Identification and characterization of a salivary adenosine deaminase from the sand fly Phlebotomus duboscqi, the vector of Leishmania major in sub-Saharan Africa Free

In their saliva, blood-feeding arthropods have potent pharmacologically active components that help them counteract the hemostatic and inflammatory system of the vertebrate host each time they attempt to take a blood meal(Ribeiro, 1987). Vasodilators,anticoagulants and inhibitors of platelet aggregation are part of this salivary mixture (Ribeiro and Francischetti, 2003). Recently, with the technological advances in DNA and protein sequencing, novel and unexpected molecules with potential biological activities have been isolated from the saliva of blood-feeding arthropods. Such molecules include hyalorunidase, nucleotidases, novel apyrases, amine-binding proteins, tissue-factor pathway inhibitors and others(Ribeiro and Francischetti,2003). Another such protein is adenosine deaminase (ADA), which was identified from transcripts of a salivary gland cDNA library of the New World sand fly Lutzomyia longipalpis(Charlab et al., 2000) and the mosquitoes Culex quinquefasciatus and Aedes aegypti(Ribeiro et al., 2001). This protein or the transcript coding for this protein have also been identified in other organisms including bacteria, fruit flies, mice and humans(Charlab et al., 2001).

Adenosine deaminase (E.C. 3.5.4.4) catalyses the conversion of adenosine and 2′-deoxyadenosine to inosine and 2′-deoxyinosine, respectively(Cristalli et al., 2001). This enzyme is evolutionarily conserved and has a beta alpha, 8 barrel structure and zinc ion in the catalytic site (Wilson et al., 1991). Adenosine deaminase deficiency in mice results in the impairment of T and B cell function(Resta et al., 1997) due to the accumulation of adenosine resulting in severe combined immunodeficiency(SCID).

The role of ADAs in insects, particularly in the saliva of blood-feeding insects, was proposed to be in the hydrolysis of adenosine, a molecule involved in pain perception (Charlab et al., 2001). The activity of this enzyme in blood-feeding insects was demonstrated in the saliva of L. longipalpis(Charlab et al., 2000), C. quinquefasciatus and Ae. aegypti(Ribeiro et al., 2001) and from the activity of the recombinant salivary ADA from L. longipalpis(Charlab et al., 2001).

Of interest, ADA enzymatic activity or the transcripts coding for this enzyme were not present in the salivary gland of the sand flies P. argentipes, P. papatasi, P. ariasi and P. perniciosus, which belong to the genus Phlebotomus(Anderson et al., 2006; Charlab et al., 2000). Instead,the saliva of P. papatasi and P. argentipes contains large amounts of adenosine and adenosine monophosphate (AMP)(Ribeiro and Modi, 2001). Therefore, it appeared that ADA activity was only present in the saliva of Lutzomyia sand flies and not in Phlebotomus sand flies. Recently, transcriptome analysis of the salivary glands of the sand fly Phlebotomus duboscqi, a sibling species of P. papatasi,resulted in the identification of a transcript with homologies to ADA(Kato et al., 2006). In the present work, we tested whether there is ADA activity in P. duboscqiand whether the identified transcript codes for this activity. Because P. papatasi does not have ADA activity, but has large amounts of adenosine and AMP in the saliva, we also tested for the presence of adenosine and AMP in the saliva of P. duboscqi sand flies.

Phlebotomus duboscqi Theodor, Mali strain, were reared using a mixture of fermented rabbit food and rabbit feces as larval food. Adult sand flies were offered a cotton swab containing 20% sucrose and were used for dissection of salivary glands at 5–7 days after emergence. Salivary glands were stored in groups of 10 pairs in 10 μl phosphate-buffered saline(PBS). Salivary glands were disrupted by ultrasonication in 1.5 ml conical tubes. Tubes were centrifuged at 10 000 g for 2 min and the resultant supernatant used for the studies.

The P. duboscqi salivary gland cDNA library was made as previously described (Kato et al., 2006). Briefly, mRNA was isolated from 55 salivary gland pairs using the Micro-FastTrack mRNA isolation kit (Invitrogen, San Diego, CA, USA). The PCR-based cDNA library was made following the instructions for the SMART cDNA library construction kit (BD-Clontech, Palo Alto, CA, USA) with some modifications (Kato et al.,2006). The P. duboscqi cDNA library was sequenced as previously described using an Applied Biosystems 3730xl DNA Analyzer (Foster City, CA, USA) and a CEQ 2000XL DNA sequencing instrument (Beckman Coulter,Fullerton, CA, USA) (Kato et al.,2006).

