About 30% of the world’s population is infected with Staphylococcus aureus, most of whom are asymptomatic carriers. At the same time, S. aureus remains a causative agent of many infections in healthcare settings and in the community. Improper use of antibiotics has led to an increase in infections caused by methicillin-resistant S. aureus (MRSA), which has become a significant clinical issue. S. aureus poses a serious threat to healthcare systems worldwide. Therefore, it is important to conduct research on the pathogenesis and antibiotic resistance of S. aureus to contribute to the development of new drug therapies.

The project is dedicated to the biochemical characterization of uracil-DNA glycosylase in S. aureus, which plays an important role in maintaining genome stability by preventing mutations that may arise from the presence of uracil in DNA. The studied enzyme is involved in the DNA repair mechanism, specifically in base excision repair (BER). All available data on BER are based on studies of the model organism Escherichia coli. However, S. aureus has its own peculiarities in the functioning of BER enzymes, and information on these, particularly on uracil-DNA glycosylase, is lacking.

Relevance

Improper use of antibiotics is the cause of the emergence of multi-drug resistant bacteria. The World Health Organization has included the ESKAPE pathogens, including S. aureus, in the list of bacteria for which the urgent development of new antibiotics is required.

Existing antibiotics target the main cellular processes of pathogens: DNA replication, RNA transcription, protein synthesis, inhibition of metabolic pathways, and cell wall biosynthesis. With improper use of these antibiotics, bacterial pathogens develop resistance. Given the importance of DNA repair in bacterial pathogenesis, BER enzymes are considered promising targets for new antibacterial drugs.

Objective

The biochemical characterization of the previously unexplored uracil-DNA glycosylase of S. aureus, which is the most common cause of hospital-acquired infections.

Expected results

The results of the project contribute to a deeper understanding of the pathogen’s resistance in the host organism and the identification of potential drug targets, which is crucial for the successful treatment of S. aureus infections.

Principal investigator

Turgimbayeva A.M. H-index – 2; ResearcherID N-6857-2017; ORCID 0000-0001-7263-1643; Scopus Author ID 57202383621.

Members of the research group

Abeldenov S.K. H-index – 3; ResearcherID F-5139-2015; ORCID 0000-0002-6974-9138; Scopus Author ID 56674705400.

Zein U.O.       H-index – 1; Scopus Author ID 57877532300

Publications and security documents of the project manager and members of the research group

1. Kirillov S, Isupov M, Paterson NG, Wiener R, Abeldenov S, Saper MA, Rouvinski A. Octahedral Iron in Catalytic Sites of Endonuclease IV from Staphylococcus aureus and Escherichia coli. Biochemistry. 2025 Jan 7;64(1):67-82. doi: 10.1021/acs.biochem.4c00447.
2. Amanzholova M., Shaizadinova A., Bulashev A., Abeldenov S. Genetic identification of Staphylococcus aureus isolates from cultured milk samples of bovine mastitis using isothermal amplification with CRISPR/Cas12a-based molecular assay. Vet Res Commun. ‒ 2023. DOI: 10.1007/s11259-023-10212-z
3. Shaizadinova A., Amanzholova M., Kirillov S., Bulashev A., Abeldenov S. Rapid and highly sensitive LAMP-CRISPR/Cas12a-based identification of bovine mastitis milk samples contaminated by Escherichia coli. Journal of Agriculture and Food Research. ‒ 2023, DOI: 10.1016/j.jafr.2023.100721
4. Zein U, Turgimbayeva A, Abeldenov S. Biochemical Assessment of the Mutant Sliding β-Clamp on Stimulation of Endonuclease IV from Staphylococcus aureus. Indian J Microbiol. 2024 Mar;64(1):165-174. doi: 10.1007/s12088-023-01148-8.
5. Turgimbayeva A, Zein U, Zharkov DO, Ramankulov Y, Saparbaev M, Abeldenov S. Cloning and characterization of the major AP endonuclease from Staphylococcus aureus. DNA Repair (Amst). 2022
6. Kirillov S, Daniyarov A, Turgimbayeva A, Ramankulov Y, Kalendar R, Abeldenov S. Draft Genome Sequence of Lactobacillus salivarius KZ-NCB, Isolated from Chicken Cecum. Microbiol Resour Announc. 2020 Dec 10;9(50):e01129-20. doi: 10.1128/MRA.01129-20.
7. Turgimbayeva A, Abeldenov S, Zharkov DO, Ishchenko AA, Ramankulov Y, Saparbaev M, Khassenov B. Characterization of biochemical properties of an apurinic/apyrimidinic endonuclease from Helicobacter pylori. PLoS One. 2018 Aug 15;13(8):e0202232. doi: 10.1371/journal.pone.0202232.
8. Sarina N, Abeldenov S, Turgimbayeva A, Zhylkibayev A, Ramankulov Y, Khassenov B, Eskendirova S. Obtaining and characterization of monoclonal antibodies against recombinant extracellular domain of human epidermal growth factor receptor 2. Hum Antibodies. 2018 Feb 5;26(2):103-111. doi: 10.3233/HAB-170327.
9. Characterization of DNA substrate specificities of apurinic/apyrimidinic endonucleases from Mycobacterium tuberculosis. Abeldenov S, Talhaoui I, Zharkov DO, Ishchenko AA, Ramanculov E, Saparbaev M, Khassenov B. DNA Repair (Amst). 2015 Sep;33:1-16. doi: 10.1016/j.dnarep.2015.05.007.

Results achieved

2024

The udg and dnaN genes, encoding uracil-DNA glycosylase and the β-subunit of DNA polymerase III, respectively, were amplified from the genomic DNA of S. aureus. The amplified genes were cloned into intermediate vectors for storage and into expression vectors for subsequent chromatographic purification. The open reading frame of the cloned genes within the intermediate and expression vectors does not contain any mutations.

Optimal conditions were selected for the most efficient expression of the udg and dnaN genes in E. coli expression strains.