2004; Wassenaar, T.M., 2005). The family of hemolysins consists of secreted, water-soluble proteins that form transmembrane, polymeric channels. They are considered a virulence factor from these microorganisms and also a member of the RTX (repeats in toxin) protein family (Welch, 2001). The presence of amphipathic and hydrophilic domains confers to the protein an overall amphiphilic character, which explains its tendency both to aggregate and to interact with membranes (Hyland et al., 2001; Schindel et al., 2001; Soloaga et al., 1999).

We found contigs with proteins containing a hemolysin domain (Fig. 10). These proteins could act as cytolytic proteins, causing erythrocyte lysis in saliva and helping the early steps of the digestion process. Also, it could be related to defense, participating in lysis of microorganisms in insect saliva.

Figure 10.

(A) ClustalW alignment of the mature forms of T. infestans hemolysin-domain containing proteins with other proteins with hemolysin-domain. The signal peptide region is not shown. (B) Phylogram. Numbers on branches indicate bootstrap value for 1000 trials. Bar indicates 20% distance in amino acid sequence.

3.5.2.4 Triatox

One of the contigs matched a toxin previously described in T. infestans saliva as triatox (|gi71725070|) (Fig. 11A). We also found this protein in the 2D gel (Fig. 12). By secondary structure prediction, this protein shows to be an amphipathic α-helix (Fig. 11B). α-helical peptides are linear molecules that exist in aqueous media and become amphipathic helices upon interaction with hydrophobic membranes, like cecropin (Christensen et al., 1988), magainins (Zasloff, 1987), and melittins (Andreu et al., 1992). The amphipathic character of antimicrobial peptides makes them surface-active products, as their biological activity occurs at lipid membrane interfaces (Maget-Dana, 1999). Their amphipathy allows them to be both soluble in an aqueous medium, such as the extracellular medium, and to diffuse towards polar/apolar interfaces such as the extracellular medium/cell membrane interface. Linear peptides that can assume an active, amphipathic, α-helical structure are among the most abundant in nature. They have evolved to act against several microbial targets and appear to represent an important role in innate defense (Giangaspero et al., 2001). The structure of triatox suggests it can function as an antimicrobial peptide and be related to the innate defense of the insect in the (SG)s.

Figure 11.

(A) triatox sequence gi71725070. (B) Prediction of triatox (TI-57) secondary structure using BioEdit program.

3.5.2.5 Thrombospondin-like

This protein shows weak similarity with larger proteins of Plasmodium annotated as “thrombospondin-related adhesive protein.” Thrombospondin domains usually bind to sulphated glycoconjugates found on cell surfaces, allowing for host-cell specificity of binding (Dubremetz, et al., 1998). The function is unknown in other genera.

3.6. 2D-Gel electrophoresis/Mass spectrometry proteomic investigation

The (SG)s of T. infestans were shown to contain proteins such as apyrase and the pore-forming protein trialysin (Amino et al., 2002; Faudry et al., 2004). To obtain further information on the salivary proteins of T. infestans, electrophoresis of saliva was performed by two-dimensional (2D) SDS-PAGE followed by mass spectrometry. Figure 12 shows the pattern of separation of T. infestans salivary proteins by 2D-gel. We were able to identify several salivary lipocalins, and the proteins named “triatox” (|gi71725070|), salivary apyrase precursor (|gi34481604|), salivary inositol polyphosphate 5-phosphate (approximately 37-kDa apparent salivary precursor |gi34481604 “salivary inositol polyphosphate 5-phophate (approximately 37-kDa apparent molecular mass) similar to pallidipin precursor, putative trialysin precursor, similar to lipocalin-like TiLipo77, and similar to pallidipin-2. Supplemental tables S1 and S2 have columns indicating the peptide sequences found in the proteomic experiment.

3.7 Salivary peptide identification by RP-HPLC/Mass spectrometry

Because the gel used for the proteomic experiment above does not well identify polypeptides below 10 kDa, we obtained a 10-kDa filtrate from 20 µL of T. infestans saliva, submitted it to RP-HPLC and performed tryptic digestion of the fractions to obtain peptide sequences by MS/MS. This allowed identification of four members of the short trialysin-like family, which have predicted mature molecular weights of 6.3 kDa. Several fragments matching the hemolysin-like proteins were also identified, indicating this protein family, whose members have more than 20 kDa, may be proteolytically processed in saliva, possibly by triapsin, the molecularly uncharacterised salivary serine protease of T. infestans (Amino et al., 2001). Supplemental tables S1 and S2 have columns indicating the peptide sequences found in this proteomic experiment.

4. Concluding remarks

In an attempt to improve our understanding of the variety of proteins and transcripts expressed in T. infestans (SG)s, we have performed a cDNA library and a 2D-gel using, respectively, mRNA and proteins from this same tissue. We described the set of cDNA present in the (SG)s of Triatoma infestans. Expression and bioassay of the novel proteins will ultimately characterise the salivary pharmacologic complexity from T. infestans evolution to blood feeding.

We believe this cDNA library should help our continuing effort to understand the evolution of blood sucking in vector arthropods and the discovery of novel pharmacologically active compounds.

Abbreviations

aa

amino acid

AMP

antimicrobial peptides

EST

expressed sequence tags

OBP

odorant-binding protein

H

putative housekeeping transcripts

S

putative secreted transcripts

U

unknown function transcripts

SG

salivary glands

Ti

Triatoma infestans

2D

two dimensional