Protists of the order Trypanosomatida possess a single multifunctional flagellum, which powers cellular displacement and mediates attachment to tissues of the arthropod vector. The kinetoplastid flagellar cytoskeleton consists of a nine-microtubule doublet axoneme; further structural elaborations, which can vary between species and life cycle stages, include the assembly of axonemal dynein complexes, a pair of singlet microtubules and the extra-axonemal paraflagellar rod. The intracellular amastigote forms of Leishmania spp. build a short, non-motile cilium whose function has remained enigmatic. Here, we used a panel of 25 barcoded promastigote cell lines, including mutants lacking genes encoding flagellar assembly proteins, axonemal proteins required for normal motility, or flagellar membrane proteins to examine how these defects impact on their virulence in macrophages and mice. Mutants lacking the intraflagellar transport (IFT) protein 88 were avirulent indicating that assembly of a flagellum is necessary to allow for Leishmania survival in a mammalian host. A similarly severe loss of virulence was observed upon deletion of BBS2, a core component of the BBSome complex, which may act as a cargo adapter for IFT. By contrast, promastigotes that were unable to beat their flagella due to loss of core axonemal proteins could establish and sustain an infection and only showed a small reduction of parasite burden in vivo compared to the parental cell lines. These results confirm that flagellar motility is not necessary for mammalian infection, but flagellum assembly and the integrity of the BBSome are essential for pathogenicity.

• Klíčová slova
• CRISPR screen, Leishmania, flagella, motility, virulence,
• MeSH
• flagella * fyziologie   genetika   metabolismus   MeSH
• Leishmania mexicana * patogenita   genetika   fyziologie   MeSH
• makrofágy parazitologie   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protozoální proteiny MeSH

The protozoan parasite Trypanosoma brucei assembles a new flagellum while maintaining the existing one in the same cell. Our group has previously proposed a model where the mature flagellum is locked after construction to full length. To test this hypothesis directly, we monitored flagellum assembly dynamics through inducible expression of tubulin marked with an intragenic tag. We found that addition of new tubulin occurs at the distal flagellum tip at a linear rate and is indeed restricted to the new flagellum in bi-flagellated cells. Depleting the locking protein CEP164C prior to induction resulted in simultaneous integration of new tubulin in both flagella. This is direct evidence that trypanosomes avoid competition between the two flagella by allowing tubulin incorporation only in the new organelle. However, by tracing flagella over several cell cycles we also found that flagella do not remain locked forever. An orthogonal approach with HaloTag-tagged radial spoke protein 4/6 (GeneID Tb927.11.4480) supported these findings. Given that flagellum length in trypanosomes is stable, this indicates regular events of transient disassembly, followed by assembly, at the distal tip.

• Klíčová slova
• Trypanosoma brucei, CEP164C, Flagellum, Grow-and-lock model, Tubulin,
• MeSH
• axonema * metabolismus   MeSH
• flagella metabolismus   MeSH
• mikrotubuly * metabolismus   MeSH
• protozoální proteiny metabolismus   genetika   MeSH
• Trypanosoma brucei brucei * metabolismus   genetika   MeSH
• tubulin * metabolismus   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• protozoální proteiny MeSH
• tubulin * MeSH

