Profiling extracellular vesicle release by the filarial nematode Brugia malayi reveals sex-specific differences in cargo and a sensitivity to ivermectin

Abstract

The filarial nematode Brugia malayi is an etiological agent of Lymphatic Filariasis. The capability of B. malayi and other parasitic nematodes to modulate host biology is recognized but the mechanisms by which such manipulation occurs are obscure. An emerging paradigm is the release of parasite-derived extracellular vesicles (EV) containing bioactive proteins and small RNA species that allow secretion of parasite effector molecules and their potential trafficking to host tissues. We have previously described EV release from the infectious L3 stage B. malayi and here we profile vesicle release across all intra-mammalian life cycle stages (microfilariae, L3, L4, adult male and female worms). Nanoparticle Tracking Analysis was used to quantify and size EVs revealing discrete vesicle populations and indicating a secretory process that is conserved across the life cycle. Brugia EVs are internalized by murine macrophages with no preference for life stage suggesting a uniform mechanism for effector molecule trafficking. Further, the use of chemical uptake inhibitors suggests all life stage EVs are internalized by phagocytosis. Proteomic profiling of adult male and female EVs using nano-scale LC-MS/MS described quantitative and qualitative differences in the adult EV proteome, helping define the biogenesis of Brugia EVs and revealing sexual dimorphic characteristics in immunomodulatory cargo. Finally, ivermectin was found to rapidly inhibit EV release by all Brugia life stages. Further this drug effect was also observed in the related filarial nematode, the canine heartworm Dirofilaria immitis but not in an ivermectin-unresponsive field isolate of that parasite, highlighting a potential mechanism of action for this drug and suggesting new screening platforms for anti-filarial drug development.

Author summary

Brugia malayi is a parasitic nematode and etiological agent of Lymphatic Filariasis (LF), a mosquito-borne Neglected Tropical Disease affecting approximately 120 million people globally. Brugia and other parasitic nematodes have the ability to modulate host biology and evade the immune response but the mechanisms by which they do this are unclear. One possibility is via immunomodulatory proteins and small RNAs packaged and released in extracellular vesicles (EV) that target host cells. Here we show that all Brugia life stages relevant to human infection release EVs that are internalized by murine macrophages. These EVs contain immunomodulatory proteins and show sex-specific differences in cargo providing insight into how B. malayi establishes and maintains infection. Critically, we show ivermectin, a drug used to treat LF and related diseases affects EV release, which provides new insight into how these drugs work and identifies a new screening paradigm to help identify future anti-parasitic drugs.