DATA

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Department of biochemistry and microbiology focus on basic research in areas of retroviral molecular biology, proteomics, plant physiology and molecular genetics, enzymology, environmental microbiology, food microbiology and bioanalytical methods. These activities create a platform for applied research aimed at developing modern therapeutic approaches, bioremediation of inorganic and organic pollutants, monitoring food safety and quality (the department also operates accredited Testing Laboratory of the Institute of Biochemistry and Microbiology) or plant-pathogen interaction. Research in our department is in many cases interdisciplinary and, in addition to close professional cooperation between the individual laboratories of the department, it would be unthinkable without cooperation with a number of national and foreign groups within the framework of joint research programs and projects. Currently, we are working on projects supported by the Czech Science Foundation, Technology Agency of the Czech Republic, Ministry of Agriculture of the Czech Republic, Ministry of Industry and Trade of the Czech Republic and the Ministry of Education and Sports.

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Research of our laboratory is mainly related to molecular endocrinology, immunology and to the study of influence of oxidative stress on the organism. At the molecular level, we study the regulatory mechanisms occurring in the immune system, adipose tissue and liver and we are interested in those influencing the development of insulin resistance and metabolic syndrome. Our intention is also to find methods and substances that can be used in the treatment of these civilization diseases. In addition, we examined selenium effect on antioxidant enzymes levels and a possible connection with infertility. The last topic is pollen and food allergies, where the aim is to find the immunological basis that causes cross-reactivity between cypress pollen and some fruits or vegetables.

More information about our lab can be found in the section: Current projects.

Current projects

Mechanism of nicotinamide phosphoribosyltransferase function

Obesity affects a high number of people and its prevalence is rising. More than 30 years ago, it was discovered that fatty tissue is not just a fat storage; it is a place of a very complex regulation and of the secretion of tissue hormones called adipokins. They affect the metabolism of lipids and carbohydrates and are often responsible for the development of insulin resistance and, consequently, of diabetes mellitus type II. This discovery identified the “mere” fat storage as the largest endocrine organ in the body. Our goal is to clarify the properties of one of the adipokins – visfatin, also known as nicotinamide phosphoribosyltransferase (Nampt). We try to characterize where Nampt is located in the cell, how it is transported and secreted into the extracellular environment. Eventually, whether Nampt participates in the development of obesity and metabolic syndrome.

Nicotinamide phosphoribosyltransferase is attributed to a number of functions, the most important of which is its role as an essential enzyme in NAD synthesis. NAD is not only a cofactor of oxidoreductases, but it also serves as a substrate for non-redox reactions that are mainly responsible for DNA repair, cell cycle control and apoptosis. Recently, Nampt has been also associated with the development of cancer, because increased amounts of Nampt were observed in many cancer cells. For this reason, our goal is to gain a better understanding of how Nampt is involved in cancer development.

To study these processes, we use cell lines of adipocytes, hepatocytes and macrophages. The research includes molecular genetic techniques, preparation of genetically engineered cells, or fluorescence and super-resolution microscopy.

In vitro cultured adipocytes with red stained fat droplets.

In vitro cultured adipocytes 3T3-L1 with red stained fat droplets.

Preadipocytes 3T3-L1 Macrophages U-937

Preadipocytes 3T3-L1 and macrophages U-937 with a stably inserted nicotinamide phosphoribosyltransferase gene with a fluorescent label. The images were obtained by fluorescence microscopy.

Cooperation

Institute for Clinical and Experimental Medicine, Prague
Institute of Biotechnology, CAS, v.v.i., Vestec
Institute of Physiology, CAS, v.v.i., Prague

Cross-reactivity of cypress allergens

Today, more and more people suffer from allergies, especially in industrialized countries, where almost 30% of the population is affected. Doctors are observing an increase in cases of so called "combination" allergies or "pollen food associated syndromes", i.e. those which occur via a cross-reaction between pollen (respiratory allergies) and food (food allergies). It is estimated that 60% of food allergies occur in combination with respiratory allergies. In collaboration with French colleagues, we study cypress pollen cross-reactions using electrophoresis and immunoblotting. Cypress pollen is a significant allergen in France and Mediterranean regions. Moreover, the allergy to this pollen is often connected with allergies to peach or citrus fruits. Demonstrating the cross-reactivity and identifying its causes contribute to the improvement of allergy diagnosis, and lead to a better patient treatment in keeping with the development of a personalized medicine.

Cooperation

Pasteur Institute a Hospital Trousseau, Paris, France

Cypress pollen grains observed in scanning electron microscopy (2200x). Orbicules or "Ubisch bodies" (300 - 600nm granules), characteristics of Cupressaceae pollen, are visible on the surface of the exine (outer membrane).

