Design
Our engineered microbe incorporates four independent modules, which are designed to realize a series of functions from CRC targeting and ultrasonic reporting to preliminary treatment and finally, removal from the digestive tract.
The CRC targeting module enables bacteria to specifically adhere to CRC lesions in the lumen. To this end, E.coli is designed to display a CRC antigen binding moiety on its surface, through which it can be tethered on the foci of CRC. For surface display vectors, we choose two engineered bacterial outer membrane proteins—OmpA (BBa_K1489002) and ice nucleation protein [1] Reference 1 [1]Bao, S., Yu, S., Guo, X., Zhang, F., Sun, Y., & Tan, L., et al. (2015). Construction of a cell-surface display system based on the n-terminal domain of ice nucleation protein and its application in identification of mycoplasma adhesion proteins. Journal of Applied Microbiology, 119(1), 236-244. , which are commonly used as carriers of protein/peptide to the outer surface of gram-negative bacteria.
As for the antigens to be targeted, there exist few choices. Despite the abundance of antigens we could think of regarding CRC, such as CEA and EGFR, the mucus layer in the gut prevents direct contact between cancer cells and microorganism. Therefore, a mucus related CRC antigen--Thomsen-Friedenreich (TF-antigen), is chosen as the target. TF-antigen is an epitope on mucin specifically secreted by most CRC cells, against which various targeting nanoprobes have been developed as optical imaging agents for early-stage CRC to assist colonoscopy [2] Reference 2 [2]Kumagai, H., Pham, W., Kataoka, M., Hiwatari, K., Mcbride, J., & Wilson, K. J., et al. (2013). Multifunctional nanobeacon for imaging thomsen-friedenreich antigen-associated colorectal cancer. International Journal of Cancer, 132(9), 2107-2117. [3] Reference 3 [3]Nakase, H., Sakuma, S., Fukuchi, T., Yoshino, T., Mohri, K., & Miyata, K., et al. (2017). Evaluation of a novel fluorescent nanobeacon for targeted imaging of thomsen-friedenreich associated colorectal cancer:. International Journal of Nanomedicine, 12, 1747-1755. .
For TF-antigen targeting, we find out two oligopeptides from a phage display library that bind specifically to TF-antigen [4] Reference 4 [4]Liu, R., Li, X., Xiao, W., & Lam, K. S. (2017). Tumor-targeting peptides from combinatorial libraries. Adv Drug Deliv Rev, 110-111, 13-37. . Moreover, these relatively hydrophilic peptides are supposed to inhibit non-specific interactions between the bacteria to the mucosa [3] Reference 3 [3]Nakase, H., Sakuma, S., Fukuchi, T., Yoshino, T., Mohri, K., & Miyata, K., et al. (2017). Evaluation of a novel fluorescent nanobeacon for targeted imaging of thomsen-friedenreich associated colorectal cancer:. International Journal of Nanomedicine, 12, 1747-1755. [5] Reference 5 [5]Lukic, J., Strahinic, I., Milenkovic, M., Nikolic, M., Tolinacki, M., & Kojic, M., et al. (2014). Aggregation factor as an inhibitor of bacterial binding to gut mucosa. Microbial Ecology, 68(3), 633-644. .
The ultrasonic reporting module contains a cluster of engineered gas vesicle proteins (ARG) that could assemble to form gas vesicles in the cytoplasm of E.coli, which are able to generate signals upon high-resolution ultrasonic inspection [6] Reference 6 [6]Bourdeau, R. W., Leegosselin, A., Lakshmanan, A., Farhadi, A., Kumar, S. R., & Nety, S. P., et al. (2018). Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts. Nature,553(7686), 86. . The expression of these gas vesicles is controlled by T7 lac promoter. After introduction into the gut, these gas vesicles would indicate the location of our engineered bacteria via ultrasonic response.
