Laboratory of Molecular Biology of Protists (Julius Lukeš)

The primary interest is functional analysis of selected mitochondrial proteins of the kinetoplastid Trypanosoma brucei. Its mitochondrion is unique in many aspects and by knocking-down or knocking-in individual genes, we are trying to establish their functions. We have focused on (i) proteins involved in RNA editing and regulation of stability of mitochondrial transcripts, (ii) subunits of respiratory complexes, (iii) iron/sulfur cluster assembly proteins, (iv) prohibitin. We are also interested in the evolution and biodiversity of parasitic kinetoplastid flagellates, as well as involved in the genotyping of European leishmaniases and designing new diagnostic approaches for their detection. Moreover, we are trying to improve a new expression system in Leishmania tarentolae.

Research projects

Endosymbionts of trypanosomatids and diplonemids

Although most trypanosomatid flagellates do not harbor endosymbiotic bacteria, members of the genera Angomonas, Strigomonas, Novymonas and Kentomonas do so. They acquired their endosymbionts by at least two independent events and seem to obtain from them various macromolecular precursors and vitamins. Diplonemids (for more information see other project abstract) contain numerous endosymbionts obtained by multiple independent (and probably repeated) acquisitions, but nothing is known about their metabolic interaction with their hosts. However, we predict that in both trypanosomatids and diplonemids, endosymbiotic bacteria have significant impact on their hosts, including their behavior, life cycle progression and ecological functions.

We are dissecting the relationship between the eukaryotic host and its bacterial symbiont using protein tagging and functional genomics. We have already identified in Novymonas a protein that is critical for the intracellular distribution of bacteria. In diplonemids, we succeeded to experimentally remove endosymbionts from the host cells and reinfect them. We believe that the protist-bacterium systems that are established in our laboratory will allow us fine dissection of their intricate relationships.

Heme: a putative master regulator in trypanosomatids

Heme is one of the most important cofactor in extant organisms, constituting a reactive core of many diverse hemoproteins. Unlike most eukaryotes, trypanosomes and leishmanias are not able to synthesize heme and thus belong to heme auxothrophs. In order to shed light on how they utilize heme acquired from their hosts, we aim to dissect the function of heme transporters in model human parasitesTrypanosoma brucei and Leishmania mexicana. Moreover, we hypothesize that heme may be the key factor driving metabolic changes throughout the life cycle of both pathogens, with important difference between the stages from insect vector and mammalian blood. One of the most common heme-containing enzymes is catalase detoxifying cell from H2O2. Surprisingly, catalase is present only in monoxenous (= insect-only) flagellates but absent from all their dixenous (insect + mammalian hosts) relatives. Our preliminary data show that when overexpressed in dixenous T.brucei and L.mexicana, catalase is active only in the insect stages and loses its activity in the vertebrate-parasitizing stages. We want to analyze inter-stagial differences in heme acquisition in trypanosomatids and the reasons and mechanism behind the intriguing multiple losses of catalase in blood-dwelling parasites.


The aim of this project is to dedicate for the first time diplonemids to systematic and multifaceted research that will address their diversity, ecology, life style, genome and transcriptome, followed by functional studies of selected mitochondrial (mt) proteins. Based on the sequences gained from the latest studies of eukaryotic diversity in world oceans, diplonemids represent the 7th most abundant and 3rd most diverse group (de Vargas et al., Science 348: 1261605), but we do not even know how these cells look like. Our aim is to:

1/ study diplonemids in as many oceanic samples as possible, with the aim to establish their abundance and diversity, and gather data about their life cycle, stages, strategy and morphology;

2/ establish a representative species/strain as a new model organism that will allow to shed light on their amazing evolutionary success in contemporary oceans.

Via these aims, we want to uncover the potentially fascinating biology of these important organisms, which are arguably the least studied group of globally abundant eukaryotes. Initially, we will analyze all aspects of the metabarcoding data from the Tara Oceans and other expeditions, including our own, with the aim to map the phylogeny and diversity of diplonemids, their distribution, abundance and morphology, and statistical analysis of their co-occurrence with other marine prokaryotes and eukaryotes. The next step will be to get representative(s) of the most dominant and widespread clades into the culture, since D. papillatum, the only species studied in some detail so far, falls into the least ecologically and evolutionary relevant clade. The cultured strain(s) will be subject to whole genome sequencing with downstream analyses, such as the assembly and annotation of both mt and nuclear genomes, and analysis of the transcriptome(s), as well as detailed morphological analyses. Possibly the most challenging part of the project will be to turn the cultured strain into a genetically tractable model. Planned functional studies of the mt RNA processing machinery will be adjusted to how successful the previous step will be. However, even if we fail to genetically modify diplonemids, protocols will be applied that allow studying this unique machinery in wild type cells. Moreover, we expect that exciting and unanticipated findings will arise from the genome information.

