The kinesin of the flagellum attachment zone in Leishmania is required for cell morphogenesis, cell division and virulence in the mammalian host

Abstract

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.

LeishIF3d is a non-canonical cap-binding protein in Leishmania

“…The morphological changes observed for the LeishIF3d(+/−) cells are in accordance with the reduced expression of the dynein light chain (LmxM.13.1180), and Kinesin (LmxM.17.1110). These regulate ciliary movement in Trypanosoma brucei ( Godar et al, 2022 ) and are essential for cell morphogenesis, cell division, and virulence ( Corrales et al, 2021 ). Reduced expression was recorded also for proteins that encode a variety of metabolic enzymes.…”

Analysis of theLeishmania mexicanapromastigote cell cycle using imaging flow cytometry provides new insights into cell cycle flexibility and events of short duration

“…Much of our current knowledge of Leishmania cell cycle events and their order and duration derives from studies where fixed promastigote parasites have been analysed manually by fluorescence microscopy (3, 5, 8, 10, 53). DNA dyes are commonly used to visualise and enumerate nuclei and kinetoplasts, or thymidine analogues such as BrdU (5-bromo-2’- deoxyuridine) or EdU (5-ethynyl-2’-deoxyuridine) are used to detect DNA synthesis.…”

“…While microscopy can provide high resolution spatial information, it can be highly labour-intensive if performed manually, for example, if rare populations of cells are being studied and many cells have to be screened to identify them, or if large numbers of cells are required for analysis or measurements of cell morphology (e.g. length, width), which are usually performed by hand using software such as ImageJ/Fiji (3, 10). Further, artefacts can be introduced if live cells are imaged for extended periods without the use of an environmental chamber, or if cells are fixed prior to staining.…”

Disruption of Leishmania flagellum attachment zone architecture causes flagellum loss

“…In Leishmania, the flagellum is only laterally attached within the flagellar pocket neck, resulting in the 'free flagellum' morphology, whereas in T. brucei, the flagellum is attached for the majority of its length Wheeler et al, 2016). Moreover, the architectural organisation of the Leishmania FAZ is more complex in comparison to the predominantly linear FAZ in trypanosomes (Corrales et al, 2021;Wheeler et al, 2016). Orthologues of FAZ proteins identified in T. brucei were shown to localise to five regions of the Leishmania flagellar pocket: (i) a ring/horseshoe at the anterior cell tip as the flagellum emerges from the cell body;…”

“…(ii) a ring associated with the flagellar pocket collar; (iii) a short line within the flagellum; (iv) a short line within the cell body parallel to the flagellum; (v) an asymmetric ring surrounding the flagellar pocket neck (Corrales et al, 2021;Halliday et al, 2020;Sunter et al, 2019;Wheeler et al, 2016).…”