Detection of potential actinobacterial inhibitors of bacterium Clostridium difficile Mur ligasesPušić, Monika (Avtor) Stres, Blaž (Mentor) Sievers, Martin (Komentor) molecular biologycloningClostridium difficileMur ligasesprotein expressionpurificationinhibitory assayClostridium difficile is one of the major public health treats nowadays as C. difficile infections (CDI) represent the majority of health-care associated infections. This is due to the standard treatment with broad-spectrum antibiotics, vancomycin and metronidazole. Treatment results in disrupted microbiota and that leads to decreased defence ability and increased reoccurrence of infections with C. difficile. Provided that, many studies in last decade are focused on finding new antimicrobial substances that would inhibit C. difficile and would not have a broad impact on gut microbiota. Mur ligases catalyse the initial steps on peptidoglycan assembly. Already known antimicrobials are mostly directed towards peptidoglycan machinery involved in the last part of peptidoglycan biosynthesis. In this reason, Mur ligase are representing a great target for new antimicrobials as this first steps of peptidoglycan assembly are not targeted by antimicrobials yet. Mur ligases could be used as multi target drug to increase the therapeutic effectiveness. This study is focused on detection of new potential inhibitors of Mur ligases from C. difficile. Genes for Mur ligases were obtained from native and synthetic DNA. Mur ligases genes were cloned into appropriate vectors and expressed in prokaryotic and eukaryotic system. The goal was to obtain active, recombinant protein. This part of the study was rather unsuccessful with native origin of C. difficile DNA. This was not surprising as C. difficile is known as genetically difficult to modify. The further work was proceeded with synthetic DNA of Mur ligases. Proteins of interest were successfully expressed and purified. After purification, MurE protein was selected for further analysis. Purified MurE ligase was concentrated and positively tested for its activity. Afterwards, several inhibitors from genus Streptomyces and clinically used antimicrobials, fosfomycin and D-cycloserin, were used in last step of the study, inhibitory assay.[M. Pušić]20192019-03-18 11:10:51Magistrsko delo/naloga106788sl