Selected publication

Nuclear transport of nicotinamide phosphoribosyltransferase is cell cycle–dependent in mammalian cells, and its inhibition slows cell growth. Svoboda P., Křížová E., Šestáková Š., Vápenková K., Knejzlík Z., Rimpelová S., Rayová D., Volfová N., Křížová I., Rumlová M., Sýkora D., Kizek R., Haluzík M., Zídek V., Zídková J., Škop V. Journal of Biological Chemistry 2019; 294(22): 8676-8689

Cypress Pollinosis: from Tree to Clinic. Charpin D., Pichot C., Belmonte J., Sutra J.P., Zidkova J., Chanez P., Shahali Y., Sénéchal H., Poncet P. Clinical Reviews in Allergy & Immunology 2017; 1-22, DOI: https://doi.org/10.1007/s12016-017-8602-y

A new allergen family involved in pollen food associated syndrome: snakin/gibberellin regulated proteins. Sénéchal H., Šantrůček J., Melčová M., Svoboda P., Zídková J., Charpin D., Guilloux L., Shahali Y., Selva M.-A., Couderc R., Aizawa T., Poncet P. Journal of Allergy and Clinical Immunology 2018; 141(1):411–414

The effect of zinc and/or vitamin E supplementation on biochemical parameters of selenium-overdosed rats. Melčová M., Száková J., Mlejnek P., Zídek V., Fučíková A., Praus L., Zídková J., Mestek O., Kaňa A., Mikulík K., Tlustoš P. Polish journal of veterinary sciences 2018; 21(4):731-740, DOI: https://doi.org/10.24425/124312

Visfatin is actively secreted in vitro from U-937 macrophages, but only passively released from 3T3-L1 adipocytes and HepG2 hepatocytes. Svoboda P., Křížová E., Čeňková K., Vápenková K., Zídková J., Zídek V., Škop V. Physiological research 2017; 66 (4):709-714

The Response of Macro-and Micronutrient Nutrient Status and Biochemical Processes in Rats Fed on a Diet with Selenium-Enriched Defatted Rapeseed and/or Vitamin E Supplementation. Rýdlová M., Růnová K., Száková J., Fučíková A., Hakenová A., Mlejnek P., Zídek V., Tremlová J., Mestek O., Kaňa A., Zídková J., Melčová M., Truhlářová K., Tlustoš P. BioMed Research International 2017, DOI: http://dx.doi.org/10.1155/2017/6759810

Effect of selenium-enriched defatted rape seeds on tissue cadmium and essential elements utilization in rats. Myška A., Száková J., Fučíková A., Mlejnek P., Zídek V., Tremlová J., Mestek O., Koplík R., Zídková J., Melčová M., Tlustoš, P. Czech Journal of Animal Science 2016; 61(11):496-505, DOI: http://dx.doi.org/10.17221/88/2015-CJAS

Biochemical and hematological response of rats on defatted rape seeds addition into the diet. Tvrdá J., Tůmová N., Fučíková A., Zídková J., Melčová M., Száková J., Mlejnek P., Zídek V., Mestek O., Kaňa A., Tlustoš P. Academia Journal of Agricultural Research 2015; 3(12): 395-401, DOI: 10.15413/ajar.2015.0162

Autophagy inhibition in early but not in later stages prevents 3T3-L1 differentiation: Effect on mitochondrial remodeling. Skop V., Cahova M., Dankova H., Papackova Z., Palenickova E., Svoboda P., Zidkova J., Kazdova L. Differentiation 2014; 87(5):220-229. DOI: https://dx.doi.org/10.1016/j.diff.2014.06.00

Impact of cadmium on the level of hepatic metallothioneins, essential elements, and selected enzymes in the experimental rat model. Zídková J., Melčová M., Bartošová K., Šestáková I., Zídek V., Száková J., Miholová D., Tlustoš P. Czech Journal of Animal Science 2014; 59(12):548-556

Female serum of immunoglobulins G, A, E and their immunological reactions to seminal fluid antigens. Brázdová A., Zídková J., Sénéchal H., Peltre G., Cibulka J., Ulčová-Galová Z. Folia Biologica-Prague 2012; 58():251-255

Autocrine effects of visfatin on hepatocyte sensitivity to insulin action. Škop V., Kontrová K., Zídek V., Sajdok J., Pravenec M., Kazdová L., Mikulík K., Zídková J. Physiological Research 2010; 59():615-618

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Home Publications

Our lab group is focused on applications of the proteomics approaches using mass spectrometry as a detection endpoint. Using tools of the mass spectrometry we can study a broad spectrum of biological systems on the molecular level. Proteomics can be characterised as a field of the biochemistry that follows up complex studying of proteins in the specific time under defined conditions and is the one of the most dynamic and evolving science fields.

For the studying of proteins we use the mass spectrometric techniques: MALDI-TOF/TOF and LC-ESI-Q-TOF.

fig1a fig1b

Examples of Research Topics

Identification of proteins involved in crop stress reaction
Studying of membrane complexes associated with signal paths of plants
Proteomics aspects of mineralized heart valves
Protein analysis of artworks and historical mortars
Proteomic analysis of alveolar bones in connection with oral surgery
Proteomic analyses of bioliquids of patients with carcinomas and neurodenerative diseases
Identification of zooplankton
Distinguishing of animal species origin of parchments and hairs
Proteomics analysis of retroviruses
Identification of pathogen microorganisms
Development of techniques for protein quantification by mass spectrometry (methods iTRAQ, dimethyl labelling, TOP3 etc)

Obrázek1 (originál) matrix (originál) umeni (originál) salmonela (originál)

Methods

Peptide mass fingerprinting → protein identification
Surface mapping → study of protein conformation
Biotyping → identification of microorganism
Protein quantification (iTRAQ, dimethyl labelling, SIM, TOP3, MeanInt)
Cross-linking and pull-down of membrane proteins

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Home Publication Projects

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In our laboratory, we work on the development and application of methods using bioaffinity interactions of molecules to detect, determine, identify, and / or separate them.

