Optimized doxycycline-inducible gene expression system for programming of tumor-targeting bacteria

Introduction: In programming of tumor-targeting bacteria, various therapeutic or reporter genes have been expressed by different gene-triggering strategies. Previously, we engineered an inducible bacterial drug delivery system, in which a therapeutic gene and a imging reporter gene was coexpressed by the bi-directional tet promoter system (pJL87) only in response to the administration of exogenous doxycycline (Doxy). The multicassette expression approach resulted in a 100-fold difference in the expression from tetA promoter (PTetA) and tetR promoter (PTetR). In the present study, we modified pJL87 to increase transcriptional activity of PTetR and engineered pJH18 in which the constitutive promoter regulates the expression of TetR repressor protein and Doxy dissociate TetR from tetO operator resulting in de-repression of PtetA and PtetR. The balance of the divergently oriented PTet promoters of pJH18 system was remarkably improved (PTetA:PTetR = 4-6:1 compared to that of pJL87 (PTetA:PTetR = 100:1) in transformed Salmonella. Gene expression such as renilla luciferase (rluc8, imaging reporter) or cytolysin A (ClyA, therapeutic gene) was identified specifically in the presence of Doxy, and was 80- and 5-fold stronger in the Salmonella transformed with pJH than in those with pJL87. Interestingly, pJH18 carrying bom sequence plays an essential role to prevent the plasmid-free population of programmed Salmonella from cell division. Overall, these optimizations of cancer-targeting Salmonella by transformation with pJH18 system could achieve balanced coexpression of imaging reporter or therapeutic genes and upregulate anticancer payload expression in tumor tissues with appropriate Doxy concentration, which would be more relevant for theranostic application.