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Description

What is the Problem?

Celiac disease affects approximately 1% of the global population with no approved therapeutic interventions outside adherence to a strict gluten-free diet1. Those with celiac disease often find themselves inadvertently exposed to gluten and having to monitor their food and environment constantly.

Celiac disease is triggered by the gluten found in wheat, barley, and rye. The main immunogenic component of gluten is gliadin, a subunit that is unable to be completely broken down by human digestive enzymes. Certain undigested fragments bind to human leukocyte antigen (HLA) molecules on the surface of antigen-presenting cells with higher affinity in people with the genetic haplotype associated with celiac disease (HLA-DQ2 or HLA-DQ8)2. These presented peptides are recognized by T-cells that release pro-inflammatory cytokines which damage the villi of the small intestine, causing side effects such as abdominal pain, diarrhea, and fatigue.

Our Inspiration

Inspired by Austin's vibrant culinary scene, with its wide range of gluten-free options and a spirit of innovation, our team saw an opportunity to use synthetic biology to develop a novel solution for alleviating the burden of celiac disease.

Moreover, previous iGEM projects exploring microbial degradation of allergens and discussions with experts in the field underscored the potential of engineered probiotics as a step towards advancing sustainable therapies for dietary disorders like celiac disease10,11. By harnessing the natural capabilities of microbes found in our digestive system and in fermented foods, we aim to utilize their genetic machinery to produce potent gliadin-degrading enzymes as a bioengineered solution to transform celiac disease management.

Our Solution

If gliadin's immunogenic epitopes are cleaved, it reduces their ability to elicit an immune response3. Our team aims to engineer probiotic bacteria that are capable of colonizing the digestive system to secrete enzymes that degrade gliadin peptides before they can trigger an immune response. This will serve as a shield against gluten contamination, and potential to provide dietary flexibility and better quality of life for individuals with celiac disease.

The Need for a Probiotic

While enzyme pills are being developed for managing celiac disease, their effectiveness is limited by factors such as stability in the environment and the need for frequent administration to maintain therapeutic levels4. Probiotic bacteria offer a potential alternative by providing sustained and localized enzymatic degradation of gliadin directly within the body.

Gliadin-degrading Microbes

By leveraging the natural resilience and enzymatic capabilities of specialized microbial communities adapted to the gastric environment, our engineered bacteria aim to provide effective gliadin degradation. Lactic Acid Bacteria (LAB), our chosen bacterial chassis, met this criteria to a tee. Renowned for their safety, utility as a probiotic species, and historical use in fermented foods, LABs can survive and function in low pH regions of the gastrointestinal tract6. Their resilience made them promising candidates for the task at hand.

Our Goals

Identifying Gliadin-degrading Microbes

By isolating microbes from diverse fermented food sources including sourdough starters and kimchi, we hoped to pinpoint naturally occuring microbial candidates demonstrating gluten or gliadin-degrading capabilities. These microbes could also provide a rich source of proteases that could contribute to further research in the field.

Transforming Gram-positive Bacteria

However, they exhibit strong resilience to the acidic and bile-rich environments of the gastrointestinal tract. Lactic acid bacteria (LAB), a type of gram-positive bacteria, are particularly well-suited for colonizing the gut. Therefore, we aimed to optimize transformation protocols specifically for these strains to enhance their potential for gut colonization and genetic modification

Protease Secretion System

A crucial aspect of our project involves developing a robust protease secretion system capable of efficiently delivering gliadin-degrading enzymes into the gut environment. We identified the broad-host-range SecB pathway as a model system found in nearly all bacterial species including LABs8. By screening a library of signal peptides that can recognize and secrete proteins through the SecB pathway, we wish to amplify LAB's ability to release glutenases into the digestive tract.

Environmental and Cellular Stability

Our research emphasizes the importance of stability and scalability in the development of our probiotics. The process of engineering bacteria to overexpress and secrete gliadin-degrading enzymes can impose a significant metabolic burden, so we will explore methods to reduce this burden to ensure consistent and efficient secretion without compromising bacterial viability or functionality in the harsh physiological conditions of the gastrointestinal environment. This involves optimizing growth conditions and genetic constructs to maintain high levels of enzyme production in our probiotic strains through successive generations of growth9.

