Download GLUTEN SENSITIVITY

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).
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
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
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
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
THE PROJECT
 Genetically modified BACTERIA: L.
A TECHNICAL DESCRIPTION
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
THE PROJECT
A TECHNICAL DESCRIPTION
 Determine the pH optimum:
 Using Z1-Gly-Pro-Z2 as a substrate and different pH values:
measure amount of released Z2
 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 Z1-Gly-Pro-Z2 (Spectrophotometry)
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)
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
Induction in E.Coli
 pH activates nhaA promoter sequences
nhaA
CRE-recombinase
STOP
AN-PEP
CRE-Lox recognition sites
 CRE-lox mechanism is used as a trigger to ensure expression of the protein
will be maximum before burst
Bacteria lysis
 From lambda phage: holin/anti-holin, endolysin, and rz / rz1 genes
nhaA
Lysis cassette
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
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)
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
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.