Basic research
A stable vector for efficient production of heterologous proteins and secondary metabolites in streptomycetes

Authors: Nováková, R., Homerová, D., Csolleiová, D., Řežuchová, B., Javorová, R., Ševčíková, B., Fecková, Ľ., Kormanec, J.

Streptomycetes are important producers of a large number of antibiotics. Their genomes contain a huge number of gene clusters of unknown antibiotics that are not expressed in laboratory conditions. One of the ways to use this potential is the heterologous expression of these antibiotics in optimized strains and expression systems. For this purpose, we optimized the strain Streptomyces lavendulae subsp. lavendulae CCM 3239 with its plasmid pSA3239 as a stable vector system. We tested this system using the mCherry and gusA reporter genes, and their production was four-fold higher in pSA3239 than in the chromosome of this strain, consistent with the higher copy number of pSA3239. We integrated the gene cluster for the antibiotic actinorhodin into pSA3239. However, the resulting strain produced very low amounts of actinorhodin. Its levels increased dramatically after integration of the gene for its positive regulator under the control of a strong promoter into this strain using a phage integration system.


Projects:
APVV-19-0009 – Príprava nových antibiotík a protinádorových látok manipuláciami génov sekundárnych metabolitov a metódami syntetickej biológie (Preparation of new antibiotics and antitumor agents by gene manipulation of secondary metabolites and synthetic biology methods)
Publications:

1. NOVÁKOVÁ, R. - HOMEROVÁ, D. - CSÖLLEIOVÁ, D. - REŽUCHOVÁ, B. - ŠEVČÍKOVÁ, B. - JAVOROVÁ, R. - FECKOVÁ, Ľ. - KORMANEC, J. A stable vector for efficient production of heterologous proteins and secondary metabolites in streptomycetes. In Applied Microbiology and Biotechnology, 2022, vol. 106, pp. 7285-7299. (2021: 5.560 - IF, Q1 - JCR, 0.987 - SJR, Q1- SJR).
2. NOVÁKOVÁ, R. - MINGYAR, E. - FECKOVÁ, Ľ. - HOMEROVÁ, D. - CSÖLLEIOVÁ, D. - REŽUCHOVÁ, B. - ŠEVČÍKOVÁ, B. - JAVOROVÁ, R. - KORMANEC, J. A new family of transcriptional regulators activating biosynthetic gene clusters for secondary metabolites. In International Journal of olecular Sciences, 2022, vol. 23, art. no. 2455. (2021: 6.208 - IF, Q1 - JCR, 1.176 - SJR, Q1 - SJR, karentované - CCC).
   

Basic research
Defining a new subfamily GH13_46 in the GH13 alpha-amylase family in the CAZy database

Authors: Janeček, Š., Mareček, F.

The CAZy database (http://www.cazy.org/) has been a globally respected database for the classification of carbohydrate-active enzymes (Carbohydrate-Active enZymes) since the end of the last century. The family of glycoside hydrolases GH13 represents the main alpha-amylase family with more than 145 thousand sequences and 30 different enzyme specificities. In 2006, the GH13 family was officially divided into the first 35 subfamilies, with more being created according to current scientific knowledge. Based on detailed in silico analysis of more than 100 proteins represented by cyclomaltodextrinase from Flavobacterium sp. Well. 92 was proposed to establish a new GH13 subfamily. Its members contain at their N-terminus an unspecified domain of the so-called of the immunoglobulin fold and the stretch of MPDLN in the region of the conserved sequence region 5. This group forms a convincing cluster on the evolutionary tree that is well separated from the other GH13 subfamilies. The new subfamily was officially assigned the number GH13_46 in the CAZy database.


Projects:
VEGA 2/0146/21 - Amylolytické enzýmy – tisíce sekvencií, stovky štruktúr, desiatky špecificít – a čo evolúcia...? (Amylolytic enzymes - thousands of sequences, hundreds of structures, dozens of specificities - and what about evolution...?)
Publications:
MAREČEK, F. - JANEČEK, Š. A Novel Subfamily GH13_46 of the alpha-Amylase Family GH13 Represented by the Cyclomaltodextrinase from Flavobacterium sp. No. 92. In Molecules. - Basel : MDPI AG, 2022, vol. 27, iss. 24, art. no 8735. (2021: 4.927 - IF, Q2 - JCR, 0.705 - SJR, Q1 - SJR, karentované - CCC).

Applied research
A sensitive protocol for the detection of SARS-CoV-2 in the wastewater environment

Authors: Rusková, M., Bučková, M.,Puškárová, A., Pangallo, D.

Virus pandemics cannot be avoided in the future, and the SARS-CoV-2 pandemic is just one example. Therefore, it is important to develop a sensitive method capable of monitoring the presence of potential pathogens. The sewage and wastewater environment has proven to be a suitable target for this kind of analysis. In this study, a model virus system containing a fragment of the SARS-CoV-2 genome encoding the Spike protein was first constructed. Encapsulated mimic S RNA (ESRM) was used to inoculate wastewater samples to evaluate the resurgence of viral particles using four different viral RNA concentration/extraction methods. The efficiency of individual approaches was evaluated using quantitative reverse transcription PCR (qRT-PCR) and one-step single-tube nested quantitative reverse transcription PCR (OSN-qRT-PCR). For the detection of viruses in wastewater samples with a low viral load, the OSN-qRT-PCR assay gave the most satisfactory results and the highest sensitivity.


