Prsentation PowerPoint

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TRANSCRIPTIONAL ANALYSIS OF PROKARYOTE-EUKARYOTE C
ROSS TALKS Thierry Pedron1, Meng Tien1,2,
Jean-Yves Coppée2, Philippe Sansonetti1 1 Unité
de Pathogénie Microbienne Moléculaire, INSERM
U389 2 Génopole INSTITUT PASTEUR

• Aim of the work to explore the faisability and
  usefulness of
• using  Transcriptomics of eukaryotic cells  to
  decipher the
• cross talks established between invasive or
  commensal bacteria
• and their target intestinal epithelial cells.
• Current model systems human colonic Caco-2 cell
  line infected by
• - an invasive Shigella flexneri strain and a
  series of its mutants
• - a commensal probiotic strain of Lactobacillus
  casei (followed
• by other commensals Bifidobacterium,
  Clostridium).
• Future model systems to extend the diversity to
  cell types
• composing the intestinal barrier
  (myo)fibroblasts, endothelial
• cells, neurons of the ENS, immune cells.
• to switch to infected tissues.

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Mechanisms of toleranceof bacterial flora at
epithelial surfaces the colonic flora
- Defective in capacity to escape trapping in
mucus - Defective in adherence/invasion -
Pentacylated LipidA
Commensals
Pathogens
- Defect in sensing molecules
Flagelline
TLR4/MD2/CD14/TLR2
TLR5
- Early warning systems of accidental effraction
Nod proteins - Permanent induction of active
anti-inflammatory systems under  pressure  of
flora IEC Nuclear receptors (PPARg, RXR, VitD
Recept.) Resident macrophages IL-10, TGFb
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HISTOPATHOLOGY OF SHIGELLOSIS ELEMENTARY LESIONS
IN HUMAN COLON
LUMEN
SUBMUCOSAL TISSUES
Shigella flexneri Endemic form Shigella
sonnei Endemic form Shigella dysenteriae 1
( Shiga toxin) Epidemic form Shigella boydii
Sporadic form
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pWR100 the 213kb- Virulence plasmid of Shigella
flexneri 5a
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Shigella Type Three Secretory System TTSS
7-8 nm
2.5 nm
60 nm
Needle
OM
Neck domain
IM
TTSS
Bulb
TTSS
44 nm
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4
Entry and cell-to cell spread of Shigella
Villin- dependent
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Gap junction
3
6
Villin- dependent
ATP
ATP
Connexin- dependent induction of permissivity
to invasion in adjacent cells (Ca2/VillinOther
factors ?)
7
7
Ca
ATP
Hemi- connexon
1
Purinergic receptor
ATP
Actin/Myosin II-dependent phagocytosisof
protrusion by adjacent cell
TTSS/Ipa-dependent entry through macropinocytosis
1
5
3
IcsA/actin-dependent motility
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TTSS/IpaB/C-dependent Lysis of the two membranes
Engagement of components of adherent
junctions (Cadherins/a,b?Catenins)
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Infection with Shigella flexneri
Plasmid-cured
uninfected
Wild-type
IpaB-
IpaA-
IpaD-
NF-kB activation
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953
NOD1
Epithelial cells
1024
CARD12
1040
NOD2
CARD
NBS
CARD
LRR(10)
Macrophages
1473
NALP1
CARD
Pyrin
NBS
LRR(12)
1062
Pyrin
NBS
NALP2
LRR(12)
1403
NBS
NAIP
BIR
BIR
BIR
LRR(5)
920
Pyrin
NBS
Cryopyrin
LRR(6)
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Inside-In Signalling for NF-kB Activation in
Epithelial Cell Cytoplasm by Shigella LPS The
Nod Short Cut
Activation of RICK
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Peptidoglycan of Gram -
Specificities
MurNAc
CH
O
GlcNAc
O
O
L-Ala
D-Glu
Meso-DAP
DAP
(D-Ala)
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FIRST STEP DEFINITION OF AN  EXTENDED
TRANSCRIPTOME  WITH AFFYMETRIX MICROARRAY
(12,000 HUMAN GENES)
PATHOGENS Shigella flexneri Inv Inv- Oth
er mutants (IpgD-, MxiE-, etc..) Kinetics 2h,
4h, 6h of incubation between bacteria and IEC.
INFLAMMATORY CYTOKINES TNFa????????treatment of
IEC).