DEVELOPMENT OF DETECTION SYSTEMS

First of all, we are engaged in the development of detection systems for determination of both, high and low molecular substances. These are single or multianalyte immunochemical techniques ELISA (Enzyme Linked Immunosorbent Assay) and LFIA (Lateral Flow Immunoassay), respectively. These methods utilize the existence of non-covalent interactions; mainly between antigen-antibody, biopolymer-solid surface, avidin-biotin and others. In recent years we have been focusing mainly on the application of immunochemical techniques for the detection and determination of environmental contaminants (e.g., pesticides), analytes from clinical biochemistry (e.g., anabolics, drugs), isoflavonoids, microorganisms (Listeria monocytogenes, genus Cronobacter), and their products (enterotoxins, Staphylococcus aureus).

Current projects

Detection of anabolic androgenic steroids (AAS) in food samples

development of quick and simple methods to reliably detect AAS in food supplements

Anabolic androgenic steroids are synthetic derivatives of testosterone. Nowadays they are used not only in medicine to support medical procedures, but also in sports as illegal doping to support rapid muscle mass growth and overall body strengthening. The free sale of these substances is prohibited, but they may be illegally sold, for example in the form of food supplements, without mentioning it on the packaging label. This behavior is primarily a threat to customers who buy a product without a declared content of anabolics, and therefore use these substances unknowingly.

New Psychoactive Substances Detection (NPS)

development of immunochemical methods for the detection of synthetic cannabinoids or tryptamines in biological fluids

NPSs are synthesized as structural analogues or chemical derivatives of already controlled substances. This is due to the efforts of manufacturers and distributors to circumvent existing legislative standards in which narcotic drugs and psychotropic substances are usually defined exhaustively.

STUDY OF CRONOBACTER BACTERIA

Image1 (originál)

Another gripping area of our interest are bacteria of the genus Cronobacter, opportunistic pathogens causing life-threatening infections, mainly in newborns and individuals with weakened immunity. We study these microorganisms from their basic properties to their interactions with the host organism. The methods used include a variety of approaches; e.g., isolation and purification of cell fractions and proteins, PCR, RFLP, MLST, mass spectrometry, work with databases and tissue cultures.

Current projects

Investigation of bacteria of the genus Cronobacter

Image2 (originál)

detailed characterization and accurate identification

Bacteria of the genus Cronobacter are very diverse in their properties and abilities. This diversity has caused a rapid development of their taxonomy and still presents us with new challenges in their accurate identification and classification. We mainly use the methods of molecular biology PCR and MLST and mass spectrometry MALDI-TOF/TOF.

interspecies variability

Currently, the genus Cronobacter includes seven species. According to the World Health Organization (WHO, 2008), the whole genus is seen as a pathogenic organism, although according to epidemiological studies, C. sakazakii and C. malonaticus are mainly associated with infections. It is therefore interesting to observe the differences in the characteristics and behavior of individual species and try to find connections with the degree of pathogenicity.

interaction of bacteria of the genus Cronobacter with cell lines

Some virulence factors have been described in the literature, but the process of pathogenesis remains unclear. In particular, protein expression in the host environment is almost non-mapped. Identification of strains capable of adhesion and invasion into human cell lines and monitoring of these interactions would be the first step to the subsequent study of virulent proteins that are expressed directly in the host by the pathogen.

identification of virulence factors in pathogenic members of the genus Cronobacter

The aim of this part of the work is to find proteins that are unique to pathogenic species. It is very likely that these are virulence factors and their identification would contribute to the understanding and description of the pathogenesis. Furthermore, these proteins could be used for the development of immunochemical methods. These methods excel in speed and low cost and would be a good tool for detecting pathogenic Cronobacter species.

Proteins are isolated from various cell fractions, characterized by SDS-PAGE and identified by LC-ESI-Q-TOF MS. To search for suitable proteins for the immunochemical detection, the immunomagnetic separation is used.

Selected publications

Šuláková, A., Fojtíková, L., Holubová, B., Bártová, K., Lapčík, O., Kuchař, M. Two immunoassays for the detection of 2C-B and related hallucinogenic phenethylamines.: J. Pharmacol.Toxicol 2019, 95, 36-46.

Holubová, B., Mikšátková, P., Kuchař, M., Karamonová, L., Lapčík, O., Fukal, L. (2019): Immunochemical techniques for anabolic androgenic steroid – matrix effects study for food supplements. Eur. Food Res. Technol. 2019, 245, 1011-1019.

Karamonová L., Holubová B., Jelínková A., Novotný J., Svobodová B. Stafylokokové enterotoxiny – superantigeny schopné ošálit imunitní systém. Chem Listy 2019, 113(11), 668-674.