Consensus protein sequences were compared to related sequences from sand flies as well as non-sand fly species obtained from GenBank. Sequences were aligned using ClustalX (Jeanmougin et al.,1998) and manually refined using BioEdit sequence editing software(http://www.mbio.ncsu.edu/BioEdit/page2.html). Phylogenetic analysis was conducted on protein alignments using Tree Puzzle version 5.2 (Schmidt et al.,2002). Tree Puzzle constructs phylogenetic trees by maximum likelihood, using quartet puzzling, automatically estimating internal branch node support (1000 replications). Derived trees were visualized using MEGA(Molecular Evolutionary Genetics Analysis) version 3.1(http://www.megasoftware.net/)(Kumar et al., 2004).

Measurement of activity was performed in quartz microcuvettes using 60μl samples (Starna Cells, Atascadero, CA, USA). 20 μmol l^–1 adenosine in PBS was added to the cuvette, followed by addition of the enzyme source. After mixing the solution by pipetting, the absorbance between 220 and 300 nm was monitored at 1.5 or 3.0 min intervals using a Lambda 18 spectrophotometer from Perkin Elmer (Norwalk, CT, USA).

Molecular sieving–high-performance liquid chromatography (MS-HPLC)was carried out using a Dionex Summit system and Chromeleon software (Dionex,Sunnyvale, CA, USA). For analysis, 20 μl of sample was applied to a Superdex Peptide PC 3.2/30 column (Amersham Biosciences, Piscataway, NJ, USA)using 10 mmol l^–1 NaPO_4, 150 mmol l^–1 NaCl, pH 6.5 as the mobile phase at a flow rate of 150μl min^–1 for 30 min of separation. Detection was performed using a photodiode array detector and a 3-D chromatogram generated using Chromeleon software. Adenosine and AMP standards (500 pmol) were applied separately. Single pairs of salivary glands from P. papatasi and P. duboscqi were sonicated in PBS, clarified by centrifugation and applied to HPLC.

DNA fragments encoding mature P. duboscqi ADA protein were amplified and inserted into the cloning site of the pCRT7/NT-TOPO vector(Invitrogen). The primers used for PCR amplification of the mature ADA encoding fragments were PDBL_P02_G04_VF(5′-GTTTTGGACATTTCGAACATTA-3′) and PDBL_P02_G09_VR(5′-TGGCTCCAAATGATTCAGACA-3′) for 2G4 (PduM73) and PDBL_P02_G09_VF(5′-CTTTGAAAATTAAACCGAAACGA-3′ and PDBL_P02_G09_VR for 2G9(PduM74). Escherichia coli strain BL21(DE3)pLysS cells (Invitrogen)were transformed with the recombinant plasmid and grown in LB broth containing ampicillin (50 μg ml^–1). Production of recombinant protein was induced by addition of IPTG to a final concentration of 1 mmol l^–1 and at 27°C for 3 h. The recombinant protein was purified from the supernatant of bacterial sonic lysate using a MagneHis Protein Purification System (Promega, Madison, WI, USA) and dialysed with Centricon Plus-20 (Millipore, Bedford, MA, USA) to remove imidazole from the elution buffer before further enzymatic analysis.

The samples were treated with NuPAGE LDS sample buffer (Invitrogen) and analysed on NuPAGE 10% Bis-Tris gels (Invitrogen) with NuPAGE MES SDS running buffer (Invitrogen). To estimate the molecular mass of the samples, SeeBlue markers from Invitrogen (myosin, bovine serum albumin, glutamic dehydrogenase,alcohol dehydrogenase, carbonic anhydrase, myoglobin, lysozyme, aprotinin, and insulin, chain B) were used. After electrophoresis, the gels were stained with SimplyBlue™ SafeStain Coomassie^® (Invitrogen) or SilverQuest™ Silver Staining (Invitrogen).

For the western blotting, the proteins in the gel were transferred to nitrocellulose membrane (Invitrogen) using NuPAGE transfer buffer(Invitrogen). After blocking with 5% milk in Tris-buffered saline containing 0.1% Tween-20, pH 8.0 (TBST), the membrane was incubated with alkaline phosphatase (AP)-conjugated anti-His₆/G antibody (Invitrogen) for 1 h at room temperature. After three washes with TBST, the blots were developed by addition of 5-bromo-4-chloro-3-indolyl-1-phosphate and nitro blue tetrazolium for visualization.

