Team:Freiburg/Delivery
Figure 1: Depiction of GST displaying spores. Conversion process of CDNB and GSH to G-SDNB by GST displaying spores.
Displaying a functional enzymatic moiety on the surface of the spores represents an essential feature for the activation of prodrugs. We evaluated glutathione-S-transferase (GST) displaying spores as potential carrier for the activation of azathioprine and verified their functionality in a colorimetric GST assay. The spores displaying GST exhibited an increased enzymatic activity in the conjugation of reduced glutathione(GSH) to 1-chloro-2,4-dinitrobenzene (CDNB) compared to unmodified spores and therefore provide evidence for their feasibility as a carrier for targeted drug delivery.
Introduction
The treatment of ulcerative colitis with the prodrug azathioprine and the conversion to its active form 6-mercaptopurine in the liver by glutathione S-transferase results in a systemic drug dispersion throughout the whole body, greatly harming healthy tissues as well as diseased cells. As a base analog 6-mercaptopurine inhibits the nucleic acid synthesis by inhibition of the purine metabolism, thus leading to apoptosis of highly proliferating cells. To ensure the local activation of the prodrug we modified the spores of B. subtilis to display a functional GST on their surface, which facilitates the activation of azathioprine. This approach provides that the enzymatic activity can be delivered to the affected sites in the gut and promotes the local treatment. Therefore, considerably lower amounts of the prodrug can be administered resulting in a reduction of systemic side effects. In order confirm the feasibility of those modification analysis of the proper display of GST and its functionality is required.
Results
To determine the functionality of the displayed GST on the spores we first verified the localization on the surface of the spore. We assembled an integration vector containing a construct with GST fused to the spore coat gene cotG and a hemagglutinin epitope tag (BBa_K2114011), which was driven by the PCotYZ-RBS promoter (BBa_K2114000) and transformed them into B. subtilis. After selection the resulting spores should display GST on their surface. We verified the proper localization of the fusion protein by immunostaining with anti-HA antibodies conjugated to Alexa Fluor 647 and flow cytometry (Figure 2). Spores bound by the conjugated antibody appeared at a higher fluorescence intensity in the scatter plot. We observed two distinguishable populations, suggesting the presence of different stages in the spores.
