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

Michael Waisberg,Ana C Leal,Michalis Kotsyfakis,Stephen F Porcella,Nidhi Gera,Jan Lukszo,Susan K Pierce,Tainá Gomes,Karine Reiter,Carolina Barillas-Mury,Daniella M Mizurini,Dongying Ma,Carlo J Oliveira,Alvaro Molina-Cruz,Robson Q Monteiro,Ivo M B Francischetti,Beatriz C Sousa,José M C Ribeiro,Kenneth D Vernick

doi:10.1371/journal.ppat.1004338

Abstract

BackgroundInvasion 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 FindingsUsing 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 (KD∼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.ConclusionsBlockade 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.

Highlights

Hematophagous animals are strictly dependent on blood feeding for survival and reproduction
Blockade of neutrophil elastase emerges as a novel antihemostatic mechanism in hematophagy; it supports the notion that neutrophils and the innate immune response are targets for antithrombotic therapy
Agaphelin is the first antihemostatic whose expression is induced by Plasmodium sp infection

Summary

Introduction

Hematophagous animals are strictly dependent on blood feeding for survival and reproduction. The mouthparts of mosquitoes canulate or lacerate arterioles and venules, or penetrate hemorrhagic pools [1,2,3]. These events cause vascular injury, and the host response is accompanied by vasoconstriction, exposure of tissue factor (TF), endothelial cell injury, and activation of platelets, monocytes, and neutrophils [4,5,6,7,8]. Evidences have been provided that neutrophils can kill pathogens in an extracellular manner that does not require phagocytic uptake This mechanism consists of weblike structures of DNA and proteins—known as neutrophil extracellular traps (NETs)—via a process called NETosis [4,5,6,7,8]. How infection modulates gene expression, and affects hematophagy remains unclear

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