By sequencing a P. duboscqi salivary gland cDNA library, we have identified two transcripts coding for a protein homologous to ADA, an enzyme that metabolizes adenosine to inosine (Kato et al., 2006). The first transcript (PduM73; NCBI accession number DQ835357) of 1846 bp codes for a secreted protein of 57.6 kDa with an isoelectric point of 5.5 (Fig. 1); the second transcript (PduM74) of 1810 bp codes for a protein of 57.2 kDa with an isoelectric point of 5.8 (data not shown). Multiple sequence comparison of P. duboscqi ADA with homologues from dipterans such as the sand fly L. longipalpis and the mosquitoes Ae. aegypti, Ae. albopictus and Culex pipiens and from mammals such as mice, rats and humans shows an overall low level of identity(Fig. 2); however, the amino acids forming part of the active site (His₁₁₆, His₁₁₈,Ala₁₂₁, Gly₃₂₈, His₃₅₅, Glu₃₅₈,Gly₃₈₁, Asp₄₄₀, Asp₄₄₁) are highly conserved(Fig. 2). The ADA from P. duboscqi and from other insects is larger than the ADA from mice, rats or humans; a large string of approximately 80 amino acids at the N-terminal region is not present in the mammalian ADA. Additionally, the signal peptide sequence is not present in the mammalian ADA(Fig. 2). Phylogenetic analysis of ADA from different organisms produced a tree with two distinct clades, one containing ADA from dipteran blood-feeders and the other clade containing other organisms including Leishmania, Plasmodium, Entamoeba, mice,rats and humans (Fig. 3). Within the dipteran blood-feeders clade, sand flies form a distinct group,separate from mosquitoes.

Because of the discovery of the ADA transcripts in the salivary gland cDNA library of P. duboscqi, we wanted to test whether the saliva of this sand fly had ADA activity. For this, SGH of P. duboscqi was incubated in the presence of adenosine and the reaction was followed spectrophotometrically by scanning from 220 nm to 300 nm every 3 min. The substrate adenosine absorbs at 265 nm, and the product of ADA activity,inosine, absorbs at 241 nm. The equivalent of 0.2 salivary gland pairs (0.2μg) of P. duboscqi converted adenosine to inosine in 30 min(Fig. 4A); by contrast, the same amount of SGH of P. papatasi had no effect on adenosine(Fig. 4B). Differential spectrum shows, in better detail, the decrease of adenosine (265 nm) and, over time, the increase of inosine (241 nm) in the presence of P. duboscqiSGH (Fig. 4C), indicating the presence of ADA activity in the salivary gland of this sand fly. To test whether this activity is secreted in the saliva of P. duboscqi, we compared ADA activity from SGH of unfed sand flies (intact saliva), from SGH of recently blood-fed sand flies (loss of secreted protein by salivation during feeding) and from punctured salivary glands (loss of all or the majority of the salivary contents and therefore any enzymatic activity). Salivary glands from unfed sand flies had the highest ADA activity while preparations from the salivary glands of recently blood-fed sand flies had approximately 70% less activity (Fig. 5). Finally, ADA activity was not detected in the preparations of punctured salivary glands (Fig. 5). Additionally, the amino-terminal sequence of the native protein was detected in the secreted fraction of the SGH of this sand fly(Kato et al., 2006). These data suggest that the molecule responsible for this activity is secreted in the saliva of this sand fly.

It was previously shown that P. papatasi and P. argentipes do not have transcripts coding for the enzyme ADA in their salivary glands or the activity was not detected within their salivary glands(Anderson et al., 2006; Charlab et al., 2000); however,it was shown that these sand flies have large amounts of adenosine and AMP within their salivary glands (Ribeiro et al., 1999). Although counterintuitive, due to the presence of ADA activity, P. duboscqi salivary glands were tested for the presence of adenosine and AMP by subjecting P. duboscqi SGH to MS-HPLC, and the eluted products were detected by photodiode array detection. As expected, and as previously shown, analysis of P. papatasi SGH resulted in the presence of two major peaks with the same retention times as adenosine (18.5 min) and AMP (14.5 min), respectively (Fig. 6). By contrast, P. duboscqi SGH showed no peaks at the retention times of adenosine and AMP (Fig. 6). Only a peak at 5 min was observed, which is the secreted proteins from the salivary glands, as determined by the retention time and the absorption spectra at 280 nm (Fig. 6). These data suggest that, in contrast to P. papatasiand P. argentipes, P. duboscqi does not have AMP or adenosine in its salivary glands and that it contains the active salivary ADA.

