Leishmania parasites possess a unique and complex cytoskeletal structure termed flagellum attachment zone (FAZ) connecting the base of the flagellum to one side of the flagellar pocket (FP), an invagination of the cell body membrane and the sole site for endocytosis and exocytosis. This structure is involved in FP architecture and cell morphogenesis, but its precise role and molecular composition remain enigmatic. Here, we characterized Leishmania FAZ7, the only known FAZ protein containing a kinesin motor domain, and part of a clade of trypanosomatid-specific kinesins with unknown functions. The two paralogs of FAZ7, FAZ7A and FAZ7B, display different localizations and functions. FAZ7A localizes at the basal body, while FAZ7B localizes at the distal part of the FP, where the FAZ structure is present in Leishmania. While null mutants of FAZ7A displayed normal growth rates, the deletion of FAZ7B impaired cell growth in both promastigotes and amastigotes of Leishmania. The kinesin activity is crucial for its function. Deletion of FAZ7B resulted in altered cell division, cell morphogenesis (including flagellum length), and FP structure and function. Furthermore, knocking out FAZ7B induced a mis-localization of two of the FAZ proteins, and disrupted the molecular organization of the FP collar, affecting the localization of its components. Loss of the kinesin FAZ7B has important consequences in the insect vector and mammalian host by reducing proliferation in the sand fly and pathogenicity in mice. Our findings reveal the pivotal role of the only FAZ kinesin as part of the factors important for a successful life cycle of Leishmania.

Department of Parasitology Charles University Prague Czech Republic

Institut de Génétique Humaine University of Montpellier CNRS Montpellier France

Research Unit Fundamental Microbiology and Pathogenicity Protist Parasite Cytoskeleton University of Bordeaux UMR 5234 CNRS Bordeaux France

Research Unit LPHI University of Montpellier CNRS Montpellier France

Research Unit MiVEGEC University of Montpellier CNRS IRD Academic Hospital of Montpellier Montpellier France

York Biomedical Research Institute and Department of Biology University of York York United Kingdom

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Wheeler RJ, Gluenz E, Gull K. Basal body multipotency and axonemal remodelling are two pathways to a 9+0 flagellum. Nat Commun. 2015;6: 8964. doi: 10.1038/ncomms9964 PubMed DOI PMC

Halliday C, de Castro-Neto A, Alcantara CL, Cunha-e-Silva NL, Vaughan S, Sunter JD. Trypanosomatid Flagellar Pocket from Structure to Function. Trends Parasitol. 2020; S1471492220303184. doi: 10.1016/j.pt.2020.11.005 PubMed DOI

Lacomble S, Vaughan S, Gadelha C, Morphew MK, Shaw MK, McIntosh JR, et al.. Three-dimensional cellular architecture of the flagellar pocket and associated cytoskeleton in trypanosomes revealed by electron microscope tomography. J Cell Sci. 2009;122: 1081–1090. doi: 10.1242/jcs.045740 PubMed DOI PMC

Wheeler RJ, Sunter JD, Gull K. Flagellar pocket restructuring through the Leishmania life cycle involves a discrete flagellum attachment zone. J Cell Sci. 2016;129: 854–867. doi: 10.1242/jcs.183152 PubMed DOI PMC

Bonhivers M, Nowacki S, Landrein N, Robinson DR. Biogenesis of the Trypanosome Endo-Exocytotic Organelle Is Cytoskeleton Mediated. PLoS Biol. 2008;6: e105. doi: 10.1371/journal.pbio.0060105 PubMed DOI PMC

Perdomo D, Bonhivers M, Robinson D. The Trypanosome Flagellar Pocket Collar and Its Ring Forming Protein—TbBILBO1. Cells. 2016;5: 9. doi: 10.3390/cells5010009 PubMed DOI PMC

Sunter JD, Gull K. The Flagellum Attachment Zone: ‘The Cellular Ruler’ of Trypanosome Morphology. Trends Parasitol. 2016;32: 309–324. doi: 10.1016/j.pt.2015.12.010 PubMed DOI PMC

Gadelha C, Rothery S, Morphew M, McIntosh JR, Severs NJ, Gull K. Membrane domains and flagellar pocket boundaries are influenced by the cytoskeleton in African trypanosomes. Proc Natl Acad Sci USA. 2009;106: 17425–17430. doi: 10.1073/pnas.0909289106 PubMed DOI PMC

