49Th Jírovec's Protozoological Days

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49th Jírovec's Protozoological Days

Conference Proceedings

Faculty of Science

Charles University

Prague, Czech Republic 2019

th 49 Jírovec's Protozoological Days

Conference Proceedings

Faculty of Science, Charles University, 2019

FOREWORD

Dear protistologists, colleagues, and friends!

Welcome to the, nearly jubilee, 49th Jírovec‟s Protozoological Days, an international protistological conference, traditionally organized by the Protozoological section of the Czech Society for Parasitology. This year„s Protodays are organized by a group from The Faculty of Science, Charles University, where 90 years ago Otto Jírovec received his doctorate and set out the foundations of Czech protistology. Since 1969, the conference has been an opportunity for graduate and postgraduate students, as well as postdocs and young scientists to present their research and results to the scientific community and the international community of protistologists. The topics presented at the conference include areas such as protistology, cell and molecular biology, genomics, phylogeny and systematics, as well as parasitology, biochemistry, and evolutionary biology and include discoveries of significant new organisms, metabolic pathways of organisms important to our understanding of the origin and evolution of life, research on the causes of serious parasitic diseases such as malaria, sleeping sickness, leishmaniasis, and many others, the development of their treatment and prevention. Thanks to English as the main language at the conference and thanks to the growing interest of foreign colleagues, students and young scientists have the opportunity to establish valuable foreign cooperation with major world scientific institutions, often leading to lifelong friendships.

We hope you fully enjoy all parts of the conference!

On behalf of the organization committee,

sincerely,

Johana Rotterová

PROGRAM SCHEDULE

Program Schedule

֎ Monday April 22nd, 2019 ֎

16:00 REGISTRATION START

18:00 WELCOME RECEPTION

֎ Tuesday April 23rd, 2019 ֎

8:00 BREAKFAST

8:50 Conference Opening

Endosymbiotic Organelles Evolution

פ Plenary lecture פ

9:00 COURTNEY STAIRS: Hacking the electron transport chain to live without oxygen

PAWEŁ HAŁAKUC: How to find things that are lost – searching 10:00 for MROs in Preaxostyla VOJTĚCH VACEK: News in iron-sulphur cluster 10:20 assembly in oxymonads LAWRENCE RUDY CADENA: Revealing an interplay between the 10:40 MICOS complex and F1Fo-ATP synthase in Trypanosoma brucei

11:00 COFFEE BREAK

PROGRAM SCHEDULE

Biodiversity, Phylogeny, and Systematics of Protozoa

ALICJA FELLS: Cell in the shell – preliminary phylogenetic 11:20 analyses of the loricate taxa of Euglenids: Trachelomonas and Strombomonas ALEXEI KOSTYGOV: Insights into the evolution of life 11:40 cycles in the genus Phytomonas KATEŘINA POLÁKOVÁ: Anaerobic scuticociliates: A diverse ciliate 12:00 lineage hosting symbiotic prokaryotes

12:20 LUNCH

Cell Biology of Protozoa

13:40 DOMINIK ARBON: Adaptive utilization of iron in Naegleria fowleri

RONALD MALYCH: Proteins interacting with oxygen 14:00 and its reactive species in Naegleria gruberi VLADIMÍRA NAJDROVÁ: The guided entry of tail-anchored 14:20 proteins pathway in Giardia intestinalis VLADIMÍR VARGA: A PCR only tagging-based system for inducible 14:40 overexpression of proteins from endogenous loci in Trypanosoma brucei ZDENĚK VERNER: Peroxisomes and Entamoeba histolytica: 15:00 To be or not to be?

15:20 COFFEE BREAK

Genomics

ZOLTÁN FÜSSY: Subcellular compartments interplay for carbon 15:40 and nitrogen allocation in Chromera velia and Vitrella brassicaformis AGNIESZKA PEŁESZ: Genome sequencing of the putatively 16:00 free-living diplomonad Trimitus sp. IT1

PROGRAM SCHEDULE

TEREZA ŃEVČÍKOVÁ: Plastid genomes and proteins illuminate 16:20 the evolution of eustigmatophyte algae and their bacterial endosymbionts MICHAŁ KARLICKI: Metagenomic insight into eukaryotic 16:40 picoplanton community of the Baltic Sea DAVID ŅIHALA: Evolution and unprecedented variants of the 17:00 mitochondrial genetic code in a lineage of green algae

17:20 POSTER SESSION

18:20 DINNER

20:00 CPS MEETING

֎ Wednesday April 24th, 2019 ֎

8:00 BREAKFAST

Endosymbiotic Organelles Evolution

פ Plenary lecture פ

ANDREW ROGER: Coevolution of genomes, organelles 9:00 and endosymbionts in anaerobic protists

LUKÁŃ V. F. NOVÁK: Comparative genomics of Preaxostyla 10:00 flagellates: insights into the evolution of amitochondriality ROMANA PETRŅELKOVÁ: Unveiling mitochondrion-related 10:20 organelles in caviomonads JOHANA ROTTEROVÁ: Where oxygen is not popular 10:40 – anaerobiosis in SAL super-group (Ciliophora)

PROGRAM SCHEDULE

11:00 COFFEE BREAK

Molecular Biology of Protozoa

MICHAELA KUNZOVÁ: Reactive oxygen species as signalling 11:20 molecules involved in RBP6 induced in vitro differentiation of Trypanosoma brucei ANSGAR GRUBER: Interplay and possible interaction 11:40 of mitochondria and nuclei in a bicosoecid JAN PYRIH: Mitochondria of some protist lineages have retained 12:00 the bacterial signal peptide recognition particle-based protein targeting machinery

12:20 LUNCH

Molecular Biology of Protozoa

JULIUS LUKEŃ: Loss of the haptoglobin-haemoglobin receptor 13:40 in Trypanosoma brucei blocks life-cycle differentiation CLARETTA BIANCHI: New species of monoxenous 14:00 trypanosomatids with catalase-encoding gene MICHALA BOUDOVÁ: The role of Q-tRNA modification 14:20 in the development of Leishmania mexicana ALEJANDRO JIMÉNEZ-GONZÁLEZ: How do I survive here? 14:40 Evolution of the metabolic and detoxification pathways in diplomonads ANZHELIKA BUTENKO: Probable endosymbiotic gene transfer 15:00 in Euglenozoa and the origin of glycosome

15:20 AFTERNOON PROGRAM – SEE WEBSITE

18:20 BANQUET

20:20 PARTY

PROGRAM SCHEDULE

֎ Thursday April 25th, 2019 ֎

8:00 BREAKFAST

Endosymbiotic Organelles Evolution

פ Plenary lecture פ

9:00 ANNA KARNKOWSKA: Exploring microbial eukaryotes to understand the evolution of the endosymbiotic organelles

KACPER MACISZEWSKI: Missing genomes, unusual structures 10:00 and other oddities: insights into the evolution of plastid genomes of Euglenophyta ANNA NOVÁK VANCLOVÁ: Novel proteins and reorganized 10:20 protein import in the secondary plastid of Euglena gracilis PETR SOUKAL: Gene transfer accompanying the secondary 10:40 endosymbiosis of euglenid plastid

11:00 COFFEE BREAK

Biodiversity, Phylogeny, and Systematics of Protozoa

ILYA UDALOV: Diversity of Fornicates in the view of environmental 11:20 V4 and V9 datasets: contrasting taxonomic composition and putative new clades MONIKA WISNIEWSKA: Phylogenomic analyses of ten 11:40 transcriptomes of secondarily free-living diplomonads ANNA GANYUKOVA: Angomonas deanei (Trypanosomatidae) life 12:00 cycle in intestines of two fly species: Calliphora vicila and Lucilia sericata (Brachycera, Calliphoridae)

12:20 LUNCH

PROGRAM SCHEDULE

Cell Biology of Protozoa

JANA PILÁTOVÁ: The purine microcrystals – particularly guanine 13:40 and uric acid – are commonly present in all main groups of protists serving as the N-rich depots INGRID SVERÁKOVÁ: Paramylon synthesis in Diplonema 14:00 papillatum HANA VÁCHOVÁ: Studying of the processes at the ciliary 14:20 tip of eukaryotic cilium

14:40 LUBOŃ VOLEMAN: Mitosomal dynamics in Giardia intestinalis

ALŅBĚTA MOTYČKOVÁ: How does ISC system work 15:00 in the mitosomes of Giardia intestinalis?

15:20 COFFEE BREAK

Molecular Biology of Protozoa

LUCIA HADARIOVÁ: Lipid analyses of E. gracilis cells, organelles 15:40 and plastid subfractions sheds light on the origin of plastid membranes KATEŘINA ŅENÍŃKOVÁ: Copper metabolism of Naegleria gruberi 16:00 and Naegleria fowleri

16:20 DANIEL SOJKA: Targetable proteolytic events in Babesia

TAMARA SMUTNÁ: Multidomain flavodiiron protein 16:40 from Trichomonas vaginalis cytosol VENDULA RAŃKOVÁ: ZapE/Afg1 is Oxa1-dependent regulator 17:00 of respiratory complexes in Trypanosoma brucei TOMÁŃ PÁNEK: Dynamin-related GTPases involved in cristae 17:20 remodelling and their evolution VOJTĚCH ŅÁRSKÝ: Mastigamoeba balamuthi 17:40 and preadaptations to parasitism of Entamoeba

PROGRAM SCHEDULE

18:00 DINNER

19:20 CONFERENCE ANNOUNCEMENTS

19:40 DEMONSTRATION OF PROTISTS

֎ Friday April 26th, 2019 ֎

8:00 BREAKFAST

Friday symposium

פ Plenary lecture פ

9:00 MATTHEW BROWN: Between a pod and a hard test: Deep evolution and development of amoeboid microbes

10:00 WORKSHOP I – Alex Tice

11:00 COFFEE BREAK

11:20 WORKSHOP II – Alex Tice

12:20 LUNCH

13:40 WORKSHOP III – Serafim Nenarokov

14:40 DEPARTURE

ABSTRACTS

Abstracts

Adaptive utilization of iron in Naegleria fowleri

Dominik Arbon1, Jan Mach1, Kateřina Ņeníńková1, Maria Grechnikova1, Ronald Malych1, Róbert Ńuťák1

1. Department of Parasitology, Faculty of Science, Charles University, BIOCEV, Czech Republic

Iron is a vital trace element for all known organisms. Its significance lies in its attribute to convert between different oxidation states, thus being able to facilitate a number of redox reactions. The availability of iron is known to be, in some cases, the limiting factor of growth in certain habitats. This phenomenon is even further augmented in the environment of another living organism, which is the case of pathogens and parasites. Naegleria fowleri is a human pathogen, the causative agent of the fatal disease primary amoebic meningoencephalitis (PAM). This condition is marked by the fast onset of symptoms such as fever, nausea, vomiting, followed by coma and death in a span of only a few weeks. Treatment is complicated and not very successful, the fatality rate is above 97%. The pathogen is found all around the world and is tied to warm, freshwater environment. Although the condition is rare, the number of undiagnosed or misdiagnosed cases is probably high since the symptoms often resemble viral or bacterial meningitis.

Our work focuses on the impact of iron ions on N. fowleri growth and metabolism. We studied the organism in iron-rich and iron-poor environments, showing that iron ions indeed affect the growth of the cells in culture. Further analysis of the proteome and transcriptome pointed out certain proteins and pathways affected by iron ion availability. Using advanced metabolomic analysis, we described the effect of iron-depleted condition on downregulation of phenylalanine degradation pathway. Determining the overall respiration showed, that alternative oxidase is utilized in iron-deficient condition, possibly compensating for the lack of iron ion components required by the respiration chain. Other attribute affected by iron ion availability was the extent of phagocytosis of bacteria. Together, this data reveals the broad spectrum of how N. fowleri is affected and underlies the requirement of iron ions.

ABSTRACTS

New species of monoxenous trypanosomatids with catalase- encoding gene

Claretta Bianchi1, Natalya Kraeva1, Alexei Y. Kostygov1, Kristína Záhonová2, Eva Horáková2, Julius Lukeń2, Vyacheslav Yurchenko1

1. Life Science Research Centre, Faculty of Science, University of Ostrava, Ostrava, Czech Republic 2. Institute of Parasitology, Biology Center, Czech Academy of Sciences and Faculty of Science, University of South Bohemia, Budweis, Czech Republic

Catalase enzyme is involved in oxidative stress protection. H2O2 is usually produced in mitochondria by a superoxide dismutase using harmful anion superoxide as a substrate. H2O2 often plays a role of secondary messenger in biochemical reactions, but in presence of iron it can be converted to a highly reactive hydroxyl radical. Most of the species use catalase to control level of H2O2. Yet, some organisms lacking this enzyme have been recently reported, presumably using other pathways. Kinetoplastid flagellates of the family Trypanosomatidae is one such odd example. The only known trypanosomatids with catalase-encoding gene was a group of monoxenous relatives of Leishmania (members of Crithidia, Leptomonas, and Novymonas). Evidently, dixenous Leishmania have secondarily lost catalase. It has been suggested that H2O2 may play a role in metacyclogenesis of these parasites (generation of infectious metacyclic promastigotes from procyclics). Thus, presence of catalase seems to be incompatible with dixenous life cycle of Leishmania. In this regard one of the overlooked trypanosomatid groups is Blastocrithidia. They came into fame for their genetic code with all stop codons encoding amino acids, with one (UAA) also as a genuine stop. Genomic analysis revealed both Blastocrithidia sp. p57 and B. triatomae possess catalase gene. A related species with standard genetic code of the so-called "jaculum" group also encodes this enzyme in its genome. We describe biochemical and phylogenetic features of catalase from Blastocrithidia and its relative, Leptomonas pyrrhocoris. We found catalase is less active in Blastocrithidia compared to Leptomonas. Phylogenetic analyses revealed that Blastocrithidia spp. acquired the gene from a different bacterium than Leishmaniinae. Blastocrithidia spp. parasitize the insect midgut, where catalase could help them survive.

Funding: OPVVV CZ.02.1.01/0.0/0.0/16_019/0000759 (Centrum výzkumu patogenity a virulence parazitů); GACR 16-18699S, 18-15962S

ABSTRACTS

Wide temperature flexibility of Crithidia thermophila and Leptomonas seymouri

Katarína Bilková1, Ľuboń Hudák1, Ingrid Sveráková1, Anton Horváth1

1. Department of Biochemistry, Faculty of Natural Sciences, Comenius University , Bratislava, Slovakia

Crithidia thermophila and Leptomonas seymouri are obligatory monoxenic parasites belonging to the family Trypanosomatidae. These species parasitize in insects at 23 °C, but as was previously shown, they are capable grow at higher temperature (34 °C). However, we have shown that they can also nicely proliferate at lower temperature (14 °C). We made growth curve for both organisms at 14 and 34 °C. Large temperature range allows studying the impact of cultivation temperature on selected parameters with more expressive trend. We isolated fractions containing mitochondria by hypotonic lysis from cells C. thermophila and L. seymouri cultivated at 14 and 34 °C. We purified mitochondrial fraction by differential centrifugation in an urografin gradient to rid of contaminating glycosomal membranes and we analysed lipid composition of mitochondria using thin-layer chromatography. We also followed the impact of cultivation temperature on morphology of mitochondria by labelling cells with Mitotracker dye. Thermotolerance is precondition for parasitism in warm- blood organisms, therefore studying of these organisms could bring more information on evolution of parasitism.

ABSTRACTS

Between a pod and a hard test: Deep evolution and development of amoeboid microbes

Matthew W. Brown1,2, Alexander K. Tice1,2

1. Department of Biological Sciences, Mississippi State University, USA 2. The Institute for Genomics, Biocomputing & Biotechnology, Mississippi State University, USA

Recent advances of both sequencing technologies and sequence library production have revolutionized the field of transcriptomics. With these new technologies, we are now able to examine the expression profiles of cell types at developmental stages. We are also able to use single cell RNAseq to robustly examine the evolutionary positions of organisms that are rare and or difficult to culture. Here we detail the methodologies, pitfalls, costs, and benefits associated with the production of such data. We also provide several case studies of the use of this technology for both types of experiments. We apply these ultra-low input RNAseq methods to phylogenomically examine the deep relationships of many protistan supergroups. Additionally, with these methods, we examine the developmental pathways using expression profiling of discrete developmental stages of life cycle of aggregatively multicellular amoebae, sporocarpic amoebae such as protosteloids, and testate amoebae. Using comparative developmental expression data, we examine differences and similarities between the opisthokont Fonticula alba and in the two distinct, distantly related amoebozoans, the well-studied dictyostelids and the lesser- known copromyxids. Copromyxa protea is a dung inhabiting sorocarpic amoeba that forms simple but macroscopic fruiting structures composed of a single cell type.

The formation of sorocarps is induced by one or a few founding amoebae, which by an unknown mechanism entice nearby trophozoites to crawl upon them and subsequently encyst causing apical growth of the sorocarp. Similarly, we are examining these types of data for the development in sporocarpic amoebae where a single cell makes a fruiting body, as well as the development of shells in testate amoebae. Using time-lapse microscopy as well as the methods in single/few cell transcriptomics, we are now able to begin to unlock the developmental program in these diverse protistans. Here we begin to unravel the developmental program of these disparate taxa to examine if these organisms use underlying homologous mechanisms.

