Team:HZAU-China/Design
In our project, we redesigned Salmonella to act as a delivery vehicle that can target tumor cells and replicate in their cytoplasm. By inducing the bacterial expression of the N-terminal domain of Gasdermin D (GSDMD-N275), bacteria are led to lysis and release this protein into the cytoplasm of tumor cell and then induce pyroptosis to the tumor cell by making membrane pores. The lysate of cell rupture during pyroptosis destroys the tumor microenvironment and attracts immune cells into tumor bed to kill tumor cells. Our project which aims to induce pyroptosis to tumor cells provides a new approach for cancer therapy (Figure 1).
Figure 1. Overall Design Circuit
This year, we chose Salmonella enterica serovar Typhimurium str. SL1344 as our chassis. The reason why we chose Salmonella as our carrier are based on following reason. First, in consideration of GSDMD-N275 induced pyroptosis only happens when delivered cytosolically but not extracellularly, Salmonella is a brilliant candidate as an intracellular parasite. Second, Salmonella is a widely used vector to cancer therapy because its natural taxis to tumor. However, feedbacks from human practice suggested that we should consider more about our safety in design and experiment. (See more details in Human Practice.) Therefore, we make efforts to improve safety of our project through knocking out sifA and displaying RGD motif on Salmonella.
sifA locates in Salmonella pathogenicity island, taking the role of maintaining the stability of Salmonella-Containing Vacuole (SCV) where Salmonella survive and replicate in host cells. Because of the unstable SCV, growth inhibition of ΔsifA mutant in macrophage is remarkable. Thus, with the view of reducing virulence of Salmonella, sifA was knocked out in our project.
RGD motif (Arg-Gly-Asp) is a well-studied tumor homing tripeptide that specifically binds to alpha v beta 3 (αvβ3) integrin, which is a biomarker of cancer cells and widely overexpressed on cancer cells and blood vessels during cancer angiogenesis. In order to enhance targeting of bacteria to tumor, RGD motif is displayed on OmpA, an outer membrane protein of bacteria (Figure 2).
Finally, the safety of our project is successfully demonstrated by a set of experiments using engineered bacteria mentioned above. (See more details in Result.)
Figure 2. Realization of tumor targeting through surface displaying RGD motif.
We use anhydrotetracycline transcriptional regulation system to regulate the expression of GSDMD-N275 in our project because of its low expression noise, high response speed and great linear relation between the inducer and the expression of upstream gene. With the presence of anhydrotetracycline (ATc), repressor TetR which under the control of tet promoter (Ptet) will integrate with ATc and Mg2+ result in expression of GSDMD-N275 (Figure 3). Finally, this system is successfully used to express GSDMD-N275 in Salmonella and induce host cell pyroptosis. (See more details in Result.)
As an intracellular parasite, some intracellular environment-dependent genes such as sifA are existed in Salmonella. Therefore, this event shows an approach for us to implement specific expression of GSDMD-N275. We utilized regulatory part from the upstream of sifA (PsifA) to control the expression of GSDMD-N275 (Figure 4). Ultimately, we successfully demonstrated the intracellular specificty of PsifA .(See more details in Result.)
Figure 4. Design of Our Circuits
1. Garmendia, J., Beuzón, C. R., Ruiz-Albert, J. & Holden, D. W. The roles of SsrA-SsrB and OmpR-EnvZ in the regulation of genes encoding the Salmonella typhimurium SPI-2 type III secretion system. Microbiology 149, 2385–2396 (2003).
2. Beuzon, C. R. Salmonella maintains the integrity of its intracellular vacuole through the action of SifA. EMBO J. 19, 3235–3249 (2000).
3. Steele-Mortimer, O. The Salmonella-containing vacuole-Moving with the times. Curr. Opin. Microbiol. 11, 38–45 (2008).
4. Nevozhay, D., Adams, R. M., Murphy, K. F., Josic, K. & Balazsi, G. Negative autoregulation linearizes the dose-response and suppresses the heterogeneity of gene expression. Proc. Natl. Acad. Sci. 106, 5123–5128 (2009).
5. Shi, H. & Wen Su, W. Display of green fluorescent protein on Escherichia coli cell surface. Enzyme Microb. Technol. 28, 25–34 (2001).
6. Earhart, C. F. Use of an Lpp-OmpA Fusion Vehicle for Bacterial Surface Display. Methods Enzymol. 326, 506–516 (2000).
7. Desgrosellier, J. S. & Cheresh, D. A. Integrins in cancer: Biological implications and therapeutic opportunities. Nat. Rev. Cancer 10, 9–22 (2010).
8. Cuthbertson, L. & Nodwell, J. R. The TetR Family of Regulators. Microbiol. Mol. Biol. Rev. 77, 440–475 (2013).
9. Kisker, C., Hinrichs, W., Tovar, K., Hillen, W. & Saenger, W. The complex formed between Tet repressor and tetracycline-Mg2+ reveals mechanism of antibiotic resistance. J. Mol. Biol. 247,260–280 (1995).
10. Nevozhay, D., Adams, R. M., Murphy, K. F., Josic, K. & Balazsi, G. Negative autoregulation linearizes the dose-response and suppresses the heterogeneity of gene expression. Proc. Natl. Acad. Sci. 106, 5123–5128 (2009).
11. Crouch, M. L. V, Castor, M., Karlinsey, J. E., Kalhorn, T. & Fang, F. C. Biosynthesis and IroC-dependent export of the siderophore salmochelin are essential for virulence of Salmonella enterica serovar Typhimurium. Mol. Microbiol. 67, 971–983 (2008).