Gheiratmand L, Brasseur A, Zhou Q, He CY. Biochemical Characterization of the Bi-lobe Reveals a Continuous Structural Network Linking the Bi-lobe to Other Single-copied Organelles in Trypanosoma brucei. J Biol Chem. 2013;288: 3489–3499. doi: 10.1074/jbc.M112.417428 PubMed DOI PMC

LaCount DJ, Barrett B, Donelson JE. Trypanosoma brucei FLA1 Is Required for Flagellum Attachment and Cytokinesis. J Biol Chem. 2002;277: 17580–17588. doi: 10.1074/jbc.M200873200 PubMed DOI

Vaughan S, Kohl L, Ngai I, Wheeler RJ, Gull K. A Repetitive Protein Essential for the Flagellum Attachment Zone Filament Structure and Function in Trypanosoma brucei. Protist. 2008;159: 127–136. doi: 10.1016/j.protis.2007.08.005 PubMed DOI

Zhou Q, Liu B, Sun Y, He CY. A coiled-coil- and C2-domain-containing protein is required for FAZ assembly and cell morphology in Trypanosoma brucei. J Cell Sci. 2011;124: 3848–3858. doi: 10.1242/jcs.087676 PubMed DOI

Hayes P, Varga V, Olego-Fernandez S, Sunter J, Ginger ML, Gull K. Modulation of a cytoskeletal calpain-like protein induces major transitions in trypanosome morphology. J Cell Biol. 2014;206: 377–384. doi: 10.1083/jcb.201312067 PubMed DOI PMC

Zhou Q, Hu H, He CY, Li Z. Assembly and maintenance of the flagellum attachment zone filament in Trypanosoma brucei. J Cell Sci. 2015;128: 2361–2372. doi: 10.1242/jcs.168377 PubMed DOI PMC

Sunter JD, Yanase R, Wang Z, Catta-Preta CMC, Moreira-Leite F, Myskova J, et al.. Leishmania flagellum attachment zone is critical for flagellar pocket shape, development in the sand fly, and pathogenicity in the host. Proc Natl Acad Sci USA. 2019;116: 6351–6360. doi: 10.1073/pnas.1812462116 PubMed DOI PMC

Halliday C, Yanase R, Catta-Preta CMC, Moreira-Leite F, Myskova J, Pruzinova K, et al.. Role for the flagellum attachment zone in Leishmania anterior cell tip morphogenesis. Hill KL, editor. PLoS Pathog. 2020;16: e1008494. doi: 10.1371/journal.ppat.1008494 PubMed DOI PMC

Lawrence CJ, Dawe RK, Christie KR, Cleveland DW, Dawson SC, Endow SA, et al.. A standardized kinesin nomenclature. J Cell Biol. 2004;167: 19–22. doi: 10.1083/jcb.200408113 PubMed DOI PMC

Wickstead B, Gull K. A “Holistic” Kinesin Phylogeny Reveals New Kinesin Families and Predicts Protein Functions. Mol Biol of the Cell. 2006;17: 10 doi: 10.1091/mbc.e05-11-1090 PubMed DOI PMC

Wickstead B, Gull K, Richards TA. Patterns of kinesin evolution reveal a complex ancestral eukaryote with a multifunctional cytoskeleton. BMC Evol Biol. 2010, 10:110. PubMed PMC

Sunter JD, Varga V, Dean S, Gull K. A dynamic coordination of flagellum and cytoplasmic cytoskeleton assembly specifies cell morphogenesis in trypanosomes. J Cell Sci. 2015;128: 1580–1594. doi: 10.1242/jcs.166447 PubMed DOI PMC

Beneke T, Madden R, Makin L, Valli J, Sunter J, Gluenz E. A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids. R Soc open sci. 2017;4: 170095. doi: 10.1098/rsos.170095 PubMed DOI PMC

Selvapandiyan A, Debrabant A, Duncan R, Muller J, Salotra P, Sreenivas G, et al.. Centrin Gene Disruption Impairs Stage-specific Basal Body Duplication and Cell Cycle Progression in Leishmania. J Biol Chem. 2004;279: 25703–25710. doi: 10.1074/jbc.M402794200 PubMed DOI

