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Identifying Mechanisms by which Escherichia coli O157:H7 Subverts Interferon-gamma Mediated Signal Transducer and Activator of Transcription-1 Activation
by
Nathan Koul-Lin Ho
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Laboratory Medicine and Pathobiology
Identifying Mechanisms by which Escherichia coli O157:H7
Subverts Interferon-gamma Mediated Signal Transducer and
Activator of Transcription-1 Activation
Nathan Koul-Lin Ho
Doctor of Philosophy
Laboratory Medicine and Pathobiology University of Toronto
2012
Abstract Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a foodborne pathogen that
causes significant morbidity and mortality in developing and industrialized nations. EHEC
infection of host epithelial cells is capable of inhibiting the interferon gamma (IFNγ) pro-
inflammatory pathway through the inhibition of Stat-1 phosphorylation, which is important for
host defense against microbial pathogens. The aim of this thesis was to determine the bacterial
factors involved in the inhibition of Stat-1 tyrosine phosphorylation. Human HEp-2 and Caco-2
epithelial cells were challenged directly with either EHEC or bacterial culture supernatants,
stimulated with IFNγ, and then protein extracts were analyzed by immunoblotting. The data
showed that IFNγ-mediated Stat-1 tyrosine phosphorylation was inhibited by EHEC secreted
proteins. Using 2D-Difference Gel Electrophoresis, EHEC Shiga toxins were identified as
candidate inhibitory factors. EHEC Shiga toxin mutants were then generated, complemented in
trans, and mutant culture supernatant was supplemented with purified Stx to confirm their ability
to subvert IFNγ-mediated cell activation. I conclude that E. coli-derived Shiga toxins represent a
novel mechanism by which EHEC evades the host immune system.
iii
Acknowledgements This thesis is dedicated my family, friends, and colleagues who have supported me through this
PhD journey. You all know who you are, and how you have contributed to my life in and outside
of the laboratory. I would not have been able to progress this far as a person without every one of
you, and I thank you all from the bottom of my heart.
Family Millie Ho Desmond Ho Serena Park-Ho Grandma Stanley Ho Eric Ho Connie Ho Kimberley Ho Hillary Ho Eddy Ho Jocelyn Ho Julius Ho Elizabeth Ho Kiara Ho Raymond Chan Anne Chan Chloe Chan Ellie Chan Sherman Group Philip Sherman Kathene Johnson-Henry Melanie Gareau Eytan Wine Juan Ossa Kevin Donato Grace Shen-Tu David Rodrigues Andrew Sousa Robert Lorentz Steve Hawley
Linköping Group Johan Söderholm Anders Carlsson Linda Gerdin Åsa Keita Ylva Braaf Maria Jönsson Cellsignals Group Richard Ellen Najib Yourish Science Rendezvous Lauren Kilgour Linda Vong Songyi Xu Ron Ammar Christopher Smith Gladys Wong Robert Lorentz Grace Shen-Tu Claire Scherzinger Yifang Liu Steve Hawley Gursonika Binepal Iwona Wenderska Kamna Singh Maher Bourbia Maisha Syeda Kelly Thickett Cherry Leung Kelsey Miller
LSCDS Group Chan-mi Lee SickKids Administration Margaret Johnson Suzanne Shek UofT Staff and Administrators Ian Crandall Martha Brown Ken Greaves Raul Cunha Peter Hurley Elissa Strome Emanuel Istrate Cynthia Goh Dwayne Miller CIHR Administrator Mary-Jo Makarchuk
Linda Vong
iv
Table of Contents Title Page ......................................................................................................................................... i
Abstract ........................................................................................................................................... ii
Acknowledgements ........................................................................................................................ iii
Table of Contents ........................................................................................................................... iv
Dissemination of Work Arising from this Thesis .......................................................................... vi
List of Tables ............................................................................................................................... viii
List of Figures ................................................................................................................................ ix
1.3.2 Locus of Enterocyte Effacement Pathogenicity Island (LEE PAI) and the Type 3 Secretion System (T3SS) ....................................................................................12
Chapter 2: Hypothesis and Objectives .......................................................................................29
Chapter 3: Identifying Mechanisms by which Escherichia coli O157:H7 Subverts Interferon-γ Mediated Signal Transducer and Activator of Transcription-1 Activation ............................................................................................................30
helveticus strain R0052 (R0052) or in combination for 3h were analyzed by (A) Principle
component Analysis (PCA), or (B) Non-Negative Matrix Factorization (NMF) to separate them
in a two-dimensional space according to genes modified significantly. Heat map analysis (C)
showed 131 out of 1,354 genes present on the ImmuneArray were modulated significantly by at
least 1.3-fold by one of the microbes. Transcriptional profiles were organized by two-
dimensional hierarchical clustering where more abundant transcripts in Caco-2 cells treated with
bacteria compared to untreated Caco-2 cells are shown in red, and less abundant transcripts are
shown in green. All analyses were done using independently collected samples (n=6, p < 0.05,
fold difference ≥ 1.3).
