Kloniranje in izražanje bakteriofagnih endolizinov ter proteinskih vezalcev toksinov Clostridioides difficile

Celan, Zala (author), Bratkovič, Tomaž (mentor), Bozovičar, Krištof (comentor)

Zdravljenje okužbe z bakterijo Clostridioides difficile zaradi odpornosti proti antibiotikom predstavlja veliko problematiko v sodobnem svetu. Do okužb prihaja zlasti v bolnišničnem okolju ob uporabi antibiotikov, ki izzovejo spremembe v sestavi črevesne mikrobiote. C. difficile povzroča vnetja in krvavitve iz debelega črevesa (kolitis), ki vodijo v hudo obliko driske in se lahko končajo tudi s smrtjo. Poglavitna virulentna dejavnika C. difficile sta eksotoksina TcdA in TcdB, glukoziltransferazi, ki delujeta na GTPaze iz družine Rho, s čimer oslabita tesne in fokalne stike v sluzničnem epiteliju. Rekombinantni probiotiki predstavljajo bioalternativni protimikrobni pristop za zdravljenje okužbe s C. difficile in so tematika mnogih (pred)kliničnih raziskav. Okužbo s C. difficile bi lahko zmanjšali ali preprečili s kombinacijo uporabe bakteriofagnih endolizinov, selektivnih do C. difficile, in specifičnih proteinskih vezalcev toksinov TcdA ter TcdB, ki bi se izražali na površini rekombinantne probiotične bakterije Lactococcus lactis. V diplomski nalogi smo se osredotočili na pridobitev vezalcev toksinov (iz skupin afimerov in DARPinov; samih ali v fuziji s C-končno domeno sidrnega proteina AcmA, ki omogočajo vezavo na celično steno L. lactis,) ter endolizina z uporabo ekspresijskega sistema Escherichia coli. V prvem delu naloge smo pripravili ekspresijske vektorje: v modificiran plazmid pET28c (pET28c-HTF), smo vstavili sintezne gene za izbrane proteine. Ekspresijski sev E. coli NiCo21(DE3) smo transformirali s plazmidi in izvedli testno izražanje rekombinantnih proteinov po indukciji z IPTG ter s poliakrilamidno gelsko elektroforezo v prisotnosti natrijevega dodecilsulfata kvalitativno ocenili količino rekombinantnih produktov. Ugotovili smo, da vezalci eksotoksinov v fuziji z AcmA niso topni. Na podlagi rezultatov smo za izražanje v povečanem obsegu izbrali le topne proteine (tj. vezalce eksotoksinov brez AcmA in endolizina). V drugem delu diplomske naloge smo rekombinantne proteine izolirali s kovinsko-kelatno afinitetno kromatografijo ter jih koncentrirali z ultrafiltracijo. Ugotovili smo, da se fuzijski proteini vezalcev eksotoksinov TcdA/TcdB s sidrno domeno AcmA nahajajo v netopni frakciji lizata in se pri uporabljenih pogojih izražanja odlagajo v inkluzijska telesca.

• Language: Slovenian
• Keywords: afimeri, Clostridioides difficile, čiščenje, DARPini, endolizini, izražanje, vezalci eksotoksinov
• Work type: Bachelor thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-152455
• Publication date in RUL: 26.11.2023

CELAN, Zala, 2023, Kloniranje in izražanje bakteriofagnih endolizinov ter proteinskih vezalcev toksinov Clostridioides difficile [online]. Bachelor’s thesis. [Accessed 11 April 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=152455

Secondary language

• Language: English
• Title: Cloning and expression of bacteriophage endolysins and protein binders of Clostridioides difficile toxins

Abstract

Treatment of the infection with Clostridioides difficile bacterium represents a major problem in the modern world due to its resistance to antibiotics. Infections occur especially in hospitalized patients treated with antibiotics which cause dysbiosis of the intestinal microbiota. C. difficile causes inflammation and bleeding from the large intestine (colitis), which leads to severe diarrhea and may even result in death. The major virulence factors of C. difficile are the TcdA and TcdB exotoxins, glucosyltransferases that act on GTPases from the Rho family, thereby weakening tight and adherense junctions in the mucosal epithelium. Recombinant probiotics represent a bioalternative antimicrobial approach for the treatment of C. difficile infection and are the subject of many (non)clinical studies. C. difficile infection could be reduced or prevented by a combination of bacteriophage endolysins, selective for C. difficile, and specific protein binders of TcdA and TcdB toxins, expressed on the surface of the recombinant probiotic bacterium Lactococcus lactis. In the diploma thesis, we focused on obtaining toxin binders (from the group of affimers and DARPins; alone or in fusion with the C-terminal domain of the anchor protein AcmA, which enables binding to the cell wall of L. lactis) and endolysins using the Escherichia coli expression system. In the first part of the thesis, we prepared expression vectors: we inserted synthetic genes for selected proteins into the modified plasmid pET28c (pET28c-HTF). We transformed the expression strain E. coli NiCo21(DE3) with the plasmids and performed test expressions of recombinant proteins after induction with IPTG, and qualitatively assessed the amount of recombinant products by sodium dodecyl sulfate polyacrylamide gel electrophoresis. We found that exotoxin binders in fusion with AcmA are not soluble. Based on the results we selected only the soluble proteins (i.e. exotoxin binders without AcmA and endolysins) for scale-up expression. In the second part of the thesis we isolated recombinant proteins using metal-chelate affinity chromatography and concentrated them by ultrafiltration. We found that fusion proteins of TcdA/TcdB exotoxin binders with the AcmA anchor domain are present in the insoluble fraction of the lysate and are deposited in inclusion bodies under the expression conditions used.

• Keywords: affimers, Clostridioides difficile, DARPins, endolysins, exotoxin binders, expression, purification