Venom vesicles from the parasitoid Ganaspis hookeri facilitate venom protein entry into host immune cells

The parasitoid wasp Ganaspis hookeri infects Drosophila melanogaster larvae, laying an egg and injecting venom directly into the host body cavity. While infected hosts mount an immune response in an attempt to eliminate the parasitoid egg, parasitoid venom proteins act to inhibit these host responses and manipulate host physiology to ensure infection success. A key immune suppressive venom protein in G. hookeri is a venom-specific isoform of the SERCA (Sarco/endoplasmic reticulum calcium-ATPase) calcium pump. However, SERCA is a large hydrophobic protein, and the mechanism by which it and other venom proteins are transported into host cells is not well understood. Electron microscopy and nanoparticle tracking analysis of G. hookeri venom revealed the presence of venom extracellular vesicles (vEVs) as a putative transport mechanism. We characterized the biophysical properties of vEVs, and found that G. hookeri venom contains multiple vesicle types that are distinguishable by size, zeta potential, protein content, and density. A subset of these vEVs contain SERCA, and we found that fluorescently labeled vEVs are able to enter into host immune cells and alter host intracellular calcium levels, and that disruption of vesicle integrity results in the loss of this SERCA activity in host cells. Together, our data support a model in which G. hookeri venom proteins, including SERCA, are packaged into an array of venom vesicles, and are transported into host cells for immune suppression.