Plasmodium falciparum infection induces expression of a mosquito salivary protein (Agaphelin) that targets neutrophil function and inhibits thrombosis without impairing hemostasis

Abstract

Background: Invasion of mosquito salivary glands (SGs) by Plasmodium falciparum sporozoites is an essential step in the malaria life cycle. How infection modulates gene expression, and affects hematophagy remains unclear.

Principal findings: Using Affimetrix chip microarray, we found that at least 43 genes are differentially expressed in the glands of Plasmodium falciparum-infected Anopheles gambiae mosquitoes. Among the upregulated genes, one codes for Agaphelin, a 58-amino acid protein containing a single Kazal domain with a Leu in the P1 position. Agaphelin displays high homology to orthologs present in Aedes sp and Culex sp salivary glands, indicating an evolutionarily expanded family. Kinetics and surface plasmon resonance experiments determined that chemically synthesized Agaphelin behaves as a slow and tight inhibitor of neutrophil elastase (K(D) ∼ 10 nM), but does not affect other enzymes, nor promotes vasodilation, or exhibit antimicrobial activity. TAXIscan chamber assay revealed that Agaphelin inhibits neutrophil chemotaxis toward fMLP, affecting several parameter associated with cell migration. In addition, Agaphelin reduces paw edema formation and accumulation of tissue myeloperoxidase triggered by injection of carrageenan in mice. Agaphelin also blocks elastase/cathepsin-mediated platelet aggregation, abrogates elastase-mediated cleavage of tissue factor pathway inhibitor, and attenuates neutrophil-induced coagulation. Notably, Agaphelin inhibits neutrophil extracellular traps (NETs) formation and prevents FeCl3-induced arterial thrombosis, without impairing hemostasis.

Conclusions: Blockade of neutrophil elastase emerges as a novel antihemostatic mechanism in hematophagy; it also supports the notion that neutrophils and the innate immune response are targets for antithrombotic therapy. In addition, Agaphelin is the first antihemostatic whose expression is induced by Plasmodium sp infection. These results suggest that an important interplay takes place in parasite-vector-host interactions.

Figure 1. Characterization of Agaphelin.

A) Clustal alignment of Agaphelin (gi| 118789673) with other proteins of the Kazal superfamily from Diptera. The boxes indicate the six conserved cysteines. Symbols below the alignment indicate: (*) identical sites; (:) conserved sites; (.) less-conserved sites. The underlined represent the signal peptide. B) Phylogram of Agaphelin (blue box) and other organisms obtained by the neighbor-joining algorithm using pairwise deletion and Poisson model. Sequences from the nonreduntant protein database of the National Center for Biotechnology Information (NCBI) are represented by the first three letters of their genus name, followed by the first three letters of the species name, followed by gi| accession number. Numbers in the phylogram nodes indicate percent bootstrap support for the phylogeny after 10,000 iterations. The bar indicates 10% amino acid divergence in the sequences. C) Secondary structure of Agaphelin , . D) Structural model of Agaphelin (red) superimposed to Infestin 4 (gray). The six cysteines and the residue at position P1 from Agaphelin are labeled in black while the P1 residue of Infestin 4 is labeled in gray. Disulfide bonds are marked in yellow. E) Mass spectrometry analysis for Agaphelin. Inset: SDS/PAGE of Agaphelin, under reducing conditions. F) Analysis by SEC-MALS-HPLC provided the molar mass distribution of the main peak compared with absorbance at 280 nm. The continuous and interrupted lines represent absorbance 280 nm and MALS results, respectively. Inset: molecular weight markers (black lines) were loaded in the same column, and elution time was compared with Agaphelin (red line). G) Circular dichroism spectrum of Agaphelin showing a negative peak maxima at 222.6 nm and 208.0 nm and a positive peak maximum at 190 nm, indicating significant α-helix content. The ellipticity (degrees cm²/dmol) was plotted as a function of wavelength (nm) composition of Agaphelin. Data show the percentage of each type of secondary structure as determined by DichroWeb server (inset).

Figure 2. Agaphelin inhibits elastase.

A) Agaphelin (1 µM) was tested against 16 different serine proteases in triplicates. Enzyme concentrations are provided in Material and Methods, (*, t-test; p≤0.05). B) Agaphelin inhibits elastase. Tight-type inhibitor: Elastase (18 nM) was incubated with Agaphelin (0–100 nM) for 30 min at RT followed by addition of chromogenic substrate MeOSuc-AAPV-pNA (600 µM). Residual activity was plotted as Vo (final velocity)/Vs (initial velocity). Inset: slow-type inhibitor: elastase (18 nM) was added to a mixture containing chromogenic substrate (600 µM) and Agaphelin (a, 0 nM; b, 150 nM, and c, 500 nM). C) Surface plasmon resonance experiments. Upper panel: Agaphelin was immobilized in a GLC chip and elastase was injected for 60 sec at 100 nM (a, blue), 50 nM (b, green), and 25 nM (c, pink). Dissociation of the Agaphelin-elastase complex was monitored for 600 sec. Representative sensograms (upper panel) are shown in black lines, and global fitting of the data points using the Langmuir equation is depicted. Lower panel: residual response.

