Transition fibres and distal appendages surround the distal end of mature basal bodies and are essential for ciliogenesis, but only a few of the proteins involved have been identified and functionally characterised. Here, through genome-wide analysis, we have identified 30 transition fibre proteins (TFPs) and mapped their arrangement in the flagellated eukaryote Trypanosoma brucei. We discovered that TFPs are recruited to the mature basal body before and after basal body duplication, with differential expression of five TFPs observed at the assembling new flagellum compared to the existing fixed-length old flagellum. RNAi-mediated depletion of 17 TFPs revealed six TFPs that are necessary for ciliogenesis and a further three TFPs that are necessary for normal flagellum length. We identified nine TFPs that had a detectable orthologue in at least one basal body-forming eukaryotic organism outside of the kinetoplastid parasites. Our work has tripled the number of known transition fibre components, demonstrating that transition fibres are complex and dynamic in their composition throughout the cell cycle, which relates to their essential roles in ciliogenesis and flagellum length regulation.
- MeSH
- bazální tělíska metabolismus MeSH
- časové faktory MeSH
- cilie genetika metabolismus MeSH
- flagella genetika metabolismus MeSH
- konzervovaná sekvence MeSH
- protozoální proteiny * genetika metabolismus MeSH
- regulace genové exprese MeSH
- transport proteinů MeSH
- Trypanosoma brucei brucei * genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Current understanding of flagellum/cilium length regulation focuses on a few model organisms with flagella of uniform length. Leptomonas pyrrhocoris is a monoxenous trypanosomatid parasite of firebugs. When cultivated in vitro, L. pyrrhocoris duplicates every 4.2 ± 0.2 h, representing the shortest doubling time reported for trypanosomatids so far. Each L. pyrrhocoris cell starts its cell cycle with a single flagellum. A new flagellum is assembled de novo, while the old flagellum persists throughout the cell cycle. The flagella in an asynchronous L. pyrrhocoris population exhibited a vast length variation of ∼3 to 24 μm, casting doubt on the presence of a length regulation mechanism based on a single balance point between the assembly and disassembly rate in these cells. Through imaging of live L. pyrrhocoris cells, a rapid, partial disassembly of the existing, old flagellum is observed upon, if not prior to, the initial assembly of a new flagellum. Mathematical modeling demonstrated an inverse correlation between the flagellar growth rate and flagellar length and inferred the presence of distinct, cell cycle-dependent disassembly mechanisms with different rates. On the basis of these observations, we proposed a min-max model that could account for the vast flagellar length range observed for asynchronous L. pyrrhocoris. This model may also apply to other flagellated organisms with flagellar length variation.IMPORTANCE Current understanding of flagellum biogenesis during the cell cycle in trypanosomatids is limited to a few pathogenic species, including Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. The most notable characteristics of trypanosomatid flagella studied so far are the extreme stability and lack of ciliary disassembly/absorption during the cell cycle. This is different from cilia in Chlamydomonas and mammalian cells, which undergo complete absorption prior to cell cycle initiation. In this study, we examined flagellum duplication during the cell cycle of Leptomonas pyrrhocoris With the shortest duplication time documented for all Trypanosomatidae and its amenability to culture on agarose gel with limited mobility, we were able to image these cells through the cell cycle. Rapid, cell cycle-specific flagellum disassembly different from turnover was observed for the first time in trypanosomatids. Given the observed length-dependent growth rate and the presence of different disassembly mechanisms, we proposed a min-max model that can account for the flagellar length variation observed in L. pyrrhocoris.
- MeSH
- buněčný cyklus * MeSH
- flagella genetika metabolismus MeSH
- hmyz parazitologie MeSH
- protozoální proteiny genetika metabolismus MeSH
- teoretické modely MeSH
- Trypanosomatina genetika růst a vývoj metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Differentiation of Trypanosoma brucei, a flagellated protozoan parasite, between life cycle stages typically occurs through an asymmetric cell division process, producing two morphologically distinct daughter cells. Conversely, proliferative cell divisions produce two daughter cells, which look similar but are not identical. To examine in detail differences between the daughter cells of a proliferative division of procyclic T. brucei we used the recently identified constituents of the flagella connector. These segregate asymmetrically during cytokinesis allowing the new-flagellum and the old-flagellum daughters to be distinguished. We discovered that there are distinct morphological differences between the two daughters, with the new-flagellum daughter in particular re-modelling rapidly and extensively in early G1. This re-modelling process involves an increase in cell body, flagellum and flagellum attachment zone length and is accompanied by architectural changes to the anterior cell end. The old-flagellum daughter undergoes a different G1 re-modelling, however, despite this there was no difference in G1 duration of their respective cell cycles. This work demonstrates that the two daughters of a proliferative division of T. brucei are non-equivalent and enables more refined morphological analysis of mutant phenotypes. We suggest all proliferative divisions in T. brucei and related organisms will involve non-equivalence.