ABSTRACTS

The role of Q-tRNA modification in the development of Leishmania mexicana

Michala Boudová1, Sneha Kulkarni1,2, Zdeněk Paris1,2

1. Institute of Parasitology, Biology Center, Czech Academy of Sciences and Faculty of Science, University of South Bohemia, Budweis, Czech Republic 2. Faculty of Sciences, University of South Bohemi a, Budweis , Czech Republic

The progression of Leishmania through their life cycle entails a series of differentiation processes that include brisk remodelling of cellular architecture as well as its physiological properties. This procedure requires rapid translational adaptation to enable survival under dynamically changing environmental conditions. As these protozoa exhibit an unusual genome organization with the protein coding genes organized in tandem arrays transcribed as polycistronic units, the regulation of gene expression occurs by posttranscriptional mechanisms exclusively.

The toolbox for fine-tuning gene expression at RNA level includes, among others, wobble base pairing that is facilitated by tRNA modifications. These can either restrict or expand codon recognition potential depending on their chemistries and thus, play a direct role in gene expression regulation. One of such modified nucleosides is queuosine (Q), a hypermodified analogue of guanosine (G), found at the anticodon wobble position of certain tRNA species. The location of Q implicates its strong influence on translation via codon usage-based mechanism. Yet, it remains to be elucidated how exactly does the presence or absence of Q-tRNAs influence global translation and primarily, the arising cellular functions.

We show that the translational requirements for Q-tRNAs alter between discrete phases of L. mexicana life cycle. Consequently, the depletion of Q-tRNAs represents difficulties for the parasite metacyclic-to-amastigote life stage transition. Furthermore, the inability of the mutant cell line to express full-spectrum amastigote-specific transcriptome and/or proteome is reflected in its decreased host cell infectivity. We hypothesize that variability in codon usage bias among the individual life stages might be correlated to the varying degree of Q modification and may account for the hampered development observed in the mutant cell line.

ABSTRACTS

Probable endosymbiotic gene transfer in Euglenozoa and the origin of glycosome

Anzhelika Butenko1,2, Fred R. Opperdoes3, Julius Lukeń1

1. Biology Centre, Institute of Parasitology, Budweis , Czech Republic 2. Faculty of Science, University of Ostrava, Ostrava, Czech Republic 3. de Duve Institute, Université Catholique de Louvain, Brussels, Belgium

Euglenozoa is a monophyletic group of protists subdivided into four main lineages: Kinetoplastea, Diplonemea, Euglenida, and Postgaardea. Euglenozoans are characterized by various lifestyles and modes of nutrition, mainly phagotrophic and osmotrophic, with the exception of the photosynthetic Euglenophyceae harboring secondary plastids of green algal origin. Several lines of evidence, including the identification of “plant-like” genes in human kinetoplastid parasites of the family Trypanosomatidae (Leishmania, Trypanosoma) and an apparent genome mosaicism in euglenids, suggest that the acquisition of Pyramimonas-like alga by the phagotrophic eukaryovorous euglenid might not have been the only event of endosymbiosis involving phototrophic organism in the euglenozoan evolution. Alternatively, “plant-like” genes could originate from the events of lateral gene transfer, possibly from food sources. The hypothesis about the presence of a photosynthetic endosymbiont in a common ancestor of euglenids and kinetoplastids was criticized for several reasons, including the scarcity of putative endosymbiont- derived genes in Leishmania and Trypanosoma and possible phylogenetic analysis artefacts due to the limited taxon representation. The sequences of diplonemids unavailable previously, might be an important missing piece of data which would help to shed light on the origin and evolution of “plant-like” genes in Euglenozoa.

We have sequenced the transcriptomes of three diplonemid species and combined this data with the available and newly sequenced genomes and transcriptomes of several euglenids and kinetoplastids and are performing a genome/transcriptome- wide screening of “plant-like” genes across Euglenozoa, accompanied by a targeted study of several gene groups. In particular, we have analyzed the genes encoding glycosome-targeted proteins, potentially involved in the transformation of peroxisomes into specialized euglenozoan-specific glycosomes.

ABSTRACTS

Revealing an interplay between the MICOS complex and F1Fo-ATP synthase in Trypanosoma brucei

Lawrence Rudy Cadena1, Alena Zíková1, Julius Lukeń1, Hassan Hashimi1

1. Institute of Parasitology, Biol ogy Center, Czech Academy of Sciences and Faculty of Science, University of South Bohemia, Budweis, Czech Republic

The intricate architecture of the mitochondria is needed for the proper orchestration of ATP generation through oxidative phosphorylation. The invaginations of the inner membrane, termed cristae, are shaped by a number of protein complexes. The Mitochondrial Contact Site and Cristae Organization System (MICOS) promotes the formation of cristae junctions, the region connecting the cristae membrane and the mitochondrial inner boundary membrane. The oligomeric F1Fo-ATP synthase is vital for the bending of cristae rims, hinting at possible antagonistic roles of these two complexes with respect to membrane curvature. While several studies have focused on how these complexes mediate proper cristae shaping individually, only a few have attempted to uncover potential links between these complexes. Moreover, these studies have been exclusive to Saccharomyces cerevisiae, leaving an enormous gap of knowledge about membrane curvature regulators outside the supergroup Opisthokonta.

Here, we attempt to address a structural interplay between the F1Fo-ATP synthase and the evolutionary diverged and recently described Trypanosome MICOS complex. We report that a core subunit of (Tb)MICOS, TbMic10-1 selectively interacts with membrane bound-subunit ATPaseTb2 of the Fo domain. Despite of obvious association, TbMic10-1 deletion does not affect steady state abundance of F1 and Fo subunits β and OSCP/ATPaseTb2, respectively. On the other hand, depletion of another core subunit, TbMic60, increases steady state levels of subunits β and OSCP, and triggers formation of higher-order oligomers of F1Fo-ATP synthase as detected by blue native electrophoresis. Interestingly this effect seems to be independent of any physical interactions between TbMic60 and F1Fo-ATP synthase subunits. We propose a model in which a subpopulation of TbMic10-1 physically links TbMICOS with F1Fo-ATP synthase while levels of TbMic60 might regulate (possibly indirectly) formation and/or stability of higher-order oligomers of F1Fo-ATP synthase.

ABSTRACTS

Leishmania oxygen-sensing adenylate cyclase (OSAC)

Arzuv Charyyeva1, Anzhelika Butenko1, Ignacio Durante2, Michaela Svobodová2, Hassan Hashimi2, Julius Lukeń2, Vyacheslav Yurchenko1

1. Life Science Research Centre, Faculty of Science, University of Ostrava, Ostrava, Czech Republic 2. Institute of Parasitology, Biology Center, Czech Academy of Sciences , Budweis, Czech Republic

Leishmania spp. belong to the family Trypanosomatidae. Many species of this family parasitize a wide range of vertebrates and cause serious health problems. Leishmania possess oxygen-sensing adenylate cyclase (OSAC), which appears to be essential for parasite surviving in hypoxic conditions. This protein has a typical heme-based sensor domain architecture. It contains a globin domain at the N- terminus, which binds oxygen and controls the catalytic activity of the C-terminal AC domain. OSACs are involved in the synthesis of the cyclic AMP (cAMP). This oxygen-dependent signaling may regulate gene transcription and cellular adaptation.

Our genome analysis demonstrated that not all trypanosomatids encode functional OSACs and members of the subgenus Viannia have pseudogenized copies in their genomes. The representatives of the genera Leishmania, „Endotrypanum‟, Blechomonas, and Blastocrithidia possess only one gene encoding OSAC. In contrast, endosymbiont-harboring trypanosomatids, monoxenous representatives of the subfamily Leishmaniinae and Wallacemonas spp. have several copies of OSAC-encoding genes. To understand the importance of this protein in the parasite virulence and to analyze parasite development in the absence of the protein, we generated OSAC-/- L. mexicana cells using CRISPR/Cas9 gene editing system. The whole genome of the OSAC-/- mutant was sequenced confirming complete ablation of the target gene. The gene knock-out is not lethal in L. mexicana, allowing us to monitor social motility, cell differentiation, growth kinetics, and perform further in vivo and in vitro experiments.

ABSTRACTS

Cell in the shell – preliminary phylogenetic analyses of the loricate taxa of Euglenids: Trachelomonas and Strombomonas

Alicja Fells1, Maja Łukomska-Kowalczyk1, Bożena Zakryś1

1. Department of Molecular Phylogenetics and Evolution, Institute of Botany, Faculty of Biology, Biological and Chemical Research Centre, University of Warsaw , Poland

Euglenids are unicellular, cosmopolitan, photosynthetic Eukaryotes. The loricate taxa of Euglenids – Trachelomonas and Strombomonas – have been under scrutiny for over a decade, due to their unusual trait of creating metal-incrusted envelopes. For many years, this exceptional quality has been used as one of their defining diagnostic features. However, as the lorica undergoes ontogenetic changes and may even be lost in laboratory strains and cultures, thus causing morphological distinguishment almost impossible, a well-established sequence database is necessary. Despite the challenging nature of DNA isolation through the envelope, we managed to obtain over 10 18S rDNA sequences of both Trachelomonas and Strombomonas species. In order to avoid misidentification due to changed cell and lorica traits, all obtained sequences come from environmental samples collected across Poland. The results of the analyses shed new light on the phylogenetic relationships between particular species and promise a possible clarification of previously unidentified taxa – with at least one new clade emerging, one sequence locating in a group with previously non-distinguished species, and several other results giving us pause.

ABSTRACTS

How many species of avian trypanosomes are transmitted by mosquitoes?

Magdaléna Fialová1, Aneņka Santolíková1, Jana Brzoňová1, Milena Svobodová1

1. Department of Parasitology, Faculty of Science, Charles University, Czech Republic

Avian trypanosomes are widespread blood parasites. Proven vectors belong to Simuliidae, Hippoboscidae, Culicidae, Ceratopogonidae, and Psychodidae. In our study, we focused on mosquitoes as vectors of avian trypanosomes. Besides Trypanosoma culicavium, T. thomasbancrofti and Trypanosoma sp. lineage III are probably transmitted by mosquitoes but experimental confirmation is lacking.

T. thomasbancrofti is cosmopolitan species recently described from Australian birds, and mosquitoes were assigned as putative vectors based on SSU rRNA sequence identity. We show that laboratory-bred Culex mosquitoes are susceptible to infection with our isolates of T. thomasbancrofti. Infections were detected in 86-94% Cx. quinquefasciatus and 44 % Cx. molestus fed on membrane. Trypanosomes firstly formed rosettes in hindgut; later free flagellates appeared. Transmission was achieved by ingestion of infected mosquito guts by two canaries (Serinus canaria) and one zebra finch (Taeniopygia guttata), as confirmed by blood culture and xenodiagnosis. Culex mosquitoes were highly succeptible to Trypanosoma sp. as well. Infections were detected in up to 80% of Cx. quinquefasciatus and 42% Cx. molestus. Trypanosomes formed rosettes in hindgut, free flagellates were in midgut,

Mosquitoes were confirmed experimentally as vectors of avian trypanosomes belonging to T. avium group. Contrary to T. culicavium, infections are located in hindgut, similarly to blackflies. However, transmission by vector ingestion seems to be a universal mode.

ABSTRACTS

Subcellular compartments interplay for carbon and nitrogen allocation in Chromera velia and Vitrella brassicaformis

Zoltán Füssy1, Tereza Faitová1, Miroslav Oborník1

1. Biology Centre, Institute of Parasitology, Czech Academy of Sciences, Budweis, Czech Republic & Department of Parasitology, Faculty of Science, Charles University, BIOCEV, Czech Republic

Endosymbioses necessitate functional cooperation of cellular compartments to avoid pathway redundancy and streamline the control of biological processes. To gain insight into the metabolic compartmentation in chromerids, phototrophic relatives to apicomplexan parasites, we prepared a reference set of proteins probably localized to mitochondria, cytosol and the plastid, taking advantage of available genomic and transcriptomic data. Training of prediction algorithms with the reference set now allows a genome-wide analysis of protein localization in C. velia and V. brassicaformis. We found that the chromerid plastids house enzymatic pathways needed for their maintenance and photosynthetic activity, but for carbon and nitrogen allocation, metabolite exchange is necessary with the cytosol and mitochondria. This indeed suggests that the regulatory mechanisms operate in the cytosol to control carbon metabolism based on the availability of both light and nutrients. We discuss that this arrangement is largely shared with apicomplexans, possibly stemming from a common ancestral metabolic architecture, and supports the mixotrophy of the chromerid algae.

ABSTRACTS

Vertebrate retortamonads share features of anaerobic metabolism with relative diplomonads

Zoltán Füssy1, Zuzana Vaitová1, Tomáń Pánek2, Ivan Čepička3, Vladimír Hampl1

1. Faculty of Science, Charles University, BIOCEV , Czech Republic 2. Faculty of Science, University of Ostrava, Ostrava, Czech Republic 3. Department of Zoology, Faculty of Science, Charles University, Prague, Czech Republic

Although the mitochondria of extant eukaryotes share a single origin, functionally these organelles diversified to a great extent, reflecting lifestyles of the organisms that host them. In anaerobic excavates of the group Metamonada, mitochondria evolved into organelles with very limited functions (also termed hydrogenosomes or mitosomes) and an amitochondriate eukaryote, Monocercomonoides exilis, has also been reported recently. Retortamonads dwelling in intestines of vertebrates form a sister group to parasitic diplomonads, Giardia and Spironucleus, and have also been hypothesized to completely lack mitochondria. In the pilot transcriptomic data from Retortamonas dobelli, we searched for the enzymes of the core energy metabolism and for the hallmark mitochondrial proteins. Among the recovered glycolytic enzymes, we found pyrophosphate-dependent analogues of phosphofructokinase and pyruvate kinase capable of reversible ATP-independent reaction steps reminiscent of the pathway arrangement in other metamonads. We found potential traces of the mitochondrial metabolism, represented by pyruvate:ferredoxin oxidoreductase, [FeFe]-Hydrogenases, a Tim14/Pam18 protein and, importantly, two hydrogenase maturase homologs (HydF and HydG), suggesting a remnant mitochondrion is still present. A more in-depth analysis is ongoing to investigate other aspects of the adaptations of retortamonads to microaerobic environments.

ABSTRACTS

Angomonas deanei (Trypanosomatidae) life cycle in intestines of two fly species: Calliphora vicila and Lucilia sericata (Brachycera, Calliphoridae)

Anna I. Ganyukova1, Marina N. Malysheva1, Andrew V. Zolotarev1,2, Alexei Y. Kostygov1,3, Alexander O. Frolov1

1. Zoological Institute of the Russian Academy of Sciences, St. Petersburg, Russia 2. St. Petersburg State University, St. Petersbur g, Russia 3. Life Science Research Centre, Faculty of Science, University of Ostrava, Ostrava, Czech Republic

We investigated life cycles of two monoxenic trypanosomatid Angomonas deanei isolates: A. deanei TCC 036E (COLPROT 044), isolated in Brazil from the intestine of the bug Zelus leucogrammus (Hemiptera, Reduviidae), and A. deanei MN, obtained in summer 2016 in the north of Leningrad region, Russia, from the intestine of the blow fly Lucilia sp. (Brachycera, Calliphoridae). Experiments on artificial infections were carried out with insects of Calliphora vicina and Lucilia sericata (Brachycera, Calliphoridae) laboratory cultures. The study shows that both isolates of A. deanei are localized in the host's hindgut (rectal ampoule). The horizontal transmission of the parasite through a wet substrate between both imagoes of the similar host species and individuals of different species is experimentally confirmed. The parasites ultrastructural organization and features of the parasite-host interaction are investigated. Moreover, a series of experiments enables us to prove the absence of infection in the intestines of experimentally infected Calliphora vicina and Lucilia sericata larvae. We are currently discussing transmission strategy, geography, and host specificity of Angomonas deanei.

Acknowledgments: The present study was supported by the project RFBR no. 18-34- 00867mol_a and project RFBR no. 18-04-00138A. The study utilized equipment of the “Taxon” Research Resource Center (http://www.ckp-rf.ru/ckp/3038/) of the Zoological Institute of the Russian Academy of Sciences.

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Interplay and possible interaction of mitochondria and nuclei in a bicosoecid

Ansgar Gruber1, Zoltán Füssy1, Dagmar Jirsová1, Miroslav Oborník1

1. Institute of Parasitology, Biology Centre, Czech Academy of Sciences , Budweis, Czech Republic

Stramenopiles comprise photosynthetic as well as non-photosynthetic groups. The plastids of the photosynthetic groups are complex plastids, which are derived from a red algal ancestor. There is quite some debate on the relationship between the different groups of algae in which such plastids are found (cryptophytes, haptophytes, plastid containing stramenopiles and most of the plastid containing alveolates), as well as on the number and sequence of plastid gains, losses or transfers between these groups.

Non-photosynthetic stramenopile groups like oomycetes or bicosoecids show no evidence of plastids. However, depending on the scenario of plastid evolution between the above-mentioned groups of algae, the photosynthetic stramenopile groups might either have lost plastids that were present in the last common ancestor of the stramenopiles, or might have never had plastids. Organelle evolution via endosymbiosis is accompanied by extensive re-targeting of proteins to intracellular compartments that are not always the same compartment as the one from which the gene originated. Therefore one key to understanding plastid evolution in organisms with complex red algal derived plastids is knowledge of the organelle/cytosol/nucleus interactions in their plastid free relatives. In a yet to be described bicosoecid, we discovered close interaction of nucleus and mitochondria in structures that were to our knowledge never observed in plastid containing stramenopiles. Here we present results of our ultrastructural and molecular biological characterizations of the interplay of mitochondria, nuclei and cytosol in this bicosoecid.

