The role of the Leishmania mexicana amastigote flagellum in parasite-host interactions

• Main Author: Valli, Jessica
• Format: Dissertation
• Language: English
• Published: University of Oxford 2017
• Subjects: Molecular biology; Parasitology

Cilia and flagella are important organelles with roles in motility and signalling across eukaryotes. Leishmania species possess both a motile 9+2 flagellum and an immotile 9+0 flagellum in different lifecycle stages. While several roles have been defined for the motile promastigote flagellum, the importance of the amastigote flagellum has long eluded understanding, but recent evidence suggests a potential role for this organelle in parasite-host interactions. In this thesis, serial block-face scanning electron microscopy and transmission electron tomography were used to build comprehensive high-resolution models of Leishmania mexicana amastigotes within parasitophorous vacuoles and to precisely define parasite-host connectivity. This provided previously unavailable three-dimensional insight into both parasite and host structures and allowed us to propose a model of amastigote positioning during vacuole maturation. Analysis of the connectivity between the amastigote flagellum and the PV membrane identified membranous extensions of the flagellar membrane as well as evidence suggestive of vesicular trafficking. In pursuit of functional insight into the amastigote flagellum, we assessed null mutants of components of the BBSome trafficking complex, and of the kinetoplastid-specific trafficking protein, KHARON1, for their ability to infect and replicate within macrophages to determine whether a correctly populated flagellar membrane is required for survival within the intracellular environment. Intriguingly, while KHARON1-null amastigotes were unable to survive within macrophages and exhibited a failure of cytokinesis as well as a cell-shape defect, knockout of individual BBSome components did not significantly impair survival of intracellular amastigotes. This thesis offers insight into the complexities of intracellular survival and provides evidence which ties in with the emerging field of flagellar vesicles as a delivery mechanism for virulence factors, and has thus contributed to the complex body of evidence towards finally defining the role of the Leishmania amastigote flagellum.