Figure 3. Agaphelin inhibits neutrophil chemotaxis in vitro.

A) Chemotaxis of HL60 cells in EZ-TAXIScan chambers. Upper panel: HL60 cells incubated with PBS and then exposed to fMLP (20 nM) concentration gradient. Middle panel: HL60 cells incubated with buffer only and not exposed to fMLP. Lower panel: HL60 cells incubated with Agaphelin (1 µM) for 1 h and exposed to fMLP (20 nM). B–E) Quantification. Cell migration was analyzed based on the results presented in (A) and plotted for the following parameters: B) Total path length; C) Cell speed; D) Directionality, and E) Cell roundness. Data are expressed as the mean ± S.E (*, p≤0.05; t-test).

Figure 4. Agaphelin inhibits elastase-mediated platelet aggregation, TFPI-cleavage by elastase, and neutrophil-induced coagulation.

A) Platelet aggregation. Washed human platelets were stimulated with elastase only (500 nM, left panel), or cathepsin G only (110 nM, middle tracing), or elastase followed by cathepsin G (right tracing). In some experiments, Agaphelin (1 or 10 µM) was added to platelets followed by addition of elastase for 1 min, and cathepsin G. B) Agaphelin does not inhibit cathepsin G (200 nM) or C) collagen (0.6 µg/ml)-induced platelet aggregation. Aggregation response was monitored by turbidimetry using a Lumi-Aggregometer. D) TFPI cleavage. Agaphelin (0.1 µM and 1 µM) was incubated with 1 µg of TFPI in the presence of PBS or human neutrophil elastase (0.8 µg/ml). After 2 h, reactions were stopped by addition of LDS loading buffer (under reducing conditions, 10 mM DTT), boiled for 5 min, and loaded in 4–12% Nu-PAGE gel. Gels were Coomassie Blue-stained. E) Neutrophil-induced coagulation. Neutrophils (5×10⁵ cells/well) were incubated with Agaphelin (1 µM), PBS (control) or DNAse (Dornase alfa, 4 µg/ml) for 1 hour, followed by addition of PMA (50 nM) for 3 hrs. Fifty µl of this suspension was added to 50 µl of plasma, and reactions were started by addition of CaCl₂ (12.5 mM, final concentration)(*, p<0.05).

Figure 5. Agaphelin inhibits NETs formation.

(A) Adherent neutrophils were incubated with Agaphelin or PBS (control) for 1 hour, and activated with PMA (5 nM, central panel), or PMA plus Agaphelin (right panel) for 3 h at 37°C. Left panel, non-activated neutrophil (no PMA). Formation of NETs was visualized under confocal microscopy, using antibody against citrullinated histone. Representative experiment is shown (4 different donors). B) Quantification. Performed as described in Methods. Sixty five per cent inhibition of NETs formation was attained with 1 µM Agaphelin (p = 0.03, t-test).

Figure 6. Agaphelin inhibits inflammation and neutrophil accumulation in vivo.

A) Paw edema in mice. Carrageenan (2%) was administered to mice, in the presence of saline or Agaphelin (1 or 10 mg/Kg). Agaphelin diluted in saline was injected as a control (without carrageenan). Edema formation was evaluated at 0, 4, and 24 h after as increase in paw thickness. B) Neutrophil recruitment in inflamed footpads was evaluated by measuring tissue myeloperoxidase activity, expressed as units of activity/g of tissue, after injection of carrageenan as above. Animals were euthanized 4 hours after. Statistical significance: *, p<0.05 or **, p<0.01 (one-way ANOVA followed by Tukey's post hoc test; n = 5 in each group), compared with carrageenan only.

Figure 7. Agaphelin inhibits thrombosis in vivo, without impairing hemostasis.

A) Arterial thrombosis. A paper filter infused with 7.5% FeCl₃ was applied to the carotid artery, and blood flow was monitored with a perivascular flow probe for 60 min or until stable occlusion took place. Fifteen minutes before injury, Agaphelin was injected into the caudal veins of the mice. Each symbol represents one animal. *, p<0.05 (ANOVA with Dunnett post-test). B) Bleeding time. Bleeding was caused by a tail transection after intravenous injection of Agaphelin at the indicated concentrations. Absorbance at 540 nm (hemoglobin concentration) was used to estimate blood loss. *, p<0.05 (ANOVA with Dunnett post-test).