Summary
We have devised Gassle, an E. coli bacteria that detects nitric oxide (NO), which is produced in large quantities during inflammation. This research explores the possibility of diagnosing Inflammatory Bowel Disease (IBD) through characteristic fart odors.
Fig.1 Diagnosing illnesses by characteristic fart odors.
- Have the E. coli detect NO, which is an inflammatory marker for IBD.
- The E. coli that detects NO produces and releases propionic acid.
- Diagnose the disease based on the intense odor of the fart.
Project Background
Current state of IBD
Inflammatory Bowel Disease (IBD) is a chronic, relapsing immune-mediated disease of the intestine. IBD is classified into two main subtypes: Ulcerative Colitis (UC) and Crohn's Disease (CD), both of which can lead to severe complications. IBD has become a global disease with increasing incidence rates across all continents [1]. The prevalence of IBD is expected to increase dramatically over the next 10 years [1]. To prepare for the growing burden of IBD, we need to develop efficient methods for rapid diagnosis.
IBD also has periods of remission and active inflammation. Due to the transition to remission and the alleviation of pain, patients may not visit the hospital, leading to delayed diagnosis[1].
Current state of IBD diagnosis
It has been found that there is a considerable delay between the initial consultation with a primary care physician and the final diagnosis of IBD [2]. Overall, the median time to diagnosis was about 10 months for CD and 3 months for UC [2].
One reason for the difficulty in early detection of IBD is that its symptoms are very similar to those of IBS, which has a global prevalence of 11.2% [1]. IBD patients with IBS-like symptoms are often misdiagnosed as IBS during medical interviews, leading to delayed discovery. [3]
Our Solution - Early detection of IBD
Early immunosuppressive therapy has been shown to improve response to treatment and reduce adverse outcomes. This is because it leads to mucosal healing, reduced hospitalizations and surgeries, and improved quality of life[4]. These can lower medical costs and increase the likelihood of the individual maintaining a stable income. It can also reduce informal care costs for family members, leading to an expected increase in family well-being.
We primarily envision two use cases:
- For undiagnosed patients: To help them distinguish between IBD (UC) and IBS before seeking medical care.
- For patients already diagnosed with IBD: To detect the transition from the remission phase to the active phase of the disease.
While IBD is an inflammatory disease, IBS is a non-inflammatory disease. Focusing on this, we aim to facilitate early detection by orally administering E. coli that can detect nitric oxide (NO), which is produced in large quantities during inflammation, to undiagnosed patients. This will help distinguish between IBD and IBS, which is one of the causes of delayed diagnosis.
Additionally, we will have patients already diagnosed with IBD use Gassle to detect the transition from remission to the active inflammatory phase.
Our Project
1. Development of E. coli that detects elevated nitric oxide concentrations during inflammation and produces propionic acid
Colonization of E. coli in the intestine
The E. coli strain Nissle 1917 (EcN) has three types of fimbriae, F1A and F1C, also known as "curli" fimbriae, which mediate adhesion to intestinal epithelial cells in cell culture experiments and to the mucus layer of the intestinal wall in vivo, promoting colonization of the intestine [5][6][7][8].
Our team transforms EcN to diagnose intestinal inflammation. While common laboratory strains like DH5α or BL21 cannot form colonies in the intestine, EcN is capable of establishing colonies in the intestinal environment.
Significant nitric oxide production at inflammation sites
We devised an experimental system to detect inflammation based on NO concentration, as NO levels in IBD patients differ significantly from healthy individuals[9].
We considered that inflammation could be detected based on NO concentration using the PnorV system by iGEM 2022_UZurich. We confirmed that it could be tested by comparing with patients' NO concentrations[10].
Fig.2 Nitric Oxide (NO) detection mechanism provided by Gassle
Promoter that detects an increase in nitric oxide concentration
We thought that the PnorV system by iGEM 2022_UZurich could detect the presence or absence of inflammation based on NO concentration.
