Description

According to the WHO GLOBOCAN database, colorectal cancer (CRC) is the third most common cancer in men and the second most common cancer in women, with 1.8 million new cases and approximately 881,000 deaths worldwide in 20181. Despite the treatment of cancer, many results have been achieved, all these approaches have their challenges and limitations in the field of cancer therapy. Nonspecific toxicity to normal cells is one of the main challenges of conventional cancer therapy. In addition, some chemotherapeutic agents can lead to the formation of multidrug-resistant cells.23

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Figure1. E. coli lactate, pH and hypoxia induce regulatory mechanisms.

Recent work has demonstrated that microorganisms injected directly intravenously can grow selectively in tumors without adverse effects on host health4 5 6. Specifically, the ability of bacteria to selectively home to tumors has increased the use of microbes as therapeutic or diagnostic possibilities7.

In order to construct a bacterial biosensor system that can distinguish the unique organ environment, oxygen, pH and lactic acid are considered as unique indicators of colorectal cancer tumor tissue. Three operons were used to specifically recognize oxygen, pH and lactic acid. pPepT is used to sense oxygen. In the absence of oxygen, FNR binds to the \([4Fe-4S]^{2+}\) cluster to generate homologous dimers. However, in the presence of oxygen, \([4Fe-4S]^{2+}\)clusters are degraded, resulting in the dissociation of FNR dimers into inactive monomers8. L-lactic acid induction is based on lldPRD operon9.The lactic acid sensing system consists of two parts. pLldP drives the expression of interested genes. LldR repressor binds to pLldP to inhibit the expression of reporter genes, unless bound to lactic acid10 11. The pH-sensitive promoter pCadC, regulated by CadC, exhibits higher activity in acidic media than in neutral pH media.

However, many challenges limit the clinical application of current biosensor systems. In order to enable living cells to perform complex signal processing operations, amplifying genetic switches and Boolean logic gates based on serine integrase are used in the design of biosensor systems12. To give the engineered strains the therapeutic ability on tumor cells, HlyE, CCL21 and CDD-iRGD are used as therapeutic parts. HlyE is testified to able to be used as a pore-forming antitumor toxin13. CCL21 and CDD-iRGD are able to activate the immune response of host1415. In addition, the YebF tag was added to the N-end of HlyE to realize bacterial exocrine16. Next, the tumor cell adhesion module was designed: HlpA can improve the penetration of engineered bacteria into colorectal tumor cells17. In order to export HlpA-binding protein to the surface of EcN, shortened INP tag was fused into the N-end of HlpA1819. In the end, φX174E was put into the module, which can lead to bacteria lysis and death2021, resulting in the release of therapeutic factors stored inside the strain22 23 24.

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  6. Zhao, M., et al. Tumor-targeting bacterial therapy with amino acid auxotrophs of GFP-expressing Salmonella typhimurium. Proc Natl Acad Sci U S A 102, 755-760 (2005). https://doi.org/10.1073/pnas.0408422102 ↩

  7. Toso, J.F., et al. Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma. J Clin Oncol 20, 142-152 (2002). https://doi.org/10.1200/jco.2002.20.1.142 ↩

  8. Yu B, Yang M, Shi L, et al. Explicit hypoxia targeting with tumor suppression by creating an "obligate" anaerobic Salmonella Typhimurium strain. Sci Rep. 2012;2:436. https://doi.org/10.1038/srep00436 ↩

  9. Crack JC, Jervis AJ, Gaskell AA, et al. Signal perception by FNR: the role of the iron-sulfur cluster. Biochem Soc Trans. Dec 2008;36(Pt 6):1144-8. https://doi.org/10.1042/bst0361144 ↩

  10. Goers L, Ainsworth C, Goey CH, Kontoravdi C, Freemont PS, Polizzi KM. Whole-cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production. Biotechnol Bioeng. 2017;114(6):1290-1300. https://doi.org/10.1002/bit.26254 ↩