Fojtíková L., Holubová B., Kuchař M., Lapčík O., Maryška M., Šuláková A. Tryptamine derivates having short linking bridge bearing carboxy functional group, and chemical processes for preparing the compounds useful for preparing immunogens by conjugation with the carrier protein. CZ Patentový spis 307719-B6, 2019.

Vlach J, Javůrková B, Karamonová L, Blažková M, Fukal L: Novel PCR-RFLP system based on rpoB gene for differentiation of Cronobacter species. Food Microbiol 2017, 62, 1-8.

Svobodová B, Vlach J, Junková P, Karamonová L, Blažková M, Fukal L: Novel method for the reliable identification of Siccibacter and Franconibacter strains: From ‘pseudo-Cronobacter’ to new Enterobacteriaceae genera. Appl Environ Microbiol 2017, 83(13), e00234-17.

Fojtíková L, Fukal L, Blažková M, Sýkorová S, Kuchař M, Mikšátková P, Lapčík O, Holubová B: Development of enzyme-linked immunosorbent assay for determination of boldenone in dietary supplements. Food Anal Methods 2016, 9(11), 3179-3186.

Jurášek M, Göselová S, Mikšátková P, Holubová B, Vyšatová E, Kuchař M, Fukal L, Lapčík O, Drašar P: Highly sensitive avidin-biotin ELISA for detection of nandrolone and testosterone in dietary supplements. Drug Test Analysis 2017, 9(4) 553-560.

Blažková M, Javůrková B, Vlach J, Göselová S, Karamonová L, Ogrodzki P, Forsythe S, Fukal L: Diversity of O-antigens within the genus Cronobacter: from disorder to order. Appl Environ Microbiol 2015, 81, 5574-5582.

Karamonová L, Junková P, Mihalová D, Javůrková B, Fukal L, Rauch P, Blažková M: The potential of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the identification of biogroups of Cronobacter sakazakii. Rapid Commun Mass Spectrom 2013, 27(3), 409-418.

Holubová B, Göselová S, Ševčíková L, Vlach M, Blažková M, Lapčík O, Fukal L: Rapid Immunoassays for Detection of Anabolic Nortestosterone in Dietary Supplements. Czech J Food Sci 2013, 31(5), 514–519.

Blažková, M., Javůrková, B., Fukal, L., Rauch, P.: Immunochromatographic strip test for detection of genus Cronobacter. Biosens Bioelectron 2011, 26(6): 2828-2834. ISSN 0956-5663.

Group members

head of the group:

prof. Ing. Ladislav Fukal, CSc.

assistant professors:

doc.Ing. Barbora Holubová, Ph.D.

Ing. Ludmila Karamonová, Ph.D.

doc. Ing. Martina Krausová, Ph.D.

Ing. Barbora Svobodová, Ph.D.

PhD students:

Ing. Judita Arnoštová

Ing. Jiří Novotný

Ing. Anna Jelínková

technician:

Hana Benadová

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Each cell within the body, despite its diverse functions, operates as an autonomous living entity and must effectively manage the resources it possesses. Mammalian cells employ various substrates, such as glucose and fatty acids, to derive energy through oxidation. This energy is then allocated to a range of tasks, including protein synthesis or the transmission of nerve signals. However, within a healthy organism, specialized cells handle energy distinctively. For instance, adipocytes specialize in storing energy substrates for times of scarcity, whereas neurons lack storage capacity and therefore require a continuous energy supply due to their substantial consumption rate. Besides the significant diversity in the functioning of individual cell types, the communication between different cells in the mammalian organism is also fascinating.

◳ AJ (png) → (šířka 450px)

The key molecule in the energy metabolism of all cells is nicotinamide adenine dinucleotide (NAD). The diagram simplifies the main processes that utilize NAD. NAD serves to transfer electrons from redox catabolic reactions to the respiratory chain for energy acquisition. The phosphorylated form of NAD (NADP) is indispensable for biosynthetic pathways. Redox reactions are characterized by converting NAD and NADP between their oxidized and reduced forms, without affecting the overall quantity of these molecules.

However, some reactions consume NAD. The most significant enzymes catalyzing these reactions are the signaling proteins from the sirtuin family (SIRT). Sirtuins deacetylate proteins by transferring the acetyl group onto NAD, releasing nicotinamide (Nam), thereby regulating the function and activity of target proteins, including histones, transcription factors, and enzymes. Sirtuins primarily act in the nucleus, where they regulate gene expression and chromatin stability, but also in the cytosol and mitochondria, where they participate in metabolism regulation.

Another group of enzymes that consume NAD are mono- and poly-ADP-ribosyltransferases (PARP), which covalently modify proteins with an ADP-ribose residue, releasing nicotinamide. This process regulates DNA repair, among other functions. Both redox and non-redox reactions utilize the same pool of NAD, which can become depleted during high sirtuin and PARP activity, leading to cellular energy metabolism collapse and cell death.

NAD replenishment in mammals primarily occurs through the nicotinamide salvage pathway. Initially, Nam combines with phosphoribosylpyrophosphate (PRPP) to form nicotinamide mononucleotide (NMN) via the enzyme nicotinamide phosphoribosyltransferase (NAMPT). NMN then combines with adenosine triphosphate (ATP) to form NAD. This pathway occurs in both the nucleus and cytosol, although it's not precisely understood if it also takes place in mitochondria or how NAD is transported into mammalian cell mitochondria. NAMPT activity determines the rate of the entire biosynthetic pathway and, consequently, the cellular NAD level.

