The unicellular parasite Leishmania has a precisely defined cell architecture that is inherited by each subsequent generation, requiring a highly coordinated pattern of duplication and segregation of organelles and cytoskeletal structures. A framework of nuclear division and morphological changes is known from light microscopy, yet this has limited resolution and the intrinsic organisation of organelles within the cell body and their manner of duplication and inheritance is unknown. Using volume electron microscopy approaches, we have produced three-dimensional reconstructions of different promastigote cell cycle stages to give a spatial and quantitative overview of organelle positioning, division and inheritance. The first morphological indications seen in our dataset that a new cell cycle had begun were the assembly of a new flagellum, the duplication of the contractile vacuole and the increase in volume of the nucleus and kinetoplast. We showed that the progression of the cytokinesis furrow created a specific pattern of membrane indentations, while our analysis of sub-pellicular microtubule organisation indicated that there is likely a preferred site of new microtubule insertion. The daughter cells retained these indentations in their cell body for a period post-abscission. By comparing cultured and sand fly derived promastigotes, we found an increase in the number and overall volume of lipid droplets in the promastigotes from the sand fly, reflecting a change in their metabolism to ensure transmissibility to the mammalian host. Our insights into the cell cycle mechanics of Leishmania will support future molecular cell biology analyses of these parasites.

• MeSH
• buněčné dělení MeSH
• buněčný cyklus MeSH
• Leishmania mexicana * genetika   MeSH
• Leishmania * MeSH
• paraziti * MeSH
• Psychodidae * parazitologie   MeSH
• savci MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Conserved histone methyltransferases of the DOT1 family are involved in replication regulation, cell cycle progression, stage differentiation, and gene regulation in trypanosomatids. However, the specific functions of these enzymes depend on the host evasion strategies of the parasites. In this study, we investigated the role of DOT1B in Leishmania mexicana, focusing on life cycle progression and infectivity. In contrast to Trypanosoma brucei, in which DOT1B is essential for the differentiation of mammal-infective bloodstream forms to insect procyclic forms, L. mexicana DOT1B (LmxDOT1B) is not critical for the differentiation of promastigotes to amastigotes in vitro. Additionally, there are no significant differences in the ability to infect or differentiate in macrophages or sand fly vectors between the LmxDOT1B-depleted and control strains. These findings highlight the divergence of the function of DOT1B in these related parasites, suggesting genus-specific adaptations in the use of histone modifications for life cycle progression and host adaptation processes.

• Klíčová slova
• DOT1, Leishmania mexicana, differentiation, histone methyltransferase, sand fly, virulence,
• MeSH
• buněčná diferenciace MeSH
• histonlysin-N-methyltransferasa * metabolismus   genetika   MeSH
• histonové methyltransferasy metabolismus   genetika   MeSH
• Leishmania mexicana * genetika   enzymologie   růst a vývoj   MeSH
• makrofágy * parazitologie   MeSH
• myši MeSH
• protozoální proteiny metabolismus   genetika   MeSH
• Psychodidae parazitologie   MeSH
• stadia vývoje * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• histonlysin-N-methyltransferasa * MeSH
• histonové methyltransferasy MeSH
• protozoální proteiny MeSH

Leishmaniasis is a parasitic vector-borne disease caused by the protistan flagellates of the genus Leishmania. Leishmania (Viannia) guyanensis is one of the most common causative agents of the American tegumentary leishmaniasis. It has previously been shown that L. guyanensis strains that carry the endosymbiotic Leishmania RNA virus 1 (LRV1) cause more severe form of the disease in a mouse model than those that do not. The presence of the virus was implicated into the parasite's replication and spreading. In this respect, studying the molecular mechanisms of cellular control of viral infection is of great medical importance. Here, we report ~30.5 Mb high-quality genome assembly of the LRV1-positive L. guyanensis M4147. This strain was turned into a model by establishing the CRISPR-Cas9 system and ablating the gene encoding phosphatidate phosphatase 2-like (PAP2L) protein. The orthologue of this gene is conspicuously absent from the genome of an unusual member of the family Trypanosomatidae, Vickermania ingenoplastis, a species with mostly bi-flagellated cells. Our analysis of the PAP2L-null L. guyanensis showed an increase in the number of cells strikingly resembling the bi-flagellated V. ingenoplastis, likely as a result of the disruption of the cell cycle, significant accumulation of phosphatidic acid, and increased virulence compared to the wild type cells.

