Robust inducible gene expression in intracellular Listeria monocytogenes in vivo

Attenuated strains of the intracellular pathogen Listeria monocytogenes can deliver genetically encoded payloads inside tumor cells. L. monocytogenes preferentially accumulates and propagates inside immune-suppressed tumor microenvironments. To maximize the payload impact in tumors and minimize damage to healthy tissues, it is desirable to induce payload synthesis when bacteria are eliminated from the healthy tissues but are grown to high numbers intratumorally. Here, we have engineered a tightly controlled gene expression system for intracellular L. monocytogenes inducible with a cumin derivative, cumate. Upon cumate addition, expression of a reporter gene is increased in L. monocytogenes growing in vitro by 80-fold, and in intracellular L. monocytogenes in murine tumors by 10-fold. This study demonstrates the feasibility of activating gene expression in intracellular bacteria in live animals using an edible inducer. The system is expected to enhance the efficacy and safety of the attenuated L. monocytogenes strains as antitumor payload delivery bacterial drones.