- MeSH
- buněčné dělení MeSH
- cytokineze MeSH
- flagella genetika metabolismus MeSH
- proliferace buněk MeSH
- protozoální proteiny genetika metabolismus MeSH
- stadia vývoje MeSH
- Trypanosoma brucei brucei cytologie genetika růst a vývoj metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
Leishmania kinetoplastid parasites infect millions of people worldwide and have a distinct cellular architecture depending on location in the host or vector and specific pathogenicity functions. An invagination of the cell body membrane at the base of the flagellum, the flagellar pocket (FP), is an iconic kinetoplastid feature, and is central to processes that are critical for Leishmania pathogenicity. The Leishmania FP has a bulbous region posterior to the FP collar and a distal neck region where the FP membrane surrounds the flagellum more closely. The flagellum is attached to one side of the FP neck by the short flagellum attachment zone (FAZ). We addressed whether targeting the FAZ affects FP shape and its function as a platform for host-parasite interactions. Deletion of the FAZ protein, FAZ5, clearly altered FP architecture and had a modest effect in endocytosis but did not compromise cell proliferation in culture. However, FAZ5 deletion had a dramatic impact in vivo: Mutants were unable to develop late-stage infections in sand flies, and parasite burdens in mice were reduced by >97%. Our work demonstrates the importance of the FAZ for FP function and architecture. Moreover, we show that deletion of a single FAZ protein can have a large impact on parasite development and pathogenicity.
- MeSH
- buněčná membrána metabolismus MeSH
- cilie genetika fyziologie ultrastruktura MeSH
- delece genu MeSH
- endocytóza MeSH
- flagella genetika fyziologie ultrastruktura MeSH
- interakce hostitele a parazita MeSH
- Leishmania genetika patogenita fyziologie ultrastruktura MeSH
- mezibuněčné spoje MeSH
- myši MeSH
- protozoální proteiny genetika metabolismus MeSH
- Psychodidae parazitologie MeSH
- virulence genetika MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Salmonella Enteritidis is the main serovar of poultry origin in humans, but its complex interaction with certain avian cells is still not fully understood. Previously we identified several genes significantly induced in chicken embryo fibroblasts (CEFs) by the wild-type strain S. Enteritidis 11 (SE 11). In the present study, we raised the question whether virulence-attenuated mutants of this strain would induce altered expression of the newly identified fibroblast genes associated with immune and non-immune functions of CEFs. Gene expression was evaluated by real-time PCR following challenge by the parental strain SE 11 and its virulence attenuated mutants lacking flagellin gene fliD only or fliD and the serovar-specific virulence plasmid pSEV. As a result, deletion mutants induced a lower expression of all immune genes, but an increased expression of the non-immune genes G0S2 and ENO2 relative to the parental strain. Our data indicate the importance of flagella and pSEV in modulation of virulence and host response in this model. We demonstrated, for the first time ever, an increased induction of survival genes G0S2 and ENO2 by virulence-attenuated mutants of S. Enteritidis.
- MeSH
- bakteriální proteiny genetika MeSH
- fibroblasty mikrobiologie MeSH
- flagella genetika MeSH
- interakce hostitele a patogenu MeSH
- kuřecí embryo MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- nemoci drůbeže imunologie mikrobiologie MeSH
- plazmidy genetika MeSH
- Salmonella enteritidis genetika patogenita MeSH
- salmonelová infekce u zvířat imunologie mikrobiologie MeSH
- virulence genetika MeSH
- zvířata MeSH
- Check Tag
- kuřecí embryo MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Azospirillum brasilense has the ability of swimming and swarming motility owing to the work of a constitutive polar flagellum and inducible lateral flagella, respectively. The interplay between these flagellar systems is poorly understood. One of the key elements of the flagellar export apparatus is the protein FlhB. Two predicted flhB genes are present in the genome of A. brasilense Sp245 (accession nos. HE577327-HE577333). Experimental evidence obtained here indicates that the chromosomal coding sequence (CDS) AZOBR_150177 (flhB1) of Sp245 is essential for the production of both types of flagella. In an flhB1:: Omegon-Km mutant, Sp245.1063, defects in polar and lateral flagellar assembly and motility were complemented by expressing the wild-type flhB1 gene from plasmid pRK415. It was found that Sp245.1063 lost the capacity for slight but statistically significant decrease in mean cell length in response to transfer from solid to liquid media, and vice versa; in the complemented mutant, this capacity was restored. It was also shown that after the acquisition of the pRK415-harbored downstream CDS AZOBR_150176, cells of Sp245 and Sp245.1063 ceased to elongate on solid media. These initial data suggest that the AZOBR_150176-encoded putative multisensory hybrid sensor histidine kinase-response regulator, in concert with FlhB1, plays a role in morphological response of azospirilla to changes in the hardness of a milieu.