We are a small team of scientists focusing on the study of NAD metabolism, particularly the enzyme NAMPT. We are working on these specific projects:

The impact of increased NAD levels on the metabolism of healthy and damaged cells. With age, chronic diseases tend to increase while NAD levels decline. By using various precursors or increasing the amount of NAMPT, it may be possible to raise NAD levels. Can boosting NAD levels be used as supportive therapy for chronic diseases such as type 2 diabetes? We are interested in understanding how increasing NAD levels will affect metabolism, gene expression, and the secretory profile of various cell types, with the aim of potentially using NAD precursors in medicine.
NAMPT secretion. An extracellular form of NAMPT has been described, but it's not clear how this enzyme is secreted from cells or what its role is in extracellular space. We are working to clarify these uncertainties.
Nuclear transport of NAMPT. NAMPT functions in both the cytosol and the nucleus, and it is present in both compartments. However, it's not clear how NAMPT is transported between the cytosol and the nucleus or how this process is regulated. Our work, which has shown how the transport of NAMPT into the nucleus is related to the cell cycle, has been featured on the cover of the prestigious Journal of Biological Chemistry, and we plan to continue investigating this topic.

Cooperation

Energy Homeostasis Section, Diabetes, Endocrinology, & Obesity Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, USA.
Laboratory of Translational and Experimental Diabetology and Obesitology, Institute for Clinical and Experimental Medicine, PragueL
Laboratory of Integrative Structural Biology, Institute of Experimental Botany of the Czech Academy of Sciences, v. v. i.
Institute Of Physiology 0f The Czech Academy of Sciences, v. v. i.
Czech Society for Atherosclerosis

Selected publications:

Škop V, Guo J, Liu N, Xiao C, Hall KD, Gavrilova O, Reitman ML: The metabolic cost of physical activity in mice using a physiology-based model of energy expenditure. Molecular metabolism. 2023, 71: 101699.
Vacurova E, Trnovska J, Svoboda P, Skop V, Novosadova V, Reguera DP, Petrezselyová S, Piavaux B, Endaya B, Spoutil F, Zudova D, Stursa J, Melcova M, Bielcikova Z, Werner L, Prochazka J, Sedlacek R, Huttl M, Hubackova SS, Haluzik M, Neuzil J: Mitochondrially targeted tamoxifen alleviates markers of obesity and type 2 diabetes mellitus in mice. Nature communications 2022, 13(1):1866.
Škop V, Guo J, Liu N, Xiao C, Hall KD, Gavrilova O, Reitman ML: Mouse thermoregulation: introducing the concept of the thermoneutral point. Cell reports 2020, 31(2):107501.
Svoboda P, Krizova E, Sestakova S, Vapenkova K, Knejzlik Z, Rimpelova S, Rayova D, Volfova N, Krizova I, Rumlova M, Sykora D, Kizek R, Haluzik M, Zidek V, Zidkova J, Skop V.: Nuclear transport of nicotinamide phosphoribosyltransferase is cell cycledependent in mammalian cells, and its inhibition slows cell growth. Journal of Biological Chemistry 2019, 294(22):8676-8689.
Svoboda P, Křížová E, Čeňková K, Vápenková K, Zídková J, Zídek V, Škop V: Visfatin is actively secreted in vitro from U-937 macrophages, but only passively released from 3T3-L1 adipocytes and HepG2 hepatocytes. Physiological Research 2017, 66(4):709-714.
Skop V, Cahova M, Dankova H, Papackova Z, Palenickova E, Svoboda P, Zidkova J, Kazdova L: Autophagy inhibition in early but not in later stages prevents 3T3-L1 differentiation: Effect on mitochondrial remodeling. Differentiation 2014, 87(5):220-229.

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Home Projects Publications

Our laboratory group is dedicated to the exploration of all aspects related to fungal genetics, ranging from the investigation of unknown genes in yeast mutant models, to the study of gene expression in fungal mycelia, and even the production of natural secondary metabolites in fungi. To achieve these objectives, we employ a broad spectrum of bioinformatic and molecular biology laboratory techniques, including polymerase chain reaction, blue-white screening, cDNA library construction, Genome Walking, cloning of unidentified genes into yeast mutants, and the isolation of proteins through metal-affinity methods. Furthermore, we collaborate closely with other scientific groups at the University of Chemistry and Technology Prague (VŠCHT Praha) and the Czech Academy of Sciences (AV ČR).

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Fungi play an important role in the ecosystem, cycling carbon and micronutrients and many even entering symbiotic relationships with plants. This can be of utmost importance for the optimization of plant growth, as research shows that mycorrhizal plants greatly benefit from the mycorrhiza due to a much larger root system and thus increased access to water and micronutrients and supposedly diminished stress from pathogens and heavy metal stress.