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Lipid analyses of E. gracilis cells, organelles and plastid subfractions sheds light on the origin of plastid membranes

Lucia Hadariová1, Aleń Tomčala2,3, Miroslav Oborník2, Vladimír Hampl1

1. Department of Parasitology, Faculty of Science, Charles University, BIOCEV, Czech Republic 2. Biology Centre, Institute of Parasitology, Czech Academy of Sciences, Budweis, Czech Republic 3. Institute of Aquaculture and Protection of Waters, Faculty of Fisheries and Protection of Waters, University of South Bohemia, Budweis , Czech Republic

Euglena gracilis is a photosynthetic flagellate possessing chlorophyte-derived secondary plastids. Resulting from eukaryote-to-eukaryote endosymbioses, secondary plastids are usually enclosed by four membranes – two membranes of the primary plastid, the plasma membrane of the symbiont and the membrane of the host phagosome. Euglenophyte plastids are, however, surrounded by three membranes.

It was generally assumed that the plastids lost one of the outermost eukaryotic membranes and that they are surrounded by two membranes of prokaryotic and a single membrane of eukaryotic origin. Recent genomic and transcriptomic analyses revealed the absence of TOC (translocon of outer chloroplast membrane) homologs in the plastids of euglenophytes which casted doubts on the prokaryotic origin of the middle membrane. We compared the lipid composition of whole cells of E. gracilis strain Z, its plastid fraction and plastid subfractions (thylakoids and plastid envelope membranes) by high performance liquid chromatography high resolution tandem mass spectrometry. We also determined the lipid composition of E. gracilis mitochondrial fraction and composition of E. gracilis bleached mutants, W10BSmL (W10) and WgmZOflL (OFL), containing probably only remnants of plastids. We focused primarily on the composition of typical structural lipids of primary plastids – glycosyldiacylglycerols (GDAGs). We detected GDAGs in bleached mutants confirming the presence of plastid remnants. The ratio among GDAGs and other phospholipids in the plastid subfractions of E. gracilis enabled us to answer the prokaryotic versus eukaryotic origin of the middle plastid membrane.

ABSTRACTS

How to find things that are lost – searching for MROs in Preaxostyla

Paweł Hałakuc1, Lukáń V.F. Novák2, Sebastian C. Treitli2, Vladimír Hampl2, Anna Karnkowska1

1. Department of Molecular Phylogenetics and Evolution, Institute of Botany, Faculty of Biology, Biological and Chemical Research Centre, University of Warsaw , Poland 2. Department of Parasitology, Faculty of Science, Charles University, BIOCEV, Czech Republic

The discovery of truly amitochondrial eukaryote, oxymonad Monocercomonoides exilis, raise the question about the presence of mitochondria-related organelles (MROs) and their possible functions in other Preaxostyla, especially Oxymonadida. To address those questions, we analysed recently sequenced draft genome of Paratrimastix pyriformis (confirmed MRO), Blattamonas nauphoetae, and Streblomastix strix, along with the partial transcriptome of Trimastix marina. Our primary goal was to verify the presence of the organelle and describe its functions. However, we also aim to standardize and simplify the pipeline allowing analyses of new assemblies in easier and replicable manner.

Validation of the absence of MRO-related genes in the genome requires very sensitive methods, which often produce false positives. We used two kinds of searches: homology-based (blast, hmmer, HHpred) and structure-based (targeting, tail-anchored and beta-barrel signals). For homology-based methods we updated databases, especially for proteins from recently described MROs. We also tested several new structure-predicting programs, such as MitoFates and NommPred. Finally, we combined updated databases and software into pipeline and automatize the process whenever it was possible.

Our preliminary results of B. nauphoetae, S. strix, and T. marina predicted proteomes homology-based searching of membrane proteins resulted in similar number of candidate proteins as in M. exilis (9, 8 and 8 vs 11), and lower than in P. pyriformis (17). However only in P. pyriformis and T. marina we identified candidates for an essential membrane transport proteins such as Tom40, Sam50 and Tim17/22/23. Furthermore, we detected several hundreds of candidates for proteins with mitochondrial targeting signal, most of them being false positives. Only for P. pyriformis we identified about dozen proteins possibly localized in MRO. Due to the high number of false positives we are currently focusing on the automatic methods for their falsification.

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Hidden diversity of Breviatea

Pavla Hanousková1, Yana Eglit2, Alastair Simpson2, and Ivan Čepička1

1. Department of Zoology, Faculty of Science, Charles University, Prague, Czech Republic 2. Department of Biology, Dalhousie University, Halifax, NS, Canada

Breviatea is a small group of free-living, anaerobic protists with an important phylogenetic position close to the supergroup Opisthokonta. The group is currently represented by only four monotypic genera of tiny amoeboid flagellates living in marine or brackish anoxic environments. Here we report that Breviatea is, in fact, a diverse and widespread lineage of anaerobic protists. We have obtained and cultured 17 strains of breviatean amoeboid flagellates from marine anoxic or hypoxic sediment worldwide. We also present a novel breviatean lineage with a markedly different morphology from all other lineages. These discoveries will enable more detail examinations of the evolution of this group of anaerobes, as well as the deep ancestry of the animal and fungi group on the Tree-of-Life.

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Tetracycline-inducible expression system in G. intestinalis

Vendula Horáčková1, Luboń Voleman1, Vladimíra Najdrová1, Pavel Doleņal1

1. Department of Parasit ology, Faculty of Science, Charles University, BIOCEV, Czech Republic

Giardia intestinalis is a protozoan parasite that infects intestine of humans and other vertebrates. There are several transfection systems which allow stable transfection and expression of genes of interest in G. intestinalis. However, there are only few inducible systems available for use in Giardia. One of them is a system based on regulatory elements from bacterial operon encoding resistance to tetracyclines which is considered one of the most efficient regulatory systems so far. Since gene expression is poorly understood in Giardia, we designed new vector for T7 RNA polymerase-driven tetracycline-inducible expression and tested our system under different conditions. Initially, we determined the optimal concentration of the inducer and the time of induction. We decided to use this system to study biology of mitosomes and the protein targeting pathways by expressing three experimental constructs. (i) a truncated version of the outer mitosomal membrane protein with BAP-tag in order to pull-down intact mitosomes (ii) human pro-apoptotic mitochondrial protein Bak was designed to permeabilize mitosomal membranes in order to test organelle function and (iii) mutated version of Get3 ATPase in order to interfere with the insertion of tail-anchored proteins into the ER membrane. Surprisingly, common presence of T7 polymerase and T7 promotor results into strong but not inducible expression in Giardia. Thus, in addition to T7 promoter, we are currently testing the combination of strong endogenous promoter of ornithine carbamoyl transferase (OCT) with Tet operator elements.

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Loss of the haptoglobin-haemoglobin receptor in Trypanosoma brucei blocks life-cycle differentiation

Eva Horáková1, Laurence Lacordier2, Paula Cahuna2, Piya Changmai1, Roman Sobotka3,4, Julius Lukeń1,4, Benoit Vanhollebeke2

1. Biology Centre, Institute of Parasitology, C zech Academy of Sciences, Budweis, Czech Republic 2. Institut de Biologie et de Medecince Moleculaires, Universite Libre de Bruxelles, Gosselies, Belgium 3. Institute of Microbiology, Třeboň, Czech Republic 4. University of South Bohemia, Budweis, Czech Republic

In its vertebrate host, Trypanosoma brucei s.l. undergoes an extensive differentiation, from a dividing long slender form to a quiescent short stumpy one, which has its haptoglobin-haemoglobin receptor (HpHbR) repressed, highly reducing the uptake of haptoglobin-haemoglobin (HpHb). Moreover, T. b. gambiense has an extremely low cellular hem, likely a consequence of a single mutation in its HbHpR. Introducing this unique mutation resulted in the same phenotype in T. b. brucei. When expressed in the wild type and HpHpR knock-out T. b. brucei, human catalase was active only in the former trypanosomes. The absence of HpHbR in T. b. brucei completely abolished differentiation, suggesting its key role in life cycle progression. We have recapitulated the same phenomenon in the wild type T. b. gambiense. Combined, we demonstrate that in the absence of HpHbR, Trypanosoma brucei s.l. fail to differentiate.

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The net-like heterotrophic amoeba Leukarachnion sp. PRA-24 (Ochrophyta, Stramenopiles) has a cryptic plastid

Karin Jańke1, Tomáń Pánek1, Aneņka Eliáńová1, Marek Eliáń1

1. Department of Biology and Ecology, Faculty of Science, University of Ostrava, Ostrava, Czech Republic

In 1942 Geitler described Leukarachnion batrachospermi, a colourless amoeboid organism forming large anastomosing networks and walled cysts. Its phylogenetic affinity remained uncertain until Grant et al. (2009) studied strain PRA-24 (maintained at ATCC) identified as a potentially new species of the genus Leukarachnion. Phylogenetic analysis of four nuclear gene sequences obtained from Leukarachnion sp. PRA-24 revealed that it is a stramenopile related to the amoeboid algae Chlamydomyxa labyrinthuloides and Synchroma grande within Ochrophyta, and thus can be classified as a secondarily non-photosynthetic representative of Synchromophyceae. This raises a possibility that it has retained a colourless plastid, but no candidates for it were found by transmission electron microscopy. We employed an alternative approach and generated transcriptomic and genomic data from Leukarachnion sp. PRA-24. Initial analyses of our transcriptome assembly revealed transcripts encoding proteins with N-terminal regions that fulfill the definition of characteristic ochrophyte bi-partite plastid-targeting presequences. Among them are enzymes of the C5 pathway of haem biosynthesis, suggesting that Leukarachnion sp. PRA-24 has a plastid-derived organelle supplying the whole cell with haem. We also identified candidates for plastid-localized components of the translation apparatus, suggesting that the hypothetical plastid has retained a genome. Searches of an assembly of DNA sequencing reads from the Leukarachnion sp. PRA-24 culture indeed retrieved two contigs that appear to represent parts of the expected plastid genome. These contigs include genes for components of the transcription and translation machineries and the ClpC protein involved in protein turnover, whereas genes related to photosynthesis are missing. Altogether, our results demonstrate that Leukarachnion sp. PRA-24 has a cryptic plastid, whose more detailed characteristics are being investigated and will be presented.

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How do I survive here? Evolution of the metabolic and detoxification pathways in diplomonads

Alejandro Jiménez-González1, Staffan G. Svärd1, Jan O. Andersson1

1. Department of Cell and Molecular Biology, Molecular Evolution program, Uppsala University, Uppsala, Sweden

Diplomonads are a group of flagellated protists that are classified within Fornicata (supergroup Excavata). Giardia intestinalis - the causative agent of giardiasis in humans - is the best-known diplomonad, but members of this group infect other mammals, as well as fishes, amphibious, and birds. In this group, we find also Trepomonas sp. PC1 that has been described as a secondary free-living. All diplomonads lack the classical aerobic mitochondria, produce energy via fermentation, and live in low-oxygen environments. However, this level of oxygen can be toxic to the cell. Although diplomonads share a central metabolism, we do not know the metabolic capacities of the different species. My Ph.D. focuses on identifying these capacities and the role they have in the adaptation to particular hosts and environments.

We reconstructed the different metabolic and detoxification pathways in diplomonads combining published experimental data with clustering and phylogenetic approaches, based on available genomes and transcriptomes. Our results showed lineage- specific adaptations from a diplomonads ancestor to the extant species. E.g., Giardia species showed clear differences in enzymes for the interaction with the host microbiota, Giardiinae species detoxify arsenic through a glutaredoxin-linked pathway, whereas Hexamitiinae species use an uncompleted methylation pathway, and we observed differences in the synthesis of non-protein thiols used in oxygen detoxification. In all the systems we observed a dynamic process of gains and losses or replacements by proteins better adapted to the environment (e.g., superoxide reductase). We showed that lateral gene transfer events have played an important role in remodelling different systems.

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Metagenomic insight into eukaryotic picoplanton community of the Baltic Sea

Michał Karlicki1, Anna Karnkowska1

1. Department of Molecular Phylogenetics and Evolution, Faculty of Biology, Biological and Chemical Research Centre, University of Warsaw, Poland

The Baltic Sea is among largest brackish water bodies in the world and is a great model to study the ecosystem structure and functions in the salinity, redox, and nutrient gradients. Despite the numerous metagenomic studies of bacterioplankton across the Baltic, studies of eukaryotic plankton still rely on metabarcoding or microscopic observations.

In this study, we re-analyzed time-series metagenomic data from the central Baltic Sea (10 km off the coast of Öland, Sweden) to study seasonal dynamics and diversity of eukaryotic picoplankton. The metagenomics datasets (37) from one year (from March till December) were processed using in-house software MetaPlastHunter. This approach allowed us to gather information about the abundance of phytoplankton taxa based on the plastid genomes and to extract rDNA sequences for further assembly.

Reads that were assigned to plastid genomes by MetaPlastHunter belonged to four groups and, seven species of phytoplankton were reported. We observed two picks of algal blooms, one during the summer months and the second during November and December. Chlorophytes and cryptophytes dominated in the summer bloom but the winter bloom, surprisingly, was overshadowed by a cryptophyte Teleaulax amphioxeia, not reported in the microscopic observations.

The assembly of rDNA resulted in 26 almost full-length 16S rDNA and 68 18S rDNA sequences. 16S rDNA were classified to Cryptophyta (4), Chlorophyta (14), Haptophyta (3), Stramenopila (4), Excavata (1) and 18S rDNA were classified to Alveolata (38), Cryptophyta (6), Chlorophyta (9), Haptophyta (2), Stramenopila (8), Excavata (2), Cercozoa (2), and Picozoa (1). Within alveolates we observed 18S rDNA sequences related to dinoflagellate Gymnodinium which might have an impact on cryptophyte population. Crucially, we identified the same groups in SSU sequences and in the MetaPlastHunter results, and dominant taxa identified by MetaPlastHunter were in agreement with the most abundant SSU sequences.

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Exploring microbial eukaryotes to understand the evolution of the endosymbiotic organelles

Anna Karnkowska1

1. Department of Molecular Phylogenetics and Evolution, Faculty of Biology, Biological and Chemical Research Centre, University of Warsaw, Poland

The diversity of eukaryotic cellular forms is often hidden among obscure and divergent lineages of microbial eukaryotes (protists). Species discovery and modern sequencing techniques allow us to uncover a lot of this diversity and highlight potential model organisms, which might help to elucidate the evolution of the eukaryotic cell. I will focus on the fundamental questions on the mechanisms of enslavement, maintaining and loss of organelles, and present two examples of fascinating microbial eukaryotes, which contribute to our understanding of endosymbiosis.

One of the unanswered questions about the early stages of endosymbiosis is which came first: gene transfer or cellular fixation. Endosymbiosis requires ingestion of a symbiont, its retention by the host, transfer of genes to the host, and evolution of a protein-targeting system. To understand the order of the events and the early stages of transition, I am studying kleptoplasty in the euglenid Rapaza viridis. Our results based on plastid genome and transcriptome analyses suggest the presence of plastid-targeted proteins encoded in the nuclear genome of R. viridis, even though its plastid is transient. This result supports the hypothesis that gene transfer came before the cellular fixation and might explain the early steps of endosymbiosis, at least in some lineages.

Endosymbiotic organelles, once established, are permanent components of the cell. However, there are more and more examples of highly reduced plastids and mitochondria among microbial eukaryotes. The cases of complete loss of organelles are still sporadic and, in case of the mitochondrial organelle, known only from one group – Metamonads. The mitochondrial organelle became dispensable in Monocercomonoides exilis due to a series of evolutionary events, including horizontal gene transfer of the Suf system. Careful investigation of M. exilis genome allowed us to understand how the endosymbiosis can be undone and how this affects the cellular complexity.

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Identification of β-1,3-glucan synthase enzyme in Euglenozoa

Martina Keńeľáková1, Juraj Krajčovič1, Dominika Veńelényiová1

1. Department of Biology, Faculty of Natural Sciences, University of Ss. Cyril and Methodius , Trnava, Slovakia

The phytoflagellate Euglena gracilis and some other Euglenozoa species produce a large amount of specific β-1,3-glucan polymer called paramylon, as a storage polysaccharide. Biosynthesis of paramylon is mediated by paramylon synthase enzyme which represents an enzyme complex of multiple subunits. Knowledge of key enzymes of metabolism of paramylon is still limited for species E. gracilis, in which only two available sequences of paramylon synthase enzyme were previously identified. In our study we detected new sequences of potential paramylon synthase enzyme in representative species of Euglenozoa. In silico approach of potential enzyme sequence identification was performed using hidden Markov model of the catalytic domain of related β-1,3-glucan synthases from different plant and ascomycota species. Sequences with these domain motifs was detected in four species of Euglenozoa. In silico analysis of protein primary structure demonstrated the evidence of three different types of identified sequences. They were found in Euglenida species (Euglena gracilis, E. longa and Eutreptiella gymnastica), which differ from the Kinetoplastida (Bodo saltans) protists. Our analysis of Euglenozoa proteomes suggest that except of Euglenida also free living Kinetoplastida may dispose genetic potential for paramylon synthase formation, while parasitic species have lost their ability to produce paramylon. The overall topology and domain architecture of enzymes from Euglenozoa differs from β-1,3-glucan synthase from plants and ascomycetes. Phylogenetic analysis also reflects that set of studied enzymes from Euglenozoa exhibits a distinct position with respect to all characterized β-1,3-glucan syntheses from different sources.