102
103
Table 5.1: Genes modified significantly in the ImmuneArray analysis.
Function EDL933 LF82 EDL933
+
R0052
LF82+
R0052
R0052
Pro-inflammation
AICDA – involved in IG class switching No
change
No
change
No
change
No
change
up
ATF4 camp response element binder. Pro-
inflammatory.
No
change
No
change
Up No
change
No
change
BCL3 NFKB associated – pro-inflamation Up Up Up Up No
change
BMP2 TGFb family of proteins – involved
in cytokine-cytokine receptor
interactions
Up Up Up Up No
change
C3 complement component C3 No
change
No
change
No
change
up up
CCL3 CC type chemokine that activates
PMN’s – pro-inflammatory
chemokine
No
change
No
change
No
change
No
change
No
change
CCL20 CC type chemokine that activates
PMN’s – pro-inflammatory
chemokine
Up Up Up Up No
change
CD1D - Antigen-presenting protein that binds
self and non-self glycolipids and
presents them to T-cell receptors on
natural killer T-cells
No
change
up No
change
No
change
No
change
CD276 – costimulatory B-cell molecule No
change
up No
change
No
change
No
change
CEBPB - transcriptional activator in the Up No Up No No
104
regulation of genes involved in
immune and inflammatory responses
– pro-inflammatory
change change change
CKLFSF4 AKA CMTM4. Chemokine like
protein – proinflammatory.
No
change
No
change
down No
change
No
change
CLCF1 cytokine of IL6 family. Phospho’s
Stat-3. Induces IL-1.
Up No
change
Up No
change
No
change
CSF1 secreted cytokine which influences
hemopoietic stem cells to
differentiate into macrophages
up No
change
Up up No
change
CSF3 Granulocyte colony-stimulating
factors – stimulates maturation of
granulocytes – pro-inflammatory
mediator.
Up No
change
Up Up No
change
CX3CL1 chemokine – pro-inflammatory No
change
No
change
Up Up No
change
CXCL1 Chemokine up up Up up no
change
CXCL2 Chemokine Up Up Up Up No
change
CXCL3 Chemokine Up Up Up Up No
change
CXCL10 Chemokine – neutrophil
chemoattractant
up No
change
Up up No
change
CXCL11 Chemokine – neutrophil
chemoattractant
No
change
No
change
Up No
change
No
change
ELF3 E74-like factor 3 (ets domain
transcription factor, epithelial-
specific Involved in mediating
vascular inflammation
Up Up Up Up No
change
105
FOS part of AP-1 complex. Pro-
inflammation
Up Up Up Up No
change
G1P2 a ubiquitin-like protein that becomes
conjugated to many cellular proteins
upon activation by interferon-alpha –
pro-inflammatory
up No
change
Up No
change
No
change
Hla-A/B MHC gene No
change
up No
change
up No
change
ICAM1 intercellular adhesion molecule for
leukocyte mediated transmigration –
pro-inflammatory
up No
Change
Up up No
Change
ICAM3 intercellular adhesion molecule for
leukocyte mediated transmigration –
pro-inflammatory
No
change
No
change
Down No
change
No
change
ICOSLG Acts as a costimulatory signal for T-
cell proliferation and cytokine
secretion
No
change
No
change
Up No
change
No
change
IFNA5
- Produced by macrophages, IFN-
alpha have antiviral activities.