Our research group focuses on heavy metal speciation in the sporocarps and mycelia of ectomycorrhizal fungi. We are especially interested in the so called hyperaccumulators, i.e. fungal species that accumulate extraordinary amounts of heavy metals, sometimes even multiple elements at once. Hyperaccumulating taxons are first discovered by measuring the total amount of metals in sporocarps from pristine and polluted habitats acquired by our collaborator Jan Borovička. If a hyperaccumulating taxon is confirmed, further molecular and biochemical analyses are performed to elucidate the biochemical background of the metal speciation (metallothioneins, compartmentalization, novel metalloproteins) and then the genetic determinants responsible for the hyperaccumulating phenotype. Our collection comprises of over twenty hyperaccumulating fungal species in the form of mycelial explants, including Amanita, Hebeloma, and Agaricus sp. This gives us a unique opportunity to study the effects of heavy metal overload and poisoning on these organisms both in vitro and in vivo.

Our other collaborative projects include speciation and transformation of arsenic in arsenic hyperaccumulators and discovering novel fungal proteins and compounds with potential technological and medicinal importance.

Current projets

Speciation of cadmium, zinc and copper in sporocarps and mycelia of fungi of the species Russula, Hebeloma, Amanita muscaria, Cystoderma carcharias, Agaricus crocodilinus – besides classic biochemical approach for the identification of metal species, we are also relying on the available public genomic sequences in which we can reveal relevant protein sequences by bioinformatic analysis and then isolate the genes coding for these proteins by homology cloning or by construction of RACE and Genome walking libraries.

Identification of bacterial communities in sporocarps and in the mycosphere – although our main field of interest are metals in fungal sporocarps, and the genes and proteins related to metal metabolism, the question of why the metal hyperaccumulation trait has evolved in fungi has lead us to the study of microbial communities, so called microbiomes, by NGS methods. Our goal is to elucidate if the bacterial communities living in the proximity of sporocarps and mycelia are different from bacterial communities living at the same locality but without a contact with the fungus.

Preparation of chiral compounds by Cunninghamella elegans – this fungus is considered very interesting for the testing of the metabolism of toxic compounds, since the enyzmatic apparatus of the fungus is similar to that of animal hepatocytes. This can be used for the prediction of metabolites of new synthetic drugs or even production of novel bioactive compounds. Currently, we are cooperating with Dr. Michal Kohout, testing if the fungus is able to keep or change the chirality of the tested compounds.

Identification of novel aluminium complex in mycelia of Oidiodendron sp. – an isolate of an unknown species of this ericoid mycorrhizal fungus was brought to us from Poland. After metal tolerance screening, it was shown that the fungus is multitolerant to many metals, including aluminium. Currently we are investigating the form of aluminium stored within the cells, including using fluorescence microscopy with an aluminium probe, and transmission electron microscopy with energy dispersive spectroscopy to determine the exact place of the Al complexes within the fungal cells.

Identification of novel cadmium complexes in sporocarps of Agaricus crocodilinus – literature search revealed an interesting article from 1983, in which German scientists found a cadmium complex with high amounts of saccharides but no sulfur amino acids, which is strange, considering cadmium has been mostly found to bind with proteins containing sulfur. This complex was called “mycophosphatin” and our goal is to isolate it and describe its biochemical properties.

Isolation and characterization of biomolecules from sporocarps and mycelia of Amanita muscaria – this fungus belongs to mildly poisonous species. Its habitat extends over the whole northern hemisphere and besides being poisonous it has been known for being psychotropic, and thus it is used in traditional shaman rituals and as a recreational drug. In our collaboration with the BAFA lab, we are investigating the potential for novel medicinal use of A. muscaria.

Speciation of arsenic in the ink stain bolete, Cyanoboletus pulverulentus – this edible fungus was studied previously by our collaborators at Graz University in Austria. Their research yielded information about very high amounts of arsenic and potentially novel arsenic species. Our goal is to identify potential novel species and to study the metabolic pathways that lead to the formation of these species both in laboratory and in the environment. This project is done in collaboration with Department of Analytical Chemistry.

List of publications

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Home Projects Publications

The tremendous diversity of microorganisms is not only astonishing; it is also a key to understanding how organisms evolve and adapt to various environmental conditions. The metabolic activity of microorganisms is essential for maintaining the functions of all ecosystems in the biosphere. Microbial ecology seeks to unveil microorganisms in their natural environments; it looks at how microbes interact with one another and their environment, and what the consequences of these interactions are for ecosystem functions.

vody In the Laboratory of Microbial Ecology, we are interested in uncovering some of the hidden gems of microbial diversity. Certain localities of the Czech Republic are very interesting from the microbial ecological point of view; being extreme (brine, radioactive), often unique, spatially isolated biotopes, they are a promising source of hitherto undiscovered microbial diversity. Such habitats include mineral water springs which are used for healing purposes in Karlovy Vary, Jáchymov or Luhačovice, or soils and mofettas from the Soos National reserve. Our objective is to analyze microbial communities in these habitats and understand their physiology, biochemistry and ecology.

exudace No less important to us is improving our understanding of plant-microbe interactions. We look at how secondary plant metabolites shape soil microbial community structure and how they induce and/or regulate biodegradative genes in soil bacteria, thereby increasing the contaminant biodegradation potential in the microbial community. In turn, we are looking at how rhizosphere microorganisms promote the growth of plants. At the same time, we are looking at how many and which of these microorganisms colonize the interior of the plants, thus becoming endophytic. The applications of this fundamental research mainly include sustainable agriculture or environmental protection.