Non Stop Blastocrithidia: the trypanosomatids with all three stop codons reassigned

Genetic code previously thought to be universal for all the life forms but soon variations from canonical code were described in many organisms all across the tree of life, most of the deviation involves reassignment of one or two stop codon to sense codons leaving 1 or 2 stop codons to confer the termination of translation. Interestingly, in 2016 two groups of protist trypanosomatid Blastocrithidia and several ciliates were shown to reassign all the tree stop codons in their nuclear genome to code amino acids. Transcriptome and proteome analysis of Blastocrithidia revealed UGA has been reassigned to code tryptophan, while UAG and UAA are reassigned to code glutamate. This finding challenges our current understanding of very fundamental process of life “Translation”.

Blastocrithidia is an ideal organisms to study this phenomenon as it belong to well studied group of kinetoplastida where all the known kinetoplastids have canonical genetic code. We have successfully optimized the genetic manipulation of Blastocrithidia which will allow us to explore different aspect of translation.

Recent development in ribosome profiling which involves deep sequencing of ribosome protected mRNA fragment is a powerful tool for monitoring in vivo translation optimization of ribosome profiling and genetic manipulation of Blastocrithidia will allow us to address following questions: Qualitative and quantitative analysis of translation process, Biased in usage of canonical and non canonical synonymous codons, how does the termination of translation work without defined stop codon and evolution of genetic code.

Evolution of trypanosomatids pathogenicity

Trypanosomatids undoubtedly belong to the most successful parasites on Earth with unique life style and ingenious adaptations to the hostile environment of the host. Because they cause devastating diseases and therefore represent major threat not only for developing countries, several dixenous (two-host) species from the genera Trypanosoma and Leishmania are subjects to thousands of studies aiming to help us understand various aspects of their molecular and cellular biology. Some of them had their genomes sequenced and now high quality reference genomes are available.

Nevertheless, it seems unlikely that genome analyses of just the dixenous species will reveal specific genetic elements responsible for successful parasite invasion. We would like to derive this information from genome sequencing and comparative analyses of monoxenous (single-host) species. For this purpose are sequencing, assembling and annotating genomes of selected monoxenous trypanosomatids belonging to phylogenetic clades, so far without a sequenced representative.

The assembly of iron-sulfur clusters in Trypanosoma brucei

Trypanosoma brucei is characterized by a number of unique cellular features. Since methods of reverse genetics are available for this flagellate, it can now be considered a model protist. Iron-sulfur (Fe-S) clusters are ancient and ubiquitous cofactors of proteins that are involved in a variety of biological functions, including enzyme catalysis, electron transport and gene expression. Nevertheless, little is known about how Fe-S clusters are assembled in T. brucei. So far, by means of RNA interference, we have down-regulated several evolutionary highly conserved components of the pathway, such as cysteine desulfurase IscS, metallochaperone IscU, frataxin, ferredoxin, and IscA. With the exception of IscA, all are essential for the parasite and their down-regulation results in reduced activity of the marker Fe-S enzyme aconitase in both the mitochondrion and cytosol. Moreover, interfering with these genes also decreased the activity of succinate dehydrogenase and fumarase, affected membrane potential of the mitochondrion and general oxygen consumption. This supports the hypothesis that the mitochondrion plays a fundamental and evolutionary conserved role in cellular Fe-S cluster assembly throughout the eukaryotes. Interestingly, we have rescued the frataxin know-down in T. brucei with its homologue from the hydrogenosome of Trichomonas vaginalis containing the hydrogenosome-targeting signal peptide. Further analyses of this rescue and the various RNAi knock-downs are under way.

Mitochondrial peptidases in T. brucei

Most mitochondrial proteins are nuclear-encoded and translocated into this organelle. Some of the pathways of protein translocation into the mitochondrion recognize a pre-sequence in the N-terminus of the nascent protein for this process to take place. Once in the mitochondrial matrix, the pre-sequence is cleaved off by mitochondrial processing peptidases, which may perform one, two or even three cuts in one single substrate. We aim to characterize these proteins in the mitochondrion of T. brucei and determine the extent of their involvement in the stabilization of the mitochondrial protein pool.

