Discussion

The Leishmania mexicana promastigote flagellum has several well-characterised functions including motility and sensing environmental stimuli, but the role of this organelle in the amastigote life cycle stage remains elusive. Some insight has been provided by the similarity in axoneme structure to that of a typical mammalian primary cilium, and by the observation that the amastigote flagellum tip is often closely associated with the parasitophorous vacuole membrane, suggesting a potential role in parasite-host interactions. We used serial block face scanning electron microscopy and transmission electron tomography to explore the interaction between the amastigote flagellum and the parasitophorous vacuole of in vitro infected macrophages. The resulting 3D models showed that amastigotes were typically tucked into the parasitophorous vacuole membrane, with the main vacuole volume bulging away from them, and revealed invaginations of the vacuole membrane at flagellum contact points. These invaginations differ from previously observed indentations of the vacuole membrane at the point of contact with the flagellum, and appear to represent vesicles either budding from or fusing with the vacuole membrane. Ongoing work addresses molecular interactions through the analysis of flagellar trafficking mutants which are unable to infect macrophages, and the detection of proteins in the amastigote flagellar membrane using a proximity-dependent biotin labelling approach.