Interferon stimulates the production
of two enzymes: a protein kinase and
an oligoadenylate synthetase pro-
inflammatory
No
change
No
change
No
change
No
change
down
IFNGR1 Interferon gamma receptor 1 – pro-
inflammatory
up No
change
Up up No
change
IFNGR2 Interferon gamma receptor 2 – pro-
inflammatory
No
change
No
change
Up up No
change
IFRD1 This gene is an immediate early gene
that encodes a protein related to
interferon-gamma
No
change
No
change
Up No
change
No
change
IKBKE dissociates inhibitors of NFkappaB up up Up up No
106
complex – pro-inflammatory change
IL1R1 IL1 cytokine receptor No
change
No
change
No
change
down No
change
IL2RG – Interleukin 2 receptor – pro-
inflammatory
No
change
No
change
No
change
up No
change
IL28RA proinflammatory cytokine No
change
No
change
Up No
change
No
change
IL8 Cytokine Up Up Up Up No
change
Il32 – cytokine that induces expression of
TNFa, il8
Up Up Up Up No
change
ILF3 Nuclear factor of activated T-cells
(NFAT) is a transcription factor
required for T-cell expression of
interleukin 2
No
change
No
change
down No
change
No
change
IRF-1 serves as an activator of interferons
alpha and beta transcription
up No
change
Up Up No
change
IRAK2 upregulates NfkappaB pro-
inflammatory
Up No
change
Up No
change
No
change
ISG20 degrades RNA – anti-viral function
of IFN against RNA viruses
No
change
No
change
Up No
change
No
change
ISGF3G – AKA IRF9 – Transcription
regulatory factor that mediates
signaling by type I IFNs (IFN-alpha
and IFN-beta). Following type I IFN
binding to cell surface receptors –
pro-inflammatory
No
change
No
change
No
change
down No
change
JUN associated with FOS to make AP-1
complex
Up Up Up Up No
change
JUNB same as JUN, associated with FOS to Up Up Up Up No
107
make AP-1 complex change
JUND same as JUN, associated with FOS to
make AP-1 complex
Up Up Up Up No
change
LIF cytokine – mitogenic - growth
promotion and cell differentiation of
different types of target cells
Up No
change
Up No
change
No
change
LTB lymphotoxin Beta – inducer of
inflammatory response system
Up Up Up Up No
change
LTBR protein encoded by this gene is a
member of the tumor necrosis factor
(TNF) family of receptors. Pro-
inflammatory
No
change
No
change
No
change
down No
change
MAP3k1 activates MAPK and JNK pathways.
Induce production of NFKappaB
up No
change
Up Up No
change
MAP3k8 activates MAPK and JNK pathways.
Induce production of NFKappaB
up No
change
Up No
change
No
change
MAP3K13 activates nfkb pathway – pro-
inflammatory.
No
change
No
change
Up No
change
No
change
MAP3K14 activates nfkb pathway – pro-
inflammatory.
No
change
No
change
Up No
change
No
change
NFATC2 member of the nuclear factors of
activated T cells transcription
complex
No
change
No
change
Up No
change
No
change
NFKB2 NF-kappa-B is a pleiotropic
transcription factor which is present
in almost all cell types and is
involved in many biological
processed such as inflammation,
immunity, differentiation, cell
growth, tumorigenesis and apoptosis
Up Up Up Up No
change
108
NR4A1 The NGFIB protein plays a key role
in mediating inflammatory responses
in macrophages
Up No
change
Up Up No
change
PELI1 Scaffold protein involved in the IL-1
signaling pathway via its interaction
with the complex containing IRAK
kinases and TRAF6. Required for
NF-kappa-B activation and IL-8 gene
expression in response to IL-1
Up No
change
Up No
change
No
change
PIM1 regulates cytokine pathwyas - These
include interleukins (IL-2, IL-3,IL-5,
IL-6, IL-7, IL12, IL-15), prolactin,
TNFα, EGF and IFNγ, among others
Up Up Up Up Up
PLA2G2A Phospholipases are a group of
enzymes that hydrolyze
phospholipids into fatty acids and
other lipophilic molecules.