• MeSH
• buněčný cyklus MeSH
• fosfatidátfosfatasa genetika   MeSH
• Leishmania guyanensis * MeSH
• Leishmaniavirus MeSH
• leishmanióza kožní * MeSH
• lipidy MeSH
• myši MeSH
• paraziti * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfatidátfosfatasa MeSH
• lipidy MeSH

Expansion microscopy (ExM) has become a powerful super-resolution method in cell biology. It is a simple, yet robust approach, which does not require any instrumentation or reagents beyond those present in a standard microscopy facility. In this study, we used kinetoplastid parasites Trypanosoma brucei and Leishmania major, which possess a complex, yet well-defined microtubule-based cytoskeleton, to demonstrate that this method recapitulates faithfully morphology of structures as previously revealed by a combination of sophisticated electron microscopy (EM) approaches. Importantly, we also show that due to the rapidness of image acquisition and three-dimensional reconstruction of cellular volumes ExM is capable of complementing EM approaches by providing more quantitative data. This is demonstrated on examples of less well-appreciated microtubule structures, such as the neck microtubule of T. brucei or the pocket, cytosolic and multivesicular tubule-associated microtubules of L. major. We further demonstrate that ExM enables identifying cell types rare in a population, such as cells in mitosis and cytokinesis. Three-dimensional reconstruction of an entire volume of these cells provided details on the morphology of the mitotic spindle and the cleavage furrow. Finally, we show that established antibody markers of major cytoskeletal structures function well in ExM, which together with the ability to visualize proteins tagged with small epitope tags will facilitate studies of the kinetoplastid cytoskeleton.

• Klíčová slova
• Leishmania major, Trypanosoma brucei, expansion microscopy, microtubule-based cytoskeleton,
• MeSH
• elektronová mikroskopie metody   MeSH
• kinetochory metabolismus   ultrastruktura   MeSH
• Kinetoplastida metabolismus   ultrastruktura   MeSH
• Leishmania major metabolismus   ultrastruktura   MeSH
• mikrotubuly metabolismus   ultrastruktura   MeSH
• protozoální proteiny metabolismus   MeSH
• Trypanosoma brucei brucei metabolismus   ultrastruktura   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protozoální proteiny MeSH

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.

• MeSH
• flagella metabolismus   MeSH
• kineziny metabolismus   MeSH
• Leishmania mexicana patogenita   fyziologie   MeSH
• leishmanióza metabolismus   MeSH
• morfogeneze MeSH
• myši MeSH
• proliferace buněk MeSH
• protozoální proteiny metabolismus   MeSH
• Psychodidae MeSH
• virulence fyziologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kineziny MeSH
• protozoální proteiny MeSH

Differentiation between distinct stages is fundamental for the life cycle of intracellular protozoan parasites and for transmission between hosts, requiring stringent spatial and temporal regulation. Here, we apply kinome-wide gene deletion and gene tagging in Leishmania mexicana promastigotes to define protein kinases with life cycle transition roles. Whilst 162 are dispensable, 44 protein kinase genes are refractory to deletion in promastigotes and are likely core genes required for parasite replication. Phenotyping of pooled gene deletion mutants using bar-seq and projection pursuit clustering reveal functional phenotypic groups of protein kinases involved in differentiation from metacyclic promastigote to amastigote, growth and survival in macrophages and mice, colonisation of the sand fly and motility. This unbiased interrogation of protein kinase function in Leishmania allows targeted investigation of organelle-associated signalling pathways required for successful intracellular parasitism.

• MeSH
• biologické modely MeSH
• buněčná diferenciace * MeSH
• CRISPR-Cas systémy genetika   MeSH
• delece genu MeSH
• flagella enzymologie   MeSH
• Leishmania mexicana cytologie   enzymologie   MeSH
• leishmanióza parazitologie   patologie   MeSH
• mutace genetika   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• protein Cas9 metabolismus   MeSH
• proteinkinasy genetika   metabolismus   MeSH
• proteom metabolismus   MeSH
• Psychodidae parazitologie   MeSH
• viabilita buněk MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protein Cas9 MeSH
• proteinkinasy MeSH
• proteom MeSH