Key words: β-1,3-glucan synthase, Euglena, paramylon, Euglenida, Kinetoplastida

Acknowledgement: This project is funded by VEGA 1/0535/17 and FPPV 2019.

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Insights into the evolution of life cycles in the genus Phytomonas

Alexei Y. Kostygov1,2, Alexander O. Frolov1, Marina N. Malysheva1, Viktoria V. Spodareva1,2, Anna I. Ganyukova1, Vyacheslav Yurchenko2,3

1. Zoological Institute of the Russian Academy of Sciences, St. Petersburg, Russia 2. Life Science Research Centre, Faculty of Science, University of Ostrava, Ostrava, Czech Republic 3. Martsinovsky Institute of Medical Parasitology, Tropical and Vector Borne Diseases, Sechenov University, Moscow, Russia

The members of the genus Phytomonas are peculiar trypanosomatids adapted to parasitism in the latex, phloem, fruits, or flowers of more than 20 different families of the vascular plants. As vectors they have phytophagous true bugs of families Coreidae, Pentatomidae, and superfamily Lygaeoidea. Some representatives of this genus affect cultural plants and therefore have economic impact. Although the genomes of four species have been sequenced and interesting peculiarities of their metabolism have been revealed, the biology of these flagellates is still essentially understudied.

Here we investigated the development of two Phytomonas spp. in their insect hosts using light and electron microscopy. One of these, P. nordicus parasitizes a predatory pentatomid bug Troilus luridus and therefore is secondarily monoxenous species. Another species, Phytomonas lipae, is a parasite of the phytophagous dock bug Coreus marginatus and proved to be a member of the second earliest diverging branch within the genus. The availability of data on the development of P. serpens (species from the tree crown) and P. oxycareni (the earliest diverging member of the genus) allowed us to conduct the comparative analysis for all four species and reconstruct the vectorial part of the life cycle of their common ancestor. The ancestral type of the development apparently included propagation of promastigotes and formation of infective endomastigotes in the intestine, the trait which was lost in P. lipae and P. oxycarenus. From the intestine, the cells migrated to haemocoel and carefully penetrated the epithelium of the salivary glands without active proliferation on the way. The promastigotes in the salivary gland lumen divided being unattached to the host cells and formed infective endomastigotes.

This work was supported by the Russian Science Foundation grant 18-14-00134 and European Regional Funds grant 16_019/0000759.

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Unveiling the FtsH (HflB) protease orientation of Trypanosoma brucei

Tomáń Kovalinka1, Tomáń Pánek2, Bianka Kováčová1, Anton Horváth1

1. Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia 2. Faculty of Science, University of Ostrava, Ostrava, Czech Republic

Trypanosoma brucei a flagellate protist belonging to a group Kinetoplastea is an important human parasite that undergoes serious cellular changes during its lifetime. The key process involved in these serious changes is protein regulation and degradation. We have focused on characterization of trypanosomal FtsH protease, whose 6 homolog genes had been found in the T. brucei genome. We have experimentally confirmed the inner mitochondrial membrane localization and orientation of all six homologs by in silico analyses, immunofluorescence and via protease assay. The evolutionary origin has been tested by comparative phylogenetic analysis, which showed two separate groups of FtsH mitochondrial proteins, potentially inherited from the last eukaryotic common ancestor (marked as mitochondrial group 1 and 2). While group 1 contains only one homolog, group 2 is much more diversified, due to various duplications of kinetoplastid genes. Some kinetoplastid FtsH expose different orientation in mitochondrial membranes apart from examined eukaryotes. Our findings clearly underline the unique character of trypanosomal/kinetoplastid mitochondria and its involvement in proteolytic homeostasis sustaining machinery.

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Diversity of monoxenous trypanosomatids from Papua New Guinea

Jana Králová1, Anastasiia Grybchuk-Ieremenko1, Jan Votýpka2,3, Julius Lukeń3,4, Petr Kment5, Alexei Kostygov1,6, Vyacheslav Yurchenko1,3,7

1. Life Science Research Centre, Faculty of Science, University of Ostrava, Ostrava, Czech Republic 2. Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic 3. Biology Centre, Institute of Parasitology, Czech Academy of Sciences, Budweis, Czech Republic 4. Faculty of Science, University of South Bohemia, Budweis, Czech Republic 5. Department of Entomology, National Museum, Prague, Czech Republic 6. Zoological Institute of the Russian Academy of Sciences, St. Petersburg, Russia 7. Institute of Environmental Technologies, Faculty of Science, University of Ostrava, Czech Republic

Here we present results of the molecular assessment of monoxenous trypanosomatids' diversity in Papua New Guinea (PNG). This region is a recognized biodiversity hotspot containing over 5% of the world's animal and plant species. For the first time, the diversity of these flagellates has been analyzed East of the Wallace's line, one of the most widely known biogeographic boundaries of the world. Out of 907 dissected insect (heteropteran and dipteran) specimens, 135 displayed trypanosomatid infection. The 18S rRNA-based molecular analysis of the samples from infected insects revealed that there were only a few widespread typing Units (TUs), while the majority have never been documented in other regions. Nearly one half of endemic trypanosomatid TUs were found in endemic and sub-endemic host species. The phylogenetic analysis of obtained sequences revealed several new clades within the subfamily Leishmaniinae and generic groups “jaculum” and Blastocrithidia and one new clade at the level of genus or infrafamily. In addition, L. pyrrhocoris, was first time recorded in a bug host belonging to a family other than Pyrrhocoridae.

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Reactive oxygen species as signaling molecules involved in RBP6 induced in vitro differentiation of Trypanosoma brucei

Michaela Kunzová1,2, Eva Doleņelová2, Alena Panicucci Zíková1,2

1. Faculty of Science, U niversity of South Bohemia, Budweis, Czech Republic 2. Biology Centre, Institute of Parasitology, Czech Academy of Sciences, Budweis, Czech Republic

The life cycle of Trypanosoma brucei includes distinct life cycle stages that differ significantly from each other in multiple aspects. One of the most prominent changes occurs in metabolic pathways providing energy. The procyclic forms (PF), found in the insect vector, depend on oxidative phosphorylation (OXPHOS) to generate ATP, while the bloodstream forms (BF) residing in the mammalian host with access to abundant glucose, generate ATP by aerobic glycolysis. Intriguingly, this metabolic rewiring from OXPHOS to aerobic glycolysis is similar to a Warburg effect described in cancer cells. Recently, reactive oxygen species (ROS) molecules were proposed to serve as signal conductors and to contribute to this metabolic switch. Hence, inspired by the similarity of these two phenomena, we decided to investigate the possible role of ROS in our model organism during in vitro differentiation from PF to metacyclic cells (MF) triggered by the overexpression of RNA binding protein 6 (RBP6). Indeed, higher levels of ROS were detected during the RBP6-induced differentiation. To eliminate the produced intracellular ROS, we took advantage of the fact, that Trypanosoma brucei genome does not encode for catalase, a ubiquitous enzyme and natural ROS scavenger. To our excitement, overexpression of catalase from a close relative of Trypanosoma species – Crithidia fasciculate hindered the ability of T. brucei to differentiate from PF to MF. The hallmarks of differentiation, e.g. higher membrane potential, higher ROS and changes in steady state of certain mitochondrial enzymes, were not detected anymore. Our findings provide insights into the emerging concept that mitochondria act as signaling organelles through the release of ROS to drive cellular differentiation.

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Anaerobic peroxisomes in Mastigamoeba balamuthi

Tien Le1, Vojtěch Ņárský1, Eva Nývltová1, Zdeněk Verner1, Ivan Hrdý1, Jan Tachezy1

1. Departm ent of Parasitology, Faculty of Science, Charles University, Czech Republic

Peroxisomes are omnipresent in aerobic eukaryotic cells. Most peroxisomes share presence of fatty acid oxidation that generates hydrogen peroxide and hydrogen peroxide degrading enzymes, namely catalase to prevent cellular oxidative damage. Other metabolic pathways are highly variable showing a remarkable versatility of peroxisomal metabolism and functions. In contrast, anaerobic protists such as Entamoeba histolytica, Giardia intestinalis, and Trichomonas vaginalis are believed to lack these organelles. Unexpectedly, analysis of the genome of Mastigamoeba balamuthi, an anaerobic free-living relative of E. histolytica revealed presence of a complete set of peroxins (Pexes) that are highly conserved among eukaryotic lineages and responsible for the biogenesis of peroxisomes. Immunofluorescence microscopy revealed co-localization of MbPex3, MbPex11, and MbPex14 in numerous vesicles that are distinct from other cellular organelles including hydrogenosomes, endoplasmic reticulum and Golgi apparatus. Combination of in silico searches in M. balamuthi genome and quantitative mass spectrometry of cellular fractions revealed fifty-seven potential peroxisomal matrix proteins. Interestingly, some of them are shared with E. histolytica. Heterologous expression of M. balamuthi proteins in Saccharomyces cerevisiae revealed that MbPex14 and eight Mb matrix proteins are specifically targeted to yeast peroxisomes. Based on our analyses a metabolic map of predicted peroxisomal pathways was constructed that includes pyrimidine biosynthesis, metabolism of CoA and carbohydrates. In conclusion, we characterized the first peroxisomes present in anaerobic eukaryote.

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The treatment of Euglena gracilis strain Z by different concentrations of xylene

Alexandra Lukáčová1, Juraj Krajčovič2, Matej Vesteg1

1. Department of Biology and Ecology, Faculty of Natural Sciences, Matej Bel University, Banská Bystrica, Slovakia 2. Department of Biology, Faculty of Natural Sciences, University of ss. Cyril and Methodius, Trnava, Slovakia

Monocyclic aromatic hydrocarbon xylene is one of the widely industrially used volatile organic compound which is usually commercially sold as a mixture of three isomers – orto-xylene (o-xylene), meta-xylene (m-xylene) and para-xylene (p- xylene). The contamination of the surface and groundwater as well as of soil ecosystems can mainly occur in industrial areas. Such a pollution can pose risks for biota of these ecosystems but also for human health. Chemical methods used for the detection of xylene in natural environments include gas chromatography and mass spectrometry. However, these methods are time-consuming, complicated and expensive, and do not provide any information about the influence of xylene on microorganisms. The aim of this study was to monitor the toxic effect of different concentrations of xylene on unicellular flagellated protist Euglena gracilis, strain Z (EGZ) in particular on its cell division, morphology and chlorophyll content. The results showed that the sensitivity of EGZ to xylene is very high. This organism responds rapidly to the exposure of xylene due to its short generation time. Lower concentrations (< 0.9 M) of xylene induce characteristic morphologic1al changes – spherical shape of cells, hypertrophied cells or formation of "gigantic cells" and special type of cell division which could be multiple rounds of mitosis without completing cytokinesis or even yet undescribed form of meiosis. Abnormal cell morphology as well as intracellular formation of lipofuscin and visible decrease of chlorophyll content was seen in cultures exposed to higher concentrations of xylene (> 0.9 M), although such high concentrations were lethal for most cells. Our results suggest that E. gracilis can be used as a suitable model organism for monitoring of the harmful influence of xylene or other aromatic hydrocarbons on the biota of natural ecosystems.

ABSTRACTS

Missing genomes, unusual structures and other oddities: insights into the evolution of plastid genomes of Euglenophyta

Kacper Maciszewski1, Nadja Dabbagh2, Aleksandra Bokus1, Matthew S. Bennett3, Richard E. Triemer3, Angelika Preisfeld2, Anna Karnkowska1

1. Department of Molecular Phylogenetics and Evolution, Faculty of Biology, Biological and Chemical Research Centre, University of Warsaw , Poland 2. Zoolog y and Didactics of Biology, Faculty of Mathematics and Natural Sciences, University of Wuppertal , Germany 3. Department of Plant Biology, Michigan State University , USA

Euglenids are unicellular marine and freshwater algae, which acquired plastids by secondary endosymbiosis with a chlorophytean alga. Despite the common origin of their plastids, a vast variability of structures can be observed in the plastid genomes, indicating their highly dynamic evolution. The quadripartite structure of plastid genomes, with two inverted repeats comprising rDNA, has been lost in euglenids at least three times independently; moreover, some species possess incomplete additional copies of the ribosomal gene cluster. The “typical” ptDNA structure is quite rare in this lineage and it remains debatable whether it was the ancestral one. To expand the current state of knowledge on the evolution of euglenid plastid genomes, we obtained three new plastid genomes of Eutreptiales, and observed more unusual ptDNA structures, such as a gene-deprived small single- copy region.

Several times euglenids secondarily lost their ability of photosynthesis. It has been shown for one of the bleached species, E. longa, that it kept reduced plastid genome, with the RuBisCo large subunit gene retained as the sole remnant of the plastid-encoded part of the photosynthetic apparatus. For several other secondarily non-photosynthetic euglenid species, the evolutionary processes affecting their plastids remain unknown. To understand the reductive evolution of plastids we investigate E. gracilis var. bacillaris W3BUL – a bleached mutant that permanently lost its photosynthetic capabilities as a result of ultraviolet irradiation. The analysis has shown contradictory results, as no trace of the plastid genome was found, but the nuclear-encoded, plastid-targeted genes were abundant. We expect to reach the conclusion on the fate of the W3BUL strain‟s plastid once its transcriptome is acquired. Furthermore, we plan to confront the aforementioned results with analyses of the non-photosynthetic E. quartana, from which the preliminary results also suggest lack of the plastid genome.

ABSTRACTS

Proteins interacting with oxygen and its reactive species in Naegleria gruberi

Ronald Malych1, Jan Mach1, Ivan Hrdý1, Róbert Ńuťák1

1. Department of Parasitology, Faculty of Science, Charles University, Prague , Czech Republic

Naegleria gruberi is a free-living nonpathogenic amoeba. It is a close relative to Naegleria fowleri, a human pathogen that causes primary amoebic meningoencephalitis (PAM). As a free-living organism, Naegleria is adapted to aerobic respiration but also has traits of anaerobic metabolism as it contains [FeFe]- hydrogenase, capable of hydrogen production. Iron metabolism is a strictly regulated process, essential for basic life functions. Excessive accumulation of this metal may result in the generation of toxic hydroxyl radicals that cause oxidative damage to the cell.

We found 3 different types of oxygen-binding, iron-containing, proteins in the genome of N. gruberi - hemerythrin, protoglobin, and rubrerythrin. Recent studies show that hemerythrin oxygen-binding domain, with its typical diiron center, is spread throughout almost all domains of life and its function vary from protection against oxidative stress in anaerobes to oxygen carrier in aerobic organisms. Protoglobin is haem containing protein that was first described in methanogenic archaea. Although it is capable of binding O2, CO and NO reversibly in vitro, its biological role is unknown. Rubrerythrins are involved in oxidative stress tolerance as peroxide scavengers. They consist of di-iron center, responsible for the reduction of H2O2 and C-terminal domain related to the rubredoxin family, responsible for electron transfer during catalysis. We have isolated and purified these proteins, produced antibodies against them and localized them in N. gruberi. In vitro characterization of these recombinant proteins included mainly fast protein liquid chromatography and spectrophotometry.

Ability to bind oxygen was shown by spectral changes of recombinant hemerythrin that was purified under anaerobic conditions and subsequently oxidized. Controlling mechanisms of oxidative stress levels are crucial for the survival of cells. These proteins could potentially play a role as a protection against oxidative stress.

ABSTRACTS

Characterization of the proteome and dynamics of encystation- specific vesicles of Giardia intestinalis

Lenka Marková1, Martina Vinopalová1, Vladimíra Najdrová1, Pavel Doleņal1

1. Department of Parasitology, Faculty of Science, Charles University, BIOCEV, Czech Republic

The process of encystation is an important part of life cycle of many pathogenic organisms. Giardia intestinalis is an intestinal parasite of various vertebrates including humans from the supergroup of Excavata. The parasite leaves the body of the host in the form of cysts, which are infectious to other hosts. The encystation is triggered by increased pH and higher concentration of bile in the lower parts of the gastrointestinal tract. During the encystation process specific endomembrane organelles are formed, called the encystation-specific vesicles (ESVs) that serve for the accumulation of material for the future cyst wall (CWM). This material is consecutively transported from the lumen of the ESVs to the surface of the cell. CWM is composed of a fibrillar matrix, containing three paralogous cyst wall proteins (CWP 1-3) and a Giardia-specific β-1,3-GalNAc homopolymer. Because all three CWPs are rich in leucine and cysteine, it´s assumed that these aminoacids are involved in protein-protein interactions between the CWPs and other probable proteins.

ESVs are also the sites of processing of CWM proteins, including several posttranslational modifications. During the maturation of ESVs, a condensed core is formed with a fluid phase at the edge, where CWP1 and the N-terminal fragment of CWP2 are present. This fragment was cleaved from pro-CWP2 when the core condensated. Compared to the fluid phase, the core of ESVs contains the C-terminal part of CWP2 and the entire CWP3.

