Discussion

The Leishmania flagellum is a multifunctional organelle used for motility, attachment to the sand fly vector and sensory functions. To discover the flagellar protein composition and enable a dissection of different flagellar functions, we developed a new deflagellation protocol for L. mexicana promastigotes. The resulting flagella and deflagellated cell bodies were separated into detergent soluble and insoluble fractions for analysis of all four fractions by mass spectrometry. We detected a total of 2491 proteins and using a label-free quantitation method we identified proteins enriched in each of the four fractions. We validated the proteomics data by fluorescence tagging and knockout using CRISPR Cas9 gene editing. 78 proteins enriched in both flagellar fractions were tagged and 66 localised to the flagellum, identifying novel components of the flagellar axoneme, PFR, matrix and flagellar membrane. To date, 18 of these 66 genes were targeted for knockout, resulting in 13 viable knockout cell lines. Four of the mutants had impaired motility; EM analysis is ongoing and flagellar ultrastructural defects for one mutant have been found. This experimental validation showed that the L. mexicana flagellar proteome defined in this study is a rich dataset that will facilitate further dissection of flagellar function in Leishmania spp. Moreover, we have shown the potential of targeted CRISPR-Cas9 screens for the discovery of mutant phenotypes.