Basic research
Genetically engineered bacterial spores as potential oral vaccines against SARS-CoV-2 and other pathogens

Authors: Vetráková, A., Kalianková Chovanová, R., Rechtoríková, R., Krajčíková, D., Barák, I.

Bacillus subtilis spores are considered to be efficient and useful tools for surface attachment of various proteins. As part of the study, recombinant spores were prepared with the bound "spike" domain of the SARS-CoV-2 protein in fusion with the spore coat proteins CotZ and CotY. When considering whether such recombinant B. subtilis spores would be suitable for the development of an oral vaccine, several factors speak in their favor. First, B. subtilis is considered a generally safe microorganism and is also used as a probiotic. Second, spores can survive the harsh environment in the gastrointestinal tract caused by stomach acids, making them an ideal vehicle for oral vaccine delivery. The results presented in the work indicate that recombinant spores with proteins bound to the envelope proteins CotY and CotZ could be widely used for the development of new oral vaccines not only against the SARS-CoV-2 virus but also against other newly emerging life-threatening viruses and bacteria.


Projects:
VEGA 2/0001/21 - Ako bunka nájde miesto asymetrického delenia počas sporulácie Bacillus subtilis.
APVV-18-0104 - Asymetrické bunkové delenie počas tvorby bakteriálnej endospóry
Publications:
VETRÁKOVÁ, A. - KALIANKOVÁ CHOVANOVÁ, R. - RECHTORÍKOVÁ, R. - KRAJČÍKOVÁ, D. - BARÁK. I. Bacillus subtilis spores displaying RBD domain of SARS-CoV-2 spike protein. In Comput Struct Biotechnol J., 2023; vol. 21, pp. 1550–1556 (2022: 6 - IF, Q1 - JCR, 1.579 - SJR, Q1 - SJR)

Basic research
Role of PDZ interactions in regulation of GABA transporter GAT1 and E protein in SARS COV, SARS-COV2 and MERS-COV viruses

Authors: Baliová, M. , Jahodová, I., Jurský, F.

PDZ interactions are an important tool in the regulation of cell signaling pathways and also play an important role in virus infection. In our work, we identified the PDZ interaction of the C-terminal section of the GABA transporter GAT1 with the PDZ protein Syntenin 1. We confirmed the interaction at the level of recombinant proteins as well as in N2a neuroblastoma cells. The interaction likely plays a role in the regulation of GAT1 activity by regulating the expression of the transporter on the neuronal surface. We also found a big difference in the intensity and promiscuity of the PDZ interactions of the E protein of the SARS COV1,2 and MERS COV viruses with the multi PDZ protein MUPP1. Significantly increased PDZ interactivity of MERS-COV correlates with significantly greater mortality, implying that PDZ interactions of protein E may contribute to increased disintegration of some cell signaling pathways and higher lethality of MERS-COV.


Projects:
VEGA 2/0127/21 - Regulácia interakčnej špecificity multi-PDZ proteínov.
Publications:

1. JAHODOVÁ, I. - BALIOVÁ, M. - JURSKÝ, F.. PDZ interaction of the GABA transporter GAT1 with the syntenin-1 in Neuro-2a cells. In Neurochemistry International, 2023, vol. 165, art. no. 105522. (2022: 4.2 - IF, Q2 - JCR, 0.958 - SJR, Q2 - SJR)
2. BALIOVÁ, M. - JAHODOVÁ, I. - JURSKÝ, F. A Significant Difference in Core PDZ Interactivity of SARS-CoV, SARS-CoV2 and MERS-CoV Protein E Peptide PDZ Motifs In Vitro. In Protein Journal, 2023, vol. 42, no. 4, p. 253-262. (2022: 3 - IF, Q3 - JCR, 0.853 - SJR, Q1 - SJR)

Basic research
Characterization of a newly identified putative initiator of DNA replication from bacteriophage phiBP

Authors: Bukovská, G., Halgašová, N., Javorová, R., Bocánová, L., Krajčíková, D., Bauer, J.A.

Phage DNA replication requires the phage's own proteins along with those of the bacterial host. We have characterized a novel replication initiation protein ROBP of phage phiBP from Paenibacillus polymyxa strain. The recombinant wild-type gpRO-HC protein exhibited low ATPase activity regardless of the presence of DNA, which increased significantly after substitution of lysine at position 8 for alanine. It is possible that the consequence of the mutation was a weakening of the phosphate hydrogen bond and an increase in the dissociation rate. The gpRO-HC protein efficiently bound to various single- and double-stranded DNA substrates and formed large oligomeric complexes with an approximate number of 12 subunits. Bioinformatics analysis revealed direct and indirect repeats in the nucleotide sequence of the ROBP gene, which often occur at the origin of DNA replication. The presence of an origin of replication within the gene sequence of the initiation protein is typical of phages with a theta mechanism of replication, which is probably also used by phage phiBP.