UNLABELLED: Flagellar motility is crucial for the swim-and-stick lifestyle and plays an important role in bacterial-algal interactions of Rhodobacterales. This alphaproteobacterial order contains three distinct types of flagellar gene clusters (FGCs) for the formation of a functional flagellum. Our phylogenetically broad taxon sampling of more than 300 genomes revealed that the most common FGC, the fla1-type, was probably already present in the common ancestor of Rhodobacterales and was strictly vertically inherited, while the other two FGC types, fla2 and fla3, were spread via horizontal operon transfers. Swimming of the marine model organism Phaeobacter inhibens DSM 17395 (Roseobacteraceae) is mediated by the archetypal fla1-type flagellum. Screening of 13,000 transposon mutants of P. inhibens on soft agar plates revealed that 40 genes, including four genes encoding conserved but not yet characterized proteins (CP1-4) within the FGC, are essential for motility. Exoproteome analyses indicated that CP1-4 are required at different stages of flagellar assembly. Only eight genes outside the FGC were identified as essential for swimming motility, including all three genes of the CtrA phosphorelay. Using comparative transcriptomics of ΔcckA, ΔchpT, and ΔctrA mutants of the distantly related model organisms P. inhibens and Dinoroseobacter shibae DSM 16493, we identified genes for the flagellum and cyclic di-GMP turnover as core targets of the CtrA phosphorelay and a conserved connection with quorum sensing across members of the Rhodobacterales. IMPORTANCE: The bacterial flagellum is a sophisticated nanomachine for swimming motility and rapid chemotactic response to gradients of attractants or repellents in the environment. It is structurally highly conserved and has been intensively studied in gammaproteobacterial model bacteria such as Escherichia coli and Salmonella enterica. However, the flagellar gene clusters of different alphaproteobacterial orders have distinct structures and compositions, as demonstrated by the three flagellar systems of Rhodobacterales investigated in the current study. The archetypal fla1-type flagellum originated in its common ancestor and evolved synchronously with the host. The universal presence of four as yet uncharacterized essential genes in fla1-type FGCs (CP1-4) reflects the order-specific adaptation of the flagellar system during bacterial evolution. Comparative transcriptome analyses of ΔcckA, ΔchpT, and ΔctrA mutants showed that the core function of the CtrA phosphorelay in Rhodobacterales is the transcriptional control of flagellar genes.

• Klíčová slova
• CtrA phosphorelay, Rhodobacterales, evolution, flagellar gene regulation, flagellar motility,
• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• flagella * genetika   fyziologie   metabolismus   MeSH
• fylogeneze MeSH
• multigenová rodina MeSH
• regulace genové exprese u bakterií MeSH
• Rhodobacteraceae * genetika   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH

A ubiquitous property of bacteria is their ability to move toward more suitable environments, which can also facilitate host-associated activities like colonization and offer the cell several benefits such as bacteria moving towards a favorable gradient or away from a harmful gradient is known as chemotaxis. Bacteria achieve this by rotating flagella in clockwise and anticlockwise directions resulting in "run" and "tumble." This ability of bacteria to sense and respond to any type of change in the environmental factors like pH, osmolarity, redox potential, and temperature is a standard signal transduction system that depends on coupling proteins, which is the bacterial chemotaxis system. There are two architectures for the coupling proteins in the chemotaxis system: CheW and CheV. Typically, a signal transduction system for chemotaxis to form a core signaling complex couples CheA activity to chemoreceptor control: two CheW coupling protein molecules span a histidine kinase CheA dimer and two chemoreceptors (also known as methyl-accepting chemotaxis protein, MCP) trimers of dimers which further transfer the signal to the flagellar motor through CheY. The current review summarizes and highlights the molecular mechanism involved in bacterial chemotaxis, its physiological benefits such as locating suitable nutrients and niches for bacterial growth, and various assay techniques used for the detection of chemotactic motility.

• Klíčová slova
• Bacteria, Chemotaxis, Motility, Run, Tumble,
• MeSH
• Bacteria * metabolismus   genetika   MeSH
• bakteriální proteiny metabolismus   genetika   MeSH
• chemotaxe * fyziologie   MeSH
• flagella fyziologie   MeSH
• fyziologie bakterií * MeSH
• MCP systém metabolismus   MeSH
• signální transdukce MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• bakteriální proteiny MeSH
• MCP systém MeSH

