Sortaz A Enzimine Karşı İnhibitör Peptitlerin Geliştirilmesi

Abstract

The emergence of pathogenic bacterial strains resistant to antibiotics is a major human health threat. In order to overcome this critical problem, it is in urgent need of developing novel antimicrobial agents those either kill or inhibit the growth of bacteria. Accordingly, the discovery of new target molecules, that will affect vital functions or the pathogenicity of the microorganisms, has been considered as an effective strategy for the development of new antimicrobial agents. Sortase enzymes, found in Gram-positive bacteria, appear as a new target molecule which can be used in the development of antimicrobial agents. Although many Gram-positive bacteria possess different isoforms of sortase, among them, sortase A (Srt A) is conserved in all pathogenic species. It has been shown that the infectivity of bacteria decreased for those having SrtA gene mutation. Agents, inhibiting the SrtA enzyme activity, have a great potential of showing antimicrobial activity against Gram-positive pathogens. Hence, the development of effective inhibitors against SrtA has become a popular subject for researchers. Phage display technology has been used frequently in the selection and development of peptides that show affinity to various target molecules. Phage display peptide libraries not only provide the development of artificial recognition molecules which selectively bind to target molecules, but also used in analysis of protein-protein interactions and mapping specific regions of the protein. Selection of enzyme inhibitors with the phage display method provides the development of new antimicrobial peptides for inhibition of the antibiotic resistant bacterial strains. In this study, it is aimed to develop novel peptide inhibitors that selectively and effectively inhibit the SrtA enzyme by using high diversity phage displayed peptide libraries. Accordingly, SrtA gene expression vector of Staphylococcus aureus was transferred into Escherichia coli, for the production and the purification of recombinant SrtA enzyme. Purified SrtA was used as the target molecule in the biopanning of phage display method. Linear and cyclic peptide libraries of 7 amino acids in length were used to select phage clones carrying the selectively binding and inhibiting peptide to SrtA. During the elution phase, free SrtA and free SrtA substrate were used and at the end of 3 cycles isolation of 88 phage clones was performed. The effects of the isolated phage clones on SrtA activity were analyzed and it was determined that 12 phage clones cause a decrease in enzyme activity. It was determined that the clone with the highest inhibitory effect among selected phage clones reduced the activity of SrtA by 30%. The variable region DNA sequences of 6 phage clones with inhibitory effect were determined and the peptide sequences of the phage clones were determined. As a result of the study, phage clones carrying peptides showing inhibitory properties against the enzyme SrtA, a significant effect on the virulence of pathogenic bacteria, were selected. It is thought that the identified peptide sequences may be used as potential SrtA inhibitors in the development of novel antimicrobial agents.