Phospholipases are ubiquitously
expressed and have diverse
biological functions including roles
in inflammation, cell growth,
signaling and death and maintenance
of membrane phospholipids.
No
change
No
change
Down No
change
No
change
PTGS2 codes for cycloxygenase-2 –
upregulated during inflammation –
produces prostaglandins – pro-
inflammatory
Up Up Up Up No
change
RELB protein binds to NFKappaB to form
complex – pro-inflammatory
Up No
change
Up No
change
No
change
RIPK1 Essential adapter molecule for the
activation of NF-kappa-B – pro-
inflammatory
No
change
No
change
No
change
down No
change
SOCS3 cytokine-inducible negative Up Up Up Up No
109
regulators of cytokine signaling. Pro-
inflammatory
change
SMAD7 blocks TGFbeta – cAMP response
element binders – pro-inflammation
Up No
change
Up No
change
No
change
TNF (AKA TNF-a) pro-inflammatory
cytokine that induces cell apoptosis.
Up No
change
Up Up No
change
TNFRSF21 This receptor has been shown to
activate NF-kappaB and
MAPK8/JNK, and induce cell
apoptosis
No
change
No
change
Up No
change
No
change
TRIP associated with TLR3/TLR4.
Activates IFNa and IFNb in
inflammatory immune response
Up Up Up Up No
change
Anti-
inflammation
Function EDL933 LF82 EDL933
+
R0052
LF82+
R0052
R0052
CD55 – AKA DAF – stops complement
cascade
No
change
No
change
Up No
change
No
change
DUSP2 negatively regulates MAPK pathway No
change
No
change
No
change
down No
change
DUSP1 negatively regulates MAPK pathway Up Up Up Up No
change
DUSP5 negatively regulates MAPK pathway Up Up Up Up No
change
DUSP6 negatively regulates MAPK pathway No
change
No
change
Up No
change
No
change
DUSP8 negatively regulates MAPK pathway Up No
change
Up No
change
No
change
110
DUSP16 negatively regulates MAPK pathway Up No
change
Up Up No
change
NFKBIA NF-kB-pathway signaling up up Up Up No
change
NFKBIE NF-kB-pathway signaling up up Up Up No
change
SIGIRR Acts as a negative regulator of the
Toll-like and IL-1R receptor
signaling pathways
No
change
No
change
No
change
down No
change
TNFAIP3 induced by TNF. Limits
inflammation by inhibiting NFKB
activation and TNF mediated
apoptosis. – anti-inflammation
Up Up Up Up No
change
TOLLIP
– ubiquitin binding protein that
interacts with TLR signal cascade
proteins. Anti-inflammatory
No
change
No
change
No
change
down No
change
Tight
Junctions
Function EDL933 LF82 EDL933
+
R0052
LF82+
R0052
R0052
CLDN4 Tight junction Up Up Up Up No
change
CLDN10 Claudin 10 – Tight Junction Protein No
change
No
change
No
change
up Up
CLDN17 Claudin 17 – Tight Junction Protein No
change
No
change
down No
change
No
change
Jam1 regulates tight junction assembly in
epithelia
No
change
No
change
No
change
up No
change
MGC16207 This gene encodes an adhesion No No Down No No
111
protein that plays a role in the
organization of adherens junctions
and tight junctions in epithelial and
endothelial cells.