kultivace We identify metabolically active microbial populations in the context of their environments without the need for their cultivation. Microorganisms whose activity is crucial for a particular bio(geo)chemical process in the ecosystem are often not very abundant in the community. Therefore, we use microbial ecological techniques that allow linking community members with specific functions, including Stable Isotope Probing (SIP) or epicPCR. We thereby identify microorganisms degrading contaminants from the soil, promoting plant growth, etc.

exudace We are also aware of the importance of pure culture in microbiology. Therefore, we are trying to modify standard extraction and cultivation procedures in order to increase the efficiency of culturing of microorganisms, be it from soil, water, plant interior or any other habitat. Resuscitation factors or signaling molecules can help us to do so, along with mimicking the conditions of natural environments in which our microbes thrive. Upon successful isolation, the novel pure cultures are classified and characterized.

In order to reach our goals, we try to apply cutting-edge methodologies and techniques, including metataxonomics, metagenomics, stable isotope probing as well as modified culturing approaches. If you are interested in more details on our research, see our current projects.

We are a group of enthusiastic, motivated and hardworking microbial ecologists. If you are a motivated and diligent student who is eager to do solid research, do not hesitate to contact us.

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Home Publications

1) Virology

In the field of virology, which deals with the study of the structure and biology of viruses and their pathogenesis, we focus mainly on retroviruses and, more recently, in cooperation, also on flaviviruses. Both Retrovirideae and Flaviviridae are enveloped RNA viruses. The aim of our research is to clarify selected steps in the life cycle of viruses, focusing mainly on the late phase of viral infection. Understanding the molecular nature of these steps might open the possibility of developing new types of drugs. Using molecular biological methods, we study the structure of viral proteins, the functional significance of their structural domains, their mutual interactions, interactions with RNA and membranes and the transport of viral proteins and whole particles in an infected cell. We also study the structure and mechanism of formation of mature and immature retroviral particles.

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2) Medicinal chemistry: Theranostics and multimodal therapy

Natural products are the basis of clinical therapy for cancer and other diseases. Their therapeutic effects can be modified with chemical derivatization in order to reduce unwanted side effects, increase cytotoxicity and selectivity towards pathological cells, change their mechanism of action, etc. In our research group, we mainly focus on basic research of toxicity and mechanism of action of novel antimitotic derivatives (e.g., colchicine and paclitaxel), inhibitors of sarco-/endoplasmic reticular Ca2+-ATPase (e.g., thapsigargin and trilobolide) and cardiac glycosides (e.g., digitoxin and digoxin). Most of the derivatives we study are developed with the aim to be utilized in multimodal and combination therapy as well as theranostics of cancer. Theranostics is based on diagnostic and therapeutic properties combined in one molecule, which often consists of a natural product conjugated to a fluorescent moiety.

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3) Cytocompatible and antimicrobial materials for research and medicinal applications

Another research topic addressed by our scientific group is the study of tailor-made polymers and metallic materials for medical applications for soft and hard tissue replacements, but also for research purposes (e.g., single cell cultivation and analysis). The aim of this research is to develop novel possibilities for functional replacement of damaged tissues but also to understand the basic mechanisms of cell-cell and cell-material interactions in dependence on its nano- and microstructured topology. We focus on evaluation of material cytocompatibility, cytotoxicity and antibacterial properties using cell biological and microbiological methods. Moreover, methods of molecular biology are utilized to study the mechanism of cell adhesion, interaction of cells with materials at the molecular level, and cell differentiation.

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4) Photodynamic therapy and molecules for photoimaging

Photodynamic therapy is a non-invasive therapy using special molecules that, when activated by light, produce highly reactive oxygen species applicable for the eradication of tumors, microorganisms, or the treatment of skin diseases. We collaborate on the development of advanced photoactive molecules and study the molecular mechanisms of their action and localization in living tumor cells in real time. We focus on these aspects in the study of the effectiveness of photoactive molecules including both inorganic materials (e.g., molybdenum clusters) and organic molecules (e.g., porphyrins, phthalocyanines, halogenated BODIPY). In addition, we focus on the development of novel fluorescent probes for imaging techniques, which are an indispensable tool for medicine and molecular biological research. Fluorescence probes allow us not only to visualize and study different cellular structures, but also to reveal the molecular basis of various diseases. Our goal is to eliminate the limitations of available probes, such as high toxicity, low specificity or insufficient photophysical properties. We use both common and highly specialized fluorescence microscopy methods (SIM, PALM / STORM) to evaluate these probes.

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5) Biologically active substances

We study the properties of plant extracts or isolated compounds with a focus on both beneficial effects and possible negative effects. Our goal is to identify biologically active substances and characterize their beneficial effects while excluding possible toxicological effects. Special emphasis is placed on biologically active substances with the ability to modulate multiple drug resistance.

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Home Projects Publications

Plants as sesile organisms are exposed to many adverse environmental effects, either abiotic such as high temperature, drought, osmotic stress, salinity or biotic caused by pathogenic microorganisms, herbivores etc. Plants can adapt to many of these external cues as they evolved complex mechanisms by which they are capable to respond to unfavorable conditions.