In this project, we strive to characterize the progressive conversion from the metabolically active cell to the resting stage of the cyst. To this aim we employ two methods, which enable high affinity purification of CWP1 and its interaction partners in the ESVs: (i) we have established in vivo biotinylation of CWP1, which is then purified upon chemical crosslinking to its interaction partners (ii) we are introducing the proximity labelling biotinylation using promiscuous mutant of biotin ligase – TurboID fused to known ESV proteins. In the combination with the live-cell microscopy we would like to improve our knowledge on the overall transformation of the parasite into the infectious cyst.

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How does ISC system work in the mitosomes of Giardia intestinalis?

Alņběta Motyčková1, Courtney Stairs2, Vladimíra Najdrová1, Vendula Horáčková1, Pavel Doleņal1

1. Department of Parasitology, Faculty of Science, Charles University, BIOCEV, Czech Republic 2. Department of Cell and Molecular Biology, Uppsala University, Sweden

Mitosomes of Giardia intestinalis are one of the most reduced mitochondria found to date. They do not have any DNA and their proteome is extensively reduced - just a few tens of proteins are identified as mitosomal. The only known metabolic pathway is the synthesis of iron-sulphur clusters (ISC pathway).

The ISC pathway consists of two phases. During the early ISC pathway the [2Fe-2S] cluster is formed by IscS, Fdx, IscU and Grx5. The late ISC pathway serves for the formation of [4Fe-4S] cluster and its transfer to target apoproteins. IscA, Nfu1 and BolA are among the proteins employed in the late phase. However, the actual role of the mitosomal ISC pathway and the exchange of metabolites between the mitosome and the cytosol remain unknown.

In this project, we tackle these unknowns by two biochemical approaches: (i) we attempt to establish affinity purification of the whole mitosomes from the cell lysate. The technique is based on specific biotinylation of the outer mitosomal membrane proteins. We have already chosen the most suitable protein candidate and now we are testing its inducible expression to minimize the experimental artifacts. (ii) we also isolate individual component of ISC pathway (Grx5, Nfu1) to co-purify missing functional components. We have identified G. intestinalis BolA homologue, which is normally present in ISC pathway of only aerobic organisms. GiBolA is localized in the mitosomal matrix and interacts with several other ISC components.

Interestingly, despite the presence of three mitosomal components of the late ISC pathway (BolA, IscA and Nfu1) no mitosomal [4Fe-4S] cluster-containing substrate has been identified yet. Our aim is to identify such substrate or to demonstrate that [4Fe-4S] cluster is directly transported across two mitosomal membranes to the cytosolic apoprotein.

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The guided entry of tail-anchored proteins pathway in Giardia intestinalis

Vladimíra Najdrová1, Luboń Voleman2, Un Seng Chio2, Shyam Saladi2, Lukáń V.F. Novák1, Bil Clemons2, Shu-ou Shan2, Pavel Doleņal1

1. Department of Parasitology, Faculty of Science, Charles University, BIOCEV , Czech Republic 2. Division of Chemistry and Chemical Engineering, California Institute of Technology , Pasadena, United States

The special class of membrane proteins, so called tail-anchored (TA) proteins, carry a single C-terminal transmembrane domain that anchors them to organelle membranes. TA proteins mediate interactions among membrane bounded compartments by their N-terminal domains during various processes such as vesicular transport, regulation of apoptosis or protein translocation.

In some eukaryotes, the specific pathway controls precise post-translational insertion of TA proteins into the endoplasmic reticulum membrane – Guided Entry of Tail- anchored proteins (GET) pathway.

Our bioinformatics analyses revealed the absence of most of the GET proteins in majority of the eukaryotic lineages except opisthokonts. However, one of the components of GET pathway (Get3) is conserved in all eukaryotic groups, excavates included. Get3 serves as ATP-dependent shuttle between cytosolic pre-targeting (Sgt2, Get4 and Get5) and ER-membrane targeting complex (Get1 and Get2). We have shown that Get3 of excavate protist Giardia intestinalis (giGet3) is a cytoplasmic protein with affinity to the endoplasmic reticulum. Using chemical crosslinking followed by affinity purification of biotinylated giGet3, the specific set of interacting proteins have been identified, including another member of GET pathway – homolog of transmembrane receptor Get2. Subsequently, we identified almost all components of the pathway in giardia by set of bioinformatics searches. Based on GRAVY and AGADIR scores we in silico determined potential giGET pathway substrates from predicted giardia TA proteins. Functional binding of TA proteins by giGet3 and giSgt2 was confirmed by in vitro binding assay. In addition to giardia- specific information, our general aim is to define the evolution of GET pathway in eukaryotes.

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Comparative genomics of Preaxostyla flagellates: insights into the evolution of amitochondriality

Lukáń V. F. Novák1, Sebastian C. Treitli1, Ondřej, Brzoň1, Laura, Eme2, Anna Karnkowski3, Shweta Pipaliya4, Petr Soukal1, Vojtěch Vacek1, Joel B. Dacks4, Marek Eliáń5, Vladimir Hampl1

1. Department of Parasitology, Faculty of Science, Charles University, BIOCEV, Czech Republic 2. Diversity, Ecology and Evolution of Microbes group (Ecology, systematics and evolution Unit), Université Paris -Sud, France 3. Department of Molecular Phylogenetics and Evolution, University of Warsa w, W arsaw, Poland 4. Division of Infectious Disease, Department of Medicine, Faculty of Medicine and Dentistry, University of Alberta , Canada 5. Faculty of Science, University of Ostrava , Ostrava, Czech Republic

The first known eukaryotic organism completely devoid of the mitochondrial compartment, Monocercomonoides exilis, belongs to Preaxostyla, a metamonad group consisting of 1) paraphyletic free-living trimastigids with excavate morphology and mitochondria-like organelles resembling hydrogenosomes, and 2) a monophyletic group of morphologically divergent, endobiotic, putatively amitochondrial oxymonads inhabiting guts of cockroaches, termites and some other metazoans. We hope to gain better insight into the radical evolutionary changes this group of protists went through (from free-living to endobiotic, from reduced mitochondria to no mitochondria) by comparative analysis and annotation of genomes and transcriptomes of selected representatives. In addition to the already available transcriptomic and genomic sequences of Trimastix marina and Monocercomonoides exilis, we have sequenced and assembled genomic sequences of Paratrimastix pyriformis, Blattamonas nauphoetae, and Streblomastix strix. Annotations of multiple cellular systems (including mitochondrion biogenesis and metabolism, amino acid metabolism, metabolite transporters, autophagy, and endomembrane transport) in the 5 organisms, as well as lateral gene transfer to Preaxostyla, is discussed in the context of endobiosis and amitochondriality evolution.

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Novel proteins and reorganized protein import in the secondary plastid of Euglena gracilis

Anna M. G. Novák Vanclová1, Martin Zoltner2, Kristína Záhonová1,3, Zoltán Füssy1, Marek Eliáń3, John M. Archibald4, Mark C. Field2, Vladimír Hampl1

1. Department of Parasitology, Faculty of Science, Charles University, BIOCEV, Czech Republic 2. School of Life Sciences, University of Dundee, Dundee, UK 3. Faculty of Science, University of Ostrava, Ostrava, Czech Republic 4. Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, Canada

Euglena gracilis is a well-studied and biotechnologically exploitable phototrophic flagellate harbouring secondary green plastids surrounded by three membranes. Several transcriptomic datasets of this organism were generated to this date, the most recent one coupled with a draft assembly of its ~0.5 Gbp genome and high-performance liquid chromatography/mass spectrometry-based proteomes of its organellar fractions. The thorough analysis of the plastid proteome revealed that the machinery for protein import across the envelope membranes has undergone major reorganization. TOC complex is either completely missing or diverged beyond recognition and TIC complex is highly reduced, possibly to the degree of constituting of only one conserved subunit (Tic21) in multiple isoforms. Strangely enough, the transit peptides of the plastid-targeted pre-proteins retain their characteristics shared with plant and other algal transit peptides (and are recognizable in a heterologous system by plant TOC), but also exhibits certain unique features which were previously overlooked. Meanwhile, the mechanism for protein import across the additional third membrane, as well as the middle one devoid of TOC, must employ novel or re-purposed translocases. We identified plastid-localized paralogs of ER- associated proteins: cpGOSR1 and cpRab5 which likely mediate the transport vesicle fusion, and two derlin-like proteins which might represent a part of a system analogous to the symbiont-specific ERAD-like machinery (SELMA) known from secondary plastids of various “chromalveolates”. Additionally, an unusual homolog of the alpha subunit of F-type ATP synthase was noticed in the plastid proteome and its phylogeny and distribution pattern in eukaryotes suggests its significance and association with plastids, and secondary plastids in particular. New insights and results regarding the evolutionary history and function of these novel plastidial proteins will be presented on the conference.

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Dynamin-related GTPases involved in cristae remodeling and their evolution

Tomáń Pánek1, Marek Eliáń1, Marie Vancová2, Julius Lukeń2, Hassan Hashimi2

1. Department of Biology and Ecology, Faculty of Science, University of Ostrava, Ostrava, Czech Republic 2. Institute of Parasitology, Biology Center, Czech Academy of Sciences, and Faculty of Sciences, University of South Bohemia, Budweis, Czech Republic

To shed light on the relationship between Mgm1 and Opa1, two dynamin-related GTPases involved in mitochondrial fusion and cristae remodeling, we carried out bioinformatic and phylogenetic analyses benefiting from the expansion of sequence data from diverse protist lineages. Our results suggest that these two proteins form two different clades of the dynamin family not directly related to each other. Moreover, C-terminal region of Opa1 has diverged considerably from Mgm1 and other dynamin-related proteins. Opa1 is not restricted to Holozoa as originally supposed, since we found it also in representatives of the supergroup CRuMs. The occurrence of Opa1 outside opisthokonts suggests that it evolved quite early in the eukaryote phylogeny and was lost independently in several lineages. On the other hand, Mgm1 remains restricted to Fungi and their closest relatives. The theoretical possibility that Mmg1 in the Holomycota is a transmogrified version of Opa1 is unlikely, as this would necessitate a reversion of its atypical C-terminal region to the more conventional form seen in Mgm1. When looking for Mgm1 and Opa1 homologs, we encountered a previously unnoticed subgroup of the dynamin family that exhibits a very peculiar distribution as it is present in one apusomonad, a few stramenopiles and, surprisingly, several giant viruses. Overall domain architecture of MidX is similar to that of Mgm1, except for the apparent absence of a transmembrane domain, which raises the possibility that MidX does not remain anchored in the IM and may be imported into the matrix. We speculate that MidX assemble into oligomers, as is typical for dynamin-related proteins, to model mitochondrial membranes. Cristae are an obvious target of the protein, as viral infections of various hosts are known to be associated with changes to cristae morphology. Perhaps manipulation of cristae may be an elusive part of the viral reproductive strategy within the host cell.

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Genome sequencing of the putatively free-living diplomonad Trimitus sp. IT1

Agnieszka Pełesz1, Monika Wiśniewska1, Petr Táborský1,2, Eric Salomaki1, Ivan Čepička2, Martin Kolísko1

1. Biology Centre, Institute of Parasitology, Czech Academy of Sciences, Budweis, Czech Republic 2. Department of Zoology, Faculty of Science, Charles University, Prague, Czech Republic

The order Diplomonadida is a group of anaerobic, flagellated, microbial eukaryotes. The most well-known representatives of this group are the infamous human parasite Giardia intestinalis that causes diarrheal disease, and a fish parasite called S. salmonicida that causes systemic infection in salmonids. There are also several free-living species within diplomonads, for example Trepomonas and Hexamita, that inhabit marine and fresh-water anaerobic sediments. However, to date, the only diplomonads studied on a genomic level are the pathogens – Giardia intestinalis and a few species of Spironucleus. This sampling bias means that we are missing interesting information regarding the genome evolution of the parasitic and free-living species within diplomonads, especially given that some of the free-living diplomonads appear to be secondarily free-living based on current phylogenies. We have sequenced the genome of putatively secondarily free-living Trimitus sp. IT1 in order to enrich our sampling of diplomonad genomes and increase our understanding of the evolution of this diverse group. We used a combination of sequencing techniques to improve assembly quality: deep sequencing of short reads generated by Illumina HiSeq X-ten and low-coverage long-reads generated by Oxford Nanopore MinION. As Trimitus sp. IT1 is co-cultured with several bacterial species, we have also obtained more or less complete genomes from the bacterial community. This will allow us to attempt to characterize the ecological relationships between the microbial eukaryote and the bacterial community.

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Nosema bombycis (Microsporidia), a model for the biological nanotube

Markéta Petrů1, Alexandra Satrapová1, Veronika Novotná1, Pavel Doleņal1

1. Department of Parasitology, Faculty of Science, Char les University, BIOCEV, Czech Republic

Microsporidia are a group of obligate intracellular parasites closely related to fungi. They are able to infect broad spectrum of animal hosts including human. Microsporidia create two different life forms – the reduced vegetative form multiplying in the host and the resistant spore determined for survival in the environment and for infection. Highly adapted invasion apparatus of the spore consists of structures unparalleled in the nature: the anchoring disc, polar tube, polaroplast and posterior vacuole. The polar tube is a hollow tube, an example of a biological nanotube, through which the sporoplasm is transported to the host cell. Neither forming mechanism nor the exact molecular composition of the tube has been elucidated yet. We have established life cycle of Nosema bombycis in our laboratory in order to investigate the polar tube of this organism. N. bombycis is a causative agent of pébrine, disease of the silkworms, which devastates silk industry. Our goal is to analyse the composition and the structure of the tube by combining molecular, biochemical and optical approaches.

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Unveiling mitochondrion-related organelles in caviomonads

Romana Petrņelková1, Pavla Hanousková2, David Ņihala1, Ivan Čepička2, Marek Eliáń1

1. Department of Biology and Ecology, Faculty of Science, University of Ostrava, Ostrava, Czech Republic 2. Department of Zoology, Faculty of Science, Charles University, Prague, Czech Republic

Fornicata (Metamonada), comprising Diplomonadida, retortamonads, “Carpedie- monas-like” organisms, and Caviomonadidae is a protist group adapted to low- oxygen environments. All fornicates studied to date lack classical mitochondria and instead possess mitochondrion-related organelles (MROs); hydrogenosomes (e.g. Spironucleus salmonicida), mitosomes (e.g. Giardia intestinalis), or a form in- between the two categories (Dysnectes brevis). However, essentially nothing is known about mitochondria in the least known fornicate group, the caviomonads. To fill in this gap, we are investigating the transcriptome assembly previously generated in our labs for the endobiotic caviomonad Iotanema spirale, in which no candidate MRO could be discerned by electron microscopy. Our search for homologs of proteins commonly associated with mitochondria/MROs so far retrieved very few candidates, but significantly they include a key subunit of the mitochondrial TOM complex and the central components of the characteristic mitochondrial Fe-S cluster assembly pathway. These data suggest that I. spirale possesses a cryptic, highly reduced MRO, perhaps similar to the mitosome of G. intestinalis. For comparison, we sequenced and assembled the transcriptome of the as-yet formally undescribed marine caviomonad strain PCS. Interestingly, this organism does not share the unique modification of the genetic code reported previously for I. spirale (i.e. the codon UAG decoded as glutamine rather than signifying translation termination). Moreover, the PCS transcriptome yielded a broader list of homologs of MRO- resident proteins, including enzymes of the redox and ATP-generating metabolism characteristic for a hydrogenosome. In accord with this, our ultrastructural investigation revealed the presence in PCS cells of structures reminiscent of hydrogenosomes in other metamonads. MROs in caviomonads thus seem to be diverse and reflect an independent trajectory of reductive evolution.

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The purine microcrystals – particularly guanine and uric acid – are commonly present in all main groups of protists serving as the N-rich depots

Jana Pilátová1, Peter Mojzeń2,3, Lu Gao3, Tatiana Ismagulova4, Ńárka Moudříková2,3, Anya Salih5, Alexei Solovchenko3,4, Ladislav Nedbal3

1. Department of Experimental Plant Biology, Faculty of Science, Charles University, Prague, Czech Republic 2. Institute of Physics, Faculty of Mathematics and Physics, Charles University, Prague, Czech Republic 3. Institute of Bio - and Geosciences/Plant Sciences (IBG -2), Germany 4. Faculty of Biology, Moscow State University, Moscow, Russia 5. Confocal Bioimaging Facility, Western Sydney Universit y, Australia

Nitrogen (N) as an essential element is a key modulator of phytoplankton photosynthetic activity that is responsible for a large share of global carbon cycle. In microalgae N depletion leads to a rapid change in the cell metabolism, such as accumulation of carbon reserves, a phenomenon which is vastly used for lipid production in algal biotechnologies. It is widely accepted that the rapid onset of N deficiency symptoms is caused by small capacity of N-reserves in algal cells that can be only partially buffered by catabolism of proteins that are not essential for survival.

Herein, we identify purine crystal inclusions, particularly guanine and uric acid, functioning as an alternative N-rich depot and at the same time we propose other potential functions that may be complementary and simultaneously divergent in various species.

