Team:UC Davis/Project

Project:
Celiac Disease:

What is Celiac Disease?
            Celiac Disease is a form of autoimmune disorder, which occurs inside the small intestine. When the body cannot digest gliadin (component of gluten) properly, this leads to an immune response (14). Furthermore, different people have different reactions to these immune responds such as pain and vomiting (14). It has been estimated that about one out of 133 Americans are currently suffering from this disease (11); including one of our teammate's friends, and that was what sparked our interest in designing this project.                                          

What happens in the normal small intestine?
            In the normal small intestine, the Brush border membrane lets small peptides and molecules like water through and into the bloodstream. The brush border membrane also lets larger molecules into the blood stream, similar to Gliadin; however, these larger molecules are converted through a transcellular route, which does not prompt an immune system response.

What happens in celiac disease small intestine?            
            The Brush border membrane in the small intestine of people suffering from Celiac Disease allows large molecules such as gliadin to go straight into the blood stream without directing them through the transcellular route. Once gliadin passes through, Antigen Presenting Cells (APCs) recognize gliadin as a foreign object and will attack. This immune response causes pain and other adverse side-effects.

The immune response not only causes abdominal bloating, pain, weight loss and vomiting, most of the time it may also damage intestinal villi, which are important for absorbing nutrition. The destruction of villi eventually leads to a lack of absorption of different nutrients, which can lead to other illnesses (14). Some people affected with this disorder can also suffer from autoimmune thyroid disease, autoimmune liver disease, rheumatoid arthritis (diseases in which body immune system attacks healthy cells/tissues) (14).

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Current treatments:

           Currently, no cure has been found for this illness. The only way to avoid some of celiac disease’s symptoms is by adopting a gluten free diet (14, 12, and 11). However, you are more likely to find gluten in your everyday diet. Therefore, avoiding gluten is not easy or pleasant from a gastronomical standpoint.
           "Oral supplementation with prolyl oligopeptidases has therefore been proposed as a potential therapeutic approach."(8). However, enzymes studied earlier were not able to degrade gluten (inside stomach before it reaches small intestine because they were "irreversibly inactivated by pepsin and acidic pH, both present in the stomach."(8)
            Nevertheless, over the past years, researchers have discovered an enzyme from Aspergillus niger, a newly identified prolyl endoprotease, that was observed to "work optimally at 4-5pH and remains stable at 2pH"(8) and maybe this enzyme will lead us to an alternative treatment for this disorder (8). Studies have shown that prolyl-endoprotease from Aspergillus niger is able to "degrade gluten in vitro and under conditions similar to the ones present in the gastrointestinal tract." (8) ; but due to licensing restrictions we have opted not to work with this protein.
           Recently (year 2007), a study has suggested an alternative approach by combining a glutamine-specific endoprotease (EP-B2 from barley) and a prolyl endopeptidase (SC PEP from Sphingomonas capsulata); with gastric activity and complementary substrate specificity there is a possibility of increasing the safe threshold of ingested gluten (12). One of the advantages of this “combination product is that both enzymes are active and stable in stomach and can therefore be administered as lyophilized powders or simple capsules or tablets” (12).

Note: Moreover, this study was first “evaluated via in vitro digestion of whole-wheat beard and then confirmed by in vivo studies in rats (12), unlike other earlier studies which were performed on synthetic gluten oligopeptides, recombinant gliadin proteins, or uncooked gluten” (12).

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Our approach:

          We are going to break down gluten inside the stomach (before it arrives to the small intestine), because gliadin does not prompt an immune system response until it is absorbed in the small intestine.  Another worry is a spread of our synthetic bug, so we engineered a ‘safety-switch’ via induction of cell death through a pH sensor system.  Due to the change of pH from the stomach to the small intestine, our pH system is induced; cell death, thus limiting our bug to the stomach.

            The advantages of our model are, that unlike other suggested treatments which require consuming capsules or tablets rapidly, by using Escherichia coli as our model chassis for this project: we can lower the number of times which this ‘cure’ needs to be consumed thus making it less troublesome, costly, and more convenient.

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Our project is split into two parts:
1.Induced Secretion

• Gene sequence for secretion system
• Adding secretion
• Assay for measuring protein localization and activity

2.Sensing pH  and inducing cell death

• Finding an appropriate biological pH sensor
• Wiring the pH sensor into our system
• Assay for measuring the behavior of the pH sensor

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