Discussion

Leishmania are transmitted between mammalian hosts by the sand fly. Upon injection into the mammalian host promastigote-form parasites are phagocytosed by macrophages, where they differentiate into amastigotes. Although virulence factors are known to modulate macrophage signalling pathways to favour infection, the delivery mechanisms are largely unknown. During differentiation to amastigotes the promastigote flagellum shortens dramatically and the fate of the excess flagellar membrane is unknown. My research investigates the possibility that during Leishmania differentiation the flagellar membrane is shed as extracellular vesicles (EVs) which provide a virulence factor delivery mechanism.  Ultracentrifugation was used to isolate EVs from differentiating parasite culture or a control promastigote parasite culture. Mass spectrometric analysis showed that known virulence factors and known EV-associated proteins were enriched in the differentiating sample in addition to many uncharacterised proteins. 34 candidate EV-associated proteins have been fluorescently tagged and localised, and work is ongoing to confirm whether these proteins are EV-associated. Isolated EVs induced changes in cytokine secretion by human macrophages, including reductions in chemokines and interferon gamma. To test whether this effect is independent of the known virulence factor LPG, ongoing research is comparing the effect of EVs from LPG1 knockout parasites with wild-type EVs.