Projects:
SAS - MOST (Taiwan) Joint Research Projects; MOST 108-2221-E-006-160-MY3; Water/wastewater epidemiology: Development of robust and reliable molecular detection systems for surveillance of disease outbreaks (PathogenTracker).
Publications:
RUSKOVÁ, M. - BUČKOVÁ, M. - ACHS, A. - PUŠKÁROVÁ, A. - WU, J.H. - KUCHTA, T. - ŠUBR, Z. W. - PANGALLO, D. Useful molecular tools for facing next pandemic events: Effective sample preparation and improved RT-PCR for highly sensitive detection of SARS-CoV-2 in wastewater environment. In International Journal of Hygiene and Environmental Health, 2022, vol. 245, art. no. 114017. (2021: 7.401 - IF, Q1 - JCR, 1.474 - SJR, Q1 - SJR)

International research projects
Blockade of TMPRSS2-mediated SARS-CoV-2 priming by lactoferricin

Authors: Ondrovičová, G., Praženicová, R., Leksa., V.

Lactoferrin, a member of the lactotransferrin family of iron-binding glycoproteins, is present in most human exocrine fluids, especially breast milk. Lactoferrin exhibits a number of biological activities, including iron sequestration and modulation of immune cells. We have recently gathered evidence that lactoferrin directly binds and blocks plasminogen, a serine protease physiologically involved in fibrinolysis and cell migration. On the other hand, it might also be involved in pathological conditions such as spread of tumor cells, invasion of bacteria or priming of viruses. We continued our research and discovered that lactoferrin can also block SARS-CoV-2 infection, which causes COVID-19. First, natural and synthetic lactoferrin-derived peptides block the activity of the transmembrane protease TMPRSS2, a plasminogen homolog responsible for the proteolytic activation of the SARS-CoV-2 spike protein. Second, lactoferrin and derived peptides block the interaction of the spike protein with heparan sulfate proteoglycans during virus attachment to the cell. This makes lactoferrin an inexpensive and widely available candidate for adjunctive therapy in the treatment of COVID-19.


Projects:

1. APVV-20-0513 - The double-edged sword of the plasminogen system: from homeostasis maintenance to COVID-19.
2. VEGA 2/0152/21- Lactoferrin and lactoferricin as natural plasmin inhibitors: from the structure resolution to therapeutic applications.

Publications:
OHRADANOVA-REPIC, A. - ŠKRABANA, R. - GEBETSBERGER, L. - TAJTI, G. - BARÁTH, P. - ONDROVIČOVÁ, G. - PRAŽENICOVÁ, R. - JANTOVA, N. - HRASNOVA, P. - STOCKINGER, H. - LEKSA, V. Blockade of TMPRSS2-mediated priming of SARS-CoV-2 by lactoferricin. In Frontiers in Immunology, 2022, vol. 13, art. no. 958581. (2021: 8.786 - IF, Q1 - JCR, 2.331 - SJR, Q1 - SJR)

International research projects
Characterization of the chromate transporter of Bacillus pseudomycoides

Authors: Chromiková, Z. Kalianková Chovanová, R., Barák. I

Chromium contaminates the environment worldwide and Cr(VI) is a Group I human carcinogen. Only some bacterial species are able to sustain vegetative growth at high Cr(VI) concentrations and thus function as bioremediation agents. One of the successful strategies for microbial Cr(VI) detoxification is the activation of chromate efflux pumps. Our results describe the transplantation of a chromate efflux pump from a potentially pathogenic but highly Cr-resistant environmental strain of Bacillus pseudomycoides into a Bacillus subtilis strain. We found that single cells of Cr-resistant B. pseudomycoides accumulate less Cr than cells of B. subtilis strain. Furthermore, we found that the survival of a B. subtilis strain during Cr stress can be increased by introducing a chromate transporter from B. pseudomycoides into its genome. Our study outlines the future direction of increasing chromium tolerance by non-pathogenic species and safe bioremediation using soil bacteria.


Projects:

1. FNSNF Swiss National Foundation SCOPES IZ 73Z0_152527/1 - The role of metal homeostasis, reduction and sporulation in the metal resistance of Gram-positive bacteria.
2. VEGA 2/0001/21 - Ako bunka nájde miesto asymetrického delenia počas sporulácie Bacillus subtilis (How a cell finds a site of asymmetric division during Bacillus subtilis sporulation)
3. APVV-18-0104 - Asymetrické bunkové delenie počas tvorby bakteriálnej endospóry (Asymmetric cell division during bacterial endospore formation)

Publications:
CHROMÍKOVÁ, Z. - KALIANKOVÁ-CHOVANOVÁ, R. - TAMINDŽIJA, D. - BÁRTOVÁ, B. - RADNOVIĆ, D. - BERNIER-LATMANI, R. - BARÁK, I. Implantation of Bacillus pseudomycoides Chromate Transporter Increases Chromate Tolerance in Bacillus subtilis. In Frontiers in Microbiology, 2022, vol. 13, art. no. 842623. (2021: 6.064 - IF, Q1 - JCR, 1.314 - SJR, Q1 - SJR)