Identifikacija molekul, ki inhibirajo aktivnost proteina LexA bakterij Escherichia coli ali Staphylococcus aureus

Halas, Barbara (avtor), Butala, Matej (mentor)

Odziv SOS pri bakterijah se sproži ob poškodbah DNA in bakteriji omogoči, da ohrani integriteto lastnega genoma. Poglavitni regulator odziva je transkripcijski faktor LexA, ki z vezavo na promotorska področja prepreči prepis genov, katerih produkti so vključeni v popravilo DNA. Genom bakteriofaga GIL01 vključuje gen za protein gp7, ki se veže s proteinom LexA bakterije Bacillus thuringiensis. Protein gp7 zveča afiniteto LexA do specifičnih nukleotidnih zaporedij in inhibira s proteinom RecA sproženo inaktivacijo LexA. Posledično protein gp7 vpliva na aktivacijo odziva SOS, kar potencialno zavre prilagoditev bakterij na stresne razmere v okolju, tudi pridobitev odpornosti proti antibiotikom. Namen magistrskega dela je bil prepoznati molekule, ki vplivajo na delovanje represorja LexA, podobno kot protein gp7. V ta namen smo izolirali protein gp8 temperatnega bakteriofaga GIL01 bakterije B. thuringiensis in protein OrbA temperatnega bakteriofaga ICP1 bakterije Vibrio cholerae. Rezultati nakazujejo, da se izolirana rekombinantna proteina ne vežeta s proteinom LexA gostiteljske bakterije in ne vplivata na cepitev proteina LexA. V nadaljevanju smo analizirali vpliv nekaterih izbranih malih molekul, pridobljenih iz laboratorija prof. dr. Stanislava Gobca iz Fakultete za farmacijo, na aktivnost LexA. Z uporabo površinske plazmonske resonance smo dokazali, da se učinkovina 6-hidroksiflavon (U5) s šibko afiniteto veže s proteinom LexA bakterij Escherichia coli in Staphylococcus aureus ter inhibira vezavo LexA bakterije E. coli na DNA. S poskusi in vivo, v tekoči kulturi, smo dokazali, da dodatek U5 upočasni rast bakterijske kulture E. coli ΔacrB in deluje sinergistično z antibiotikom ciprofloksacin. Učinkovina U5 je šibko zavrla tudi rast seva E. coli RW542, ki ima okvarjen gen za LexA in okvarjen promotorski element gena sulA, zato predvidevamo, da U5 inhibitorno vpliva tudi na procese, ki niso del odziva SOS.

• Jezik: Slovenski jezik
• Ključne besede: LexA represor, odziv SOS, OrbA, gp8, SPR, Escherichia coli, Staphylococcus aureus, Vibrio cholerae, Bacillus thuringiensis
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: BF - Biotehniška fakulteta
• Založnik: [B. Halas]
• Leto izida: 2023
• PID: 20.500.12556/RUL-144833
• UDK: 577
• COBISS.SI-ID: 146399235
• Datum objave v RUL: 16.03.2023

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Identification of molecules that inhibit activity of Escherichia coli or Staphylococcus aureus LexA protein

Izvleček

SOS response in bacteria is triggered upon DNA damage. It enables bacteria to maintain the integrity of their genome. The key regulator of the SOS response is the transcription factor LexA. LexA binds to promoter regions and represses the induction of SOS genes involved in DNA damage repair. The bacteriophage GIL01 encodes the gp7 protein, which interacts with LexA from the bacterium Bacillus thuringiensis. The gp7 protein increases the affinity of LexA for target DNA sites and inhibits RecA-induced self-cleavage of LexA. Therefore, the gp7 protein affects the induction of the SOS response which presumably inhibits the adaptation of bacteria to harsh environmental conditions, such as antibiotic stress. The aim of this work was to identify molecules that have the potential to affect the activities of LexA, similar to the gp7 protein. Therefore, we purified the recombinant proteins: the gp8 protein of the temperate bacteriophage GIL01, which infects the bacterium B. thuringiensis and the recombinant protein OrbA of the bacteriophage ICP1 that infects bacterium Vibrio cholerae. Our results indicate that the OrbA and gp8 proteins do not interact with the protein LexA of the host bacterium and do not affect the self-cleavage activity of LexA. Next, we tested the effect of selected small compounds that we obtained from the laboratory of Prof. Dr. Stanislav Gobec of the Faculty of Pharmacy, on LexA activity. Using surface plasmon resonance spectroscopy, we showed that the molecule 6-hydroxyflavone (U5) has a low affinity for the LexA protein of Escherichia coli and Staphylococcus aureus and inhibits the binding of LexA from E. coli to the target DNA sites. Using in vivo experiments, we demonstrated that U5 inhibits the growth of the E. coli ΔacrB strain and acts synergistically with the antibiotic ciprofloxacin. Furthermore, the molecule U5 weakly inhibited the growth of strain E. coli RW542, which carries the frameshift mutation in the lexA gene and the deleted promoter element of the sulA gene. Therefore, we hypothesize that compound U5 also affects processes other than the SOS response.

• Ključne besede: LexA repressor, SOS response, OrbA, gp8, SPR, Escherichia coli, Staphylococcus aureus, Vibrio cholerae, Bacillus thuringiensis