Laboratory of Molecular Biology of Protists (J. Lukes)

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.

http://www.paru.cas.cz/cs/oddeleni/oddeleni-molekularni-patazitologie/laborator-molekularni-biologie-prvoku/

 

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).

Diagnostics and epidemiology of European leishmaniasis

In a collaborative effort sponsored by the EU, we would like to map the species composition and population structure of the Leishmania donovani complex. Our focus is mostly on strains isolated from patients in various countries of the Mediterranean, but we are extending the analysis also to African and Indian strains. To this end, we have sequenced hundreds of kilobases of protein-coding genes and performed extensive RAPD analyses that provide novel information about geographic correlation, recombination and diversity of these pathogens. We have also designed species-specific and highly sensitive PCR assays that shall highly improve diagnostics of leishmaniases.

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.

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.

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