Intact salivary gland pairs were collected from adult female X. cheopis fleas. Individual fleas (anesthetized by chilling on ice) were dissected in 10 μl of PBS on a glass microscope slide on the stage of a dissecting stereomicroscope. By grasping the dorsal half of the flea above the forelegs with forceps, pressing down on the abdomen just posterior to the midgut with a bent dissecting needle, and pulling, the two pairs of salivary glands and the midgut would usually remain attached to the head and be pulled free of the rest of the body. The common lateral salivary ducts were cut to release each pair, which were then hooked with a dissecting pin and placed in PBS at 4°C. Pools of 40 pairs of glands in 20 μl PBS were frozen at 70°C. Glands used for apyrase assays were dissected and stored in 10 mM TrisHCl and 150 mM NaCl, pH 7.4 rather than PBS.
Salivary gland isolation and library construction
X. cheopis salivary gland mRNA was isolated from 200 salivary gland pairs from adult fleas using the Micro-FastTrack mRNA isolation kit (Invitrogen, SanDiego, CA). The PCR-based cDNA library was made following the instructions for the SMART cDNA library construction kit (Clontech, Palo Alto, CA). This system utilizes oligoribonucleotide (SMART IV) to attach an identical sequence at the 5' end of each reverse-transcribed cDNA strand. This sequence is then utilized in subsequent PCR reactions and restriction digests.
First strand synthesis was carried out using PowerScript reverse transcriptase at 42°C for 1 hr in the presence of the SMART IV and CDS III (3') primers. Second strand synthesis was performed by a long distance (LD) PCR-based protocol, using Advantage™ Taq Polymerase (Clontech) mix in the presence of the 5' PCR primer and the CDS III (3') primer. The cDNA synthesis procedure resulted in the creation of SfiI A &B restriction enzyme sites at the ends of the PCR products that are used for cloning into the phage vector. The PCR conditions were: 95°C for 20 sec; 24 cycles of 95°C for 5 sec., 68°C for 6 min. A small portion of the cDNA obtained by PCR was analysed on a 1.1% agarose gel to check for the quality and range of cDNA synthesised. Double stranded cDNA was immediately treated with proteinase K (0.8 μg/ml) at 45°C for 20 min and the enzyme was removed by ultrafiltration though a Microcon (Amicon) YM100 centrifugal filter device. The cleaned, doublestranded cDNA was then digested with SfiI at 50°C for 2 hrs, followed by size fractionation on a ChromaSpin-400 column (Clontech, Palo Alto, CA). The profile of the fractions was checked on a 1.1% agarose gel and fractions containing cDNAs of more than 400 bp were pooled and concentrated using a Microcon YM100.
The cDNA mixture was ligated into the λ TriplEx2 vector (Clontech, Palo Alto, CA) and the resulting ligation mixture was packaged using the GigaPack® III Plus packaging extract (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The packaged library was plated by infecting log phase XL1 Blue E. coli cells (Clontech, Palo Alto, CA). The percentage of recombinant clones was determined by performing a blue-white selection screening on LB/MgSO4 plates containing X-gal/IPTG. Recombinants were also determined by PCR, using vector primers (5' λ TriplEx2 Sequencing Primer and 3' λ TriplEx2 Sequencing) flanking the inserted cDNA and visualising the products on a 1.1% agarose/EtBr gel.
Sequencing of the X. cheopis cDNA library
The X. cheopis salivary gland 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 selected and transferred to 96-well MICROTEST™ U Bottom plates (BD BioSciences, Franklin Lakes, NJ), containing 100 μl of SM buffer [0.1 M NaCl; 0.01 M MgSO4; 7H2 O; 0.035 M TrisHCl (pH 7.5); 0.01% gelatin] per well. The plates were covered and placed on a gyrating shaker for 30 min at room temperature. The phage suspension was either immediately used for PCR or stored at 4°C for future use.
To amplify the cDNA using a PCR reaction, four microliters of the phage sample was used as a template. The primers were sequences from the λ TriplEx2 vector and named pTEx2 5 seq (5'TCC GAG ATC TGG ACG AGC 3') and pTEx2 3 LD (5' ATA CGA CTC ACT ATA GGG CGA ATT GGC 3'), positioned at the 5' end and the 3' end of the cDNA insert, respectively. The reaction was carried out in 96 well flexible PCR plates (Fisher Scientific, Pittsburgh, PA) using the TaKaRa EX Taq polymerase (TAKARA Mirus Bio, Madison, WI), on a Perkin Elmer GeneAmp® PCR system 9700 (Perkin Elmer Corp., Foster City, CA). The PCR conditions were: one hold of 95°C for 3 min; 25 cycles of 95°C for 1 min, 61°C for 30 sec; 72°C for 2 min. The amplified products were analysed on a 1.5% Agarose/EtBr gel. 1100 cDNA library clones were PCR amplified and the ones showing single band were selected for sequencing. Approximately 200–250 ng of each PCR product was transferred to Thermo-Fast 96-well PCR plates (ABgene Corp., Epsom, Surray, UK) and frozen at 20°C, before cycle sequencing using an ABI3730 XL machine.
