Abstract
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.
Highlights
We present here the first protocol to quantify the ability of activated prodrugs to exit a bacterial activator cell, based on coculture of an activator strain expressing a prodrug converting enzyme with a recipient strain that can quantify drug-induced damage
The ability of activated prodrug metabolites to transfer from activating cells to neighbouring cells is a critical aspect of enzyme-prodrug therapies [1]
We developed a model that employs bacterial activator cells in co-culture with nitroreductase-null SOS-GFP recipient cells
Summary
The ability of activated prodrug metabolites to transfer from activating cells to neighbouring cells (i.e., the bystander effect) is a critical aspect of enzyme-prodrug therapies [1]. An effective model to assay the abilities of different prodrugs to exit an activating bacterial cell has not previously been described. We developed a model that employs bacterial activator cells (over-expressing a prodrug-converting nitroreductase) in co-culture with nitroreductase-null SOS-GFP recipient cells The 7NT activator strain was transformed with pUCX expressing a prodrug-converting nitroreductase, and transformed with empty pCDFDuet plasmid to confer spectinomycin resistance. The recipient strain was SOS-R4 (7NT pre-transformed with pANODuet, a spectinomycin resistant plasmid expressing a gfp reporter gene under control of the SOS-responsive sfiA promoter), further transformed with empty pUCX to confer ampicillin resistance. For nitroreductase assays metronidazole has a negligible bacterial bystander effect [3] and can be used as a nil bystander control to compare to other prodrugs
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