A recently redescribed two-flagellar trypanosomatid Vickermania ingenoplastis is insensitive to the classical inhibitors of respiration and thrives under anaerobic conditions. Using genomic and transcriptomic data, we analyzed its genes of the core metabolism and documented that subunits of the mitochondrial respiratory complexes III and IV are ablated, while those of complexes I, II, and V are all present, along with an alternative oxidase. This explains the previously reported conversion of glucose to acetate and succinate by aerobic fermentation. Glycolytic pyruvate is metabolized to acetate and ethanol by pyruvate dismutation, whereby a unique type of alcohol dehydrogenase (shared only with Phytomonas spp.) processes an excess of reducing equivalents formed under anaerobic conditions, leading to the formation of ethanol. Succinate (formed to maintain the glycosomal redox balance) is converted to propionate by a cyclic process involving three enzymes of the mitochondrial methyl-malonyl-CoA pathway, via a cyclic process, which results in the formation of additional ATP. The unusual structure of the V. ingenoplastis genome and its similarity with that of Phytomonas spp. imply their relatedness or convergent evolution. Nevertheless, a critical difference between these two trypanosomatids is that the former has significantly increased its genome size by gene duplications, while the latter streamlined its genome.

• Klíčová slova
• Phytomonas, Vickermania ingenoplastis, genome sequencing, metabolism,
• Publikační typ
• časopisecké články MeSH

The shape and form of the flagellated eukaryotic parasite Leishmania is sculpted to its ecological niches and needs to be transmitted to each generation with great fidelity. The shape of the Leishmania cell is defined by the sub-pellicular microtubule array and the positioning of the nucleus, kinetoplast and the flagellum within this array. The flagellum emerges from the anterior end of the cell body through an invagination of the cell body membrane called the flagellar pocket. Within the flagellar pocket the flagellum is laterally attached to the side of the flagellar pocket by a cytoskeletal structure called the flagellum attachment zone (FAZ). During the cell cycle single copy organelles duplicate with a new flagellum assembling alongside the old flagellum. These are then segregated between the two daughter cells by cytokinesis, which initiates at the anterior cell tip. Here, we have investigated the role of the FAZ in the morphogenesis of the anterior cell tip. We have deleted the FAZ filament protein, FAZ2 and investigated its function using light and electron microscopy and infection studies. The loss of FAZ2 caused a disruption to the membrane organisation at the anterior cell tip, resulting in cells that were connected to each other by a membranous bridge structure between their flagella. Moreover, the FAZ2 null mutant was unable to develop and proliferate in sand flies and had a reduced parasite burden in mice. Our study provides a deeper understanding of membrane-cytoskeletal interactions that define the shape and form of an individual cell and the remodelling of that form during cell division.

• MeSH
• buněčná membrána MeSH
• cytokineze MeSH
• cytoskelet metabolismus   MeSH
• flagella fyziologie   ultrastruktura   MeSH
• interakce hostitele a parazita * MeSH
• Leishmania růst a vývoj   ultrastruktura   MeSH
• leishmanióza parazitologie   MeSH
• morfogeneze * MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• Psychodidae parazitologie   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

Kinetoplastid flagellates are known for several unusual features, one of which is their complex mitochondrial genome, known as kinetoplast (k) DNA, composed of mutually catenated maxi- and minicircles. Trypanosoma lewisi is a member of the Stercorarian group of trypanosomes which is, based on human infections and experimental data, now considered a zoonotic pathogen. By assembling a total of 58 minicircle classes, which fall into two distinct categories, we describe a novel type of kDNA organization in T. lewisi. RNA-seq approaches allowed us to map the details of uridine insertion and deletion editing events upon the kDNA transcriptome. Moreover, sequencing of small RNA molecules enabled the identification of 169 unique guide (g) RNA genes, with two differently organized minicircle categories both encoding essential gRNAs. The unprecedented organization of minicircles and gRNAs in T. lewisi broadens our knowledge of the structure and expression of the mitochondrial genomes of these human and animal pathogens. Finally, a scenario describing the evolution of minicircles is presented.

• MeSH
• adenosintrifosfatasy genetika   MeSH
• editace RNA MeSH
• fylogeneze MeSH
• genom mitochondriální MeSH
• guide RNA, Kinetoplastida genetika   MeSH
• mitochondrie genetika   MeSH
• podjednotky proteinů genetika   MeSH
• protozoální DNA genetika   MeSH
• RNA protozoální genetika   MeSH
• Trypanosoma lewisi genetika   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfatasy MeSH
• guide RNA, Kinetoplastida MeSH
• podjednotky proteinů MeSH
• protozoální DNA MeSH
• RNA protozoální 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.

• Klíčová slova
• Leptomonas pyrrhocoris, flagellar length regulation, flagellum disassembly, trypanosomatids,
• 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
• Názvy látek
• protozoální proteiny MeSH