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

Beneke, T. et al. (2019) Genetic dissection of a Leishmania flagellar proteome demonstrates requirement for directional motility in sand fly infections. PLoS Pathogens, 15(6), e1007828. (doi: 10.1371/journal.ppat.1007828) (PMID:31242261) (PMCID:PMC6615630)

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Abstract

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.

Item Type:Articles
Additional Information:Funding: EG and JSm were jointly funded by the UK Medical Research Council (MRC) and the UK Department for International Development (DFID) under the MRC/DFID Concordat agreement; grant no. MR/R000859/1 (https://mrc.ukri.org/). EG was supported by a Royal Society University Research Fellowship (UF100435 and UF160661; https://royalsociety.org/). RW was supported by the Wellcome Trust, grant nos. [211075/Z/18/Z, 103261/Z/13/Z, 104627/Z/14/Z] (https://wellcome.ac.uk/). PV, JSa and TL were supported by European Regional Development (ERD) Funds, project CePaViP (CZ.02.1.01/0.0/0.0/ 16_019/0000759; https://ec.europa.eu/regional_policy/en/funding/erdf/). TB was supported by MRC PhD studentship (15/16_MSD_836338; https://mrc.ukri.org/) and an Erasmus grant (https://www.erasmusplus.org.uk/study-abroad). JV was supported by MRC PhD studentship (13/14_MSD_OSS_363238; https://mrc.ukri.org/) HJ was supported by by BBSRC Interdisciplinary Biosciences DTP studentship (https://www.ox.ac.uk/admissions/graduate/courses/interdisciplinary-bioscience?wssl=1) and an Oxford Radcliffe Scholarship. SS was supported by by BBSRC Interdisciplinary Biosciences DTP studentship (https://www.ox.ac.uk/admissions/graduate/courses/interdisciplinary-bioscience?wssl=1). FD was supported by an Erasmus grant (https://www.erasmusplus.org.uk/study-abroad). NA was funded by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre (BRC; https://oxfordbrc.nihr.ac.uk/).
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Gluenz, Dr Eva
Creator Roles:
Gluenz, E.Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review and editing
Authors: Beneke, T., Demay, F., Hookway, E., Ashman, N., Jeffery, H., Smith, J., Valli, J., Becvar, T., Myskova, J., Lestinova, T., Shafiq, S., Sadlova, J., Volf, P., Wheeler, R. J., and Gluenz, E.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:PLoS Pathogens
Publisher:Public Library of Science
ISSN:1553-7366
ISSN (Online):1553-7374
Copyright Holders:Copyright © 2019 Beneke et al.
First Published:First published in PLoS Pathogens 15(6):e1007828
Publisher Policy:Reproduced under a Creative Commons license

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