change change change change
PVRL2 This protein is one of the plasma
membrane components of adherens
junctions
No
change
No
change
Up No
change
No
change
THBS1 adhesive glycoprotein that mediates
cell-cell and cell-matrix interactions
Up No
change
Up No
change
No
change
VCL Actin filament (F-actin)-binding
protein involved in cell-matrix
adhesion and cell-cell adhesion
No
change
No
change
Up No
change
No
change
Cytoskeletal
Genes
Function EDL933 LF82 EDL933
+
R0052
LF82+
R0052
R0052
TUBB2B Tubulin Up No
change
Up No
change
No
change
TUBB2C Tubulin No
change
No
change
No
change
down No
change
VAV3 – Guanine nucleotide exchange
factor – involved in actin cytoskeletal
rearrangements
No
change
up No
change
No
change
No
change
Cellular
Growth/Prote
ction
Function EDL933 LF82 EDL933
+
R0052
LF82+
R0052
R0052
ADM Pro cell survival – increase tolerance
of cells to stress, injury, promotes
angiogenesis
up up Up Up No
change
112
AKT2 kinase – pro-cell survival No
change
No
change
No
change
down No
change
AREG part of epidermal growth factor
family
up up Up Up up
ASH1L associated to tight junctions No
change
No
change
down No
change
No
change
ATF1 encodes transcription factor that is
involved in growth, survival, and
other cellular activities
No
change
down No
change
No
change
No
change
BIRC3 inhibits apoptosis by inhibiting
capases
up up Up Up No
change
BNIP3 an apoptotic protector No
change
No
change
Up No
change
No
change
CCND1 Cell growth No
change
No
change
Up No
change
No
change
CCND3 Cell growth No
change
No
change
Up No
change
No
change
EVI1 AKA MECOM – anti apoptosis. No
change
No
change
Down No
change
No
change
EPHA2 encodes receptor involved in
angiogenesis
Up No
change
Up No
change
No
change
GADD45A activated by environmental stresses
and activates p38/JNK pathway. Cell
protection
Up Up Up Up No
change
GADD45B activated by environmental stresses
and activates p38/JNK pathway. Cell
protection
Up No
change
Up Up No
change
GDF15 regulating inflammatory and
apoptotic pathways in injured tissues
Up Up Up Up No
change
113
and during disease processes
GPR109B counteracts prolipolytic influences
due to oxidation stress
Up Up Up Up No
change
HBEGF cell growth No
change
No
change
Up No
change
No
change
HSPA8 Heatshock protein 8 No
change
No
change
Down No
change
No
change
ID1 This protein may play a role in cell
growth, senescence, and
differentiation
No
change
down Up Down No
change
ID3 This protein may play a role in cell
growth, senescence, and
differentiation
No
change
No
change
Up No
change
No
change
ITIH2 a family of structurally related
plasma serine protease inhibitors
involved in extracellular matrix
stabilization
No
change
Down Down Down No
change
PLK2 – involved in cell division No
change
No
change
Up No
change
No
change
MAP3K3 – activates SEK, MEK1/2, not p38. No
change
No
change
No
change
down No
change
MNT This protein is likely a transcriptional
repressor and an antagonist of Myc-
dependent transcriptional activation
and cell growth.
No
change
No
change
Up Down No
change
NDRG1 The protein encoded by this gene is a
cytoplasmic protein involved in
stress responses, hormone responses,
cell growth, and differentiation
No
change
No
change
Up No
change
No
change
PDGFA Platelet derived growth factor – Up No Up No No
114
mitogen for cells- wound healing change change change
PDGFRA
Platelet derived growth factor –
mitogen for cells- wound healing
No
change
No
change
No
change
down No
change
PIK3R1 proliferation, cell survival,
degranulation, vesicular trafficking
and cell migration. Anti-apoptosis
No
change
No
change
down No
change
No
change
PTPNS1 AKA SIRPA – his protein was found
to participate in signal transduction
