Molecular biology a genomics

The research programme consists of 8 main activities

Main activity 1 Identification and characterisation of new or poorly studied viruses

Building on the research already started, viruses of selected protist groups, mainly trypanosomatids and unicellular algae, will be studied to elucidate their diversity, evolution and interaction with the host. We expect to discover entirely new viruses through the analysis of genomic data. Metagenomic data will also be analyzed to map the true diversity of selected virus groups and their gene repertoire, with special attention to the PLV and NCLDV virus groups.

The activity will be implemented in sub-phases with defined objectives, expected likely outputs, benefits and lessons learned:

• Bioinformatic analysis of RNA diversity of trypanosomatid viruses and unicellular algae in transcriptomic databases
  • Generation of new transcriptomic data from trypanosomatid and unicellular algal cultures
  • Systematic survey of trypanosomatid and unicellular algal transcriptomic databases targeted to identify the presence of genes characteristic of RNA viruses (RNA-dependent, RNA polymerase)
  • Detailed comparative and phylogenetic analyses of candidate viral sequences found
  
  
• Detailed comparative and phylogenetic analyses of the candidate viral sequences found
  • Generation of new genomic data from trypanosomatid and unicellular algal cultures
  • Systematic survey of trypanosomatid and unicellular algal sequence databases targeted to identify genes characteristic of target viruses (capsid proteins, DNA polymerases, etc.)
  • Detailed comparative and phylogenetic analyses of the candidate viral sequences found
  
  
• Targeted search for selected virus groups in metagenomes and metatranscriptomes
• Targeted in vitro and in vivo studies of newly discovered viruses

Main activity 2 Identification and characterization of virulence factors of leishmaniases

A very well run research programme will continue towards understanding the molecular determinants of virulence of human pathogens of the genus Leishmania, particularly using the model species L. mexicana. Additional candidates for virulence factors will be tested using a wide arsenal of in vitro and in vivo functional genomics methods. This line of research will include further study of the physiological function of the enzyme catalase in trypanosomatids to understand what role the loss of this enzyme has played in the evolution of the dixenic life cycle of Leishmania.

The activity will be carried out in sub-phases with defined objectives, expected likely outputs, benefits and findings:

• Bioinformatic analysis of potential virulence factors of leishmaniases
  • Selection of potential leishmania virulence factors from the literature and our own databases
  • Analysis of expression levels of selected genes using the TriTrypDB database
  
  
• Testing the effects of potential antiparasitics on leishmania physiology
  • Evaluation of the effect of the compound callunene on in vitro cultures of procyclic promastigotes and amastigotes of L. mexicana
  • Evaluation of the effect of a callunene compound on the in vivo propagation of L. mexicana in insect vectors and mouse hosts
  • Similar tests of another candidate antiparasitic (defined during the course of the research)
  
  
• Functional analysis of leishmania virulence potentiation factors
  • Detailed phenotypic characterization of knock-out lines
  • In vitro characterization of target gene products
  • In vivo characterization of target gene products
  
  
• Study of the physiological function of the enzyme catalase in trypanosomatids
  • In vitro characterization of target gene products
  • Comparative analysis of biochemical characteristics of trypanosomatid catalases: localization, expression level and stability
  • Analysis of catalase roles in monoxenic trypanosomatids of the genus Leptomonas: knock-out and tagging of catalase in Leptomonas seymouri
  
  
• Publication of results

Main activity 3 Comprehensive research on protist endosymbionts

The biology of prokaryotic endosymbionts of selected groups of protists, mainly trypanosomatids and unicellular algae, will be studied using genomic and other methods. In the case of kinetoplastids it will be mainly the systems "Novymonas esmeraldas - Ca. Pandoraea novymonadis" and "Angomonas deanei - Ca. Kinetoplatobacterium sp." In particular, this research should reveal the molecular mechanisms behind the respective endosymbiotic relationships.

