Protimikrobni potencial aktinobakterijskih inhibitorjev ligaz MurA in MurB v bakteriji Clostridium difficile

Enzymes 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.Encimi MurA, MurB in MurC katalizirajo začetne stopnje citoplazmatske biosinteze peptidoglikana. Za Mur ligaze je značilno, da ohranjene med različnimi vrstami bakterij, kar predstavlja velik potencial, da postanejo tarče za nove razrede antimikrobnih sredstev. Glavni cilj te študije je bila produkcija rekombinantnih MurA in MurB ligaz iz C. difficile in testiranje potencialnih inhibitorjev, izoliranih iz rodu Streptomyces sp. V ta namen smo murA in murB gene klonirali iz Clostridium difficile DNA, pomembnega javnega zdravstvenega patogena. Klonirani proteini so bili izraženi v žuželčjih celicah Spodoptera frugiperda in Escherichia coli. Dobljene proteine smo očistili z uporabo His tag afinitetne kromatografije in kot drugo stopnjo čiščenja z izključitveno kromatografijo. Vzporedno smo klonirali tudi kodon optimizirane sintetične MurA, MurB in MurC, za primerjavo kloniranja iz naravne in sintetične DNA. Slednje se je izkazalo za uspešnejše v tem raziskovalnem delu zaradi optimizacije DNA za ekspresijo v celicah žuželk in E. coli. Vsebnost beljakovin smo analizirali z elektroforezo v natrijevem dodecil sulfatu in poliakrilamidnem gelu SDS-page in Western blot. Za določitev aktivnosti očiščenega rekombinantnega MurA proteina smo uporabili inhibitorni test in potrdili katalitično aktivnost MurA. Ekstrakti iz rodu Streptomyces sp. so bili testirani, kot potencialni inhibitorji proti prekomerno izraženi MurA ligazi iz C. difficile . Kot pozitivno kontrolo smo uporabili fosfomicin, znan antibiotik proti MurA ligazi. Glede na rezultate Streptomyces ekstrakti niso pokazali inhibitorno aktivnost proti MurA ligazi