AI-2 quorum sensing controlled delivery of cytolysin-A by tryptophan auxotrophic low-endotoxic Salmonella and its anticancer effects in CT26 mice with colon cancer

Ram Prasad Aganja; Chandran Sivasankar; John Hwa Lee

doi:10.1016/j.jare.2023.09.003

AI-2 quorum sensing controlled delivery of cytolysin-A by tryptophan auxotrophic low-endotoxic Salmonella and its anticancer effects in CT26 mice with colon cancer

J Adv Res. 2023 Sep 7:S2090-1232(23)00238-2. doi: 10.1016/j.jare.2023.09.003. Online ahead of print.

Authors

Ram Prasad Aganja¹, Chandran Sivasankar¹, John Hwa Lee²

Affiliations

¹ Department of Veterinary Public Health, College of Veterinary Medicine, Jeonbuk National University, Iksan 54596, South Korea.
² Department of Veterinary Public Health, College of Veterinary Medicine, Jeonbuk National University, Iksan 54596, South Korea. Electronic address: johnhlee@jbnu.ac.kr.

PMID: 37689243
DOI: 10.1016/j.jare.2023.09.003

Abstract

Introduction: The limitations of conventional cancer therapies necessitate target-oriented, highly invasive, and safe treatment approaches. Hence, the intrinsic anti-tumor activity of Salmonella can offer better options to combat cancers.

Objectives: This study aims to utilize attenuated Salmonella and deliver cytolytic protein cytolysin A (ClyA) under quorum sensing (QS) signaling for precise localized expression in tumors but not in healthy organs.

Methods: The therapeutic delivery strain was imposed with tryptophan auxotroph for selective colonization in tumors by trpA and trpE deletion, and lipid-A and O-antigen were altered by pagL and rfaL deletions using lambda red recombination method. The strain was transformed with the designed QS-controlled ClyA expression vector which was validated by western blot. The in vivo passaged therapeutic strain was used for treatment four times at a weekly interval, with a dose of 5 × 10⁶ CFU/mouse for cancer therapy.

Results: The attenuated strain induced minimal endotoxicity-related cytokines TNF-α, IL-1β, and IFN-γ and exhibited adequate colonization despite earlier exposure in mice. The QS-controlled ClyA expression was confirmed by western blot from bacterial cultures grown at different cell densities. The results demonstrated that the in vivo passaged strain preferentially colonized the tumor after vacating the spleen, liver, and lung, leaving no outward histological scars. The anti-cancer effect of the designed construct was evaluated in the murine CT26 colon cancer model. The expression of ClyA increased tumoricidal activity by 67 % compared to vector control.

Conclusion: Hence, the anti-tumor effect of the engineered Salmonella strain was improved by ClyA expression via QS activation after achieving the threshold bacterial cell density. Further, immunohistochemical staining of the tumor and other organs corroborated the QS-controlled tumor-specific expression of ClyA. Overall, the results imply that the developed anti-cancer Salmonella has low endotoxicity and QS-controlled expression of ClyA as beneficial safety elements and supports recurrent Salmonella inoculation by O-antigen deficiency.

Keywords: Cytolysin A; Quorum sensing; Reduced endotoxicity; Salmonella; Tryptophan auxotroph; Tumoricidal activity.