Targeted deletion of thearaoperon ofSalmonella typhimuriumenhances L-arabinose accumulation and drives P_BAD-promoted expression of anti-cancer toxins and imaging agents

Abstract

Tumor-specific expression of antitumor drugs can be achieved using attenuated Salmonella typhimurium harboring the PBAD promoter, which is induced by L-arabinose. However, L-arabinose does not accumulate because it is metabolized to D-xylulose-5-P by enzymes encoded by the ara operon in Salmonellae. To address this problem, we developed an engineered strain of S. typhimurium in which the ara operon is deleted. Linear DNA transformation was performed using λ red recombinase to exchange the ara operon with linear DNA carrying an antibiotic-resistance gene with homology to regions adjacent to the ara operon. The ara operon-deleted strain and its parental strain were transformed with a plasmid encoding Renilla luciferase variant 8 (RLuc8) or cytolysin A (clyA) under the control of the PBAD promoter. Luciferase assays demonstrated that RLuc8 expression was 49-fold higher in the ara operon-deleted S. typhimurium than in the parental strain after the addition of L-arabinose. In vivo bioluminescence imaging showed that the tumor tissue targeted by the ara operon-deleted Salmonella had a stronger imaging signal (~30-fold) than that targeted by the parental strain. Mice with murine colon cancer (CT26) that had been injected with the ara operon-deleted S. typhimurium expressing clyA showed significant tumor suppression. The present report demonstrates that deletion of the ara operon of S. typhimurium enhances L-arabinose accumulation and thereby drives PBAD-promoted expression of cytotoxic agents and imaging agents. This is a promising approach for tumor therapy and imaging.

Metabolomics of primary cutaneous melanoma and matched adjacent extratumoral microenvironment

“…The attenuated strain is engineered to exclude the ara operon that is responsible for metabolizing arabinose to D-xylulose-5-phosphate. When affected mice were treated with the operon-deleted strain, arabinose accumulation resulted, facilitating expression of the cytotoxic protein cytolysin A in the tumors [ 33 ]. In conjunction with arabinose supplementation, such therapies may show promise among earlier staged melanomas, as well as in the treatment of metastatic melanomas that accumulate high levels of arabinose.…”

Salmonella-Mediated Cancer Therapy: Roles and Potential

“…In our study, the pBAD promoter controlled toxin gene expression until the administration of L-arabinose by intravenous or intraperitoneal injection [7]. Deletion of the ara operon of Salmonella inhibited L-arabinose metabolism, leading to the activation of the pBAD promoter following induction [71].…”

“…Overcoming these obstacles is important to move BMCT closer to clinical application in the future. [7,10,71,72] pTet Tetracylin [30] RecA Radiation [12] Quorum sensing Bacterial density [72,73] Hypoxia-inducing promoters Low oxygen condition [11,20,72,74,75] …”

Tuning Salmonella for cancer therapy

“…[1][2][3] In this issue of Cell Cycle, Min and colleagues report the enhanced expression of inducible cytoxins and imaging agents in Salmonella typhimurium, thereby increasing the sensitivity in visualization of tumors and therapeutic efficacy. 4 For tumor-specific expression of cytotoxins with reduced toxicity against normal cells, they used P BAD promoter for the attenuated Salmonella typhimurium as an inducible system, which can be activated by L-arabinose. 5 However, L-arabinose does not accumulate because it is metabolized by enzymes encoded by the ara operon in Salmonellae.…”