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

T. Beneke, F. Demay, E. Hookway, N. Ashman, H. Jeffery, J. Smith, J. Valli, T. Becvar, J. Myskova, T. Lestinova, S. Shafiq, J. Sadlova, P. Volf, RJ. Wheeler, E. Gluenz,

. 2019 ; 15 (6) : e1007828. [pub] 20190626

Jazyk angličtina Země Spojené státy americké

Typ dokumentu časopisecké články, práce podpořená grantem

Perzistentní odkaz   https://www.medvik.cz/link/bmc19044722

Grantová podpora
Wellcome Trust - United Kingdom
MR/R000859/1 Medical Research Council - United Kingdom
211075/Z/18/Z Wellcome Trust - United Kingdom
103261/Z/13/Z Wellcome Trust - United Kingdom
104627/Z/14/Z Wellcome Trust - United Kingdom
15/16_MSD_836338 Medical Research Council - United Kingdom
13/14_MSD_OSS_363238 Medical Research Council - United Kingdom
Biotechnology and Biological Sciences Research Council - United Kingdom
Department of Health - United Kingdom

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.

Citace poskytuje Crossref.org

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