Ticks and SG preparation
Ticks were removed from zebu cows located on Point G in Bamako, Mali, in December 2008. The SGs were dissected by one of us (JMA) and transferred to RNAlater (Ambion, Austin, Texas, USA). The vials were kept at 4°C for 24 hours then stored at 30°C until use. Tick carcasses were saved and analyzed by Dr. Dmitry A. Apanaskevich (Assistant Curator, U.S. National Tick Collection, Institute of Arthropodology and Parasitology, Georgia Southern University, Statesboro, Georgia, USA). They were all identified to be adult female specimens of H. m. rufipes Koch, 1844.
cDNA library construction and sequencing
H. m rufipes mRNA from one pair of SGs was isolated using the Micro-FastTrack mRNA isolation kit (Invitrogen, San Diego, California, USA). The PCR-based cDNA library was made following the instructions for the SMART cDNA library construction kit (Clontech, Palo Alto, California, USA). 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 hour in the presence of the SMART IV and CDS III (3′) primers. Second-strand synthesis was performed using 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 and B restriction enzyme sites at the ends of the PCR products that are used for cloning into the phage vector. PCR conditions were as follows: 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 analyzed on a 1.1% agarose gel to check quality and range of cDNA synthesized. 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 YM-100 centrifugal filter device (Amicon Inc., Beverly, California, USA). The cleaned, double-stranded cDNA was then digested with SfiI at 50°C for 2 hours, followed by size fractionation on a ChromaSpin–400 column (Clontech). 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 YM-100.
The cDNA mixture was ligated into the λ TriplEx2 vector (Clontech), and the resulting ligation mixture was packaged using the GigaPack® III Plus packaging extract (Stratagene, La Jolla, California, USA) 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 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, with subsequent visualization of the products on a 1.1% agarose/EtBr gel.
The H. m. rufipes 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 selected and transferred to 96-well MICROTEST ™ U-bottom plates (BD BioSciences, Franklin Lakes, New Jersey, USA) containing 100 μl of SM buffer [0.1 M NaCl; 0.01 M MgSO4; 7 H2O; 0.035 M Tris-HCl (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, 4 μl of the phage sample was used as a template. The primers were sequences from the λ TriplEx2 vector and named pTEx2 5seq (5′-TCC GAG ATC TGG ACG AGC-3′) and pTEx2 3LD (5′-ATA CGA CTC ACT ATA GGG CGA ATT GGC-3′), positioned at the 5′ and the 3′ end of the cDNA insert, respectively. The reaction was carried out in 96-well flexible PCR plates (Fisher Scientific, Pittsburgh, Pennsylvania, USA) using the TaKaRa EX Taq polymerase (TAKARA Mirus Bio, Madison, Wisconsin, USA), on a Perkin Elmer GeneAmp® PCR system 9700 (Perkin Elmer Corp., Foster City, California, USA). 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 6 min. Approximately 200–250 ng of each PCR product was transferred to Thermo-Fast 96-well PCR plates (ABgene Corp., Epsom, Surrey, UK) and frozen at −20°C. Samples were shipped on dry ice to the Rocky Mountain Laboratories Genomics Unit with primer and template combined together in an ABI 96-well Optical Reaction Plate (P/N 4306737) following the manufacturer’s recommended concentrations. Sequencing reactions were set up as recommended by Applied Biosystems BigDye® Terminator v3.1 cycle sequencing kit by adding 1 μl ABI BigDye® Terminator ready reaction mix v3.1 (P/N 4336921), 3 μl 5× ABI sequencing buffer (P/N 4336699), and 2 μl of water for a final volume of 10 μl. Cycle sequencing was performed at 96°C for 10 sec, 50°C for 5 sec, 60°C for 4 min for 27 cycles on either a Bio-Rad Tetrad 2 (Bio-Rad Laboratories, Hercules, California, USA) or ABI 9700 (Applied Biosystems, Inc., Foster City, California, USA) thermal cycler. Fluorescently labeled extension products were purified following Applied Biosystems BigDye® XTerminator™ purification protocol and subsequently processed on an ABI 3730xL DNA Analyzer (Applied Biosystems, Inc.). The AB1 file generated for each sample from the 3730xL DNA analyzer was provided to researchers in Rockville, Maryland, USA, through a secure network drive for all subsequent downstream sequencing analysis. In addition to the sequencing of the cDNA clones, primer extension experiments were performed in selected clones to further extend sequence coverage.