COMMENSALS Lactobacillus casei (2h, 6h, 24h of
incubation with IEC).
Comparison of the various transcriptional profiles
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Affymetrix microarray analysis (12,000
genes). Up-regulation of gene transcription in
human colonic Caco-2 cells (i) infected for 2 h
by invasive (M90T), or non-invasive (BS176)
S.flexneri, (ii) or treated for 2 h with TNF-a.
Ratios are calculated over the transcriptome of
uninfected/untreated cells.
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Impact de L. Casei (DN -114 001) sur
l expression des gènes des cellules Caco-2
Technologie Affymetrix
Cinétiques de coculture Caco-2/L. Casei pendant
t0, 2h, 6h et 24 h Impact du surnageant de L.
Casei (2 réplicats par point)
Up-Regulation Lacto/NIgt2 Down-regulation
NI/Lactogt2
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 EXTENDED TRANSCRIPTOME  OF CaCo-2
CELLS STUDIED BY AFFYMETRIX MICROARRAYS.
- Clear differences can be observed between the
various transcriptomes (wt Shigella vs Shigella
mutants vs Lactobacillus vs inflammatory
cytokines) in spite of basic activation of NF-kB
under all circumstances. - This opens the door
to - the definition of transcriptional
regulons responding to certain transcriptional
activators, or combinations of transcriptional
activators, differentially expressed according
to the response of Caco-2 cells to microbial
PAMPs as well as virulence and commensalism
factors that may express activating or
inhibiting functions. Concept of each
microorganism  carving its own transcriptional
pattern . - the conception of a Macroarray
exploring a  Restricted transcriptome 
correspondig to, at least, all the transcripts
that are up- and down-regulated during exposure
to all the microbes and stimuli studied.
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SECOND STEP DEVELOPMENT OF A MACROARRAY
REFLECTING THE  RESTRICTED TRANSCRIPTOME 
1050 PCR products spotted on nylon membranes
(Génopole) corresponding to - ca. 400 human
genes whose transcription has been modified in
the conditions described before (identified by
AFFYMETRIX). - ca. 600 human genes related to
inflammation, cell death, cell
cycle, oncogenes, cell adherence and
structure, immune system, intestinal
epithelial physiology. - Called  Human
Inflammation Chip .
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Experiences en cours et projets (Meng-Tsung TIEN)
- Cinétique L. casei-Caco-2 différenciées. -
Impact du surnageant de L.casei - Impact L.
casei lors d une infection par Shigella -
Impact de différentes souches de
probiotiques Bifidobacterium animalis DN-173
010 Bifidobacterium breve DN-156
007 Streptococcus thermophilus DN-001
147 Lactobacillus bulgaricus DN-100
107 Lactobacillus plantarum DN-121
022 Bacteroides thetaiomicron VPI-5482
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Ongoing experiments  from pathogenesis to
commensalism 
1 - Continuing fine transcriptional mapping in
CaCo2 cells exposed to wt Shigella and mutants,
inflammatory cytokines and commensals. Validation
of new Macroarray. 2 - Identification of
bacterial factors achieving specificity
( carving ) of transcriptomes (bacterial
mutants, purified products, expressed
prokaryotic genes in eukaryotic cells). Major
candidates IpaH/Osp proteins of Shigella. 3 -
 Anti-pro-inflammatory signalling  expressed by
commensal Lactobacillus casei against wt Shigella
flexneri. 4 - Intercellular cross-talks between
infected epithelial cells and other cells of
the intestinal barrier (myo)fibroblasts,
endothelial cells, neurons. 5 - Opening on
tissue analysis in HuSCID mice.
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Openings 1 - Developing new Macroarrays with
oligonucleotides. 2 - Studying other pathogens
in collaboration Rotavirus and other
enterovirus, Cryptococcus neoformans. 3 -
Development of the  commensalism  project. 4
- Development of a clinical project on severe
sepsis and septic shock.