Aedes albopictus D7 Salivary Protein Prevents Host Hemostasis and Inflammation

Abstract

Mosquitoes inject saliva into the host skin to facilitate blood meal acquisition through active compounds that prevent hemostasis. D7 proteins are among the most abundant components of the mosquito saliva and act as scavengers of biogenic amines and eicosanoids. Several members of the D7 family have been characterized at the biochemical level; however, none have been studied thus far in Aedes albopictus, a permissive vector for several arboviruses that causes extensive human morbidity and mortality. Here, we report the binding capabilities of a D7 long form protein from Ae. albopictus (AlboD7L1) by isothermal titration calorimetry and compared its model structure with previously solved D7 structures. The physiological function of AlboD7L1 was demonstrated by ex vivo platelet aggregation and in vivo leukocyte recruitment experiments. AlboD7L1 binds host hemostasis agonists, including biogenic amines, leukotrienes, and the thromboxane A2 analog U-46619. AlboD7L1 protein model predicts binding of biolipids through its N-terminal domain, while the C-terminal domain binds biogenic amines. We demonstrated the biological function of AlboD7L1 as an inhibitor of both platelet aggregation and cell recruitment of neutrophils and eosinophils. Altogether, this study reinforces the physiological relevance of the D7 salivary proteins as anti-hemostatic and anti-inflammatory molecules that help blood feeding in mosquitoes.

Keywords: D7 proteins; arthropods; blood feeding; isothermal titration calorimetry; leukocyte recruitment; leukotrienes; mosquito; platelet aggregation; saliva; salivary glands.

Figure 1

Sequence alignment of AlboD7 and AeD7 salivary proteins. Comparison of AlboD7 and AeD7 long form salivary proteins: AlboD7L1 (GenBank: AAV90665.1), AeD7L2 (GenBank: AAL16049), AlboD7L2 (GenBank: AAV90666.1), and AeD7L1 (GenBank: AAA29347). Sequences without a signal peptide were aligned with Clustal Omega and refined using the BoxShade server. Black background shading represents identical amino acids, while grey shading shows similar amino acids. The magenta boxes show the predicted amino acids involved in the U-46619, thromboxane A₂ analog; the cyan box indicates the Tyr-52 predicted to be involved in the TxA₂ binding, based on the solved crystal structure of the AnSt-D7L1 protein [37]; the yellow boxes reflect the amino acids involved in the norepinephrine binding according to the crystal structure obtained from AeD7L1 bound to norepinephrine [35].

Figure 2

Purification of recombinant AlboD7L1. (A) Purification of AlboD7L1 by cation-exchange chromatography using a MonoS 5/50 GL column. Gradient of NaCl (%) is indicated by the blue line. (B) Purification of AlboD7L1 by size exclusion chromatography using a Superdex 200 Increase 10/300 GL column. (C) Coomassie-stained NuPAGE 4–12% Bis-Tris gel electrophoresis of recombinant protein AlboD7L1. SeeBlue Plus2 Pre-stained protein ladder was used as protein standards.

Figure 3

Binding of AlboD7L1 to biogenic amines by isothermal titration calorimetry. Binding experiments were performed on a VP-ITC microcalorimeter. The upper curve in each panel shows the measured heat for each injection, while the lower graph shows the enthalpies for each injection and the fit to a single-site binding model for calculation of thermodynamic parameters. Binding of AlboD7L1 to dopamine (A), norepinephrine (B), epinephrine (C), histamine (D), tryptamine (E), and serotonin (F). The insets show the names and chemical formulas for these compounds.

Figure 4

Binding of AlboD7L1 to biolipids by isothermal titration calorimetry. Binding experiments were performed on a VP-ITC microcalorimeter. The upper curve in each panel shows the measured heat for each injection, while the lower graph shows the enthalpies for each injection and the fit to a single-site binding model for calculation of thermodynamic parameters. Binding of AlboD7L1 to LTB₄ (A), LTC₄ (B), LTD₄ (C), LTE₄ (D), and U-46619 (E). The insets show the names and chemical formulas for these compounds.

Figure 5

Structural model of AlboD7L1. (A) Secondary structure prediction of AlboD7L1 using I-TASSER software. Prediction for coils (C), helixes (H), and strands (S) are indicated. (B) Tertiary structure model of AlboD7L1 predicted by I-TASSER and visualized in Chimera software. N-terminal is indicated by ALA-1, and C-terminal is shown as VAL-311 of the mature protein sequence. Coils and helixes are depicted in black and magenta, respectively. The three amino acids predicted to form a strand are represented in cyan. (C) Superposition of AeD7L1 (PDB ID: 3DXL, shown in grey) and protein structure model of AlboD7L1, represented in cyan, shows an overall similar helix structure. (D) Hydrophobicity surface potential of AeD7L1 (PDB ID: 3DXL) generated by Chimera software with blue being positive and red being negative. (E) Hydrophobicity surface potential of the AlboD7L1 model.

Figure 6

AlboD7L1 inhibits pro-aggregatory effects of serotonin, but not epinephrine. (A) AlboD7L1 inhibits 5-HT-induced shape change and 5-HT-induced potentiation of platelet aggregation triggered by collagen. Platelets were incubated with Tyrode (vehicle) or AlboD7L1 (1 μM), followed by the addition of serotonin (5-HT, 0.8 μM) alone or plus collagen (0.75 μg/mL), as indicated. From left to right, traces show shape change with 5-HT alone, inhibition of 5-HT-induced shape change in the presence of AlboD7L1, potentiation of the collagen response by co-addition of 5-HT resulting in a full aggregation response, and loss of potentiation by 5-HT in the presence of AlboD7L1. (B) AlboD7L1 does not inhibit epinephrine-mediated potentiation of platelet aggregation induced by collagen. Platelets were incubated with Tyrode (vehicle) or AlboD7L1 (1 μM), followed by the addition of epinephrine (EPI 0.8 μM) plus collagen (0.75 μg/mL), as indicated. From left to right, traces show no response of platelets to AlboD7L1 and epinephrine, shape change induced by collagen alone, potentiation of the collagen response by co-addition of epinephrine resulting in a full aggregation response, and no loss of potentiation by epinephrine in the presence of AlboD7L1. A Chrono-Log aggregometer model 500-CA was used.

Figure 7

AlboD7L1 prevents cell recruitment caused by Saccharomyces cerevisiae β-glucan to the peritoneal cavity in mice. (A) Total cell number, (B) neutrophils, (C) eosinophils, and (D) mononuclear cells were collected from the peritoneal cavity of mice after i.v. pretreatment with PBS or AlboD7L1 (100 μg/kg), followed by a i.p. injection of 200 μg of β-glucan from S. cerevisiae. Cell numbers are expressed as cells/cavity (×105). Results are shown as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) followed by Bonferroni post hoc test was used to compare the groups (*p ≤ 0.05; **p ≤ 0.01; ****p ≤ 0.0001).