The activity will be carried out in phases with defined objectives, expected likely outputs, benefits and findings:

• Selection of promising host-endosymbiont model systems
  • Survey of historical literature on protists with potential endosymbionts
  • Systematic screening of genomic and transcriptomic data
  
  
• Basic characterization of hosts and their endosymbionts
• Taxonomic and phylogenetic characterization
• Morphological and ultrastructural characterization
• Genomic characterization of hosts and their endosymbionts
  • Generation and assembly of genomic and transcriptomic data
  • Bioinformatic analyses of genomic and transcriptomic data
  
  
• Functional characterization of endosymbiont-host relationships
  • Physiological, biochemical and proteomic analyses
  • Targeted genetic manipulations
  
  
• Publication of results

Main activity 4 Stablishing the possibility of targeted genetic manipulations in selected protist models

The introduction of targeted genetic manipulation techniques, primarily based on the CRISPR-Cas system, will substantially expand the possibilities of functional genomic research in selected important model unicellular eukaryotes. First of all, some representatives of trypanosomatids, e.g. Vickermania ingenoplastis or Novymonas esmeraldas, characterized by unique biological features revealed by existing genomic and transcriptomic analyses. The unique translational system of trypanosomatids of the genus Blasthocrithidia with position-dependent significance of some codons will also be further studied within this research direction.  

The activity will be implemented in sub-phases with defined objectives, expected likely outputs, benefits and findings:

• Optimisation of cultivation and selection conditions
  • Testing optimal conditions for cultivation of target organisms
  • Testing appropriate strategies and agents for selection of genetically modified lines
  
  
• Development of protocols for the preparation of targeted knock-in mutants
  • Testing alternative uses of CRISPR-Cas9 technology in new experimental models
  • Selection of mutants, verification of specificity and stability of induced mutations
  
  
• Development of protocols for regulatable expression of transgenes
  • Preparation of transgenic lines with an integrated cassette for regulated expression (Tet-On or Tet-Off system)
  • Preparation and validation of transgenic lines with their own target transgene with regulated expression
  
  
• Subsequent experiments with the prepared mutants
  • Preparation of multiple mutants, complementation assays, etc.
  
  
• Publication of results

Main activity 5 Characterization of unusual functional complexes and pathways of endosymbiotic organelles

Building on previous results, selected components of mitochondria and plastids of non-standard model systems will be studied in detail, representing new and previously unstudied functional aspects of these organelles. Examples include the newly discovered mitochondrial system based on the bacterial T2SS or a previously uncharacterized metabolic pathway encoded by a specific operon in the plastid genome of some eustigmatophyte algae.

The activity will be implemented in sub-phases with defined objectives, expected likely outputs, benefits and findings:

• In silico characterization of organellar proteomes
  • In silico prediction of organellar proteomes in selected taxa
  • Functional annotation of predicted organellar proteomes
  
  
• Experimental characterization of organellar proteomes
  • Optimization of procedures for organelle purification in selected newly defined organismal models
  • Identification of organellar proteins by mass spectrometry
  • Functional annotation of experimentally defined organellar proteomes
  
  
• Functional characterization of selected organellar components
  • In vitro functional characterization of target proteins using biochemical and biophysical methods
  • In vivo functional characterization of target proteins using reverse genetics and cell biology methods
  
  
• Evolutionary and phylogenetic analyses
  • Defining the evolutionary origin of the studied proteins using molecular phylogenetic methods
  • Comparative genomic analyses defining the relationship between the evolution of individual organismal groups, organelles and organellar components under study
  
  
• Publication of results

Main activity 6 Genomic characterization of new organismal lineages

Genomic data will be obtained from organisms representing poorly studied groups of microbial eukaryotes, including previously unknown and newly described species. The results will improve understanding of the phylogenetic diversity of eukaryotes and the evolution of their gene repertoire.