Projects:
VEGA 2/0079/22 - Príprava mutantných lytických a replikačných proteínov bakteriofágov a ich antibakteriálny potenciál.
Publications:
HALGAŠOVÁ, N. - JAVOROVÁ, R. - BOCÁNOVÁ, L. - KRAJČÍKOVÁ, D. - BAUER, J. - BUKOVSKÁ, G. Characterization of a newly discovered putative DNA replication initiator from Paenibacillus polymyxa phage phiBP. In Microbiological Research, 2023, vol. 274, art. no. 127437. (2022: 6.7 - IF, Q1 - JCR, 1.168 - SJR, Q1 - SJR)

Applied research
Strategy of molecular and enzymatic protection of our cultural heritage

Authors: Pavlović, J., Puškárová, A., Planý, M., Farkas, Z., Rusková, M., Kvalová, K., Kraková, L., Bučková, M., Pangallo, D.

In order to preserve cultural heritage for future generations, it is important to find new ways of protecting historical and artistic objects from the process of biodeterioration. We used molecular approaches based on third-generation sequencing (MinION) and bioinformatics procedures to gain a deeper understanding of the microbial pigmentation of historical books and archival documents. Sequencing has revealed complex biological relationships between fungi and bacteria present on objects and in the ambient air, expanding our understanding of the contributors to the pigmentation and deterioration of our written heritage. Another important aspect regarding the preservation of our cultural heritage is the development of strategies for the restoration of damaged items. We have developed a practical method using an extracellular enzymatic mixture of microorganisms (EEM) to remove stamps from books. This discovery is especially valuable for conservators in libraries, where the removal of institutional stamps is the first step in the restoration process.


Projects:
APVV-19-0059 - Farebné škvrny na historických papieroch: biologická a chemická charakterizácia spojená s ich odstraňovaním.
Publications:

1. PAVLOVIĆ, J. - PUŠKÁROVÁ, A. - PLANÝ, M. - FARKAS, Z. - RUSKOVÁ, M. - KVÁLOVÁ, K. - KRAKOVÁ, L. - BUČKOVÁ, M. - PANGALLO, D. Colored stains: Microbial survey of cellulose-based and lignin rich papers. In International Journal of Biological Macromolecules, 2023, vol. 241, art. no. 124456. (2022: 8.2 - IF, Q1 - JCR, 1.187 - SJR, Q1 - SJR)
2. FARKAS, Z. - PUŠKÁROVÁ, A. - OPÁLKOVÁ ŠIŠKOVÁ, A. - POLJOVKA, A.- ZÁMOCKÝ, M. - VADKERTI, E. - URÍK, M. - FARKAS, B. - BUČKOVÁ, M. - KRAKOVÁ, L. - PANGALLO, D. Evaluation of enzymatic stamp removal startegies on handmade (cellulose-based) and machine-made (lignin-containing) papers. In International Journal of Biological Macromolecules, 2023, vol. 242, art. no. 124599. (2022: 8.2 - IF, Q1 - JCR, 1.187 - SJR, Q1 - SJR)

International research projects
Detection of two functionally distinct luminal Ca2+-binding sites in ryanodine receptors: elucidation of differential regulation of RyR1 and RyR2 channel opening and closing

Authors: Bauerová-Hlinková, V., Bauer, J.

Ryanodine receptors (RyR) are calcium channels that are regulated by Ca2+ binding on the cytoplasmic and luminal side (oriented to the inside of the sarcoplasmic reticulum). Disruption of this regulation causes a change in the function of RyR, which conditions several diseases of the skeleton (malignant hyperthermia, disease of the central nuclei) and heart muscle (arrhythmias). In this work, we elucidated the luminal regulation of RyR1 and RyR2 isoforms by Eu3+ and proposed the localization of Ca2+ binding sites (Figure). The results show a different mode of luminal regulation in RyR1 and RyR2; at a higher concentration of Ca2+ (also Eu3+), RyR2 is activated, while RyR1 is inhibited. A study of the RyR1 Y524S mutation, which is associated with malignant hyperthermia (MH), showed the same sensitivity of luminal Ca2+ regulation as native RyR1, suggesting that MH in this mutation is not caused by altered luminal Ca2+ regulation. These results were obtained in an international collaboration with János Almássy, Semmelweis University, Budapest, Hungary.


Projects:
VEGA 2/0131/20 - Štúdium vplyvu mutácií asociovaných so srdcovými arytmiami na štruktúru a funkciu ľudského ryanodínového receptora 2.
Publications:
MAGYAR, Z. E. - BAUER, J. - BAUEROVÁ-HLINKOVÁ, V. - JÓNA, I. - GABURJÁKOVÁ, J. - GABURJÁKOVÁ, M. - ALMÁSSY, J. Eu3+ detects two functionally distinct luminal Ca2+ binding sites in ryanodine receptors. In Biophysical Journal, 2023, vol. 122, no. 17, pp. 3516-3531. (2022: 3.4 - IF, Q2 - JCR, 1.216 - SJR, Q1 - SJR)