Bioinformatics tools used
ESTs were trimmed of primer and vector sequences. The BLAST suite of programs [33
], CAP3 assembler [34
], and ClustalW [35
] software were used to compare, assemble, and align sequences, respectively. For functional annotation of the transcripts, we used blastx [33
] to compare the nucleotide sequences with the NR protein database of the NCBI and to the Gene Ontology database [36
]. The program reverse position-specific BLAST (RPS-BLAST) [33
] was used to search for conserved protein domains in the Pfam [37
], SMART [38
], Kog [39
], and conserved domains databases [40
]. 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, ENSEMBL, or VectorBase and to the assembled EST salivary database described before [3
], and found in http://exon.niaid.nih.gov/transcriptome/tickreview/Sup-Table-1.xls
from where the fasta set can also be recovered at http://exon.niaid.nih.gov/transcriptome/tick_review/tick_proteins_fasta.zip
. Segments of the three-frame translations of the EST (because the libraries were unidirectional, six-frame translations were not used) starting with a methionine found in the first 300 predicted amino acids, or the predicted protein translation in the case of complete coding sequences, were submitted to the SignalP server [41
] to help identify translation products that could be secreted. O-glycosylation sites on the proteins were predicted with the program NetOGlyc [42
]. Functional annotation of the transcripts was based on all the comparisons above.
For sequence comparisons and phylogenetic analysis, we retrieved tick sequences from GenBank, and we have also deducted protein sequences from ESTs deposited in Dbest, as described and made accessible in a previous review article [3
]. Phylogenetic analysis and statistical neighbour-joining bootstrap tests of the phylogenies were done with the Mega package [43
] after sequence alignment performed by Clustal [44
]. Codon volatility was calculated as previously described [45
Proteomic characterization using one-dimensional gel electrophoresis and tandem mass spectrometry (MS)
The soluble protein fraction from salivary gland homogenates from H. marginatum corresponding to approximately 200 μg of protein was brought up in reducing Laemmli gel-loading buffer. The sample was boiled for 10 min and applied to two lanes (~50 and ~150 μg in each lane) and resolved on a NuPAGE 4–12% Bis-Tris precast gel. The separated proteins were visualized by staining with SimplyBlue (Invitrogen). The gel was sliced into 20 individual sections that were destained and digested overnight with trypsin at 37°C. Peptides were extracted and desalted using ZipTips (Millipore, Bedford, MA) and resuspended in 0.1% TFA prior to S analysis.
Nanoflow reversed-phase liquid chromatography tandem MS (RPLS-MS/MS) was performed using an Agilent 1100 nanoflow LC system (Agilent technologies, Palo Alto, CA) coupled online with a linear ion-trap (LIT) mass spectrometer (LTQ, ThermoElectron, San José, CA). NanoRPLC columns were slurry-packed in-house with 5 μm, 300-Å pore size C-18 phage (Jupiter, Phenomenex, CA) in a 75-μm i.d. × 10-cm fused silica capillary (Polymicro Technologies, Phoenix, AZ) with a flame-pulled tip. After sample injection, the column was washed for 30 min with 98% mobile phase A (0.1% formic acid in water) at 0.5 μL/min, and peptides were eluted using a linear gradient of 2% mobile phase B (0.1% formic acid in acetonitrile) to 42% mobile phase B in 40 min at 0.25 μL/min, then to 98% B for an additional 10 min. The LIT-mass spectrometer was operated in a data-dependent MS/MS mode in which each full MS scan was followed by seven MS/MS scans where the seven most abundant molecular ions were dynamically selected for collision-induced dissociation (CID) using a normalized collision energy of 35%. Dynamic exclusion was applied to minimize repeated selection of peptides previously selected for CID.
Tandem mass spectra were searched using SEQUEST on a 20-node Beowulf cluster against the H. marginatum rufipes
described in this paper and the Bos taurus
proteome (downloaded from ftp://ftp.ncbi.nih.gov/genomes/Bos_taurus/protein/
) with methionine oxidation included as dynamic modification. Only tryptic peptides with up to two missed cleavage sites meeting a specific SEQUEST scoring criteria [delta correlation (ΔCn
) ≥ 0.08 and charge-state-dependent cross correlation (Xcorr
) ≥ 1.9 for [M + H]1+
, ≥ 2.2 for [M + 2H]2+
, and 3.5 for [M + 3H]3+
] were considered as legitimate identifications. The peptides identified by MS were converted to Prosite block format [46
] by a program written in Visual Basic. This database was used to search matches in the Fasta-formatted database of salivary proteins, using the program Seedtop, which is part of the Blast package. The result of the Seedtop search is piped into the hyperlinked spreadsheet to produce a text file as shown in supplemental table S2
. Notice that the ID lines indicate, for example, 18_73, which means that one match was found for fragment number 73 from gel band 18. Because the same tryptic fragment can be found in many gel bands, another program was written to count the number of fragments for each gel band, displaying a summarized result in an Excel table. The summary in this form of 11 →18 | 12 →18 | 13→2 | indicates that 18 fragments were found in Fraction 11, while 18 and 2 peptides were found in fractions 12 and 13, respectively. Furthermore, this summary included protein identification only when two or more peptide matches to the protein were obtained from the same gel slice.