Confirmation that it can be tested in comparison with the patient's NO concentration
Propionic Acid Production - Sleeping Beauty Mutase
We considered activating the Sleeping Beauty Mutase (sbm operon) to produce propionic acid. For details, refer to Engineering Success)
The reasons for choosing propionic acid include its low olfactory threshold, making it easy to detect by smell, as well as its anti-inflammatory properties. For details, refer to Human Practices)
When producing propionic acid, we simulated the concentration of propionic acid in intestinal gas. We used a compartment model for this. For details, refer to Dry Lab Model)
2. E. coli growth control using the HSV-TK/GCV system
Addressing safety concerns
The Gassle technology is currently in the experimental stage, with ongoing research on odor detection and capsule design. When considering its introduction into the intestinal microbiome, we will be incorporating genetically modified E. coli into the body, so we must be able to appropriately control the number of live bacteria. Additionally, there is a need to regulate the production of propionic acid in the colon. As a solution to these issues, we have implemented a control system using thymidine kinase (HSVTK) and ganciclovir (GCV).
Mechanism of the HSV-TK/GCV system
We have introduced a system that specifically kills only the introduced E. coli without eliminating the intestinal bacteria by administering GCV.
Fig.3 HSVTK/GCV system
DNA replication is inhibited, allowing specific control of proliferation.
Development of a capsule to deliver E. coli to the colon
Approach to capsule design
To ensure that the genetically modified E. coli reaches the colon safely, we designed a capsule that protects the bacteria from stomach acid and releases them in the colon. This capsule design is crucial for the effective delivery of our diagnostic E. coli, Gassle, to the target area.
Capsule release mechanism
The capsule is designed to gradually dissolve as it moves through the digestive tract, with complete dissolution occurring in the colon. This ensures targeted release of the E. coli in the area where IBD inflammation typically occurs. For more details, please refer to Hardware experiments.
References
[1] Inflammatory Bowel Diseases, Volume 23, Issue 10, 1 October 2017, Pages 1825–1831
[2] Nahon S, Lahmek P, Lesgourgues B, Poupardin C, Chaussade S, Peyrin-Biroulet L, Abitbol V. Diagnostic delay in a French cohort of Crohn's disease patients. J Crohns Colitis. 2014 Sep;8(9):964-9. doi: 10.1016/j.crohns.2014.01.023. Epub 2014 Feb 13. PMID: 24529604.
[3] Burgmann T, Clara I, Graff L, Walker J, Lix L, Rawsthorne P, McPhail C, Rogala L, Miller N, Bernstein CN. The Manitoba Inflammatory Bowel Disease Cohort Study: prolonged symptoms before diagnosis--how much is irritable bowel syndrome? Clin Gastroenterol Hepatol. 2006 May;4(5):614-20. doi: 10.1016/j.cgh.2006.03.003. Epub 2006 Apr 17. PMID: 16630762.
[4] Vu Q. Nguyen, Dingfeng Jiang, Sharon N. Hoffman, Srikar Guntaka, Jessica L. Mays, Anthony Wang, Joseph Gomes, Dario Sorrentino, Impact of Diagnostic Delay and Associated Factors on Clinical Outcomes in a U.S. Inflammatory Bowel Disease Cohort, *Inflammatory Bowel Diseases*, Volume 23, Issue 10, 1 October 2017, Pages 1825–1831, doi.org/10.1097/MIB.0000000000001257
[5] Sonnenborn, U., & Schulze, J. (2009). The non-pathogenic *Escherichia coli* strain Nissle 1917 – features of a versatile probiotic. *Microbial Ecology in Health and Disease*, *21*(3–4), 122–158. https://doi.org/10.3109/08910600903444267
[6] Sonnenborn, U., & Schulze, J. (2009). The non-pathogenic *Escherichia coli* strain Nissle 1917 – features of a versatile probiotic. *Microbial Ecology in Health and Disease*, *21*(3–4), 122–158. https://doi.org/10.3109/08910600903444267
[7] Sonnenborn, U., & Schulze, J. (2009). The non-pathogenic *Escherichia coli* strain Nissle 1917 – features of a versatile probiotic. *Microbial Ecology in Health and Disease*, *21*(3–4), 122–158. https://doi.org/10.3109/08910600903444267
[8] Sonnenborn, U., & Schulze, J. (2009). The non-pathogenic *Escherichia coli* strain Nissle 1917 – features of a versatile probiotic. *Microbial Ecology in Health and Disease*, *21*(3–4), 122–158. https://doi.org/10.3109/08910600903444267
[9] Lundberg JO, Hellström PM, Lundberg JM, Alving K. Greatly increased luminal nitric oxide in ulcerative colitis. Lancet. 1994 Dec 17;344(8938):1673-4. doi: 10.1016/s0140-6736(94)90460-x. PMID: 7996962.
[10] UZurich iGEM. (2022). Team/Engineering. iGEM 2022. Retrieved October 1, 2024, from