Isogenic bacterial populations can display probabilistic cell-to-cell variation in response to challenges. This phenotypic heterogeneity can affect virulence in animals, but its impact on plant pathogens is unknown. Previously, we showed that expression of the type III secretion system (T3SS) of the plant pathogen Pseudomonas syringae displays phenotypic variation in planta. Here we use flow cytometry and microscopy to investigate single-cell flagellar expression in relation to T3SS expression, showing that both systems undergo phenotypic heterogeneity in vitro in apoplast-mimicking medium and within apoplastic microcolonies throughout colonization of Phaseolus vulgaris. Stochastic, spatial and time factors shape the dynamics of a phenotypically diverse pathogen population that displays division of labour during colonization: effectors produced by T3SS-expressing bacteria act as 'common goods' to suppress immunity, allowing motile flagella-expressing bacteria to increase and leave infected tissue before necrosis. These results showcase the mechanisms of bacterial specialization during plant colonization in an environmentally and agriculturally relevant system.

• MeSH
• bakteriální proteiny metabolismus   genetika   MeSH
• fazol * mikrobiologie   MeSH
• fenotyp MeSH
• flagella * metabolismus   MeSH
• nemoci rostlin * mikrobiologie   MeSH
• průtoková cytometrie MeSH
• Pseudomonas syringae * patogenita   genetika   metabolismus   fyziologie   MeSH
• regulace genové exprese u bakterií MeSH
• sekreční systém typu III * metabolismus   genetika   MeSH
• virulence MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• sekreční systém typu III * MeSH

This article explores the use of expansion microscopy, a technique that enhances resolution in fluorescence microscopy, on the autotrophic protist Euglena gracilis A modified protocol was developed to preserve the cell structures during fixation. Using antibodies against key cytoskeletal and organelle markers, α-tubulin, β-ATPase, and Rubisco activase, the microtubular structures, mitochondria, and chloroplasts were visualised. The organisation of the cytoskeleton corresponded to the findings from electron microscopy while allowing for the visualisation of the flagellar pocket in its entirety and revealing previously unnoticed details. This study offered insights into the shape and development of mitochondria and chloroplasts under varying conditions, such as culture ages and light cycles. This work demonstrated that expansion microscopy is a robust tool for visualising cellular structures in E. gracilis, an organism whose internal structures cannot be stained using standard immunofluorescence because of its complex pellicle. This technique also serves as a complement to electron microscopy, facilitating tomographic reconstructions in a routine fashion.

• MeSH
• chloroplasty ultrastruktura   MeSH
• cytoskelet * ultrastruktura   MeSH
• Euglena gracilis * ultrastruktura   MeSH
• flagella ultrastruktura   MeSH
• fluorescenční mikroskopie * metody   MeSH
• mitochondrie ultrastruktura   MeSH
• mitóza MeSH
• protilátky chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• protilátky MeSH

The phylum Heterolobosea Page and Blanton, 1985 is a group of eukaryotes that contains heterotrophic flagellates, amoebae, and amoeboflagellates, including the infamous brain-eating amoeba Naegleria fowleri. In this study, we investigate the deep evolutionary history of Heterolobosea by generating and analyzing transcriptome data from 16 diverse isolates and combine this with previously published data in a comprehensive phylogenomic analysis. This dataset has representation of all but one of the major lineages classified here as orders. Our phylogenomic analyses recovered a robustly supported phylogeny of Heterolobosea providing a phylogenetic framework for understanding their evolutionary history. Based on the newly recovered relationships, we revised the classification of Heterolobosea to the family level. We describe two new classes (Eutetramitea cl. nov. and Selenaionea cl. nov) and one new order (Naegleriida ord. nov.), and provide a new delimitation of the largest family of Heterolobosea, Vahlkampfiidae Jollos, 1917. Unexpectedly, we unveiled the first two cases of genetic code alterations in the group: UAG as a glutamine codon in the nuclear genome of Dactylomonas venusta and UGA encoding tryptophan in the mitochondrial genome of Neovahlkampfia damariscottae. In addition, analysis of the genome of the latter species confirmed its inability to make flagella, whereas we identified hallmark flagellum-specific genes in most other heteroloboseans not previously observed to form flagellates, suggesting that the loss of flagella in Heterolobosea is much rarer than generally thought. Finally, we define the first autapomorphy of the subphylum Pharyngomonada, represented by a fusion of two key genes for peroxisomal β-oxidation enzymes.