mediated by various growth factor
receptors -
No
change
No
change
down No
change
No
change
PTPRB phosphatase involved in cell growth No
change
No
change
No
change
up No
change
RPS6KA4 regulate diverse cellular processes
such as cell growth, motility, survival
and proliferation
No
change
No
change
down No
change
No
change
TGFBR2 This receptor/ligand complex
phosphorylates proteins, which then
enter the nucleus and regulate the
transcription of a subset of genes
related to cell proliferation
No
change
No
change
down down No
change
TNFRSF21A Promotes angiogenesis and the
proliferation of endothelial cells
No
change
No
change
Up No
change
No
change
VEGF stimulates angiogenesis Up Up Up Up No
change
Cell Death Function EDL933 LF82 EDL933
+
R0052
LF82+
R0052
R0052
DDIT3 pro-apoptotic factor Up No
change
Up No
change
No
change
115
FADD pro-apoptosis signal. No
change
No
change
No
change
down No
change
TAOK2 involved in apoptotic morphological
changes – pro-apoptosis
down No
change
No
change
down No
change
Cell Arrest Function EDL933 LF82 EDL933
+
R0052
LF82+
R0052
R0052
CDKN1A cell cycle inhibitor No
change
No
change
Up Up No
change
CDKN1B cell cycle inhibitor up No
change
Up up No
change
INHBB inhibin – inhibits cell growth and
differentiation
No
change
No
change
Up No
change
No
change
KLF10 inhibits cell growth Up No
change
Up No
change
No
change
Other Genes Function EDL933 LF82 EDL933
+
R0052
LF82+
R0052
R0052
ADCY3 enzyme that makes cAMP. No
change
No
change
Down No
change
No
change
CACNA1A calcium channel, voltage-dependent,
P/Q type, alpha 1A subunit
up No
change
Up No
change
No
change
EGR1 – transcription factor – target genes
involved in differentiation and
mitogenesis
Up No
change
Up Down No
change
EMP3 epithelial membrane protein 3 up No No No No
116
change change change change
ENDOG encodes a DNAse/RNAse protein No
change
No
change
down No
change
No
change
GRM1 – activates phospholipase C - No
change
No
change
Up No
change
No
change
KLF6 functions as a tumor suppressor Up Up Up Up No
change
MUC13
produce mucins. No
change
up Up No
change
up
MUC12 No
change
up No
change
No
change
No
change
NTRK1 – encodes receptor that binds to
neutrophin, activates MAPK pathway
No
change
No
change
No
change
up Up
RGS3 - inhibits G-protein mediated signal
transduction
No
change
No
change
No
change
down No
change
SNAI1 downregulates expression of
ectodermal genes within mesoderm.
Up No
change
Up No
change
No
change
117
Table 5.2: Primer pairs used in the qRT-PCR analysis.
Gene
Name
Function GenBank
Accession
Number
Primer Sequence
Normalizing Genes
ACTB
Involved in various types of cell
motility and ubiquitously expressed
in all eukaryotic cells used as a
reference gene
NM_00110
1
F: GTTGTCGACGACGAGCG
R: GCACAGAGCCTCGCCTT
B2M
Serum protein involved in a
complex with another protein on the
surface of nearly all nucleated cells
used as a reference gene
NM_00404
8
F: TCTCTGCTGGATGACGTGAG
R: TAGCTGTGCTCGCGCTACT
RPLP0 Ribosomal protein P0 used as a
reference gene
NM_00100
2
F: GGCGACCTGGAAGTCCAACT
R: CCATCAGCACCACAGCCTTC
Pro-inflammation
PIM1 regulates cytokine pathways such as
interleukins, prolactin, TNFα, EGF
and IFNγ. Plays a role in signal
transduction in blood cells.
Contributes to both cell proliferation
and survival
NM_00264
8.3 F: ATGGTAGCGGATCCACTCTG
R: CTCAAGCTCATCGACTTCGG
CXCL1 Chemokine NM_00151
1.2
F: CTTCCTCCTCCCTTCTGGTC
R: GAAAGCTTGCCTCAATCCTG
IL8 One of the major mediators of the NM_00058 F: AAATTTGGGGTGGAAAGGTT
118
inflammatory response 4 R: TCCTGATTTCTGCAGCTCTGT
JUN associated with FOS to make AP-1
complex
Transcription factor that regulates
gene expression
NM_00222
8.3
F: TCTCACAAACCTCCCTCCTG
