Our project Gut Matters is a comprehensive solution that covers multiple current issues in the fight against IBD. We briefly divided our project into three parts: a Diagnostic Tool, a Treatment Approach, and a Delivery Strategy.
Diagnosis Tool
Currently, the diagnosis of IBD relies on a combination of clinical, endoscopic, radiologic, and histologic criteria[1]. A molecular diagnosis method, therefore, will facilitate the early diagnosis and clinical intervention of IBD, which can greatly reduce patients' sufferings. We here propose an RNA biomarker-based molecular diagnosis method named Proximity-labelling Assisted and CRISPR-Cas Inspired RNA Targeting (Pro-LAC) system . Pro-LAC utilizes a gRNA-guided fusion protein with a miniSOG domain that, upon blue light activation, can specifically label the guanosine on biomarker RNA with biotin-NH2 probes, thus making them detectable through a simple lateral flow assay. Hopefully, Pro-LAC can become a fast, easy-to-use household tool for IBD early diagnosis.
Fig.1 The progress of Pro-LAC specific RNA detection system
Treatment Approach
This is the key component of our project. We identified three critical therapeutic targets in IBD: damage of the intestine surface, dysbacteriosis of the intestinal microflora, and imbalance of intestinal metabolites. Our treatment approach accordingly consists of: Therapeutic Proteins Secretion Project to promote intestinal tract surface recovery, Microcin Regulation Project to modulate intestinal microflora composition, and Bile acid Balance Regulation Project to regain intestinal metabolic balance.
Therapeutic Proteins Secretion Project (Mucosal healing)
Many cytokines and growth factors are known to promote the healing process of the intestine surface. So the basic idea here is to make E. coli Nissle 1917 (EcN) produce these proteins inside the patient's intestine when there is inflammation caused by IBD, through the construction of a conditional protein secretion system. The system mainly consists of 4 parts: Sensor Element, Secretion Peptide, Linker, and Therapeutic proteins.
Fig.2 The construction of a conditional protein secretion system
This protein secretion system is modular, in that there are many alternative choices for its basic parts. We have found several kinds of sensors, secretion peptides and treatment proteins compatible with it. Thus, these modules can generate a great variety of combinations. We expect that, for example, when sensing the inflammation signal NO, thiosulfate or tetrathionate, the engineered E. coli Nissle 1917 would produce a specific kind of treatment proteins (e.g. trefoil factors, aka TFF) and secrete them into the intestinal tract with the help of secretion peptides. Hopefully, the treatment proteins produced by this quaternary system will be able to alleviate IBD in situ.
Fig.3 Stimulation of EcN causes transcription of therapeutic proteins
Microcin Regulation Project (Microbiota regulation)
Adherent-invasive Escherichia coli (AIEC) is one of the pathogenic bacteria that may be the causing agents of IBD. They can adhere to intestinal epithelial cells and then invade them. Microcins, a group of secretion peptides generated by certain E. coli strains, can bind to the ATPase on other related strains’ plasma membrane and inhibit their growth - including AIECs, which is why a lot of these strains are considered probiotic. In addition, the antimicrobial microcins can also keep its producer from being outcompeted by other bacteria. E. coli Nissle 1917 (EcN) already possesses a microcin expression system that can produce microcin MccH47, but it is only turned on under certain nutrient depletion conditions[2],such as iron starvation. The idea here, therefore, is to endow EcN with the ability to controllably produce MccH47, by introducing into it the microcin expression system of E.coli CA46, which has a more stable production and has been better characterized previously. This engineered EcN can then inhibit pathogenic bacteria in intestine, including AIEC. Hopefully, it will help IBD patients recover by regulating the composition of their gut microflora.
Bile acid Balance Regulation Project (Metabolic balancing)
It is now widely accepted that microbial metabolites play an important intermediate role in the crosstalk between intestinal bacteria and the human body. Bile acids are among the key metabolite groups that may greatly affect many physiological processes, but are typically dysregulated in IBD patients. Specifically, IBD pateints often exhibit an imbalance between primary and secondary bile acids in the gut, which is caused by reduced activity of bile salt hydrolase (BSH), an enzyme that hydrolyses the conjugated primary bile acids into free bile acids. The idea here, then, is to engineer a Lactococcus lactis strain so that it can reside in the intestine and supplement BSH. This would be achieved by expressing a Lactobacillian BSH and a Listerian adhesion protein in L. lactis. Hopefully, our engineered L.lactis can restore the balance between primary and secondary bile acids, thus improving the intestinal metabolic environment to facilitate IBD treatment.
Delivery Strategy
A prerequisite for our engineered bacteria to carry out their jobs in the intestine is that they must first survive in the environment of the digestive tract, for instance low pH and multiple digestive enzymes. In fact, the efficiency of delivery has always been a major issue in designing orally-administered probiotics. Here, we plan to utilize a novel cell-coating technique to increase the survival ability of our engineered bacteria through the digestive tract. This new technique works by co-depositing chitosan with polydopamine on the cell surface through chemical self-assembly, and has been reported to be a convenient and effective way to protect engineered bacteria against harsh physical environment. In our project, hopefully, this delivery strategy can effectively protect our engineered bacteria on their way to their final workplace.
