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 circuit design.
This year, we chose Salmonella enterica serovar Typhimurium str. SL1344 as our chassis. Why we chose Salmonella as our carrier is based on the following reasons. First, GSDMD-N275 can only induce pyroptosis from the inside of a cell, therefore Salmonella is a brilliant candidate as an intracellular parasite. Second, Salmonella is a widely used carrier to cancer therapy because its natural taxis to tumor (Figure 2). However, feedbacks from human practice suggested that we should consider more about the safety in our 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.
Figure 2. Salmonella has natural taxis to tumor.
sifA locates in Salmonella pathogenicity island, taking the role of maintaining the stability of Salmonella-Containing Vacuole (SCV) when Salmonella survive and replicate in host cells. Because of the unstable SCV, growth inhibition of ΔsifA mutant in macrophage is remarkable1. Thus, to reduce virulence of Salmonella, sifA was knocked out in our project (Figure 3).
Figure 3. Schematic diagram of sifA mutant in macrophage.
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 in cancer cells and blood vessels during cancer angiogenesis2. In order to enhance targeting of bacteria to tumor, RGD motif is displayed on OmpA, an outer membrane protein of bacteria (Figure 4).
Finally, the safety of our project is successfully demonstrated by a set of experiments using engineered bacteria mentioned above. (See more details in Results.)
Figure 4. 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 downstream gene3. With the presence of anhydrotetracycline (ATc), the repressor TetR which is under the control of tet promoter (Ptet) will combine with ATc and Mg2+, resulting in expression of GSDMD-N275 (Figure 5). Finally, this system is successfully used to express GSDMD-N275 in Salmonella and induces host cell pyroptosis. (See more details in Results.)
Figure 5. Schematic diagram of ATc-dependent expression of GSDMD-N275.
As an intracellular parasite, some intracellular environment-dependent genes such as sifA exist in Salmonella4. Therefore, this feature gives an approach for us to implement specific expression of GSDMD-N275. We utilized the regulatory part from the upstream of sifA (PsifA) to control the expression of GSDMD-N275 (Figure 6). Ultimately, we successfully demonstrated the intracellular specificty of PsifA. (See more details in Results.)
Figure 6. Schematic diagram of intracellular environment-dependent expression of GSDMD-N275.
1. Thurston, T. L. et al. Growth inhibition of cytosolic Salmonella by caspase-1 and caspase-11 precedes host cell death. Nature communications 7, 13292, doi:10.1038/ncomms13292 (2016).
2. Danhier, F., Le Breton, A. & Preat, V. RGD-based strategies to target alpha(v) beta(3) integrin in cancer therapy and diagnosis. Mol Pharm 9, 2961-2973, doi:10.1021/mp3002733 (2012).
3. Nevozhay, D. Negative autoregulation linearizes the dose–response and suppresses the heterogeneity of gene expression. PNAS 106 5123-5128, doi:10.1073/pnas.0809901106 (2008).
4. Garmendia, J., Beuzon, 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, doi:10.1099/mic.0.26397-0 (2003).
Overview
Chassis
ATc-dependent expression of GSDMD-N275
Intracellular environment-dependent expression of GSDMD-N275
Reference
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