Date Published: June 26, 2019
Publisher: Public Library of Science
Author(s): Tom Beneke, François Demay, Edward Hookway, Nicole Ashman, Heather Jeffery, James Smith, Jessica Valli, Tomas Becvar, Jitka Myskova, Tereza Lestinova, Shahaan Shafiq, Jovana Sadlova, Petr Volf, Richard John Wheeler, Eva Gluenz, Kent L. Hill.
The protozoan parasite Leishmania possesses a single flagellum, which is remodelled during the parasite’s life cycle from a long motile flagellum in promastigote forms in the sand fly to a short immotile flagellum in amastigotes residing in mammalian phagocytes. This study examined the protein composition and in vivo function of the promastigote flagellum. Protein mass spectrometry and label free protein enrichment testing of isolated flagella and deflagellated cell bodies defined a flagellar proteome for L. mexicana promastigote forms (available via ProteomeXchange with identifier PXD011057). This information was used to generate a CRISPR-Cas9 knockout library of 100 mutants to screen for flagellar defects. This first large-scale knockout screen in a Leishmania sp. identified 56 mutants with altered swimming speed (52 reduced and 4 increased) and defined distinct mutant categories (faster swimmers, slower swimmers, slow uncoordinated swimmers and paralysed cells, including aflagellate promastigotes and cells with curled flagella and disruptions of the paraflagellar rod). Each mutant was tagged with a unique 17-nt barcode, providing a simple barcode sequencing (bar-seq) method for measuring the relative fitness of L. mexicana mutants in vivo. In mixed infections of the permissive sand fly vector Lutzomyia longipalpis, paralysed promastigotes and uncoordinated swimmers were severely diminished in the fly after defecation of the bloodmeal. Subsequent examination of flies infected with a single paralysed mutant lacking the central pair protein PF16 or an uncoordinated swimmer lacking the axonemal protein MBO2 showed that these promastigotes did not reach anterior regions of the fly alimentary tract. These data show that L. mexicana need directional motility for successful colonisation of sand flies.
Eukaryotic flagella / cilia are complex multifunctional organelles conserved from protists to humans . Protists use flagella for swimming, feeding, cell-to-cell communication, adherence to substrates and morphogenesis . Single-celled organisms, most prominently among them the green algae Chlamydomonas reinhardtii, have served as important model organisms to study molecular mechanisms of ciliogenesis and ciliary function , spurred on by the recognition that ciliary defects cause human genetic disorders collectively termed “ciliopathies” . The eukaryotic flagellum is a complex, highly structured organelle and dissection of the molecular mechanisms underpinning its diverse functions requires detailed knowledge of its component parts. Proteomic studies of isolated flagella or axonemes from diverse species typically identified at least 300 distinct proteins [5–9] and phylogenetic profiling identified a set of 274 evolutionarily conserved ciliary genes . All of these datasets comprise many “hypothetical” proteins still awaiting functional characterisation in addition to well-characterised core components of the microtubule axoneme, associated motor proteins and regulatory complexes.
This study demonstrates the power of high-throughput CRISPR-Cas9 knockout screens to discover mutant phenotypes in Leishmania. We first defined a flagellar proteome by pursuing a flagellar isolation protocol yielding a defined section of intact flagella and comparing both the flagella and the deflagellated cell body fractions to define a relative enrichment score for each protein. The SINQ method [25,27,28] eliminated from our analysis abundant cell body proteins that were likely cross-contaminants in the flagellar fractions. The flagellar proteins (Fig 2) defined by this method showed similarities in numbers and types of proteins to other analyses of eukaryotic flagella and cilia (S5 Fig, S4 Table, ). We then used these high-confidence flagellar proteome data in conjunction with transcriptomics data and prior knowledge of conserved axonemal proteins to demonstrate a role in motility for >50 genes from a set of one hundred. We also show the importance of directional flagellar motility in the colonisation of sand flies. The data from the pooled mutant population show a progressive loss of paralysed or uncoordinated swimmers over nine days from infection. Because these data report total abundance of each genotype in the whole fly without discriminating between regions of the gut, we probed this question further in infections with the ΔPF16 mutant, which is essentially paralysed and incapable of sustained directional motility due to a defined defect in the central pair complex of the axoneme . The results show that ΔPF16 Leishmania were rapidly lost from most of the dissected flies, consistent with the depletion of this mutant from the mixed pool, and additionally shows that none of the few remaining parasites reached anterior parts of the alimentary tract. A similarly severe defect in colonisation was observed in the ΔMBO2 mutant. MBO2 is an evolutionarily conserved axonemal protein  and derives its name from Chlamydomonas mutants that move backwards only because the algal flagella remain locked in a flagellar beat and cannot readily switch to a ciliary beat . Whilst the precise function of MBO2 remains unknown, it is likely that the uncoordinated swimming behaviour of Leishmania ΔMBO2 mutants (Fig 3B, S13 Fig) is also the result of defective waveform control.