Confocal Raman microscopy combined with 15N labeling and transmission electron microscopy revealed dynamics of formation and catabolism of purine crystals in algal cells over periods of starvation and rich supply. The purine crystals were formed preferentially during lag phase upon re-feeding by various N-sources when the cells were not dividing and then disintegrated in rapid growth periods mainly upon N starvation as we show in Amphidinium carterae.

In addition, we demonstrate the general presence of purine crystals in various protist species both photosynthetic and nonphotosynthetic coming from various types of habitats ranging throughout the diversity of eukaryotes including all the major supergroups: Archaeplastida, Stramenopiles, Alveolata, Rhizaria, Amoebozoa, Excavata. The common presence of purine crystals in microalgae may indicate their early evolutionary emergence which may be either mutual for all of them or came into existence independently.

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Endo/exo/phosphatase in Leishmania mexicana

Ester Poláková1, Sara Zimmer2, Vyacheslav Yurchenko1

1. Life Science Research Centre, University of Ostrava, Ostrava, Czech Republic 2. University of Minnesota, Department of Biomedical Sciences, Duluth, USA

EEP is an endo/exo/phosphatase domain containing protein, which has been suggested to act as a ribonuclease in Trypanosoma brucei. This protein is developmentally regulated (reduced expression in bloodstream form) and can potentially play an important role in mRNA turnover during differentiation process.

We study this protein in another model organism from the family Trypanosomatidae – Leishmania mexicana. Parasites with exogenously tagged EEP were analyzed for the kinetics of cell division, stability of the expressed protein, and its localization. To determine its biological function, we are trying to ablate the EEP-encoding gene. To this end, we have obtained a Leishmania population with EEP knock-down. The ability of these cells to differentiate and divide are being examined in comparison with wild type L. mexicana cells.

Acknowledgement:

This work was supported by ERD Founds, project OPVVV CZ.02.1.01/0.0/0.0/16- 019/0000759 (Centrum výzkumu patogenity a virulence parazitů).

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Anaerobic scuticociliates: A diverse ciliate lineage hosting symbiotic prokaryotes

Kateřina Poláková1, Johana Rotterová1, William Bourland2, Ivan Čepička1

1. Department of Zoology, Faculty of Science, Charles University, Prague, Czech Republic 2. Department of Biological Sciences, Boise State University, Boise, Idaho, USA

Anaerobic ciliated protists, eukaryotes that thrive under anoxic conditions, represent a significant part of the microbial diversity in both freshwater and marine anoxic habitats. In fact, anaerobic lifestyle is a widespread phenomenon among ciliates, some of the ciliate classes being solely anaerobic. Ciliates from the subclass Scuticociliatia (class Oligohymenophorea) are common ciliates found in both soil and aquatic environments with hundreds of aerobic species described, yet only a few scuticociliate species have been reported from anoxic habitats. However, we have discovered that anaerobic scuticociliates are common in both freshwater and marine anoxic sediments. We are successfully maintaining over 60 strains of mostly marine representatives of this group in long-term cultures, allowing us the privilege to study this diverse anaerobic lineage in detail. We have performed a phylogenetic study using 18S rRNA gene sequences of more than 40 representatives and have confirmed that anaerobic scuticociliates constitute a novel and diverse lineage. We studied their morphology using light microscopy and various staining methods, including protargol, silver carbonate, and silver nitrate, and employed both scanning and transmission microscopy methods.

A common feature of most of the marine anaerobic scuticociliates is to harbor ectosymbiotic prokaryotes attached to the cell surface. Interestingly, it seems that the presence/absence of the ectosymbionts is dependent on the ciliate lineage. According to our preliminary results from CARD–FISH method, the ectosymbionts are sulfate-reducing bacteria. Moreover, some anaerobic scuticociliates have methanogenic Archaea as endosymbionts in their cytoplasm. Although symbioses are widespread in anoxic world, we still do not know the identity of most of the symbionts and lack information about the metabolic nature of the host-symbiont interactions, particularly those involving ectosymbionts. Studying these symbioses can also be important for understanding the ecology of anoxic environments since some of the symbionts identified are ecologically important prokaryotes.

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Mitochondria of some protist lineages have retained the bacterial signal peptide recognition particle-based protein targeting machinery

Jan Pyrih1,2, Tomáń Pánek3, Kristýna Cimrhanzlová1, Vendula Rańková1, Eva Kriegová1, Anastasios Tsaousis4, Marek Eliáń3, Julius Lukeń1

1. Institute of Parasitology, Biology Centre, Czech Academy of Sciences, Budweis, Czech Republic 2. Department of Parasitology, Faculty of Science, Charles University, BIOCEV , Czech Republic 3. Life Science Research Centre, Faculty of Science, University of Ostrava, Ostrava, Czech Republic 4.School of Biosciences, University of Kent, Canterbury,UK

The bacterial signal peptide-based targeting machinery is comprised of signal recognition particle-docking protein FtsY, signal peptide recognition particle protein Ffh, and RNA component. It targets proteins into, or across, the inner bacterial membrane via SecYEG and/or YidC protein complexes. Although mitochondria are of bacterial origin, up till now no such targeting system was identified in this organelle. Surprisingly, we identified homologs of both bacterial Ffh and FtsY in the four protist taxa representing distantly related deeply diverged lineages: Heterolobosea, Alveida, Hemimastigophora, and the genus Goniomonas in Cryptista. Most of the retrieved are strongly predicted as mitochondrion-targeted and the nucleus-encoded Ffh and FtsY from the heterolobosean Naegleria gruberi show mitochondrial localization when expressed in a heterologous system. Phylogenetic analysis confirmed that the monophyly of the mitochondrial Ffh and FtsY proteins among their respective bacterial homologs, and at least in case of Ffh α- proteobacterial origin can be inferred with confidence. It indicates common origin of this pathway (named mtSRP) in all four lineages and its presence in the last eukaryotic common ancestor (LECA). Furthermore, we have analysed the N-termini of several proteins encoded by the N. gruberi mitochondrial DNA in search for possible substrates of mtSRP. We found out that only the hydrophobic ones carry a signal peptide resembling sequence. These signal peptides targeted a reporter mNeonGreen fluorescent protein into the endoplasmic reticulum of Trypanosoma brucei, and hence were likely recognized by the cytosolic (i.e. eukaryotic) signal recognition particle. In summary, we have identified an ancestral mitochondrial protein targeting system that further enriches the spectrum of original bacterial traits that have survived in modern mitochondria.

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Identification and characterization of Trypanosoma brucei mitochondrial LECA related genes

Vendula Rańková1,2, Ignacio Durante1, Jeremy Wideman3, Julius Lukeń1,2

1. Biology Centre, Institute of Parasitology, Czech Academy of Sciences, Budweis, Czech Republic 2. Faculty of Science, University of South Bohemia, Budweis, Czech Republic 3. Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada

Pioneering work by Woese and Fox grouped all cellular life into three evolutionary well-defined domains: Eubacteria, Archaea, and Eukarya. Subsequent phylogenetic analysis indicated that Eukarya and Archaea are sister groups, and that eukaryotic cells possess a mixture of archaeal and bacterial, in addition to eukaryotic-specific genes. Studying the last eukaryotic common ancestor (LECA) is key in eukaryogenesis. Extensive phylogenetic analysis identified 10 Trypanosoma brucei LECA-related putatively mitochondrial proteins (LMPs). T. brucei is a parasitic protist that serves as an important model organism not only due to the possession of unique features such as kinetoplast DNA, trans-splicing and RNA editing, but also for the availability of a wide range of tools for forward and reverse genetics. We show here that the identified LMPs exhibit structural similarity with yet unidentified mitochondrial carriers, Oxa-1 and SAM35/37. Using the fidelity of pPOT-derived endogenous tagging system, we showed expression of most of LMPs in the T. brucei procyclic stage. We also confirmed their mitochondrial localization both by immunofluorescence and western blot analysis. We are now in the process of characterizing RNAi stable transfected cell lines in order to establish the functional roles of these mitochondrial proteins in T. brucei.

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ZapE/Afg1 is Oxa1-dependent regulator of respiratory complexes in Trypanosoma brucei

Vendula Rańková1,2, Jan Pyrih1, Ingrid Ńkodová-Sveráková3, Julius Lukeń1,2

1. Biology Centre, Institute of Parasitology, Czech Academy of Sciences, Budweis, Czech Republic 2. Faculty of Science, University of South Bohemia, Budweis, Czech Republic 3. Faculty of Sciences, Comenius University, Bratislava, Slovakia

ZapE/Afg1 protein was identified either in the inner membrane of bacteria or alternatively in the inner mitochondrial membrane. According to various studies, this protein is involved in FtsZ-dependent division of bacterial cells and facilitates the degradation of mitochondrially-encoded subunits of respiratory complexes III and IV. Moreover, loss of this protein leads to increased apoptotic resistance, decreased oxidative stress tolerance, and impacts mitochondrial protein homeostasis. Herefore, ZapE is either a central multifunctional protein, or some of its described functions could possibly be explained as a secondary consequence of respiratory chain disruption. Trypanosoma brucei is an important model organism, especially thanks to unique features, such as kinetoplast DNA, trans-splicing and RNA editing. We analysed the functions of ZapE in T. brucei and confirmed its mitochondrial localization. Using a newly developed BioID2 proximity-dependent biotinylation approach, we identified Oxa1 as its direct interacting partner. Furthermore, RNAi- based depletion of both ZapE homologs affected the function of respiratory complexes I and IV.

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Coevolution of genomes, organelles and endosymbionts in anaerobic protists

Andrew J. Roger1

1. Centre for Comparative Genomics and Evolutionary Bioinformatics, Department of Biochemistry and Molecular Biology, Dalhousie University , Halifax, NS, Canada

Anaerobic protists are abundant in diverse low-oxygen environments ranging from marine sediments to animal guts. Although the parasitic anaerobic protists collectively infect >2 billion people worldwide, comparatively little is known about the diversity in anaerobic protists with respect to cellular structure and function, their genomes and interactions with other microbes. With the development of inexpensive long-read sequencing technologies, we can now characterize the genomes of diverse anaerobic protists to understand the mechanisms by which they have evolved to inhabit low oxygen, become parasites and influence the prokaryotic microbiota around them. Unexpectedly, we found that symbiotic prokaryotes have an important role in influencing the physiology and evolutionary trajectories of anaerobic protists. I will discuss these findings and their implications for understanding the evolutionary history of eukaryote cells and organelles.

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Where oxygen is not popular – anaerobiosis in SAL supergroup (Ciliophora)

Johana Rotterová1, William Bourland2, Virginia P. Edgcomb3, Roxanne Beinhart4, Martin Kolísko5, Petr Táborský5, Eric Salomaki5, David Ņíhala6, Ivan Čepička1

1. Department of Zoology, Faculty of Science, Charles University, Prague, Czech Republic 2. Department of Biological Sciences, Boise State University, Boise, Idaho, USA 3. Department of Geology and Geophysics, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts, USA 4. Graduate School of Oceanography, University of Rhode Island, Kingston, Rhode Island, USA 5. Institute of Parasitology, Biology Centre Czech Academy of Sciences, Budweis, Czech Republic 6. Department of Biology and Ecology, Faculty of Science, University of Ostrava, Ostrava, Czech Republic

Although ciliates are one of the most studied protists in general, much remains unknown about the phylogenetic relationships among the major lineages, as well as about their mitochondrial metabolism and the nature of their symbioses, albeit their known diversity and distribution are striking. As our study reveals, anaerobiosis is greatly common among ciliates and particularly within the SAL (Spirotrichea, Armophorea, Litostomatea, Odontostomatea, and other lineages) super-group. After the recent discovery of several novel lineages within SAL, we selected several representatives to sequence their metagenomes and transcriptomes in order to expand our knowledge of the evolution of ciliates, phylogenetic relationships within lineages of SAL, the mechanisms of adaptation to life in anoxia, including the mitochondrial metabolism and various symbioses. Analyses of metagenomic and transcriptomic data from metopids and two newly discovered anaerobic ciliate lineages revealed traces of the classical mitochondrial pathways including amino- acid metabolism, fatty acid metabolism, and Fe-S cluster biogenesis. However, partial mitochondrial genomes, containing genes of mitochondrial Complex I, were also detected among some. Representative of one of the newly discovered lineages shows remarkable metabolic capacity, as several genes for the structure and assembly of respiratory complexes II and III and a complete F1F0 ATP synthase (Complex V) were retained. Additionally, a putatively mitochondrial-targeted pyruvate:ferredoxin oxidoreductase (PFO), which is likely involved in the production of hydrogen for ATP-synthesis, was identified.

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Deep transcriptomic analysis of Chromera velia under Mercury- stress condition

Abdoallah Sharaf1, Roberto De Michele2, Ayush Sharma1, Miroslav Oborník1

1. Institute of Parasitology, Biology Centre, Czech Academy of Sciences, Budweis, Czech Republic 2. Institute of Biosciences and Bioresources (IBBR ), National Research Council (CNR) of Italy, Palermo, Ital y

The heavy metal pollution in ecosystems is of increasing global concern. The main sources of the metal toxicity are the industrial waste, mining and the use of pesticides containing heavy metals. Out of all heavy metals, mercury (Hg) is considered to be the one, easily accumulated in terrestrial plants as well as aquatic organism. Hg can induce more severe oxidative stress by triggering production of reactive oxygen species (ROS) and damage macro-molecules. ROS serve not only as dangerous molecules that damage proteins, lipids and DNA but also as signaling molecules in the regulation of biological processes such as biotic and abiotic stress responses, growth and development. This study shows the change in the Chromera velia transcriptome due to Hg stress. C. velia is the recently discovered closest known relative of apicomplexans. Our study shows the first deep transcriptomic analysis of C. velia, specifically focusing on the expression level of the genes involved in detoxification defense systems under heavy metal stress. The results show that in total 1239 genes are differentially expressed. Differentially expressed genes (DEGs) showed overall up-regulation (1,070 genes) and only 169 up- regulated genes in cultures under Mercury stress (Cvel_mer) when compared to the control level (Cvel_cont). The qPCR expression analysis exhibited the positive correlation with the findings of RNA-seq data.

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Role of MICOS in long slender bloodstream acristate stage Trypanosoma brucei

Shaghayegh Sheikh1,2, Jiří Heller2, Julius Lukeń1,2, Hassan Hashimi1,2

1. Biology Centre, Institute of Parasitology, Czech Academy of Sciences, Budweis, Czech Republic 2. Faculty of Science, University of South Bohemia, Budweis, Czech Republic

Mitochondria have two membranes: an outer and inner boundary membranes. The space between them is called the intermembrane space. The inner membrane surrounds the internal matrix compartment. It is highly folded to form cristae, which contains proteins of the respiratory chain. The mitochondrial contact site and cristae organization system (MICOS) is essential for biogenesis and maintenance of mitochondrial cristae junctions. MICOS had been well studied in Opisthokonts supergroup but we have recently characterized it in discoidal cristae bearing procyclic stage of Trypanosoma brucei. In contrast, bloodstream stages in the mammalian host do not rely on oxidative phosphorylation for ATP production. The mammalian stages have two forms: long slender (LS) and short stumpy (SS). LS is proliferative, while SS is arrested in G1/G0 and express Protein Associated with Differentiation 1 (PAD1). We have engineered the AnTat1.1 pleomorphic cell line for Dox-inducible, T7-RNA-polymerase-mediated expression in order to facilitate the study of MICOS in these bloodstream stages. We call this cell line AnSMOX. We can recapitulate in vitro differentiation from LS to SS to study how MICOS can influence the biogenesis of tubular cristae in SS. Furthermore, we would like to see what MICOS, a complex responsible for shaping cristae, is doing in acristate LS.

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Multidomain flavodiiron protein from Trichomonas vaginalis cytosol

Tamara Smutná1, Ivan Hrdý1

1. Department of Parasitology, Faculty of Science , Charles University, Prague , Czech Republic

Flavodiiron proteins (FDPs) represent large family of proteins widespread among anaerobic and some aerobic prokaryotes (Archaea, Bacteria, and cyanobacteria) and some anaerobic prostists harbouring modified mitochondria. All FDPs consist of two-domain minimal core, a metallo β-lactamase domain with diiron catalytic center and a flavodoxin domain, harbouring flavine mononucleotide (FMN). In addition to this minimal core, many other recently described FDP members exhibit additional C-terminal extension with up to several functional domains that participate in intramolecular electron transfer. All these proteins are able to reduce O2 or/and NO, which plays important role in the protection against oxidative and nitrosative stress.

T. vaginalis is a microaerophilic parasitic protist inhabiting mucosal surface of vagina. In their natural environment trichomonads are confronted with toxic NO molecules arising from host immune system responding to the pathogen. To survive in these conditions, trichomonads must be endowed with effective mechanisms to cope with NO. Although NO reduction and detoxification were previously documented in T. vaginalis cell lysate, the protein responsible for this activity has not been detected to date.

Here we describe multidomain FDP from T. vaginalis cytosol with NO reductase activity that potentially functions in the protection of the parasite against host defense.