Tull D, Naderer T, Spurck T, Mertens HDT, Heng J, McFadden GI, et al.. Membrane protein SMP-1 is required for normal flagellum function in Leishmania. J Cell Sci. 2010;123: 544–554. doi: 10.1242/jcs.059097 PubMed DOI

Kelly BL, Stetson DB, Locksley RM. Leishmania major LACK Antigen Is Required for Efficient Vertebrate Parasitization. Journal of Experimental Medicine. 2003;198: 1689–1698. doi: 10.1084/jem.20031162 PubMed DOI PMC

Wheeler RJ, Gluenz E, Gull K. The cell cycle of Leishmania: morphogenetic events and their implications for parasite biology: The cell cycle of Leishmania. Mol Microbiol. 2011;79: 647–662. doi: 10.1111/j.1365-2958.2010.07479.x PubMed DOI PMC

Ambit A, Woods KL, Cull B, Coombs GH, Mottram JC. Morphological Events during the Cell Cycle of Leishmania major. Eukaryot Cell. 2011;10: 1429–1438. doi: 10.1128/EC.05118-11 PubMed DOI PMC

Florimond C, Sahin A, Vidilaseris K, Dong G, Landrein N, Dacheux D, et al.. BILBO1 Is a Scaffold Protein of the Flagellar Pocket Collar in the Pathogen Trypanosoma brucei. Hill KL, editor. PLoS Pathog. 2015;11: e1004654. doi: 10.1371/journal.ppat.1004654 PubMed DOI PMC

Albisetti A, Florimond C, Landrein N, Vidilaseris K, Eggenspieler M, Lesigang J, et al.. Interaction between the flagellar pocket collar and the hook complex via a novel microtubule-binding protein in Trypanosoma brucei. PLoS Pathog. 2017;13: e1006710. doi: 10.1371/journal.ppat.1006710 PubMed DOI PMC

Halliday C, Billington K, Wang Z, Madden R, Dean S, Sunter JD, et al.. Cellular landmarks of Trypanosoma brucei and Leishmania mexicana. Mol Biochem Parasitol. 2019;230: 24–36. doi: 10.1016/j.molbiopara.2018.12.003 PubMed DOI PMC

Wang Z, Wheeler RJ, Sunter JD. Lysosome assembly and disassembly changes endocytosis rate through the Leishmania cell cycle. Microbiol Open. 2020;9. doi: 10.1002/mbo3.969 PubMed DOI PMC

Tinevez J-Y, Perry N, Schindelin J, Hoopes GM, Reynolds GD, Laplantine E, et al.. TrackMate: An open and extensible platform for single-particle tracking. Methods. 2017;115: 80–90. doi: 10.1016/j.ymeth.2016.09.016 PubMed DOI

Bates P, Rogers M. New Insights into the Developmental Biology and Transmission Mechanisms of Leishmania. CMM. 2004;4: 601–609. doi: 10.2174/1566524043360285 PubMed DOI

Demonchy R, Blisnick T, Deprez C, Toutirais G, Loussert C, Marande W, et al.. Kinesin 9 family members perform separate functions in the trypanosome flagellum. J Cell Biol. 2009;187: 615–622. doi: 10.1083/jcb.200903139 PubMed DOI PMC

Douglas RL, Haltiwanger BM, Albisetti A, Wu H, Jeng RL, Mancuso J, et al.. Trypanosomes have divergent kinesin-2 proteins that function differentially in flagellum biosynthesis and cell viability. J Cell Sci. 2020;133: jcs129213. doi: 10.1242/jcs.129213 PubMed DOI

Marande W, Kohl L. Flagellar kinesins in protists. Future Microbiol. 2011;6: 231–246. doi: 10.2217/fmb.10.167 PubMed DOI

Sunter J, Gull K. Shape, form, function and Leishmania pathogenicity: from textbook descriptions to biological understanding. Open Biol. 2017;7: 170165. doi: 10.1098/rsob.170165 PubMed DOI PMC

Wheeler RJ, Gluenz E, Gull K. The Limits on Trypanosomatid Morphological Diversity. Li Z, editor. PLoS ONE. 2013;8: e79581. doi: 10.1371/journal.pone.0079581 PubMed DOI PMC