In order to determine if the ADA activity detected in P. duboscqiSGH was related to the transcript coding for this enzyme, we cloned the two transcripts coding for this protein into the PCRT7NT-TOPO bacterial expression vector. The soluble expressed proteins were purified from the supernatant of bacterial lysate by nickel magnetic beads, and an aliquot was subjected to western blot analysis and detected using anti-histidine antibody. This revealed a protein of approximately 60 kDa, which is the estimated molecular mass of the predicted ADA including the 4-kDa N-terminal addition that includes His₆G and Xpress™ peptide epitopes(Fig. 7A, lanes 1 and 2). No protein of this molecular mass was detected in the supernatant of bacteria expressing the empty vector (Fig. 7A, lane 3). Furthermore, a soluble expressed protein with the same migration pattern was detected by Coomassie blue staining(Fig. 7B, lanes 1 and 2), and this protein was not detected in samples from bacteria transformed with control plasmid (Fig. 7B, lane 3). The purified soluble recombinant proteins were then tested for the presence of ADA activity. Both sand fly recombinant proteins had a high level of ADA activity as detected spectrophotometrically by the conversion of adenosine to inosine (Fig. 8A,B). This activity was not detected in the supernatant of bacteria transformed with control plasmid(Fig. 8C). These data suggest that the P. duboscqi transcript coding for an ADA is responsible for the ADA activity detected in the saliva of this sand fly.

Phlebotomus duboscqi is a proven vector of Leishmania major in sub-Saharan Africa. It belongs to the subgenus Phlebotomus, together with the sand fly vector P. papatasi. Knowledge of the repertoire of salivary activities or molecules from the saliva of P. duboscqi is very limited. We recently sequenced a large number of transcripts from the salivary gland of P. duboscqi and identified a transcript coding for the enzyme ADA(Kato et al., 2006). This is the first report of ADA in a sand fly from the genus Phlebotomus,including data from transcriptome analysis from the salivary glands of P. papatasi, P. ariasi, P. argentipes and P. perniciosus sand flies(Anderson et al., 2006).

In the present work, we have demonstrated the presence of ADA activity in the saliva of P. duboscqi and we also demonstrated that the soluble recombinant protein produced from the transcript coding for this enzyme exhibited ADA activity. Phylogenetic analysis placed P. duboscqi ADA in the same clade with ADA from other blood-feeding arthropods. This group belongs to the ADGF/CECR1 family of proteins identified previously in Sarcophaga peregrina, Lutzomyia longipalpis, Drosophila, Aplysia and humans (Dolezelova et al.,2005). This sub-family of ADAs has an extended N-terminus region and is targeted for secretion. These data suggest that P. duboscqiacquired this activity independent of other Phlebotomus sand flies. The question remains as to what the role of this protein is in blood feeding. It was previously speculated that the activity may be related to the hydrolysis of adenosine, an important component in pain perception and in immunity (Charlab et al.,2001). What is puzzling is that other Phlebotomus sand flies do not have ADA in their salivary glands but they have large amounts of adenosine and AMP, very active vasodilators and platelet inhibitors. Neither adenosine nor AMP was present in the saliva of P. duboscqi, as demonstrated in here. Lutzomyia longipalpis also lacks adenosine and AMP in its saliva; however, it has maxadilan, a very potent vasodilator(Ribeiro et al., 1989). Maxadilan was not identified in the P. duboscqi cDNA library(Kato et al., 2006). Therefore,it appears that P. duboscqi may contain a novel vasodilator that will replace the lack of vasodilatory activities exerted by AMP and adenosine in other Phlebotomus sand flies. The fact that ADA is present only in P. duboscqi and not in other Phlebotomus sand flies examined to date emphasizes the ability of blood-feeding arthropods to acquired independent strategies to overcome or modulate the host hemostatic,inflammatory and immune system.

ADA has an important role in immunity as a result of the effects of adenosine, 2-deoxyadenosine and the hydrolytic product of these compounds(Cristalli et al., 2001). Further work will be necessary to determine the effect of this enzyme in parasite transmission. It may be possible that this enzyme changes the environment in the skin where the Leishmania parasite is deposited by the sand fly. Inosine is the primary metabolite of adenosine by ADA. Inosine has been shown to inhibit the production of proinflammatory cytokines including TNF-α, IL-1, IL-12, MIP1-α and INFγ in stimulated macrophages and spleen cells (Hasko et al.,2000). Additionally, adenosine and inosine can alter cutaneous vasopermeability by activating A₃ receptors on mast cells(Tilley et al., 2000). Then it may be possible that inosine may favor a Th2 environment that will benefit parasite establishment in the skin of the mammalian host.

We would like to thank Dr J. M. C. Ribeiro for critical discussion on this work, Dr Fabiano Oliveira and Dr Jennifer Anderson for reviewing this manuscript, Dr Robert Gwadz for his continuous support and Nancy Shulman for editorial assistance. This project was funded by Grant 1Z01AI00093202 to J.G.V. from the Division of Intramural Research, National Institutes of Allergy and Infectious Diseases, National Institutes of Health.