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Targetable proteolytic events in Babesia

Daniel Sojka1, 2. Marie Jalovecká1, Luise Robbertse1, Dominika Reichensdorferová2, Anthony J. O´Donoghue3

1. Biology Centre, Institute of Parasitology, Czech Academy of Sciences, Budweis, Czech Republic 2. Faculty of Science, University of South Bohemia, Budweis, Czech Republic 3. Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, USA

Although Babesia represent an important worldwide veterinary threat and an emerging risk to humans, these parasites have been poorly studied as compared to Plasmodium, their malaria-causing relative. Since the proteasome of Plasmodium has been validated by our collaborators as a potential target for anti-malarial drug development our experimental investigated the effect of epoxyketone (carfilzomib, ONX-0914 and epoxomicin) and boronic acid (bortezomib and ixazomib) proteasome inhibitors on the growth and survival of Babesia. Testing the compounds against Babesia divergens ex vivo revealed suppressive effects on parasite growth with activity that was higher than the cytotoxic effects on a non-transformed mouse macrophage cell line. Furthermore, we showed that the most-effective compound, carfilzomib, significantly reduces parasite multiplication in a Babesia microti infected mouse model without noticeable adverse effects. Treatment with carfilzomib lead to an ex vivo and in vivo decrease in proteasome activity and accumulation of polyubiquitinated proteins compared to untreated control confirming the on-target effect. In addition, we identify four Babesia microti aspartyl peptidases (tagged as BmASP 2, 3, 5, 6) homologous to malarial plasmepsins, that have been recently demonstrated as crucial enzymes for parasite egress, invasion and intracellular survival. We analyze the relation to plasmepsins, perform qPCR based dynamic expression profiling over the B. microti lifecycle and propose functional analogies in Babesia. In conclusion, our results demonstrate that the Babesia proteasome and aspartyl proteases are valid targets for drug development and warrant the design of potent proteasome inhibitors for the treatment of babesiosis.

Acknowledgements: Czech Science Foundation (GA CR) No. 17-14631S

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Gene transfer accompanying the secondary endosymbiosis of euglenid plastid

Petr Soukal1, Ńtěpánka Hrdá1, Naoji Yubuki1, Gordon Lax2, Alastair Simpson2 Kristína Záhonová1, Marek Eliáń3, Rafal Milanovski4, Vladimír Hampl1

1. Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic 2. Dalhousie University, Department of Biology, Halifax, Canada 3. Faculty of Science, University of Ostrava, Ostrava, Czech Republic 4. University of Warsaw, Department of Molecular Phylogenetics and Evolution, Warsaw, Poland

Autotrophic euglenids (Euglenozoa: Euglenophyceae) form a monophyletic group with secondary chloroplasts derived from a green alga (probably closely related to Pyramimonadales), which was most probably acquired by a common ancestor of autotrophic euglenids (plastid-late hypothesis). However, the acquisition of the plastid earlier in the evolution of euglenids lineage (plastid-early hypothesis) cannot be ruled out. The process of organelle acquisition is accompanied by the transfer of genes from the endosymbiont to host nucleus (EGT), the presence of such genes in euglenids provides a footprint of past endosymbioses. To test the plastid-early hypothesis and to learn more about the contribution of EGT to euglenid genomes, we are analyzing transcriptomes of four autotrophic and five osmotrophic euglenids for the amount of EGT derived genes. Using an automatic pipeline, we performed BLAST searches against custom database followed by phylogenetic analyses (using RAxML on Metacentrum), which enable us to select transcripts of genes putatively related to algae (ARG). The contribution of green algal genes in autotrophic euglenids is higher than in primarily osmotrophic one slightly supporting the plastid- late hypothesis. Surprisingly we observed a high number of genes related to other algal groups (especially secondary red algae) in osmotrophic and autotrophic euglenids. We perform mapping of entries of ARG into the phylogeny of euglenids using orthoMCL. Astonishingly, we are not able to pinpoint a single endosymbiotic event in the history of euglenids.

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Hacking the electron transport chain to live without oxygen

Courtney Stairs1

1. Institute for Cell and Molecular Biology, Uppsala University, Sweden

Anaerobic eukaryotes contribute to the ecology of many of Earth‟s microbiomes including marine sediments, anoxic dead zones and the animal gut. Eukaryotic microbes that occupy such low-oxygen environments often have drastically different mitochondrial metabolisms compared to their aerobic relatives. How these different metabolic strategies have evolved in eukaryotes remains a hotly-debated topic of biology. A common theme among many of anaerobic protists is the loss of different components of the respiratory chain. Genomic and molecular studies of protist lineages across the tree of Eukaryotes has revealed patterns of how the respiratory chain can be remodelled over time. Here, I will discuss the diversity of respiratory chains found in protists that experience transient or permanent anoxia and how this fits in the larger context of eukaryotic evolution.

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Paramylon synthesis in Diplonema papillatum

Ingrid Sveráková1,2, Galina Prokopchuk2, Priscila Peña-Diaz1, Anton Horváth1, Julius Lukeń2

1. Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia 2. Institute of Parasitology, Biology Centre, and Faculty of Sciences, University of South Bohemia, Budweis, Czech Republic

Until recently, diplonemids had been considered an insignificant group of protists. However, current worldwide ecological studies revealed that diplonemids are one of the most genetically diverse, cosmopolitan and abundant marine eukaryotic groups, and hence belong to the key players of the oceanic ecosystem. Diplonema papillatum is the best-known representative, which possesses many common features with the sister groups of kinetoplastids and euglenids. We have found that under certain in vitro conditions, D. papillatum is capable of storing a saccharide polymer similar to paramylon, the main storage polysaccharide in Euglena gracilis. We have assessed the growth of D. papillatum under carbon-rich and carbon- depleted conditions using immunofluorescence microscopy. A specific antibody against beta 1,3 glucan detected its presence in vesicle-like bodies throughout the cell only under carbon-depleted conditions in vitro. Next, the polymer was isolated and submitted to acid hydrolysis and thin layer chromatography to assess its composition. So far, the dynamics of paramylon and its precursor synthesis have not been described in any other Euglenozoan apart from Euglena. The conditions under which D. papillatum accumulates paramylon differ from those in euglenids, as it appears to be synthesized once the carbon source in the medium reaches a critical threshold for cell survival. This phenomenon raises the question about the unusual adaptation of diplonemids to changes in growth conditions.

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Plastid genomes and proteins illuminate the evolution of eustigmatophyte algae and their bacterial endosymbionts

Tereza Ńevčíková1,Tatiana Yurchenko2, Karen P Fawle3, Raquel Amaral4, Hynek Strnad5, Lilia M A Santos4, Marvin W Fawley3,6, Marek Eliáń1,2

1. Department of Biology and Ecology , Faculty of Science, University of Ost rava, Ostrava, Czech Republic 2. Faculty of Science, Institute of Environmental Technologies, University of Ostrava, Ostrava, Czech Republic 3. Division of Sciences and Mathematics, University of the Ozarks, Clarksville, Arkansas 4. Coimbra Collection of A lgae (ACOI), Department of Life Sciences, University of Coimbra, Coimbra, Portugal 5. Laboratory of Genomics and Bioinformatics, Institute of Molecular Genetics of the CAS, v.v.i., Prague, Czech Republic 6. School of Mathematical and Natural Sciences, Univ ersity of Arkansas at Monticello, Monticello, Arkansas

Eustigmatophytes, a class of stramenopile algae (ochrophytes), include not only the extensively studied biotechnologically important genus Nannochloropsis but also a rapidly expanding diversity of lineages with much less well characterized biology. Recent discoveries have led to exciting additions to our knowledge about eustigmatophytes. Some proved to harbor bacterial endosymbionts representing a novel genus, Candidatus Phycorickettsia, and an operon of unclear function (ebo) obtained by horizontal gene transfer from the endosymbiont lineage was found in the plastid genomes of still other eustigmatophytes. To shed more light on the latter event, as well as to generally improve our understanding of the eustigmatophyte evolutionary history, we sequenced plastid genomes of seven phylogenetically diverse representatives (including new isolates representing undescribed taxa). A phylogenomic analysis of plastid genome-encoded proteins resolved the phylogenetic relationships among the main eustigmatophyte lineages and provided a framework for the interpretation of plastid gene gains and losses in the group. The ebo operon gain was inferred to have probably occurred within the order Eustigmatales, after the divergence of the two basalmost lineages (a newly discovered hitherto undescribed strain and the Pseudellipsoidion group). When looking for nuclear genes potentially compensating for plastid gene losses, we noticed a gene for a plastid-targeted acyl carrier protein that was apparently acquired by horizontal gene transfer from Phycorickettsia. The presence of this gene in all eustigmatophytes studied, including representatives of both principal clades (Eustigmatales and Goniochloridales), is a genetic footprint indicating that the eustigmatophyte–Phycorickettsia partnership started no later than in the last eustigmatophyte common ancestor.

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Evolution of bacterial endosymbionts in marine diplonemids

Emma E. George1, Filip Husník1, Daria Tashyreva2, Galina Prokopchuk2, Aleń Horák2, Waldan Kwong1, Julius Lukeń2, Patrick Keeling1

1. University of British Columbia, Faculty of Science, Department of Botany, Vancouver, Canada 2. Biology Centre, Institute of Parasitology, Czech Academy of Sciences, Budweis, Czech Republic

Protists host a large diversity of bacterial endosymbionts that are in various stages of integration and evolutionary trajectories. Recently, we documented the first case of endosymbiosis in diplonemids, which are the most diverse and abundant marine planktonic eukaryotes. Three newly described diplonemid species were shown to harbor cytoplasmic or intranuclear bacterial symbionts. The 16S rRNA gene and multigene phylogenies showed that these alphaproteobacterial endosymbionts belonged to Holosporales and Rickettsiales, for which we established three new genera. To determine the functional role of the bacterial symbiosis and gain insight into their genome evolution, we sequenced, assembled and annotated genomes from four different diplonemid symbiont species. Their genomes undergone major reduction resulting in remarkably similar gene content and metabolic potential. A large portion of the genomes were dedicated to metabolite transporters including ATP/ADP translocases, and protein secretion by modified Type II, Flagellar Type III and Type VI Secretion Systems. While metabolic pathways involved in cell wall and membrane synthesis were preserved, the energy metabolism such as glycolysis, TCA and pentose phosphate pathways were completely missing or severely reduced. The bacteria have the smallest genomes (616 - 632 kbp) reported from protist symbionts, with the most reduced genome containing only 505 protein-coding genes. The massive loss of metabolic pathways and retention of secretion systems and effectors provided evidence of a possible defensive symbiosis.

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Diversity of Fornicates in the view of environmental V4 and V9 datasets: contrasting taxonomic composition and putative new clades

Ilya Udalov1,2, Martin Kolísko1

1. Biology Centre, Institute of Parasitology, Czech Academy of Sciences, Budweis, Czech Republic 2. Zoological Institute of the Russian Academy of Sciences, St. Petersburg, Russia

Fornicata (Excavates, Metamonada) is a monophyletic group of both host-associated and free-living flagellates that inhabit anaerobic environments, predominantly anoxic sediments or within the gut of diverse animals. This taxon unites the diplomonads and retortamonads, which are sister lineages, with the paraphyletic assemblage of “Carpediemonas-like” organisms which occupies a basal position in the Fornicata clade. Despite the fact that Fornicates were first documented in the 19th century and amongst them are medically and economically important parasites, their diversity remains poorly studied and online databases contain fewer than 200 18S rRNA sequences for the group.

The aim of this study is to expand our knowledge of fornicate biodiversity by identifying previously unrecognized fornicates from large-scale high-throughput sequencing efforts. We have compiled data from amplicon surveys using the hypervariable V4 and V9 regions of 18S rRNA gene of microbial eukaryotic communities living in anoxic biotopes (anoxic fresh-water and marine sediments, deep-sea, or soil), which are likely to contain our sequences of interest. Putative Fornicata sequences were searched using BLASTn and their true phylogenetic assignment was confirmed using the Evolutionary Placement Algorithm (EPA). Our analyses demonstrated strong V4 primer bias against the diplomonads and retortamonads, as we have recovered no sequences from these groups in V4 datasets, while we recovered representatives from all groups in the V9 datasets. Most of the newly identified Fornicata V4 amplicons branch with the “Carpediemonas-like” organisms and our analyses revealed the existence of putative new lineages within Fornicata.

Supported with GAČR 18-28103S research grant.

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News in iron-sulphur cluster assembly in oxymonads

Vojtěch Vacek1, Beatrice Py2, Vladimír Hampl1

1. Department of Parasitology, Faculty of Science, Charles University, BIOCEV, Czech Republic 2. Laboratoire de Chimie Bactérienne, Institut de Microbiologie de la Méditerranée, CNRS UMR, Marseille, Fra nce

Monocercomonoides exilis was reported to be first amitochondriate eukaryote and a potential preadaptation for loss of mitochondrion was the acquisition of SUF pathway for Fe-S cluster synthesis in the common ancestor of Preaxostyla. The pathway is well conserved in this group and it composes of SufB, C, and DSU genes. Interestingly, genes SufDSU represents a Preaxostyla specific fusion of three proteins encoded individually in prokaryotic systems. Repertoire of the downstream CIA pathway enzymes is consistent with that of closely related species that retain ISC pathway, indicating that the switch from ISC to SUF did not affected the process of cytosolic and nuclear Fe-S proteins maturation and suggesting potential interactions between SUF and CIA pathway.

We have performed experiments validating the functionality of the SUF proteins using Escherichia coli knock-out cell lines. Strains of E. coli deficient in particular SUF gene was transfected with its homologue from M. exilis cloned into pTrc99A vector. Subsequently, the cells were exposed to oxidative stress and iron starvation by addition of phenazine methosulfate and 2,2,-bipyridyl respectively. SufB, S and U showed slightly positive growth phenotype and are possibly capable of partial complementation of its E. coli homologue function. Complementation of SufB was furthermore verified by β-galactosidase assay. Fusion protein SufDSU and SufC showed no growth phenotype and overexpression of SufC seemed to be toxic for the E. coli. To elucidate potential interaction between SUF and CIA pathways, we performed a series of experiments using Bacterial Adenylate-Cyclase Two Hybrid system (BACTH) between Nbp35 protein of CIA pathway on one side and SufB, C and U proteins on the other. No combination revealed positive protein-protein interaction.

Our experiments provide first experimental evidence for the functionality of M. exilis SUF pathway but its connection to CIA has not been revealed yet.

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A PCR only tagging-based system for inducible overexpression of proteins from endogenous loci in Trypanosoma brucei

Hana Váchová1, Miroslava Ńedinová1, Glenda Alquicer-Barrera1, Vladimír Varga1

1. Institute of Molecular Genetics of the Czech Academy of Sciences , Czech Republic

Overexpressing endogenous proteins is often informative as to their cellular function and mechanisms determining their subcellular localization. Overexpressing a protein in cell culture in addition facilitates its purification and subsequent biochemical characterization. However, some proteins, in particular the cytoskeletal ones, are large and repetitive, which makes cloning of their ORFs into expression vectors challenging. In the protozoan parasite Trypanosoma brucei a very potent machinery for homologous recombination operates, which enabled development of the pPOT system for tagging proteins in endogenous loci by PCR products only (Dean et al., Open Biology, 2015). We introduced a T7 polymerase promoter and tet operators from an expression vector pDEX (Poon et al., Open Biology, 2012) into pPOT, enabling insertion of this tet inducible overexpression cassette, together with an affinity tag for purification and a fluorescent tag into the trypanosome genome upstream of an ORF of interest. We demonstrate that this approach indeed enables rapid and efficient generation of transgenic cell lines. These, when not induced with tetracycline, show a wild type-like level of expression of fluorescently tagged proteins, which is advantageous when screening for transgenic clones. Upon induction with tetracycline high levels of overexpression of tagged proteins are achieved. We demonstrate this for a number of large proteins, including dynein heavy chains, and present how overexpression data contribute to understanding their biology. Furthermore, we show that these transgenic cell lines can indeed be used as a good source for protein purification. We believe that this approach, which bypasses the need for cloning ORFs will prove to be a valuable tool to study function of large proteins in particular.

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Studying of the processes at the ciliary tip of eukaryotic cilium

Hana Váchová1, Luděk Ńtěpánek1, Miroslava Ńedinová1, Peter Gorilák1, Bill Wickstead2, Vladimír Varga1

1. Institute of Molecular Genetics of the Czech Academy of Sciences, Czech Republic 2. University of Nottingham, School of Life Sciences, Nottingham NG7 2UH, UK

Cilia and flagella, highly evolutionary conserved organelles, can be found on the surface of many eukaryotic cells from protists to mammals. Cilia have critical motility, sensory and signalling functions and their malfunctions in humans lead to a complex spectrum of hereditary diseases called ciliopathies. Ciliary structure is well conserved and consists of a basal body, transition zone, axoneme and ciliary tip. Very little is known about the molecular composition of the ciliary tip, yet this is the site of axonemal growth, turnover of proteins and localization of signalling molecules. To identify tip proteins we combine powerful approaches with high experimental tractability of the unique flagellated model organism Trypanosoma brucei. Leveraging the Tryptag project (Tryptag.org) and the structural immunoprecipitation approach (Varga et al., PNAS, 2017) we have so far identified over 30 proteins at the tip of Trypanosoma brucei flagellum. We characterized the majority of them by inducible RNAi revealing that some of them function in axoneme construction. We also performed evolutionary analysis showing that more than half of the identified proteins have orthologs in humans. We demonstrated that the depletion of a human ortholog protein causes severe defects in T. brucei, and indeed is localized to the ciliary tip in mammalian cells. In conclusion, we show that Trypanosoma brucei is an excellent model to characterize the poorly understood flagellar/ciliary tip.