  11. Aguilera L, Campos E, Giménez R, Badía J, Aguilar J, Baldoma L. Dual role of LldR in regulation of the lldPRD operon, involved in L-lactate metabolism in Escherichia coli. J Bacteriol. Apr 2008;190(8):2997-3005. https://doi.org/10.1128/jb.02013-07 ↩

  12. Weghoff MC, Bertsch J, Müller V. A novel mode of lactate metabolism in strictly anaerobic bacteria. Environ Microbiol. Mar 2015;17(3):670-7. https://doi.org/10.1111/1462-2920.12493 ↩

  13. Schlundt A, Buchner S, Janowski R, et al. Structure-function analysis of the DNA-binding domain of a transmembrane transcriptional activator. Sci Rep. Apr 21 2017;7(1):1051. https://doi.org/10.1038/s41598-017-01031-9 ↩

  14. Viala JP, Méresse S, Pocachard B, Guilhon AA, Aussel L, Barras F. Sensing and adaptation to low pH mediated by inducible amino acid decarboxylases in Salmonella. PLoS One. 2011;6(7):e22397. https://doi.org/10.1371/journal.pone.0022397 ↩

  15. Lee YH, Kim JH, Bang IS, Park YK. The membrane-bound transcriptional regulator CadC is activated by proteolytic cleavage in response to acid stress. J Bacteriol. Jul 2008;190(14):5120-6. https://doi.org/10.1128/jb.00012-08 ↩

  16. Courbet A, Endy D, Renard E, Molina F, Bonnet J. Detection of pathological biomarkers in human clinical samples via amplifying genetic switches and logic gates. Sci Transl Med. May 27 2015;7(289):289ra83. https://doi.org/10.1126/scitranslmed.aaa3601 ↩

  17. Ryan RM, Green J, Williams PJ, et al. Bacterial delivery of a novel cytolysin to hypoxic areas of solid tumors. Gene Ther. Mar 2009;16(3):329-39. https://doi.org/10.1038/gt.2008.188 ↩

  18. Loeffler M, Le'Negrate G, Krajewska M, Reed JC. Salmonella typhimurium engineered to produce CCL21 inhibit tumor growth. Cancer Immunol Immunother. May 2009;58(5):769-75. https://doi.org/10.1007/s00262-008-0555-9 ↩

  19. Ho CL, Tan HQ, Chua KJ, et al. Engineered commensal microbes for diet-mediated colorectal-cancer chemoprevention. Nature biomedical engineering. Jan 2018;2(1):27-37. https://doi.org/10.1038/s41551-017-0181-y ↩

  20. Li Q, Yan Q, Chen J, et al. Molecular characterization of an ice nucleation protein variant (inaQ) from Pseudomonas syringae and the analysis of its transmembrane transport activity in Escherichia coli. Int J Biol Sci. 2012;8(8):1097-108. https://doi.org/10.7150/ijbs.4524 ↩

  21. Li R, Zhou T, Khan A, et al. Feed-additive of bioengineering strain with surface-displayed laccase degrades sulfadiazine in broiler manure and maintains intestinal flora structure. J Hazard Mater. 2021/03/15/ 2021;406:124440. https://doi.org/10.1016/j.jhazmat.2020.124440 ↩

  22. Prindle A, Samayoa P, Razinkov I, Danino T, Tsimring LS, Hasty J. A sensing array of radically coupled genetic'biopixels'. Nature. Dec 18 2011;481(7379):39-44. https://doi.org/10.1038/nature10722 ↩

  23. Young KD, Young R. Lytic action of cloned phi X174 gene E. J Virol. Dec 1982;44(3):993-1002. https://doi.org/10.1128/jvi.44.3.993-1002.1982 ↩

  24. Marguet P, Tanouchi Y, Spitz E, Smith C, You L. Oscillations by minimal bacterial suicide circuits reveal hidden facets of host-circuit physiology. PLoS One. Jul 30 2010;5(7):e11909. https://doi.org/10.1371/journal.pone.0011909 ↩