Infections with shiga toxin-producing bacteria, like enterohemorrhagic Escherichia coli and Shigella dysenteriae, represent a serious medical problem. No specific and effective treatment is available for patients with these infections, creating a need for the development of new therapies. Recombinant lactic acid bacterium Lactococcus lactis was engineered to bind Shiga toxin by displaying novel designed albumin binding domains (ABD) against Shiga toxin 1 B subunit (Stx1B) on their surface. Functional recombinant Stx1B was produced in Escherichia coli and used as a target for selection of 17 different ABD variants (named S1B) from the ABD scaffold-derived high-complex combinatorial library in combination with a five-round ribosome display. Two most promising S1Bs (S1B22 and S1B26) were characterized into more details by ELISA, surface plasmon resonance and microscale thermophoresis. Addition of S1Bs changed the subcellular distribution of Stx1B, completely eliminating it from Golgi apparatus most likely by interfering with its retrograde transport. All ABD variants were successfully displayed on the surface of L. lactis by fusing to the Usp45 secretion signal and to the peptidoglycan-binding C terminus of AcmA. Binding of Stx1B by engineered lactococcal cells was confirmed using flow cytometry and whole cell ELISA. Lactic acid bacteria prepared in this study are potentially useful for the removal of Shiga toxin from human intestine.
- MeSH
- albuminy metabolismus MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- ELISA MeSH
- HeLa buňky MeSH
- imobilizované proteiny metabolismus MeSH
- Lactococcus lactis metabolismus MeSH
- lidé MeSH
- metody zobrazení buněčného povrchu MeSH
- podjednotky proteinů metabolismus MeSH
- povrchová plasmonová rezonance MeSH
- proteinové domény MeSH
- průtoková cytometrie MeSH
- rekombinace genetická genetika MeSH
- rekombinantní proteiny metabolismus MeSH
- ribozomy metabolismus MeSH
- sekvenční homologie aminokyselin MeSH
- shiga toxin 1 chemie metabolismus MeSH
- transport proteinů MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The virulence of the Candida pathogens is enhanced by the production of secreted aspartic proteases, which therefore represent possible targets for drug design. Here, the crystal structure of the secreted aspartic protease Sapp2p from Candida parapsilosis was determined. Sapp2p was isolated from its natural source and crystallized in complex with pepstatin A, a classical aspartic protease inhibitor. The atomic resolution of 0.83 Å allowed the protonation states of the active-site residues to be inferred. A detailed comparison of the structure of Sapp2p with the structure of Sapp1p, the most abundant C. parapsilosis secreted aspartic protease, was performed. The analysis, which included advanced quantum-chemical interaction-energy calculations, uncovered molecular details that allowed the experimentally observed equipotent inhibition of both isoenzymes by pepstatin A to be rationalized.
- MeSH
- aspartátové proteasy chemie genetika izolace a purifikace metabolismus MeSH
- Candida chemie enzymologie genetika MeSH
- exprese genu MeSH
- fungální proteiny chemie genetika izolace a purifikace metabolismus MeSH
- inhibitory proteas chemie MeSH
- izoenzymy chemie genetika izolace a purifikace metabolismus MeSH
- katalytická doména MeSH
- kinetika MeSH
- krystalografie rentgenová MeSH
- kvantová teorie MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- pepstatiny chemie MeSH
- sekundární struktura proteinů MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- strukturní homologie proteinů MeSH
- substrátová specifita MeSH
- termodynamika MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