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Our laboratory is interested in topics related to the response of plants to various stress factors, focusing primarily on the role of various proteins in stress responses. We are studying plant stress response taking place both at the level of the whole plant and at the level of the cell.

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As a model organism, we use Arabidopsis thaliana and oilseed rape (Brassica napus). We use modern biochemical, microscopic and molecular biological techniques to solve our projects.

Collaboration

Institute of Experimental Botany of the Czech Academy of Sciences: Institute of Experimental Botany of the Czech Academy of Sciences (doc. Ing. Lenkou Burketová, CSc.)
Institute of Experimental Botany of the Czech Academy of Sciences: Laboratory of Signal Transduction ( RNDr. Jan Martinec, CSc.)
Institute of Experimental Botany of the Czech Academy of Sciences: Laboratory of Hormonal Regulations in Plants (vedená RNDr. Janem Petráškem, Ph.D.)
Department of Experimental Plant Biology, Faculty of Science, University of South Bohemia
Dr. Eric Ruelland CNRS, Institut d’Ecologie et des Sciences de l’Environnement de Paris, UMR 7618, Créteil, FRANCE; Université Paris-Est; UPEC, Institut d’Ecologie et des Sciences de l’Environnement de Paris, Créteil, FRANCE

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Home Projects Publications

Proteins play an important role in the living world. Their diversity is huge and fascinating. In our laboratory we study proteins in a complex manner. We are mainly interested in the relationship between the structure and catalytic activity of enzymes, looking for new proteins with interesting properties, optimizing the production of recombinant proteins and developing new molecular modeling methods to simulate slow and computationally demanding processes such as protein folding or interactions of protein with ligands.

Current projets

Cold-active enzymes

Enzymes from organisms living in permanently cold environments (e.g. mountain and polar regions) have significantly higher activity at low temperatures compared to enzymes from meso- and thermophilic sources. This property makes them interesting biocatalysts for low temperature applications. In our group we study cold-active enzymes (specifically chitinase, amylase, cellulase, β-galactosidase), which have a potential for use in the paper, food or pharmaceutical industries.

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Enzymes with therapeutical potential

Asparaginases have found application in medicine in the treatment of acute myeloid leukemia. It uses their natural ability to cleave L-asparagine, which is depleted in tumor cells, as a result of which protein biosynthesis is stopped and apoptosis occurs. Healthy cells are able to synthesize L-asparagine due to the presence of L-asparagine synthetase and are therefore not affected by this type of treatment. Preparations containing recombinant L-asparaginase from E. coli and Dickeya dadantii are currently used in practice, however, the application of both enzymes has a number of complications, and therefore it is necessary to look for other sources of L-asparaginase with more suitable properties for clinical applications.

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Nucleases have been studied for a long time because of their anti-tumor effects. So far, the main focus has been on animal nuclases. However, in our group we are focusing on enzymes from other sources, such as plants or bacteria. These enzymes are studied using molecular biology and protein engineering methods. Biological studies and crystallographic experiments are carried out in cooperation with the Institute of Plant Molecular Biology, the Institute of Physiology and Animal Genetics and the Biotechnology and Biomedical Center of the Academy of Sciences and Charles University in Vestec (BIOCEV).

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Structural protein of lipid droplets

We are interested in cellular lipid droplets and their life cycle, with special emphasis on the characterization of proteins associated with lipid droplets permanently or within a specific stage of the life cycle. Our goal is to contribute to the description of the life cycle of these specialized organelles and thus to the biotechnological use of both lipid droplets themselves (lipid and biofuel production, oil degradation, drug transport and immobilization systems) and their associated proteins (fusion anchors for industrial recombinant protein expression). As a model organisms we use both prokaryotic (marine bacteria Alcanivorax borkumensis capable to break down oil) and eukaryotic (plant Arabidopsis thaliana and yeast Saccharomyces cerevisiae) organisms.

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Proteins involved in the synthesis of ladderans in anammox bacteria

So far not well-known anammox bacteria (from the English anaerobic ammonium oxidation) were discovered in the mid-1980s and seem to play an important role in the nitrogen cycle on Earth. Anammox bacteria directly use ammonia as a source of energy, which they convert into molecular nitrogen . They do not need oxygen and, instead of producing carbon dioxide, they consume it. for this purpose they contain an organelle called the anammoxosome, in which energy metabolism is located. The membrane of the anammoxosome is composed of very interesting phospholipids, which have very specific lipids attached – ladderans. During the processing of ammonia in the anammoxosome, a highly reactive and toxic hydrazine is formed as an intermediate, which is just held inside the organelle by ladderans, which, unlike common fatty acids, give the membrane lower permeability to various substances. The biosynthesis of ladderans has not yet been elucidated and is the subject of our research.

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Molecular modelling

Computer simulations make it possible to study complex dynamic behavior of proteins, saccharides, other biomolecules and their assemblies. The main drawback of molecular simulations is their high computational cost. To study procesess that take place in long time scales it is necessary to apply special simulation methods. We use and metadynamics and other methods and we endeavour to contribute to their development, for example by application of artificial neural networks.

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