Paratrypanosoma confusum is a monoxenous kinetoplastid flagellate that constitutes the most basal branch of the highly diverse parasitic trypanosomatids, which include human pathogens Trypanosoma and Leishmania This makes Paratrypanosoma uniquely informative for the evolution of obligatory parasitism from free-living lifestyle and the evolution of human parasitism in some trypanosomatid lineages. It has typical promastigote morphology but also forms surface-attached haptomonads and amastigotes. Haptomonads form by attachment to a surface via a large bulge at the base of the flagellum, which is then remodeled into a thin attachment pad associated with flagellum shortening. Promastigotes and haptomonads multiply by binary division, and the progeny of a haptomonad can either remain attached or grow a flagellum and resume swimming. Whole genome sequencing and transcriptome profiling, in combination with analysis of the cell ultrastructure, reveal how the cell surface and metabolism are adapted to parasitism and how characteristic cytoskeletal features are conserved. Our data demonstrate that surface attachment by the flagellum and the flagellar pocket, a Leishmania-like flagellum attachment zone, and a Trypanosoma cruzi-like cytostome are ancestral features, while evolution of extant trypanosomatids, including the human parasites, is associated with genome streamlining and diversification of membrane proteins.
- MeSH
- cytoskelet genetika MeSH
- flagella genetika MeSH
- fylogeneze MeSH
- genom protozoální genetika MeSH
- Leishmania genetika MeSH
- lidé MeSH
- protozoální proteiny genetika MeSH
- stadia vývoje genetika MeSH
- stanovení celkové genové exprese metody MeSH
- Trypanosoma cruzi genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The transition zone (TZ) of eukaryotic cilia and flagella is a structural intermediate between the basal body and the axoneme that regulates ciliary traffic. Mutations in genes encoding TZ proteins (TZPs) cause human inherited diseases (ciliopathies). Here, we use the trypanosome to identify TZ components and localize them to TZ subdomains, showing that the Bardet-Biedl syndrome complex (BBSome) is more distal in the TZ than the Meckel syndrome (MKS) complex. Several of the TZPs identified here have human orthologs. Functional analysis shows essential roles for TZPs in motility, in building the axoneme central pair apparatus and in flagellum biogenesis. Analysis using RNAi and HaloTag fusion protein approaches reveals that most TZPs (including the MKS ciliopathy complex) show long-term stable association with the TZ, whereas the BBSome is dynamic. We propose that some Bardet-Biedl syndrome and MKS pleiotropy may be caused by mutations that impact TZP complex dynamics.
- MeSH
- Bardetův-Biedlův syndrom genetika metabolismus MeSH
- bazální tělíska metabolismus ultrastruktura MeSH
- cilie genetika metabolismus MeSH
- ciliopatie genetika metabolismus MeSH
- cytoskelet metabolismus ultrastruktura MeSH
- encefalokéla genetika metabolismus MeSH
- flagella genetika metabolismus ultrastruktura MeSH
- fluorescenční mikroskopie MeSH
- kompartmentace buňky MeSH
- lidé MeSH
- mutace MeSH
- polycystická choroba ledvin genetika metabolismus MeSH
- poruchy ciliární motility genetika metabolismus MeSH
- proteom genetika metabolismus MeSH
- protozoální proteiny genetika metabolismus MeSH
- RNA interference MeSH
- transmisní elektronová mikroskopie MeSH
- Trypanosoma genetika metabolismus ultrastruktura MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The cell shape of Trypanosoma brucei is influenced by flagellum-to-cell-body attachment through a specialised structure - the flagellum attachment zone (FAZ). T. brucei exhibits numerous morphological forms during its life cycle and, at each stage, the FAZ length varies. We have analysed FLAM3, a large protein that localises to the FAZ region within the old and new flagellum. Ablation of FLAM3 expression causes a reduction in FAZ length; however, this has remarkably different consequences in the tsetse procyclic form versus the mammalian bloodstream form. In procyclic form cells FLAM3 RNAi results in the transition to an epimastigote-like shape, whereas in bloodstream form cells a severe cytokinesis defect associated with flagellum detachment is observed. Moreover, we demonstrate that the amount of FLAM3 and its localisation is dependent on ClpGM6 expression and vice versa. This evidence demonstrates that FAZ is a key regulator of trypanosome shape, with experimental perturbations being life cycle form dependent. An evolutionary cell biology explanation suggests that these differences are a reflection of the division process, the cytoskeleton and intrinsic structural plasticity of particular life cycle forms.
- MeSH
- cilie genetika metabolismus MeSH
- cytokineze genetika MeSH
- cytoskelet genetika metabolismus MeSH
- flagella genetika metabolismus MeSH
- mikrotubuly genetika MeSH
- protozoální proteiny genetika metabolismus MeSH
- stadia vývoje genetika MeSH
- Trypanosoma brucei brucei genetika růst a vývoj MeSH
- tvar buňky genetika MeSH
- vývojová regulace genové exprese MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
