Genetic dissection of a Leishmania flagellar proteome demonstrates requirement for directional motility in sand fly infections.

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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.", "claims": [{"id": "claim-method", "statement": "To discover proteins enriched in each of the four fractions, we used a label-free normalized spectral index quantitation method (SINQ, [25]; S1, S2 and S3 Tables) to generate a SINQ enrichment plot (Fig 2A).", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:31242261", "locator": "sections[3] \"Defining the promastigote flagellar proteome\" paragraph 2", "quote": "To discover proteins enriched in each of the four fractions, we used a label-free normalized spectral index quantitation method (SINQ, [25]; S1, S2 and S3 Tables) to generate a SINQ enrichment plot (Fig 2A)."}], "state": "complete"}, {"id": "claim-data-access", "statement": "The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD011057.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:31242261", "locator": "sections[26] \"Data Availability\" paragraph 2", "quote": "The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD011057."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "complete", "objective": null, "steps": [{"id": "read_published_estimate", "order": 1, "name": "Read the published estimate from the source", "kind": "other", "state": "complete", "blocked_on": null, "description": "The claim, method and evidence span published with the source article.", "duration_hours": null, "parameters": {}, "inputs": ["source:pmid:31242261"], "outputs": ["the published estimate and its evidence"], "instruments": [], "reagents": []}, {"id": "obtain_source_data", "order": 2, "name": "Obtain the source data from the named repository", "kind": "data-preparation", "state": "complete", "blocked_on": null, "description": "The GEO series accession resolves and its sample accessions are listed. 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This information was used to generate a CRISPR-Cas9 knockout library of 100 mutants to scre", "the BlastR PCR product was confirmed for all transfected cell lines; no BlastR PCR product was amplif", ") Strategy for deletion of PFR2 array, using CRISPR-Cas9 to insert a pTNeo cassette with homolog"], "order_basis": "paper_text_order:724", "id": "assay:3"}, {"role": "data_generation", "definition_id": "cell_culture", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.99, "evidence": ["cell line L. mexicana SMP1:TYGFPTY [17] was cultured in supplemented M199 with the addition of 40 μg", "/ml G-418 Disulfate. L. mex Cas9 T7 [23] was cultured in supplemented M199 with the addition of 50 μg", "hat the corresponding knockout cell line was passaged through sand flies (see Figs 6 and 7).(PDF)C"], "order_basis": "paper_text_order:2251", "id": "assay:4"}, {"role": "data_generation", "definition_id": "western_blot", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.93, "evidence": ["nd expected PCR amplicons are indicated. (D) western blots of parental and PFR2 KO protein lysates. Upp"], "order_basis": "paper_text_order:14442", "id": "assay:5"}]}