The suicide module is for removing the engineered bacteria from the digestive tract right after the examination, in a bid to eliminate the risk of, for example, interfering gut flora as much as possible. Considering that arabinose would serve as a safe and effective inducer of bacterial gene expression in the gut, we place the phage protein φX174E (BBa_K2500006), which triggers bacteria cell lysis under control of arabinose operon, to achieve an arabinose-governed bacterial cell death.
The therapeutic module aims for a preliminary bio-treatment of CRC after its diagnosis. This module is to be introduced into another strain, which is employed for treatment only, together with the CRC targeting module and suicide module. As a minor part of our project, the therapeutic module mainly consists of a well established anticancer agent—a peptide derived from azurin (BBa_K2500002), used by ETH Zurich 2017. In order to produce the agent in a CRC specific manner, a nitric oxide responsive promoter (pYeaR/pNorV) manipulates its expression, since nitric oxide is an unstable molecule overproduced by CRC cells and polyps, with its concentration highest around these lesions [7] Reference 7 [7]Cianchi, F., Cortesini, C., Fantappiè, Messerini, L., Schiavone, N., & Vannacci, A., et al. (2003). Inducible nitric oxide synthase expression in human colorectal cancer: correlation with tumor angiogenesis. American Journal of Pathology, 162(3), 793-801. [8] Reference 8 [8]Keum, D. H., Jung, H. S., Wang, T., Shin, M. H., Kim, Y. E., & Kim, K. H., et al. (2015). Microneedle Biosensor for Real-Time Electrical Detection of Nitric Oxide for In Situ Cancer Diagnosis During Endomicroscopy. Advanced Healthcare Materials, 1153-1158. . The targeting module would work in synergy, and the suicide module releases the agent from the cells.
Reference
[1]Bao, S., Yu, S., Guo, X., Zhang, F., Sun, Y., & Tan, L., et al. (2015). Construction of a cell-surface display system based on the n-terminal domain of ice nucleation protein and its application in identification of mycoplasma adhesion proteins. Journal of Applied Microbiology, 119(1), 236-244.
[2]Kumagai, H., Pham, W., Kataoka, M., Hiwatari, K., Mcbride, J., & Wilson, K. J., et al. (2013). Multifunctional nanobeacon for imaging thomsen-friedenreich antigen-associated colorectal cancer. International Journal of Cancer, 132(9), 2107-2117.
[3]Nakase, H., Sakuma, S., Fukuchi, T., Yoshino, T., Mohri, K., & Miyata, K., et al. (2017). Evaluation of a novel fluorescent nanobeacon for targeted imaging of thomsen-friedenreich associated colorectal cancer:. International Journal of Nanomedicine, 12, 1747-1755.
[4]Liu, R., Li, X., Xiao, W., & Lam, K. S. (2017). Tumor-targeting peptides from combinatorial libraries. Adv Drug Deliv Rev, 110-111, 13-37.
[5]Lukic, J., Strahinic, I., Milenkovic, M., Nikolic, M., Tolinacki, M., & Kojic, M., et al. (2014). Aggregation factor as an inhibitor of bacterial binding to gut mucosa. Microbial Ecology, 68(3), 633-644.
[6]Bourdeau, R. W., Leegosselin, A., Lakshmanan, A., Farhadi, A., Kumar, S. R., & Nety, S. P., et al. (2018). Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts. Nature,553(7686), 86.
[7]Cianchi, F., Cortesini, C., Fantappiè, Messerini, L., Schiavone, N., & Vannacci, A., et al. (2003). Inducible nitric oxide synthase expression in human colorectal cancer: correlation with tumor angiogenesis. American Journal of Pathology, 162(3), 793-801.
[8]Keum, D. H., Jung, H. S., Wang, T., Shin, M. H., Kim, Y. E., & Kim, K. H., et al. (2015). Microneedle Biosensor for Real-Time Electrical Detection of Nitric Oxide for In Situ Cancer Diagnosis During Endomicroscopy. Advanced Healthcare Materials, 1153-1158.