The activity will be implemented in sub-phases with defined objectives, expected likely outputs, benefits and lessons learned:

• Isolation and cultivation of new eukaryotic microorganisms
  • Sampling and establishment of laboratory cultures of microorganisms
  • Basic phylogenetic characterization of cultures using standard molecular markers to select organisms of interest for further investigation
• Basic biological characterization of new organisms
  • Cytological studies using light, fluorescence and electron microscopy
  • Study of physiological manifestations (growth requirements, behavioural manifestations, modes of reproduction, etc.)
  • Formal taxonomic descriptions of new taxa
• Generation of reference genomic data
  • Production of raw sequencing data using a combination of methods (Illumina and long-read methods)
  • Generation of reference sequences of genomes and transcriptomes of study organisms (assembly and polishing)
  • Structural and functional annotation of genome sequences
• Phylogenomic analyses
  • Phylogenomic reconstructions of the relationships of the studied organism groups
  • Definition of characteristic and diagnostic features of newly studied organisms at the genomic level
• Publication of results

Main activity 7 Comparative genomic analyses of novel genes

The history of the gene repertoire in selected groups of eukaryotes will be reconstructed in order to understand the role of gene duplications, horizontal gene transfer and de novo origin of genes in the evolution of these groups. Special attention will be paid to the reconstruction of metabolic pathways and the identification of candidate proteins with novel enzyme activities that can then be characterized by experimental approaches.

The activity will be carried out in sub-phases with defined objectives, expected likely outcomes, benefits and findings:

• Comparative genomic analysis of gene repertoire
  • Reconstruction of the evolution of the gene repertoire of the studied organismal groups
  • Detailed evolutionary analyses of selected genes and gene families
  
  
• In silico reconstruction of metabolic and other functional maps
  • Bioinformatic predictions of components of metabolic and other functional pathways and cellular modules of selected groups of eukaryotic microorganisms
  • Identification of unusual aspects of these pathways ("pathway holes", paralog multiplication, translational fusions, etc.) in order to find targets of interest for experimental characterization
  
  
• Identification of new targets for experimental investigation using phylogenetic profiling methods
  • Identification of candidates for new functional partners of genes of specific interest using the "phylogenetic profiling" method
  • Subsequent detailed bioinformatic analyses to validate candidates for experimental characterization
  
  
• Experimental characterization of newly defined target genes
  • In vitro experiments and experiments using heterologous systems (enzymology essays, expression in E. coli, etc.)
  • In vivo experiments using functional genomics and cell biology methods
  
  
• Publication of results

Key activity 8 New study and academic programmes

A new specialisation NMgr. degree programme and a new habilitation and professorship programme in the field thematically related to the Research Programme "Molecular Biology and Genomics".

The activity will be implemented in sub-phases with defined objectives, expected likely outputs, benefits and findings

• Accreditation of the new specialization NMgr. degree programme; working title "Genomic Biology and Bioinformatics"
  • Preparation of the accreditation dossier
  • Submission of the accreditation dossier and settlement of objections
  
  
• Accreditation of a new habilitation and professorship program; working title "Molecular Biology and Genomics"
  • Preparation of the accreditation file
  • Submission of accreditation file and settlement of objections

Cooperation with other VPs

Due to the very strong interdisciplinary links, the VP1 research team collaborates with research teams on some sub-activities:

• VP2 in the field of biophysical and chemical analyses and characterization, analytical use of developed methods - identification and quantification of selected metabolites, determination of enzyme activity and other sample analyses for VP1 HA2, 3, 4, 5 and 7
• VP3 for simulation and modelling of genomic data, modelling of viral protein and capsid structures (VP1 HA1), modelling of structure and function of organellar proteins (VP1 HA5), modelling of structure and function of novel proteins, joint preparation and submission of accreditation dossier, settlement of objections - NMgr. Genome Biology and Bioinformatics (VP1 HA8), cooperation consists mainly in the use of expert staff
• VP6 cooperation in the preparation of the new PhD programme Biomedicine (working title)