Antimicrobial potencial of acinobacterial inhibotors of MurA and MurB ligases in bacterium Clostridium difficileRustemi, Elmedine (Avtor) Stres, Blaž (Mentor) Sievers, Martin (Komentor) molecular biologycloningClostridium difficileMur ligasesactinobacterial inhibitorsprotein expressioninsect cellspurificationinhibitory assayEnzymes MurA, MurB and MurC catalyze the initial cytoplasmic steps of peptidoglycan biosynthesis. Mur ligases are highly conserved among various bacterial species with a high potential to become targets for new classes of antimicrobial agents. Main aim of this study was production of recombinant MurA and MurB ligases from C. difficile and screening of potential inhibitors isolated from Streptomyces sp. For this purpose, murA and murB genes from native Clostridium difficile DNA, a major public health care pathogen, were cloned and the corresponding proteins were overproduced in insect cells Spodoptera frugiperda and Escherichia coli. Obtained proteins were purified using His tag affinity chromatography and as second step of purification size exclusion chromatography was performed. In parallel codon optimized synthetic MurA, MurB and MurC were purchased in order to compare cloning from native and synthetic DNA. The latter was proven to be more successful in this research work, due to codon optimization for expression in insect cells and E. coli. Protein contents were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-page and Western blot. To determine the activity of the purified recombinant MurA protein Inhibitory assay kit was used and catalytic activity of MurA was confirmed. Potential inhibitors isolated from Streptomyces sp. against overexpressed C. difficile MurA were tested. As a positive control fosfomycin a known antibiotic against MurA ligase was used. Results indicated no inhibitory potential of Streptomyces extracts against MurA.[E. Rustemi]20192019-08-06 07:45:12Magistrsko delo/naloga108923sl