Detection of potential actinobacterial inhibitors of bacterium Clostridium difficile Mur ligases

Pušić, Monika (author), Stres, Blaž (mentor), Sievers, Martin (co-mentor)

Clostridium 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.

• Language: English
• Keywords: molecular biology, cloning, Clostridium difficile, Mur ligases, protein expression, purification, inhibitory assay
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [M. Pušić]
• Year: 2019
• PID: 20.500.12556/RUL-106788
• UDC: 579.61:579.852.13:577.2
• COBISS.SI-ID: 5034872
• Publication date in RUL: 18.03.2019

Secondary language

• Language: Slovenian
• Title: Odkrivanje potencialnih aktinobakterijskih inhibitorjev za ligaze Mur bakterije Clostridium difficile

Abstract

Bakterija Clostridium difficile v današnjem času predstavlja eno izmed večjih groženj javnemu zdravju, saj so infekcije s C. difficile eden od glavnih razlogov bolnišničnih okužb. Vzrok za to je predvsem zdravljenje infekcij s široko spektralnima antibiotikoma, vankomicinom in metronidazolom. Uporaba omenjenih antibiotikov podre ravnovesje črevesne mikrobiote in zmanjša zmožnost obrambe, ter posledično poveča možnost za ponoven pojav okužbe s C. difficile. Veliko zanimanje je zato usmerjeno v iskanje novih potencialnih tarč protimikrobnih sredstev, ki bi delovali bolj ozkospektralno in ne bi imeli velikega vpliva na črevesno mikrobioto. Mur ligaze so encimi, ki katalizirajo začetne korake sinteze peptidoglikana. Do sedaj znana antimikrobna sredstva so usmerjena predvsem na zadnje stopnje sinteze peptidoglikana, prvi koraki pa so še redko ciljani. V ta namen Mur ligaze predstavljajo velik potencial v iskanju novih protimikrobnih učinkovin. Ta študija se osredotoča na odkrivanje potencialnih inhibitorjev Mur ligaz iz C. difficile. Geni za Mur ligaze so bili pridobljeni iz naravne in sintetične DNA. Geni za Mur ligaze so bili klonirani v ustrezne vektorje za ekspresijo v prokariontskem in evkariontskem sistemu. Cilj je bil pridobiti aktivne rekombinantne Mur ligaze. Ta del študije je bil precej neuspešen z nativno DNA C. difficile. Nadaljnje delo je tako potekalo zgolj s sintetično DNA Mur ligaz. Ligaze so bile uspešno izražene in prečiščene. Po proteinski ekspresiji smo študijo osredotočili na MurE protein. Očiščeno MurE ligazo smo koncentrirali in testirali njeno aktivnost. S tem smo potrdili naše predvidevanje, saj smo pridobili aktivno, rekombinantno MurE ligazo. V zadnjem koraku študije smo izvedli test inhibicije z več različnimi inhibitorji iz rodu Streptomyces, ter dva antibiotika v klinični uporabi, fosfomicin in D-cikloserin.

• Keywords: molekularna biologija, kloniranje, Clostridium difficile, Mur ligaze, izražanje proteinov, čiščenje proteinov, test inhibicije