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Peroxisomes and Entamoeba histolytica: To be or not to be?

Zdeněk Verner1, Tien Le1, Ravi Kumar Narayanasamy1, Abhijith Makki1, Petr Rada1, Ivan Hrdý1, Iris Bruchhaus2, Jan Tachezy1

1. Department of Parasitology, Faculty of Science, Charles University, BIOCEV, Czech Republic 2. Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany

Peroxisomes are a single membrane organelles of various metabolic functions. Up to now, they have been found only in aerobic organisms. Entamoeba histolytica is an anaerobic amoeba, a parasite of human gut. Through genomic search based on sequences from a free-living sister group Mastigamoeba balamuthi, we identified a set of peroxisome biogenesis factors in E. histolytica. As Entamoeba is amenable to genetic manipulation, we overexpressed the tagged versions of the identified genes, localized them within a cell and attempted isolation of microbodies-like organelles coupled with MS identification of their protein content to shed a light on their identity and possible function(s).

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In silico analysis of calpains in flagellates Euglena gracilis and Euglena longa

Dominika Veńelényiová1, Lenka Raabová1, Juraj Krajčovič1

1. Department of Biology, Faculty of Natural Sciences, University of Ss. Cyril and Methodius , Trnava, Slovakia

Calpains create a superfamily of calcium-dependent cysteine proteases. They are well conserved and are present in a wide range of organisms, from bacteria to humans. The number of calpains, as well as their structure varies among different species, however, they share a common functional and structural motif – their catalytic domain, CysPc. Calpain superfamily is further divided into families, based on domains associated with CysPc and the presence or absence of transmembrane helices in proteins. We decided to investigate calpains in euglenoid flagellates Euglena gracilis and E. longa, since previous studies of calpains in these species had not been done. We used available sequences of CysPc to create hidden Markov model (HMM), which we then applied on the transcriptomes, identifying potential calpain sequences. We then used in silico tools to analyze their domain structure, catalytic residues, transmembrane helices and to predict cell localization. We also attempted to sort these calpains into existing calpain families, sorting out sequences with an unusual domain structure. Our results show, that euglenas possess surprisingly high number of calpains, especially in contrast to land plants with only a single calpain. We also found, that not all calpains found can be divided into existing families, because of their unique structure. Despite the fact, that calpains are primarily cytosolic, we predicted, that calpains in euglenoid flagellates are localized in the nucleus, as well as mitochondria and cytosol.

Key words: calpains, cysteine proteases, CysPc, E. gracilis, E. longa

Acknowledgements: This project is funded by VEGA 1/0535/17 and FPPV 2019

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Novel protein of Giardia intestinalis links the function of the mitosomes and the encystation specific vesicles

Martina Vinopalová1, Luboń Voleman1, Josef Pelc1, Lenka Marková1, Vladimíra Nadjrová1, Pavel Doleņal1

1. Departm ent of Parasitology, Faculty of Science, Charles University, BIOCEV, Czech Republic

Majority of Giardia intestinalis genes encode proteins of unknown function. However, it is mainly these unknowns, which represent unique biology of this important intestinal parasite. The highly reduced mitochondria known as mitosomes belong among such Giardia distinctive features and multiple mitosomal proteins of unknown function have been described to date.

One such protein, referred to as GiMlf1IP (GL50803_16424), has drawn our attention as the interacting partner of known mitosomal proteins from all tested mitosomal subcompartments - the inner and the outer membrane and the mitosomal matrix. GiMlf1IP has atypical phylogenetic distribution, as outside of the Hexamitidae family it can be found only in Viridiplantae and Metazoa, where it localizes to the nucleus as putative transcription factor of Mlf1IP (Myelodysplasia-myeloid leukemia factor 1- interacting protein) family.

Using specific polyclonal antibody we show that, in contrast to previous reports, the protein is not in the mitosomes but predominantly at discrete foci right next to the organelles. In addition, the protein was found at the rim of the adhesive disc and, interestingly, it colocalizes with the encystation specific vesicles (ESVs) in the later stages of encystation. GiMlf1IP possesses C-terminal intrinsically disordered region, which have been shown to mediate dynamic protein-protein interactions and enable creation of “membraneless compartments” and allow quick response to the current state of the cell. We are characterising these “membraneless compartments” by affinity purification of chemically crosslinked complexes and by CRISPRi technique, which has been recently introduced to Giardia. In addition, we are studying the dynamics of GiMlf1IP localization upon the environmental stimuli and during the life cycle of the parasite.

ABSTRACTS

Mitosomal dynamics in Giardia intestinalis

Luboń Voleman1, Pavla Tůmová2, Pavel Doleņal1

1. Department of Parasitology, Faculty of Science, Charles University, BIOCEV, Czech Republic 2. Department of Tropical Medicine, First Faculty of Medicine, Charles Universit y, Prague, Czech Republic

Mitosomes are the smallest evolutionary forms of mitochondria that evolved in eukaryotes adapted to anaerobic environments. This adaptation manifests as the absence of the mitochondrial genome and vast majority of the mitochondrial proteome, including the components of the mitochondrial division machinery. Here, we studied the dynamics of mitosomes in Giardia intestinalis using FIB-SEM technique. This method showed that the mitosomes are actually asymmetrical. They are partially flattened on the side where the two membranes surrounding the organelle get closer to each other. We also found that mitosomal division is restricted to mitosis, when both central and peripheral organelles divide in a unique and synchronized manner. During the segregation of the divided mitosomes, the subset of the organelles between two G. intestinalis nuclei has a prominent role. These central mitosomes are physically connected to the flagella via specialized fibril during the whole process, thus ensuring proper inheritance of these prominent organelles to the daughter cells. Moreover, despite the absence of the ERMES components, mitosomal division involves association with the endoplasmic reticulum, a relationship commonly seen during the division of mammalian and fungal mitochondria.

ABSTRACTS

Phylogenomic analyses of ten transcriptomes of secondarily free-living diplomonads

Monika Wiśniewska1, Agnieszka Pełesz1, Petr Táborský1, Eric Salomaki1, Martin Kolisko1, Eva Mazancova2, Vasana Jinatham3, Eleni Gentekaki3, Ivan Čepicka2

1. Biology Centre, Institute of Parasitology, Czech Academy of Sciences, Budweis, Czech Republic 2. Department of Zoology, Faculty of Science, Charles University, Prague, Czech Republic 3. School of Science, Mae Fah Luang Univers ity, Chiang Rai, Thailand

Diplomonads are flagellated protists that inhabit oxygen-poor environments and lack conventional mitochondria. They are classified within Fornicata and include both free-living organisms that inhabit freshwater and marine anoxic sediments (e.g., Hexamita inflata), and host-associated organisms that reside in the intestinal tract of animals, including humans (e.g., Giardia intestinalis). The group is particularly interesting as the free-living taxa appear to be nested within a clade of host- associated species, suggesting a reversal from a host dependent to a free-living lifestyle. This reversal represents an extremely rare evolutionary event as most parasites evolve they become increasingly reliant on their hosts and lose genes for metabolic pathways essential for a free-living lifestyle. Previously, a transcriptomic investigation of the putatively secondarily free-living diplomonad Trepomonas sp. suggested that Trepomonas acquired several genes by horizontal gene transfer (HGT), widening its metabolic capacity and allowing a reversal back to free-living lifestyle. This finding was striking, however all prior studies suffer from low taxon and gene sampling, especially within the free-living diplomonads. In this study we sequenced the transcriptomes of 12 additional diplomonad isolates, (10 of them putatively free-living and 2 host-associated) for phylogenomic and comparative analysis.

A 248-gene phylogenomic analysis providing a well resolved evolutionary history of diplomonads which provides a framework for subsequent comparative analyses. Additionally, we present findings from the 12 newly sequenced transcriptomes that were investigated for genes identified as originating from HGT and putatively functioning in free-living lifestyle in the previously published Trepomonas transcriptome.

ABSTRACTS

Bacterial endosymbionts of Novymonas

Alexandra Zakharova1, Daria Tashyreva2, Jorge Morales3, Eva Nowack3, Julius Lukeń2, Vyacheslav Yurchenko1

1. Life Science Research Centre, University of Ostrava, Ostrava, Czech Republic 2. Biology Centre, Institute of Parasitology, Czech Academy of Sciences, Budweis, Czech Republic 3. Heinrich Heine University, Düsseldorf, Germany

Protists of the family Trypanosomatidae have been intensively studied because some of them are agents of human diseases, such as Chagas‟ disease, African sleeping sickness, and leishmaniasis. In the current classification system, 4 trypanosomatid genera from 2 subfamilies [Leishmaniinae (genus Novymonas) and Strigomonadinae (genera Angomonas, Strigomonas, and Kentomonas)] were found to harbor bacterial symbionts. The case of Novymonas is of particular interest since none of the partners of this endosymbiotic system has close relatives involved in such relationships, implying its independent and relatively recent origin.

Genomic and proteomic analyses of Novymonas and its endosymbiont revealed numerous host-encoded proteins enriched in the endosymbiont fraction: enzymes for β-oxidation, transporters, transmembrane proteins and hypothetical proteins. To dissect functions of some of these proteins, we are tagging them endogenously and exogenously. Here we report successful exogenous tagging of 2 proteins (NESM_000205400.1 Transmembrane protein, NESM_000666800.1 hypothetical protein) after their integration into the SSU rRNA locus of Novymonas esmeraldas.

ABSTRACTS

Mitochondrial metabolism of anaerobic amoeba Pelomyxa schiedti

Kristína Záhonová1, Sebastian C. Treitli1, Anna Karnkowska2, Joel B. Dacks3, Ivan Čepička4, Vladimír Hampl1

1. Faculty of Science, Charles University, BIOCEV, Czech Republic 2. Department of Molecular Phylogenetics and Evol ution, University of Warsaw, Poland 3. Department of Medicine, University of Alberta, Edmonton, Alberta, Canada 4. Department of Zoology, Faculty of Science, Charles University, Prague, Czech Republic

Pelomyxa schiedti (Mastigamoebaea, Archamoebae, Amoebozoa) is a flagellated amoeba living in anaerobic environments, and most likely bearing an anaerobic derivate of mitochondria, similarly to its relatives Mastigamoeba balamuthi and Entamoeba histolytica. We have generated genome and transcriptome reads by Illumina HiSeq and MiSeq and Oxford Nanopore platforms, and the genome was assembled by SPAdes assembler. After separation of prokaryotic sequences and improving the assembly using the P_RNA_scaffolder tool, we obtained a genomic assembly of 52.3 Mb (5,340 contigs, N50=51,552). Our initial analyses revealed the presence of two sets of NIF system subunits to mediate FeS cluster assembly. In contrast to M. balamuthi, in which one set localizes in mitochondria and one in the cytosol, both sets of P. schiedti are predicted to be cytosol-localized. We found a few proteins involved in translocation across the mitochondrial membranes (Tom40, Sam50, and Sam37) and chaperonins Cpn60 and Cpn10, the latter containing predicted mitochondrial targeting signals (MTS). Complete set of proteins of the mitochondrial glycine cleavage system was also predicted to possess MTS. Taken together, these results suggest that P. schiedti indeed possesses an anaerobic derivate of mitochondrion.

ABSTRACTS

Trypanosomatids diversity is Northern Eurasia: who lives in taiga and tundra?

Andrew V. Zolotarev1,2, Marina N. Malysheva1, Alexei Y. Kostygov1,2, Alexander O. Frolov1, Anna I. Ganyukova1

1. Zoological Institute of the Russian Academy of Sciences, St. Petersburg, Russia 2. St. Petersburg State University, St. Petersburg , Russia 3. Life Science Research Centre, Faculty of Science, University of Ostrava, Ostrava, Czech Republic

One of the least studied regions in terms of trypanosomatid diversity is Northern Eurasia, especially the Far North regions. In 2016-2018 we conducted field researches in the following northern regions of Russia (58°51′ N – 67°35′ N): Leningrad Region, Karelia Republic, Komi Republic, Yamalo-Nenets Region, Chukotka Region and Kamchatka Region. A total of 2189 insects were collected and dissected, including Brachycera (N = 1342; infected – 98), Nematocera (N = 657; infected – 0), Hemiptera (N = 103; infected – 25) and Hymenoptera ( N = 87; infected – 2). The total percentage of infected insects was 5.7%. We obtained 63 trypanosomatid laboratory cultures and identified trypanosomatid isolates using the 18S SSU rDNA molecular marker. Among the extracted laboratory samples, 39 isolates are now reliably identified. Much of the unidentified isolates are the result of parasitic mixed infections. Crithidia brevicula (8 isolates), Angomonas deanei (6 isolates) and Trypanosoma theileri (9 isolates) most frequently occur in the Russian North. In addition, 13 isolates of trypanosomatids, which have no analogues in the GeneBank and are potentially new species, were distinguished into the cultures.

Acknowledgments: The present study was supported by the project RFBR no. 18-34- 00867mol_a. The study utilized equipment of the “Taxon” Research Resource Center (http://www.ckp-rf.ru/ckp/3038/) of the Zoological Institute of the Russian Academy of Sciences.

ABSTRACTS

Mastigamoeba balamuthi and preadaptations to parasitism of Entamoeba

Vojtěch Ņárský1, Jan Tachezy1

1. Department of Parasitology, Faculty of Science, Charles University, BIOCEV, Czech Republic

We utilize the genome sequence of Mastigamoeba balamuthi, a free living relative of Entamoeba histolytica, to infer on the genome level characteristics of the common ancestor of M. balamuthi and Entamoeba. We further search for traits that possibly enabled the free-living ancestor of Entamoeba to transition to parasitic lifestyle.

ABSTRACTS

Copper metabolism of Naegleria gruberi and Naegleria fowleri

Kateřina Ņeníńková1, Maria Grechnikova1, Jan Mach1, Róbert Ńuťák1

1. Department of Parasitology, Faculty of Science, Charles University , Prague, Czech Republic

Copper is an important trace element, essential for all organisms. As a cofactor, it takes part in key biochemical reactions. It conveys electron transfer in complex IV of the respiratory chain, as an essential part of superoxide dismutase it helps to detoxify superoxide radicals and also participates on the import of iron ions into the cell as a cofactor of oxidase-permease system Fet3-Ftr1. Homeostasis of copper ions must be strictly regulated, to prevent their accumulation in the organism. Excess of these ions is highly toxic and may lead to cell death caused by copper-catalyzed reactions producing hydroxide radicals, a dangerous member of reactive oxygen species, that damages the structure of lipids, proteins, and DNA. Many studies also describe the importance of copper ions as a limiting factor for virulence of pathogens, where these ions induce the immune response of the host organism.

Our project is focused on the impact of the availability of copper for free-living microorganisms Naegleria gruberi and its close pathogenic relative Naegleria fowleri. Our previous results from proteomic analysis and cell respiration measurement confirmed that N. gruberi is able to alter the energy metabolism in relation to copper ion availability. Furthermore, we confirmed that the rate of iron uptake is also dependent on accessible copper. Another part of our work is focused on copper trafficking in the cell. We identified several copper transporters as well as putative copper exporting ATPase of N. fowleri, which is probably the key protein in copper detoxification pathway. Other protein of interest is CutC. CutC belongs to the group of Cut proteins that play a role in the acquisition, intracellular storage and export of copper ions. The exact biological function of CutC is not yet fully understood, however, we have shown high-affinity binding of copper to native CutC using microscale thermophoresis. Altogether we have described that the availability of copper ions affects many biological processes, including basic energy metabolism, making it an essential element for cell life.

ABSTRACTS

Evolution and unprecedented variants of the mitochondrial genetic code in a lineage of green algae

David Ņihala1, Marek Eliáń1

1. Department of Biology and Ecology & Institute of Environmental Technologies, Faculty of Science, University of Ostrava, Ostrava, Czech Republic

Mitochondria of diverse eukaryotes have evolved various departures from the standard genetic code, but the breadth of possible modifications and their phylogenetic distribution are known only incompletely. Furthermore, it is possible that some codon reassignments in previously sequenced mitogenomes have been missed, resulting in inaccurate protein sequences in databases. Here we show, considering the distribution of codons at conserved amino acid positions in mitogenome-encoded proteins, that mitochondria of the green algal order Sphaeropleales exhibit a diversity of codon reassignments, including previously missed ones and some that are unprecedented in any translation system examined so far, necessitating redefinition of existing translation tables and creating at least seven new ones. We resolve a previous controversy concerning the meaning the UAG codon in Hydrodictyaceae, which beyond any doubt encodes alanine. We further demonstrate that AGG, sometimes together with AGA, encode alanine instead of arginine in diverse sphaeroplealeans. Further newly detected changes include Arg-to-Met reassignment of the AGG codon and Arg-to-Leu reassignment of the CGG codon in particular species. Analysis of tRNAs specified by sphaeroplealean mitogenomes provides direct support for and molecular underpinning of the proposed reassignments. Furthermore, we point to unique mutations in the mitochondrial release factor mtRF1a that correlate with changes in the use of termination codons in Sphaeropleales, including the two independent stop-to-sense UAG reassignments, the reintroduction of UGA in some Scenedesmaceae, and the sense-to-stop reassignment of UCA widespread in the group. Codon disappearance seems to be the main drive of the dynamic evolution of the mitochondrial genetic code in Sphaeropleales.

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