Future Outlook

While we are confident in the conceptual integrity of our project design, we acknowledge its limitations and hope our work may serve as a gateway to an enhanced synthetic biology-based approach for probiotic therapeutics. Our team's long-term goals are to create an application of synthetic biology effective enough to accommodate ingesting the standard amount of gluten found in the American diet. In addition to this, our project hopes to spread awareness about the importance of gluten-free options and the challenges faced by individuals with celiac disease. We hope our work in this area will bring attention to celiac disease community as we continue to optimize our modular gliadin-degradation system.

Summary

Our project is comprised of several key research areas that are intended to work synergistically to prevent an immune response to gluten. We have deliberately and strategically chosen elements of this framework to maximize gluten-degradation prior to reaching the site of immune response in the small intestine. This system utilizes LABs, a category of probiotic gram-positive bacteria, as a bacterial chassis for the secretion of gliadin-degrading enzymes to break down immunogenic residues of gluten. As it currently stands, this work aims to mitigate the immune response from incidental gluten ingestion due to cross contamination. Future plans for our team include projects to create a more effective probiotic which can completely degrade undigested gliadin and improve quality of life for those with celiac disease.

References

  1. Wagh, S. K., Lammers, K. M., Padul, M. V., Rodriguez-Herrera, A., & Dodero, V. I. (2022). Celiac disease and possible dietary interventions: From enzymes and probiotics to Postbiotics and viruses. International Journal of Molecular Sciences, 23(19), 11748. https://doi.org/10.3390/ijms231911748
  2. Kupfer, S. S., & Jabri, B. (2012). Pathophysiology of celiac disease. Gastrointestinal Endoscopy Clinics of North America, 22(4), 639–660. https://doi.org/10.1016/j.giec.2012.07.003
  3. Van Buiten, C. B., & Elias, R. J. (2021). Gliadin sequestration as a novel therapy for celiac disease: A prospective application for polyphenols. International Journal of Molecular Sciences, 22(2), 595. https://doi.org/10.3390/ijms22020595
  4. Yoosuf, S., & Makharia, G. K. (2019). Evolving therapy for celiac disease. Frontiers in Pediatrics, 7. https://doi.org/10.3389/fped.2019.00193
  5. Zamakhchari, M., Wei, G., Dewhirst, F., Lee, J., Schuppan, D., Oppenheim, F. G., & Helmerhorst, E. J. (2011). Identification of rothia bacteria as gluten-degrading natural colonizers of the upper gastro-intestinal tract. PLoS ONE, 6(9). https://doi.org/10.1371/journal.pone.0024455
  6. Castellone, V., Bancalari, E., Rubert, J., Gatti, M., Neviani, E., & Bottari, B. (2021). Eating fermented: Health benefits of lab-fermented foods. Foods, 10(11), 2639. https://doi.org/10.3390/foods10112639
  7. Sheridan, P. O., Odat, M. A., & Scott, K. P. (2023). Establishing genetic manipulation for novel strains of human gut bacteria. Microbiome Research Reports, 2(1), 1. https://doi.org/10.20517/mrr.2022.13
  8. Green, E. R., & Mecsas, J. (2016). Bacterial secretion systems: An overview. Microbiology Spectrum, 4(1). https://doi.org/10.1128/microbiolspec.vmbf-0012-2015
  9. Grob, A., Di Blasi, R., & Ceroni, F. (2021). Experimental tools to reduce the burden of bacterial synthetic biology. Current Opinion in Systems Biology, 28, 100393. https://doi.org/10.1016/j.coisb.2021.100393
  10. Ma, J., Lyu, Y., Liu, X., Jia, X., Cui, F., Wu, X., Deng, S., & Yue, C. (2022). Engineered probiotics. Microbial Cell Factories, 21(1). https://doi.org/10.1186/s12934-022-01799-0
  11. Gordon, S. R., Stanley, E. J., Wolf, S., Toland, A., Wu, S. J., Hadidi, D., Mills, J. H., Baker, D., Pultz, I. S., & Siegel, J. B. (2012). Computational design of an α-gliadin peptidase. Journal of the American Chemical Society, 134(50), 20513–20520. https://doi.org/10.1021/ja3094795