Razvoj rekombinantnih sevov bakterije Lactococcus lactis s sposobnostjo dostave proteinskih zaviralcev citokinske osi IL-23/IL-17

Klemenčič, Kaja (author), Berlec, Aleš (mentor), Plavec, Tina Vida (comentor)

Patogeneza kronične vnetne črevesne bolezni (KVČB) temelji na prekomerni aktivaciji imunskega odziva zaradi neuravnotežene aktivnosti celic T pomagalk in regulatornih celic T. V črevesni sluznici bolnikov so odkrili povečano število celic Th17, za katere je značilno izločanje citokina IL-17. Njihovo ekspanzijo spodbuja citokin IL-23, celotna signalna pot pa se imenuje citokinska os IL-23/IL-17. Za zdravljenje hudih oblik bolezni so zato na voljo monoklonska protitelesa proti citokinom ali drugim vnetnim molekulam. Zaradi velike in nestabilne strukture, oteženega pridobivanja ter imunogenosti protiteles so v razvoju tudi majhni proteinski vezalci neimunoglobulinskega izvora. V izogib razgradnji proteinskih vezalcev lahko kot dostavni sistem uporabimo mlečnokislinske bakterije (MKB), saj številni sevi preživijo prehod skozi gastrointestinalni trakt. S tem lahko hkrati dosežemo sinergistično delovanje probiotičnih MKB in dostavljenih terapevtskih molekul v črevesu. V okviru magistrske naloge smo želeli razviti rekombinantne seve bakterije Lactococcus lactis s sposobnostjo dostave proteinskih vezalcev (ARS) receptorja za IL-17 (IL-17RA). Z metodami genskega kloniranja smo pripravili genske konstrukte ARS12, ARS14, ARS19 in ARS14CO v plazmidu pSDBA3b za predstavitev na bakterijski površini ali za izločanje v gojišče. Plazmid je vseboval gena za signalni peptid Usp45 in peptidno oznako Flag, v primeru površinske predstavitve pa še zapis za sidrno domeno cAcmA. Gene smo v bakterijah nadzorovano izražali z nizinom in izražene vezalce analizirali s poliakrilamidno gelsko elektroforezo v prisotnosti NaDS (NaDS-PAGE) ter prenosom western - s protitelesi proti oznaki Flag. Pri tem smo zaznali le vezalce v fuziji s sidrno domeno cAcmA. S pretočno citometrijo smo nato potrdili površinsko predstavitev proteinov ARS12, ARS14 in ARS19. Nadalje smo želeli razviti še multifunkcionalne bakterije L. lactis, zmožne hkratnega izražanja vezalca IL-17A (finomera) in vezalcev IL-17RA (ARS14 ter ARS19) z namenom dosega sinergističnega učinka na vnetni proces v črevesu. Izražanje smo preverili z NaDS-PAGE in prenosom western - s protitelesi proti oznaki Flag, s katero so bili označeni vezalci ARS, ter protitelesi proti oznaki cMyc, s katero so bili označeni finomeri. Zaznali smo vezalce v fuziji s sidrno domeno cAcmA, pa tudi vezalec ARS14, ki so ga bakterije izločile v gojišče. V raziskovalnem delu smo torej dokazali sposobnost bakterij L. lactis za dostavo vezalcev IL-17A in IL-17RA, v nadaljevanju pa bo potrebno ovrednotiti še njihovo funkcionalnost ter jih testirati v mišjem modelu KVČB.

• Language: Slovenian
• Keywords: Lactococcus lactis, proteinski vezalci, površinska predstavitev, kronična vnetna črevesna bolezen, citokinska os IL-23/IL-17
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-145090
• Publication date in RUL: 05.04.2023

Secondary language

• Language: English
• Title: Development of recombinant Lactococcus lactis strains with the ability to deliver protein blockers of IL-23/IL-17 cytokine axis

Abstract

The pathogenesis of inflammatory bowel disease (IBD) depends on excessive activation of the immune response due to the unbalanced activity of T helper cells and regulatory T cells. The expansion of Th17 cells is promoted by IL-23, and the entire cascade is referred to as the IL-23/IL-17 cytokine axis. Therefore, monoclonal antibodies with immuno-regulatory effects are used in the treatment of a severe form of the disease. Due to their large and unstable structure, difficult production and immunogenic potential, non-immunoglobulin binding proteins are already being developed. Lactic acid bacteria (LAB) can be used as a delivery vehicle to avoid protein digestion, as many of the strains are able to survive the passage through the gastrointestinal tract. Probiotic LAB and delivered therapeutic molecules could have a synergistic effect on the inflammatory response in the intestine. As a part of the master's thesis, we wanted to develop recombinant Lactococcus lactis strains capable of delivering protein blockers (ARS) of the IL-17 receptor (IL-17RA). Gene cloning methods were used to prepare ARS12, ARS14, ARS19 and ARS14CO genetic constructs in plasmid pSDBA3b for either binding to the bacterial surface, or for secretion into the medium. The plasmid contained genes for the Usp45 secretion signal, the Flag-tag, and the cAcmA surface anchor in the case of the surface display. Gene expression was controlled by the addition of nisin and ARS binders were analyzed by SDS-PAGE and western blot, using anti-FLAG antibodies. Only variants anchored to the bacterial wall were detected. Surface display of ARS12, ARS14 and ARS19 proteins was then confirmed by flow cytometry. Furthermore, we wanted to develop dual functionalized L. lactis strains capable of co-expressing IL-17A binders (Fynomers) and IL-17RA binders (ARS14 and ARS19) to achieve a synergistic effect on the immune response. Co-expression was analyzed by SDS-PAGE and western blot, using anti-Flag antibodies for detection of flag-tagged ARS binders, and anti-cMyc antibodies for cMyc-tagged Fynomers. We detected anchor-containing fusion proteins as well as secreted ARS14 protein. In this work we demonstrated the ability of L. lactis bacteria to deliver IL-17A and IL-17RA binders. In the future it is necessary to evaluate their functionality and test them in a mouse model of IBD.

• Keywords: Lactococcus lactis, protein binders, surface display, inflammatory bowel disease, IL-23/IL-17 cytokine axis