A deep insight into the sialotranscriptome of the gulf coast tick, Amblyomma maculatum

Abstract

Background: Saliva of blood sucking arthropods contains compounds that antagonize their hosts' hemostasis, which include platelet aggregation, vasoconstriction and blood clotting; saliva of these organisms also has anti-inflammatory and immunomodullatory properties. Perhaps because hosts mount an active immune response against these compounds, the diversity of these compounds is large even among related blood sucking species. Because of these properties, saliva helps blood feeding as well as help the establishment of pathogens that can be transmitted during blood feeding.

Methodology/principal findings: We have obtained 1,626,969 reads by pyrosequencing a salivary gland cDNA library from adult females Amblyomma maculatum ticks at different times of feeding. Assembly of this data produced 72,441 sequences larger than 149 nucleotides from which 15,914 coding sequences were extracted. Of these, 5,353 had >75% coverage to their best match in the non-redundant database from the National Center for Biotechnology information, allowing for the deposition of 4,850 sequences to GenBank. The annotated data sets are available as hyperlinked spreadsheets. Putative secreted proteins were classified in 133 families, most of which have no known function.

Conclusions/significance: This data set of proteins constitutes a mining platform for novel pharmacologically active proteins and for uncovering vaccine targets against A. maculatum and the diseases they carry.

Figure 1. The salivary Kunitz family of proteins in Amblyomma maculatum.

Bootstrapped phylogram (1,000 iterations) resulting from the alignment of 101 full-length protein sequences containing one or more Kunitz domains. The numbers on the nodes indicate the bootstrap support, and the bar at the bottom indicates 20% amino acid divergence. Clades with more than 70% bootstrap support are indicated by Roman numerals.

Figure 2. The salivary TIL domain family of proteins in Amblyomma maculatum.

Bootstrapped phylogram (1,000 iterations) resulting from the alignment of 57 full-length protein sequences containing the Kunitz domain. The numbers on the nodes indicate the bootstrap support, and the bar at the bottom indicates 20% amino acid divergence. Clades with more than 80% bootstrap support are indicated by Roman numerals.

Figure 3. The tick salivary carboxypeptidase inhibitor family of proteins.

(A) Alignment of Amblyomma maculatum, Dermacentor andersoni, and Rhipicephalus bursa proteins. (B) Bootstrapped phylogram (1,000 iterations) resulting from the alignment in (A). The numbers on the nodes indicate the bootstrap support above 50%. The clade indicated by CPI contains the functionally characterized R. bursa protein. The bar at the bottom indicates 10% amino acid divergence. The Dermacentor proteins were deducted from ESTs deposited in DBEST.

Figure 4. Phylogram of the phosphatidylethanolamine binding protein (PEBP) family of selected arthropods.

The bootstrapped phylogram (1,000 iterations) was obtained from the alignment of deducted Amblyomma maculatum proteins with homologs found in the non-redundant protein database of the NCBI. The Am. maculatum protein names start with Am- and the remaining proteins are named by the first three letters of the genus name followed by the first three letters of the species name followed by their NCBI gi| accession number. The number at the nodes indicates the bootstrap support, and the bar at the bottom indicates 10% amino acid divergence. Two tick clades are indicated by Roman numerals.

Figure 5. Phylogram of the tick salivary metalloproteases of the reprolysin family.

The bootstrapped phylogram (1,000 iterations) was obtained from the alignment of deducted Amblyomma maculatum proteins with homologs found in the non-redundant protein database of the NCBI. The Am. maculatum protein names start with Am- and are recognized by a red circle marker; the remaining proteins are named by the first three letters of the genus name followed by the first three letters of the species name followed by their NCBI gi| accession number. The number at the nodes indicates the bootstrap support, and the bar at the bottom indicates 20% amino acid divergence. Clades and superclades with strong bootstrap support are indicated with Roman numerals.

Figure 6. The salivary disintegrins 40–270 family of Amblyomma maculatum.

(A) Alignment indicating conserved cysteines in black background, RGD motif in blue background, and blue bar above the sequences indicating the signal peptide indicative of secretion. The symbols at the bottom indicate (*) identity of residues, (:) conserved, and (.) less conserved residues. (B) Bootstrapped phylogram of the alignment in (A). The numbers at the nodes indicate the bootstrap support, and the bar at the bottom indicates 2% amino acid divergence.

Figure 7. Phylogram of tick proteins from the 23-kDa family.

The bootstrapped phylogram (1,000 iterations) was obtained from the alignment of deducted Amblyomma maculatum proteins with homologs found in the non-redundant protein database of the NCBI. The Am. maculatum protein names start with Am- and are recognized by a red circle marker; the remaining proteins are named by the first three letters of the genus name followed by the first three letters of the species name followed by their NCBI gi| accession number. The number at the nodes indicates the bootstrap support above 50%, and the bar at the bottom indicates 20% amino acid divergence. Clades and superclades with strong bootstrap support are indicated with Roman numerals.

Figure 8. The one-of-each family of ticks.

(A) Alignment indicating conserved cysteines in black background, conserved residues in blue background. Sequences starting with Am are from A. maculatum and are recognized by a red circle, those starting with IXOSCA are from Ixodes scapularis retrieved from GenBank and are recognized by a blue triangle, those from Rhipicephalus were assembled from DBEST ESTs as described before and are recognized by a green square . The symbols at the bottom indicate (*) identity of residues, (:) conserved and (.) less conserved residues. (B) Bootstrapped phylogram of the alignment in (A). The numbers at the nodes indicate the bootstrap support and the bar at the bottom indicates 20% amino acid divergence.

Figure 9. Phylogram of tick proteins from the novel 40-33 family.

The bootstrapped phylogram (1,000 iterations) was obtained from the alignment of deducted Amblyomma maculatum proteins with homologs deducted from DBEST sequences described in a previous publication . The Am. maculatum protein names start with Am- and are recognized by a red circle marker; the Ixodes scapularis protein is named IXOSCA followed by the first three letters of the species name followed by their NCBI gi| accession number. The number at the nodes indicates the bootstrap support above 50%, and the bar at the bottom indicates 20% amino acid divergence. Clades are indicated with Roman numerals.

Figure 10. The evasin family of metastriate ticks.

The bootstrapped phylogram (1,000 iterations) was obtained from the alignment of deducted Amblyomma maculatum proteins with homologs deducted from DBEST sequences described in a previous publication , and the R. sanguineus evasins from GenBank. The Am. maculatum protein names start with Am- and are recognized by a red circle marker. Other name conventions are as described in the previous figures. The number at the nodes indicates the bootstrap support above 50%, and the bar at the bottom indicates 20% amino acid divergence. Clades and superclades with strong bootstrap support are indicated with Roman numerals.

Figure 11. The salivary 40–279 novel family of metastriate ticks.

(A) Clustal alignment. The symbols at the bottom indicate (*) identity of residues, (:) conserved and (.) less conserved residues. Identical residues are represented with red letters and yellow background. Conserved residues are shown in blue background. Glycines are shown in yellow background. The red boxes mark the glycine rich regions of 3 Rhipicephalus sequences. (B) Bootstraped phylogram of the alignment in A. The numbers at the nodes indicate the bootstrap support and the bar at the bottom indicates 10% amino acid divergence. The Amblyomma maculatum protein names start with Am- and are recognized by a red circle marker. Remaining sequences are from Rhipicephalus microplus, R. appendiculatus and A. variegatum. Clades with strong bootstrap support are indicated with Roman numerals.