• Klíčová slova
• Alternative genetic code, Cryptic flagella, Mitochondrial genome, Molecular phylogenomics, Naegleria,
• MeSH
• Bayesova věta MeSH
• Eukaryota * genetika   klasifikace   MeSH
• flagella genetika   MeSH
• fylogeneze * MeSH
• genetický kód * MeSH
• molekulární evoluce MeSH
• transkriptom MeSH
• Publikační typ
• časopisecké články MeSH

Cilia assembly and function rely on the bidirectional transport of components between the cell body and ciliary tip via Intraflagellar Transport (IFT) trains. Anterograde and retrograde IFT trains travel along the B- and A-tubules of microtubule doublets, respectively, ensuring smooth traffic flow. However, the mechanism underlying this segregation remains unclear. Here, we test whether tubulin detyrosination (enriched on B-tubules) and tyrosination (enriched on A-tubules) have a role in IFT logistics. We report that knockout of tubulin detyrosinase VashL in Chlamydomonas reinhardtii causes frequent IFT train stoppages and impaired ciliary growth. By reconstituting IFT train motility on de-membranated axonemes and synthetic microtubules, we show that anterograde and retrograde trains preferentially associate with detyrosinated and tyrosinated microtubules, respectively. We propose that tubulin tyrosination/detyrosination is crucial for spatial segregation and collision-free IFT train motion, highlighting the significance of the tubulin code in ciliary transport.

• MeSH
• axonema * metabolismus   MeSH
• biologický transport MeSH
• Chlamydomonas reinhardtii * metabolismus   genetika   MeSH
• cilie metabolismus   MeSH
• flagella * metabolismus   MeSH
• mikrotubuly * metabolismus   MeSH
• tubulin * metabolismus   MeSH
• tyrosin * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• tubulin * MeSH
• tyrosin * MeSH

Leishmania species, members of the kinetoplastid parasites, cause leishmaniasis, a neglected tropical disease, in millions of people worldwide. Leishmania has a complex life cycle with multiple developmental forms, as it cycles between a sand fly vector and a mammalian host; understanding their life cycle is critical to understanding disease spread. One of the key life cycle stages is the haptomonad form, which attaches to insect tissues through its flagellum. This adhesion, conserved across kinetoplastid parasites, is implicated in having an important function within their life cycles and hence in disease transmission. Here, we discover the kinetoplastid-insect adhesion proteins (KIAPs), which localise in the attached Leishmania flagellum. Deletion of these KIAPs impairs cell adhesion in vitro and prevents Leishmania from colonising the stomodeal valve in the sand fly, without affecting cell growth. Additionally, loss of parasite adhesion in the sand fly results in reduced physiological changes to the fly, with no observable damage of the stomodeal valve and reduced midgut swelling. These results provide important insights into a comprehensive understanding of the Leishmania life cycle, which will be critical for developing transmission-blocking strategies.

• MeSH
• buněčná adheze MeSH
• flagella * metabolismus   MeSH
• hmyz - vektory parazitologie   MeSH
• hmyzí proteiny metabolismus   genetika   MeSH
• interakce hostitele a parazita MeSH
• Leishmania * fyziologie   genetika   metabolismus   MeSH
• leishmanióza parazitologie   přenos   MeSH
• protozoální proteiny metabolismus   genetika   MeSH
• Psychodidae * parazitologie   MeSH
• stadia vývoje MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• hmyzí proteiny MeSH
• protozoální proteiny MeSH

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.

• Klíčová slova
• Trypanosoma, Cilia, Ciliogenesis, Distal appendages, Flagella, Transition fibres,
• 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
• práce podpořená grantem MeSH
• Názvy látek
• protozoální proteiny * MeSH