Protocol for evaluating the abilities of diverse nitroaromatic prodrug metabolites to exit a model Gram negative bacterial vector

Bacterial-directed enzyme-prodrug therapy (BDEPT) uses tumour-tropic bacteria armed with a genetically-encoded prodrug-converting enzyme to sensitise tumours to a systemically-administered prodrug. A strong bystander effect (i.e., efficient bacteria-to-tumour transfer of activated prodrug metabolites) is critical to maximise tumour cell killing and avoid bacterial self-sterilisation. To investigate the bystander effect in bacteria we developed a sensitive screen that utilised two Escherichia coli strains grown in co-culture. The first of these was an activator strain that overexpressed the E. coli nitroreductase NfsA, and the second was a nitroreductase null recipient strain bearing an SOS-GFP DNA damage responsive gene construct. In this system, induction of GFP by genotoxic prodrug metabolites can only occur following their transfer from the activator to the recipient cells. This can be monitored both in fluorescence based microtitre plate assays and by flow-cytometry, enabling modelling of the abilities of diverse nitroaromatic prodrug metabolites to exit a Gram negative vector.