1. GLUTEN SENSITIVITY MEETING POINT FOR GENETICS, PROTEIN CHEMISTRY AND IMMUNOLOGY  Western societies: 1% of the population Coeliac disease : caused by a genetically determined, specific immune response to antigens present in wheat gluten, focused on a limited region of the α-gliadin. The antigenic 33-mer peptide generated by digestion with intestinal enzymes produce a highly stimulatory antigen for CD4+ T cells. Moreover, this peptide is resistant to further digestion by intestinal brush border enzymes, because of it’s high proline and glutamine content. The epitopes’ recognition by CD4+ T cells previously requires the deamination of the glutamine residues by the tissue Transglutaminase (TTG).

3. THE PROJECT A GLOBAL DESCRIPTION Inspiring from probiotic yogurts, we would chose an oral supplementation of these probiotic bacteria, choosing the Lactobacillus Acidophilus. This bacteria support the acidic conditions of our stomach As well as it support the higher pH of the intestine Naturally present in our flora, so that it won’t induce an immune response BUT: for the pH sensing, introducing such a network (PAC sensing pathway) : not sure if it is possible to completely clone it and the secretion is also a problem Escherichia coli is one of the many species of bacteria present in our gut, and this would represent many advantages: Well understood genetics, manipulations quite easier For this organism, we can use known pH sensor  nhaA encodes an Na1/H1 antiporter in E. coli which is essential for adaptation to high salinity and alkaline pH in the presence of Na.  MOLECULAR PHYSIOLOGY OF THE Na+/H+ ANTIPORTER IN E. COLI

4. THE PROJECT A GLOBAL DESCRIPTION This bacteria should so produce an enzyme that will degrade these resistant α-gliadin: we will use the prolyl endoprotease of the Aspergillus niger (a fungi) for this purpose This enzyme works optimally at pH 4-5 and remain stable at pH 2, the pH of the stomach where it would be optimal to begin with the proteins degradation Moreover, this enzymes is completely resistant to digestion with pepsin, produced naturally by the chief cells in the stomach Another advantages, it has been shown that this enzyme is capable of degrading all T cell stimulatory peptides as well as intact gluten molecules, a very good point for us, because this degradation will go on in the intestine. The rapidity of degradation of this enzyme is 60 times faster than a prolyl oligopeptidase found in our body

5. THE PROJECT A GLOBAL DESCRIPTION After the oral ingestion of the yogurt, the bacteria will remain for a while in the stomach. Under these low pH conditions, the genetic network will be induced and it will begin the production of the enzyme for a first degradation of the dietary food taken.  cf inducible genetic network We think that it is important that the secretion of the enzyme can be inducible, in fact that it only begins in the stomach, and thus for different reasons: conservation and stability of the product, survival of the bacteria The enzyme should then be produced all along the intestinal tract to ensure a sufficiently good degradation of all gluten proteins present

6. THE PROJECT A GLOBAL DESCRIPTION It would be another good idea to induce the bacteria to commit “suicide” when the “work” is finished, but this is only optional and it would probably occur naturally in the gut. The bacteria has to live long enough to degrade with a certain efficiency all proteins found, and particularly the gluten’s one, so the rate of degradation is important, and the life time is something we have to check: so a population dynamics analysis would also be a good thing for this project

7. THE PROJECT A TECHNICAL DESCRIPTION Genetically modified BACTERIA: L. Acidophillus DNA cloning strategies: Genes of interest: put restriction sites by polymerase chain reaction PCR Use 2 enzymes to check the differences: Prolyl oligopeptidase from F. Meningosepticum FM-POP Prolyl endoprotease from A. Niger AN-PEP Controls: Analysis of PCR products by agarose gel electrophoresis, DNA ligation, transformation of plasmid DNA into bacteria Inoculation of bacterial cultures quantitation and analysis of DNA by UV spectrophotometry Analysis of plasmid DNA by restriction enzyme digestion and DNA sequence analysis Protein quantification by spectrophotometric assay, SDS-Polyacrylamide gel electrophoresis

8. THE PROJECT A TECHNICAL DESCRIPTION Determine the pH optimum: Using Z 1 -Gly-Pro-Z 2 as a substrate and different pH values: measure amount of released Z 2 Check the stability at low pH + resistance to pepsin degradation our secreted enzyme: Mixing with pepsin in a first step, neutralizing it’s activity with inhibitor, pepstatin: measure left enzymatic activity Make an activity assay in solutions that mimic stomach and intestinal conditions, in terms of pH, enzymatic contents etc.. Using a fluorogenic substrate Z 1 -Gly-Pro-Z 2 (Spectrophotometry)

9. THE PROJECT A TECHNICAL DESCRIPTION Real Enzymatic digestions + Degradation rate measurement: Same type of experiment but with synthetic peptides dissolved, all containing the this 33-mer resistant peptide: LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF To do that: produce recombinant gliadins (such α and γ-gliadins) : take the gene, use a plasmid to transform into E. coli Have to mimic real digesting conditions: Make a brush border enzyme preparation using (if possible) rat small intestine (jejunum)

10. Genetic Network, E.Coli K12 Expression of en enzyme to process gluten FM-POP AN-PEP Induction of expression (in response to pH decrease) Na1-induced transcription of nhaA, which encodes an Na1/H1 antiporter in Escherichia coli, is positively regulated by nhaR and affected by hns NYMU-Taipei, iGEM 2008 Burst of the bacteria to release the enzymes Lysis cassette including λ phage lysis genes. Lysis occurs 40-45 minutes after induction Caltech, iGEM 2008

11. Induction in E.Coli pH activates nhaA promoter sequences CRE-recombinase nhaA CRE-Lox recognition sites CRE-lox mechanism is used as a trigger to ensure expression of the protein will be maximum before burst AN-PEPSTOP

12. Bacteria lysis From lambda phage: holin/anti-holin, endolysin, and rz / rz1 genes Lysis cassette nhaA

13. Need to investigate Induction working in a strain of E.Coli compatible with conditions in gut. Is lysis using genes from lambda phage working in the same strain? What pH induces the cascade? Delay before lysis

14. Help of bioinformatics Tune a model to link the amount of enzymes needed to digest gluten in a normal life. Diffusion model: the enzyme has to cut efficiently most of the gluten present in a low concentration inside a high volume. We have to know how many bacteria can produce these enzymes What concentration of gluten can be reached after degradation? Try to quantify the number of bacteria triggered at pH from gut (or any induction system)

16. Lactobacillus acidophilus Advantages : Gram positive (easier for secretion) Grow at low pH Occur naturally in the gastrointestinal tract Culture : Anaerobic conditions Grow on MRS Work with a limited range of plasmid A protocol exists for plasmid pNZ123

17. L. Acidophilus (2) MIT worked in 2008 with L. bulgaricus L. bulgaricus would also be a possible bacteria The y wrote the protocols for electroporation for L. bulgaricus and L. acidophilus Difficulties because these bacteria aren’t much used.