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Title: Dissecting the function of the Leishmania flagellum in a CRISPR-Cas9 knockout screen
Author: Beneke, Tom
ISNI:       0000 0004 8507 2555
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2020
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Eukaryotic cilia and flagella are conserved organelles with specialized structures and functions. Cilia and flagella can be well studied in parasites of the genus Leishmania, which possess at different stages in their life cycle either a motile 9 + 2 or immotile 9 + 0 flagellum. Much remains unknown about Leishmania-specific flagellar biology, for example whether flagellar motility is required throughout the Leishmania life cycle. More fundamentally, it is still not fully known how flagellar bending is controlled at a molecular level in motile cilia and flagella. To dissect flagellar function in Leishmania with regard to these open questions, the Cas9 gene editing toolbox LeishGEdit was developed, enabling a knockout screen of ~450 proteins identified in the promastigote flagellum of L. mexicana. This identified 14 mutants with a flagellar curling phenotype. A systematic analysis of the curling phenotype showed that flagellar bending in Leishmania is supported by the extra-axonemal paraflagellar rod structure and regulated through central pair projections and inner dynein arm (IDA) f/l1 components. Further analysis of the IDA f/l1 tether/tether head (T/TH) complex identified LAX28 as a novel IDA f/l1 assembly factor. However, using the CRISPR-Cas9 toolbox not only allowed a dissection of the regulation of flagellar bending in vitro, but also enabled assessing the requirement for motility in vivo. By passaging a range of barcoded Leishmania motility mutants in pooled populations through sand flies and mice, flagellar-driven directional motility was shown to be essential for colonialization of the sand fly vector, but dispensable for survival in the mammalian host. Thus, this thesis shows the implementation of a powerful Cas9 toolbox that provided new insights into the regulation of flagellar bending and the importance of Leishmania motility in vivo.
Supervisor: Gluenz, Eva Sponsor: Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available