Fig.3 Delivery strategies protect our engineered bacteria from digestion
Team:Tsinghua/Design
Design
Reference
[1] Wang, P., Tang, W., Li, Z., Zou, Z., Zhou, Y., Li, R., Xiong, T., Wang, J., & Zou, P. (2019). Mapping spatial transcriptome with light-activated proximity-dependent RNA labeling. Nat. Chem. Biol., 15(11), 1110–1119. https://doi.org/10.1038/s41589-019-0368-5
[2] Ding, T., Zhu, L., Fang, Y., Liu, Y., Tang, W., & Zou, P. (2020). Chromophore-Assisted Proximity Labeling of DNA Reveals Chromosomal Organization in Living Cells. Angew. Chem. Int. Ed., 59(51), 22933–22937. https://doi.org/10.1002/anie.202005486
[3]Schultz, M., Watzl, S., Oelschlaeger, T. A., Rath, H. C., Göttl, C., Lehn, N., Schölmerich, J., & Linde, H. J. (2005). Green fluorescent protein for detection of the probiotic microorganism Escherichia coli strain Nissle 1917 (EcN) in vivo. Journal of microbiological methods, 61(3), 389–398. https://doi.org/10.1016/j.mimet.2005.01.007
[4]Joeres-Nguyen-Xuan, T. H., Boehm, S. K., Joeres, L., Schulze, J., & Kruis, W. (2010). Survival of the probiotic Escherichia coli Nissle 1917 (EcN) in the gastrointestinal tract given in combination with oral mesalamine to healthy volunteers. Inflammatory bowel diseases, 16(2), 256–262. https://doi.org/10.1002/ibd.21042
[5]Vanz, A. L. , Renard, G. , Palma, M. S. , Chies, J. M. , Dalmora, S. L. , & Basso, L. A. , et al. (2008). Human granulocyte colony stimulating factor (hg-csf): cloning, overexpression, purification and characterization. Microbial Cell Factories, 7(1), 1-12.
[6]Malekian, R., Jahanian-Najafabadi, A., Moazen, F., Ghavimi, R., Mohammadi, E., & Akbari, V. (2019). High-yield Production of Granulocyte-macrophage Colony-stimulating Factor in E. coli BL21 (DE3) By an Auto-induction Strategy. Iranian journal of pharmaceutical research : IJPR, 18(1), 469–478.
[7]Koeninger, L. , Armbruster, N. S. , Brinch, K. S. , Kjaerulf, S. , & Wehkamp, J. . (2020). Human β-defensin 2 mediated immune modulation as treatment for experimental colitis. Frontiers in Immunology, 11, 93.
[8]Mauro V. P. (2018). Codon Optimization in the Production of Recombinant Biotherapeutics: Potential Risks and Considerations. BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy, 32(1), 69–81. https://doi.org/10.1007/s40259-018-0261-x
[9]Vanz, A. L. , Renard, G. , Palma, M. S. , Chies, J. M. , Dalmora, S. L. , & Basso, L. A. , et al. (2008). Human granulocyte colony stimulating factor (hg-csf): cloning, overexpression, purification and characterization. Microbial Cell Factories, 7(1), 1-12.
[10]Malekian, R., Jahanian-Najafabadi, A., Moazen, F., Ghavimi, R., Mohammadi, E., & Ak
[11] Lavelle, A. , & Sokol, H. . (2020). Gut microbiota-derived metabolites as key actors in inflammatory bowel disease. Nature Reviews Gastroenterology & Hepatology, 17(4).
[12]Damian, R., Plichta, Daniel, B., & Graham, et al. (2019). Therapeutic opportunities in inflammatory bowel disease: mechanistic dissection of host-microbiome relationships - sciencedirect. Cell, 178(5), 1041-1056.【3】Franzosa, E. A. et al. Gut microbiome structure and metabolic activity in inflammatory bowel disease. Nat. Microbiol. 4, 293–305 (2019).
[13] Joyce, S. A. , Shanahan, F. , Hill, C. , & Gahan, C. G. . (2014). Bacterial bile salt hydrolase in host metabolism: potential for influencing gastrointestinal microbe-host crosstalk. Gut Microbes, 5(5), 669-674.
[14] Bi, J. , Liu, S. , Du, G. , & Chen, J. . (2015). Bile salt tolerance of lactococcus lactis is enhanced by expression of bile salt hydrolase thereby producing less bile acid in the cells. Biotechnology Letters, 38(4), 659-665.
[15] Dong, Z. , Zhang, J. , Lee, B. H. , Li, H. , Du, G. , & Chen, J. . (2013). Secretory expression and characterization of a bile salt hydrolase from lactobacillus plantarum in escherichia coli. Journal of Molecular Catalysis B Enzymatic, 93, 57-64.
[16] Drolia, R. , Amalaradjou, M. , V Ryan, Tenguria, S. , & Bhunia, A. K. . (2020). Receptor-targeted engineered probiotics mitigate lethal listeria infection. Nature Communications, 11(1).
[17]Lee, J. G., Han, D. S., Jo, S. V., Lee, A. R., Park, C. H., Eun, C. S., & Lee, Y. (2019). Characteristics and pathogenic role of adherent-invasive Escherichia coli in inflammatory bowel disease: Potential impact on clinical outcomes. PloS one, 14(4), e0216165. https://doi.org/10.1371/journal.pone.0216165
[18]Vassiliadis G, Destoumieux-Garzón D, Lombard C, Rebuffat S, Peduzzi J. Isolation and characterization of two members of the siderophore-microcin family, microcins M and H47. Antimicrobial Agents and Chemotherapy. 2010 Jan;54(1):288-297. DOI: 10.1128/aac.00744-09. PMID: 19884380; PMCID: PMC2798501.
[19]Sassone-Corsi, M., Nuccio, SP., Liu, H. et al. Microcins mediate competition among Enterobacteriaceae in the inflamed gut. Nature 540, 280–283 (2016). https://doi.org/10.1038/nature20557
[20]Palmer, J. D., Piattelli, E., McCormick, B. A., Silby, M. W., Brigham, C. J., & Bucci, V. (2018). Engineered Probiotic for the Inhibition of Salmonella via Tetrathionate-Induced Production of Microcin H47. ACS infectious diseases, 4(1), 39–45. https://doi.org/10.1021/acsinfecdis.7b00114