Disruption of Leishmania flagellum attachment zone architecture causes flagellum loss

Abstract Leishmania are flagellated eukaryotic parasites that cause leishmaniasis and are closely related to the other kinetoplastid parasites such as Trypanosoma brucei. In all these parasites there is a cell membrane invagination at the base of the flagellum called the flagellar pocket, which is tightly associated with and sculpted by cytoskeletal structures including the flagellum attachment zone (FAZ). The FAZ is a complex interconnected structure linking the flagellum to the cell body and has critical roles in cell morphogenesis, function and pathogenicity. However, this structure varies dramatically in size and organisation between these different parasites, suggesting changes in protein localisation and function. Here, we screened the localisation and function of the Leishmania orthologues of T. brucei FAZ proteins identified in the genome‐wide protein tagging project TrypTag. We identified 27 FAZ proteins and our deletion analysis showed that deletion of two FAZ proteins in the flagellum, FAZ27 and FAZ34 resulted in a reduction in cell body size, and flagellum loss in some cells. Furthermore, after null mutant generation, we observed distinct and reproducible changes to cell shape, demonstrating the ability of the parasite to adapt to morphological perturbations resulting from gene deletion. This process of adaptation has important implications for the study of Leishmania mutants.

Class 5 -FAZ29 and FAZ1 localised to the FAZ within the cell body and the collar region.
From left, an overlay of the phase contrast (grey), mNeonGreen (mNG) tagged protein (green), mCherry (mCh) tagged marker (red) and Hoechst DNA (blue) then mNG only, and mCh only, the far right is an overlay of mNG and mCh.Scale 5 μm.Other FAZ proteins are shown in Figure 2.

Figure S3
Figure , and U) FAZ9.gDNA was extracted from null mutants and tested alongside the parental gDNA for confirmation of gene deletion.LmxM.10.0620 (FLABP) ORF was used as a positive control for amplification except for the FLABP null mutant, for which FAZ29 ORF was amplified.The primers were designed to amplify a 500 bp region from the ORF except for FAZ32, in which a 400 bp region was amplified.P: Parental cell line.N: Null mutant cell line.(Morriswood et al., 2013) Table S1: FAZ proteins in Trypanosoma brucei .Gene IDs and protein information, including predicted molecular weight (MW), TMHMM (transmembrane predictions) and PFAM domains were obtained from TriTrypDB and InterPro (Aslett et al., 2009;Blum et al., 2021).L. mexicana orthologs were identified by OrthoMCL in January 2020 (Fischer et al., 2011).†Tb927.9.13880 gene is currently annotated Tb927.9.13860.References for proteins confirmed to be localised in the FAZ of T. brucei are shown.

Figure S2 :
Figure S2: FAZ proteins in Leishmania mexicana are classified into five classes based on their localisation patterns.

Figure S4 :
Figure S4: Examples of L. mexicana orthologs that do not localise to the FAZ.

Figure S6 :
Figure S6: Cell cycle and morphological analysis of FAZ gene deletions in L. mexicana.

FLAM3
Figure S3: Leishmania FAZ proteins with complex FAZ localisations.Widefield fluorescence images for each FAZ protein in L. mexicana, with a complex localisation pattern.From left, an overlay of the phase contrast (grey), mNeonGreen (mNG) tagged protein (green), mCherry (mCh) tagged marker (red) and Hoechst DNA (blue), scale 5 μm.Then enlarged images of the FAZ region with mNG only, mCh only, and an overlay of mNG, mCh, and DNA in the far right.Additional localisations are indicated beneath protein names, with arrows indicating specific features.