Other evolutionarily conserved proteases with known moonlighting functions have evolved new roles in the mitochondrion of T. brucei. The presence of certain Leucine Amino Peptidases (LAP) known to be involved in n-terminal cleavage of amino acids as well as glutathione metabolism are in T. brucei distributed in diverse cellular compartments involved in tasks related to cytokinesis and mitochondrial stability.

RNA editing, mitochondrial biogenesis and dynamics

Our laboratory is interested in various aspects of trypanosome mitochondrial biology, such as organellar gene expression. In this respect, we have been focused on the unique process of kinetoplastid U-insertion/deletion RNA editing, a vital process that is required for decrypting mitochondrial RNAs into bona fide templates for translation. These RNAs mainly encode subunits of the respiratory chain, which are vital for mitochondrial physiology. The membrane potential generated from this process is needed for various other pathways underlying mitochondrial biogenesis, such as protein import and the maintenance of mitochondrial volume. To this end, we have begun to investigate factors that are involved in shaping the organelle.

Mitochondria are very dynamic organelles which are constantly reshaped by fission and fusion processes. T. brucei parasites have a single continuous mitochondrion throughout their life cycle which needs to divide only once during the cell cycle just prior to cytokinesis. The only protein identified to date that was shown to play a role in trypanosome mitochondrial division is a dynamin-like protein (DLP). We are interested in identifying other proteins involved in mitochondrial dynamics such as potential adaptors that interact with DLP in both bloodstream and procyclic life stages.

Functional analysis of RNA editing in Trypanosoma brucei

We have generated knock-down cell lines of T. brucei procyclics, in which RNA binding proteins MRP1 and MRP2 are down-regulated by RNA interference. We have shown that these proteins exist in a complex composed of two MRP1 and MRP2 molecules each, which is involved in RNA editing and stabilization of mitochondrial transcripts. In a collaborative effort, we would like not only to decipher the 3D structure of this complex, but also to understand how RNA molecules are bound to it. Moreover, we are using the newly developed TAP-purification method to detect binding partners of the MRP proteins and another RNA-binding protein, TbRGG1. Furthermore, we will dissect the function of individual MRP protein domains.

Evolution and biodiversity of Kinetoplastida

Kinetoplastida are omnipresent and serious parasites of animals and plants. Using conserved rRNA and protein-coding gene sequences, we are mapping biodiversity of species infecting insects and vertebrates. Moreover, we would like to shed light on the evolution of hallmark features of the kinetoplastid flagellates, such as the extremely complex mitochondrial (= kinetoplast) genome and RNA editing. Insect trypanosomatids from North, Central, and South America that are collected will be used not only for phylogenetic analyses, but also to study components of their respiratory complexes. We would also like to understand the process of dyskinetoplastidy (loss of kinetoplast DNA), in particular its extent in species pathogenic for horses (Trypanosoma equiperdum) and possibly also for humans (Trypanosoma evansi).

Analysis of dyskinetoplastic trypanosomes

Trypanosoma brucei is a kinetoplastid flagellate, the agent of human sleeping sickness and ruminant nagana in Africa. Kinetoplastid flagellates contain their eponym kinetoplast DNA (kDNA), consisting of two types of interlocked circular DNA molecules: scores of maxicircles (each ~23 kb) and thousands of minicircles (~1.0 kb). Maxicircles have typical mt genes, most of which are translatable only after RNA editing. Minicircles encode guide (g) RNAs, required for decrypting the maxicircle transcripts. The life cycle of T. brucei involves a bloodstream stage (BS) in vertebrates and a procyclic stage (PS) in the tsetse fly vector. Partial (dyskinetoplastidy, Dk) or total loss (akinetoplastidy, Ak) of kDNA locks the trypanosome in the BS form. Transmission between vertebrates becomes mechanical without PS and tsetse mediation, allowing the parasite to spread outside the African tsetse belt. Trypanosoma equiperdum and Trypanosoma evansi are agents of dourine and surra, diseases of horses, camels, and water buffalos. We have characterized representative strains of T. equiperdum and T. evansi by numerous molecular and classical parasitological approaches. We show that both species are actually strains of T. brucei, which lost part (Dk) or all (Ak) of their kDNA. These trypanosomes are not monophyletic clades and do not qualify for species status. They should be considered two subspecies, respectively T. brucei equiperdum and T. brucei evansi, which spontaneously arose recently. Dk/Ak trypanosomes may potentially emerge repeatedly from T. brucei.


Biology Centre CAS
Institute of Parasitology
Branišovská 1160/31
370 05 České Budějovice

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