Ticks from diverse genera encode chemokine-inhibitory evasin proteins

Abstract

To prolong residence on their hosts, ticks secrete many salivary factors that target host defense molecules. In particular, the tick Rhipicephalus sanguineus has been shown to produce three salivary glycoproteins named "evasins," which bind to host chemokines, thereby inhibiting the recruitment of leukocytes to the location of the tick bite. Using sequence similarity searches, we have identified 257 new putative evasin sequences encoded by the genomes or salivary or visceral transcriptomes of numerous hard ticks, spanning the genera Rhipicephalus, Amblyomma, and Ixodes of the Ixodidae family. Nine representative sequences were successfully expressed in Escherichia coli, and eight of the nine candidates exhibited high-affinity binding to human chemokines. Sequence alignments enabled classification of the evasins into two subfamilies: C₈ evasins share a conserved set of eight Cys residues (four disulfide bonds), whereas C₆ evasins have only three of these disulfide bonds. Most of the identified sequences contain predicted secretion leader sequences, N-linked glycosylation sites, and a putative site of tyrosine sulfation. We conclude that chemokine-binding evasin proteins are widely expressed among tick species of the Ixodidae family, are likely to play important roles in subverting host defenses, and constitute a valuable pool of anti-inflammatory proteins for potential future therapeutic applications.

Keywords: bioinformatics; chemokine; evasin; fluorescence anisotropy; host-pathogen interaction; inhibitor; recombinant protein expression; tick protein.

Figure 1.

Schematic workflow for identification of evasins by sequence similarity searches. DB, database; Local Transcr, locally obtained I. holocyclus and R. microplus transcriptome data sets; Rs, R. sanguineus; TSA, Transcriptome Shotgun Assembly.

Figure 2.

Features of the evasin protein family. A, multiple sequence alignment (obtained using MUSCLE) of selected evasin candidates with R. sanguineus evasin-1 and -4. Features indicated are conserved cysteines (bold and boxed) and glycines (green), potential N-linked glycosylation sites (cyan), and putative tyrosine sulfation sites (magenta). The secondary structure and regions of evasin-1 that interact with CCL3 in the crystal structure are shown above the alignment; yellow arrows indicate β-strands and the rose wavy line indicates the α-helix. Abbreviations use for tick species are: RSA, R. sanguineus; RPU, R. pulchellus; AAM, A. americanum; ACA, A. cajennense; AMA, A. maculatum; APA, A. parvum; ATR, A. triste; IRI, I. ricinus; IHO, I. holocyclus. B, conserved sequence motifs of the C₈ and C₆ evasins showing (in red) the disulfide bond connectivity observed in the structure of R. sanguineus evasin-1.

Figure 3.

Purification and characterization of recombinant R. sanguineus evasin-4. A, size exclusion chromatogram for purification of evasin-4 (after His₆ tag removal) with non-reducing SDS-PAGE of fractions (Fr.) spanning the main peak (boxed on the chromatogram); the molecular weight marker used was Bio-Rad Precision Plus Protein^TM unstained standards. B, fluorescence anisotropy-binding curves showing displacement of a fluorescent CCR2-derived sulfopeptide from each of six chemokines using purified recombinant evasin-4. Data plotted are the averages of three independent experiments, each recorded in duplicate, and error bars represent the S.E. Solid lines are fitted binding displacement curves.

Figure 4.

Purification and characterization of representative evasins from three genera. For evasins, ACA-01 from A. cajennense (A), RPU-01 from R. pulchellus (B), IRI-01 from I. ricinus (C), and IHO-01 from I. holocyclus (D) are shown. Top left, the size exclusion chromatogram for purification of the evasin protein (with a C-terminal His₆ tag); bottom left, non-reducing SDS-PAGE of fractions (Fr.) spanning the main peak (boxed on the chromatogram); right, competitive fluorescence anisotropy curves for binding of the purified evasin to each of five CC chemokines. The molecular weight marker used for SDS-PAGE was Bio-Rad Precision Plus Protein unstained standards. Binding data represent the average ± S.E. (error bars) of values from three independent experiments, each recorded in duplicate. Solid lines are fitted binding displacement curves.

Figure 5.

Inhibition of chemokine activity by purified evasins. Shown are concentration-response curves for inhibition of the chemokines MCP-1 (10 nm) (A) and MCP-2 (100 nm) (B) acting at the receptor CCR2 and for inhibition of the chemokines eotaxin-1 (100 nm) (C) and eotaxin-2 (100 nm) (D) acting at the receptor CCR3. Chemokine activity was detected as the ability of the chemokine to inhibit forskolin (FSK)-induced production of cAMP as detected via a BRET sensor (see “Experimental procedures” for details); thus, evasins inhibit the cAMP-inhibitory activity of the chemokines. Data represent the average ± S.E. (error bars) of values from three independent experiments, each recorded in duplicate.