Jovana Ćurčić1*, Milka Malešević1, Branko Jovčić2
1 Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia
2 Faculty of Biology, University of Belgrade
jcurcic [at] imgge.bg.ac.rs
Abstract
Antibiotic resistance represents a serious global health threat. Available antibiotics progressively lose efficacy against many bacterial pathogens. This phenomenon underscores the urgent need for alternative strategies to combat bacterial infections. Progress in understanding the regulation of bacterial pathogenicity has prompted researchers to explore potential antivirulence drugs as a promising alternative. Quorum quenching enzymes capable of degrading N-acyl homoserine lactones can impede bacterial virulence and biofilm formation by disrupting cell-to-cell communication. In this study, we employed in silico structural characterization of YtnP lactonase originating from Stenotrophomonas maltophilia 6960, utilizing various online software tools, including AlphaFold2, I-TASSER, PSIPRED, Phyre2, and SWISS-MODEL algorithms. The results obtained from these programs were compared to each other.
The analysis revealed a 278 amino acid residue protein with a molecular weight of 31.02 kDa, predicted to be a transmembrane protein with an N-terminal extracellular domain and a C-terminal cytoplasmic domain, predominantly comprised of extracellular amino acid residues. Experimental validation demonstrated the quorum quenching activity of S. maltophilia 6960 towards exogenous AHLs, supporting the predicted role of YtnP lactonase in modulating quorum sensing of the surrounding bacteria. Furthermore, the observation of QQ
activity in the crude extract and not in the cell-free supernatant of bacterial strain 6960 indicates that the YtnP lactonase is active within the bacterial cells. Secondary structure predictions revealed a balanced distribution of alpha-helices and beta-sheets, while tertiary structure predictions suggested a homodimeric configuration with four Zn2+ binding sites.
These findings, which combine in silico predictions with experimental validation, provide a solid foundation for further exploration in the development of effective antivirulence therapeutics. Leveraging in silico methodologies to predict and characterize the functional properties of potential antivirulence agents holds promise for accelerating the translation of research findings into clinical applications.
Keywords: bioinformatics, in silico prediction, quorum quenching, antivirulence therapeutics, lactonase
Acknowledgement: This study was supported by the Ministry of Science, Technological Development and Innovation of the Republic of Serbia (Agreement no – 451-03-47/2023-01/ 200042