Bioinformatic tools and procedures used
Expressed sequence tags (EST) were trimmed of primer and vector sequences, clusterized, and compared with other databases as described [44
]. The BLAST tool [77
], CAP3 assembler [78
], ClustalW [79
], and Treeview software [80
] were used to compare, assemble, and align sequences and to visualise alignments. For functional annotation of the transcripts we used the tool BlastX [33
] to compare the nucleotide sequences to the NR protein database of the National Center for Biotechnology Information (NCBI) and to the Gene Ontology (GO) database[34
]. The tool RPSBlast [33
] was used to search for conserved protein domains in the Pfam [81
], SMART [82
], Kog [83
] and Conserved Domains Databases (CDD) [35
]. 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 (Drosophila melanogaster
), or ENSEMBL (An. gambiae
). Segments of the three-frame translations of the EST (because the libraries were unidirectional we did not use six-frame translations), starting with a methionine found in the first 100 predicted AA, or to the predicted protein translation in the case of complete coding sequences, were submitted to the SignalP server [36
] to help identify translation products that could be secreted. O-glycosylation sites on the proteins were predicted with the program NetOGlyc ([84
]. Functional annotation of the transcripts was based on all the comparisons above. Following inspection of all these 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. Phylogenetic analysis and statistical Neighbor Joining (NJ) bootstrap tests of the phylogenies were done with the Mega package [85
Gel electrophoresis studies
Flea salivary proteins representing approximately 100 gland pairs were resolved by one-dimensional (1D) sodium dodecylsulfate polyacrylamide gel electrophoresis (4–12% gradient gels) and visualised with Coomassie blue staining (Pierce, Rockford, IL). Excised gel bands were destained using 50% acetonitrile in 25 mM NH4HCO3, pH 8.4 and vacuum dried. Trypsin (20 μg/mL in 25 mM NH4HCO3, pH 8.4) was added and the mixture was incubated on ice for one hr. The supernatant was removed and the gel bands were covered with 25 mM NH4HCO3, pH 8.4. After overnight incubation at 37°C, the tryptic peptides were extracted using 70% acetonitrile, 5% formic acid, and the peptide solution was lyophilised and desalted using ZipTips (Millipore, Bedford, MA).
Low MW fractionation of flea salivary proteins
A low molecular protein sample was prepared by resuspending 51 μg of total flea protein salivary homogenate into 2 mL of 100 mM NH4HCO3, pH 8.4, containing 10% acetonitrile. Low MW proteins were obtained by centrifugal ultrafiltration using Centriplus 30 kDa ultrafilters (Millipore, Billerica, MA) spun at 750 × g at 4°C. The low MW filtrate was lyophilised and resuspended in 50 μL of 25 mM NH4HCO3, pH 8.4, and half of the solution was digested with trypsin (enzyme:protein ratio of 1:50) for 16 h at 37°C. The undigested and digested samples were desalted using C18 ZipTips (Millipore, Bedford, MA), lyophilised to dryness and resuspended in 14 μL 0.1% TFA for subsequent nanoRPLCMS/MS analysis.
Nanoflow reversedphase liquid chromatography tandem mass spectrometry (nanoRPLCMS/MS)
The tryptic peptides were analyzed using nanoRPLCMS/MS. A 75 μm i.d. × 360 μm o.d. × 10 cm long fused silica capillary column (Polymicro Technologies, Phoenix, AZ) was packed with 3 μm, 300 Å pore size C-18 silica bonded stationary RP particles (Vydac, Hysperia, CA). The column was connected to an Agilent 1100 nanoLC system (Agilent Technologies, Palo Alto, CA) that was coupled online with a linear iontrap (LIT) mass spectrometer (LTQ, ThermoElectron, San Jose, CA). The peptides were separated using a gradient consisting of mobile phase A (0.1% formic acid in water) and B was (0.1% formic acid in acetonitrile). The peptide samples were injected and gradient elution was performed under the following conditions: 2% B at 500 nL/min in 30 min; a linear increase of 2–42% B at 250 nL/min in 110 min; 42–98% in 30 min including the first 15 min at 250 nL/min and then 15 min at 500 nL/min; 98% at 500 nL/min for 10 min. The LIT-MS was operated in a datadependent tandem MS (MS/MS) mode in which the five most abundant peptide molecular ions in every MS scan were selected for collision induced dissociation (CID) using a normalized collision energy of 35%. Dynamic exclusion was applied to minimize repeated selection of previously analyzed peptides. The capillary temperature and electrospray voltage were set to 160°C and 1.5 kV, respectively. Tandem MS spectra from the nanoRPLCMS/MS analyses were searched against a protein fasta database derived from the flea salivary gland, using SEQUEST operating on an 18 node Beowulf cluster. For a peptide to be considered legitimately identified, it had to achieve stringent charge state and proteolytic cleavage-dependent cross correlation (Xcorr) and a minimum correlation (ΔCn) score of 0.08.
Measurement of apyrase activity
Apyrase activity was measured as described previously [86
]. Specific conditions are given in the legend accompanying Figure .