Field MC, Carrington M. The trypanosome flagellar pocket. Nat Rev Microbiol. 2009;7: 775–786. doi: 10.1038/nrmicro2221 PubMed DOI

Sinclair-Davis AN, McAllaster MR, de Graffenried CL. A functional analysis of TOEFAZ1 uncovers protein domains essential for cytokinesis in Trypanosoma brucei. J Cell Sci. 2017;130: 3918–3932. doi: 10.1242/jcs.207209 PubMed DOI PMC

Wheeler RJ, Gull K, Sunter JD. Coordination of the Cell Cycle in Trypanosomes. Annu Rev Microbiol. 2019;73: 133–154. doi: 10.1146/annurev-micro-020518-115617 PubMed DOI

Robinson DR, Sherwin T, Ploubidou A, Byard EH, Gull K. Microtubule polarity and dynamics in the control of organelle positioning, segregation, and cytokinesis in the trypanosome cell cycle. J Cell Biol. 1995;128: 1163–1172. doi: 10.1083/jcb.128.6.1163 PubMed DOI PMC

Gluenz E, Ginger ML, McKean PG. Flagellum assembly and function during the Leishmania life cycle. Curr Opin Microbiol. 2010;13: 473–479. doi: 10.1016/j.mib.2010.05.008 PubMed DOI

Kelly FD, Tran KD, Hatfield J, Schmidt K, Sanchez MA, Landfear SM. A cytoskeletal protein complex is essential for division of intracellular amastigotes of Leishmania mexicana. J Biol Chem. 2020;295: 13106–13122. doi: 10.1074/jbc.RA120.014065 PubMed DOI PMC

Tran KD, Vieira DP, Sanchez MA, Valli J, Gluenz E, Landfear SM. Kharon1 Null Mutants of Leishmania mexicana Are Avirulent in Mice and Exhibit a Cytokinesis Defect within Macrophages. PLoS ONE. 2015;10: e0134432. doi: 10.1371/journal.pone.0134432 PubMed DOI PMC

Beneke T, Demay F, Hookway E, Ashman N, Jeffery H, Smith J, et al.. Genetic dissection of a Leishmania flagellar proteome demonstrates requirement for directional motility in sand fly infections. PLoS Pathog. 2019;15: e1007828. doi: 10.1371/journal.ppat.1007828 PubMed DOI PMC

Skalický T, Dobáková E, Wheeler RJ, Tesařová M, Flegontov P, Jirsová D, et al.. Extensive flagellar remodeling during the complex life cycle of Paratrypanosoma, an early-branching trypanosomatid. Proc Natl Acad Sci USA. 2017;114: 11757–11762. doi: 10.1073/pnas.1712311114 PubMed DOI PMC

Berens RL, Brun R, Krassner SM. A Simple Monophasic Medium for Axenic Culture of Hemoflagellates. J Parasitol. 1976;62: 360. doi: 10.2307/3279142 PubMed DOI

Tetaud E, Lecuix I, Sheldrake T, Baltz T, Fairlamb AH. A new expression vector for Crithidia fasciculata and Leishmania. Mol Biochem Parasitol. 2002;120: 195–204. doi: 10.1016/s0166-6851(02)00002-6 PubMed DOI

Gazanion E, Garcia D, Silvestre R, Gérard C, Guichou JF, Labesse G, et al.. The Leishmania nicotinamidase is essential for NAD+ production and parasite proliferation: Leishmania NAD+ metabolism. Mol Microbiol. 2011;82: 21–38. doi: 10.1111/j.1365-2958.2011.07799.x PubMed DOI

Volf P, Volfova V. Establishment and maintenance of sand fly colonies. J Vector Ecol. 2011;36: S1–S9. doi: 10.1111/j.1948-7134.2011.00106.x PubMed DOI

Myskova J, Votypka J, Volf P. Leishmania in Sand Flies: Comparison of Quantitative Polymerase Chain Reaction with Other Techniques to Determine the Intensity of Infection. J Med Entomol. 2008;45: 133–138. doi: 10.1603/0022-2585(2008)45[133:lisfco]2.0.co;2 PubMed DOI