R: GAGGGGGTTACAAACTGCAA
Anti-inflammation
DUSP1 negatively regulates MAPK pathway NM_00441
7
F: GGCCCCGAGAACAGACAAA
R: GTGCCCACTTCCATGACCAT
NFKBI
A
Inhibits the activity of the NF-
kappa-B (NFKB) protein complex
NM_02052
9
F: CCGCACCTCCACTCCATCC
R:
ACATCAGCACCCAAGGACACC
TNFAI
P3
induced by TNF. Limits
inflammation by inhibiting NFKB
activation and TNF mediated
apoptosis. – anti-inflammation
NM_00629
0.2
F: TCACAGCTTTCCGCATATTG
R: GGACTTTGCGAAAGGATCG
Tight Junctions
CLDN4 Tight junction NM_00130
5.3
F: ATAAAGCCAGTCCTGATGCG
R: TAACTGCTCAACCTGTCCCC
CLDN1
0
Tight Junction Protein NM_18284
8.3
F: GCTGACAGCAGCGATCATAA
R: AGGGTCTGTGGATGAACTGC
THBS1 adhesive glycoprotein that mediates
cell-cell and cell-matrix interactions
NM_00324
6.2
F:
CACAGCTCGTAGAACAGGAGG
R: CAATGCCACAGTTCCTGATG
119
Cellular Growth/Protection
AREG part of epidermal growth factor
family
NM_00165
7.2
F:
TGGAAGCAGTAACATGCAAATG
TC
R:
GGCTGCTAATGCAATTTTTGATA
A
ADM Pro cell survival – increase tolerance
of cells to stress, injury, promotes
angiogenesis
NM_00112
4.1
F: ACGGAAACCAGCTTCATCC
R: GCCAGTGGGACGTCTGAG
BIRC3 inhibits apoptosis by inhibiting
capases
NM_00116
5.3
F: TGTTGGGAATCTGGAGATGA
R: CGGATGAACTCCTGTCCTTT
Cell Death
DDIT3 pro-apoptotic factor NM_00408
3.5
F: TGGATCAGTCTGGAAAAGCA
R: AGCCAAAATCAGAGCTGGAA
FADD pro-apoptosis signal NM_00382
4.3
F: TCTCCAATCTTTCCCCACAT
R: GAGCTGCTCGCCTCCCT
Cell Arrest
CDKN
1A
cell cycle inhibitor NM_07846
7.2
F:
TGGAGACTCTCAGGGTCGAAA
R:
GGCGTTTGGAGTGGTAGAAATC
120
CDKN
1B
cell cycle inhibitor NM_00406
4.3
F: CGCCATATTGGGCCACTAA
R: CGCAGAGCCGTGAGCAA
121
Industrially prepared probiotics do not alter pathogen mediated genetic responses. qRT-
PCR analysis of the 11 genes generally showed higher fold changes compared to microarray
analysis (Figure 5.2). qRT-PCR also showed that both enteric pathogens (EHEC O157:H7,
strain EDL933 and AIEC, strain LF82) induced higher gene expression changes in Caco-2 cells
compared to the probiotic L. helveticus R0052. Co-incubation or pre-incubation with the
probiotics did not affect changes in gene expression caused by the pathogens (Figure 5.2).
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Figure 5.2. Microarray and qRT-PCR analysis of Caco-2 cells infected alone, co-incubated, or
pre-incubated with probiotics and pathogens. Caco-2 cells were incubated with either probiotics
or pathogens alone for 3h, co-incubated with probiotic and pathogen for 3h, or pre-incubated
with probiotics for 3h before the addition of a pathogen for 3h. Pro-inflammatory genes (Panels
A to C), anti-inflammatory genes (Panels D and E), cellular growth/protection genes (Panels F
to H), cell arrest/death genes (Panels I and J) and tight junction genes (Panel K) were analyzed
by both microarray and qRT-PCR. qRT-PCR fold expression were normalized to three reference
genes: ACTB, B2M, and RPLP0. All analyses were performed using independently collected
samples (n=6).
123
124
Growth of probiotics in MRS medium enables the suppression of pathogen gene mediated
effects. Previous studies demonstrated that preparation conditions can affect probiotic activity
(Sherman et al., 2005). Therefore, the effects of L. helveticus R0052 and L. rhamnosus GG
cultured in MRS broth on gene expression changes in Caco-2 cells either alone or as a 3h
pretreatment before challenge with E. coli EDL933 and E. coli LF82 was tested. As shown in
Figure 5.3, probiotics grown in MRS broth affected Caco-2 gene expression in a manner
distinctly different from the same strain prepared by using industrial means. Furthermore, MRS
cultured probiotics also prevented pathogen mediated changes in Caco-2 cell gene expression
(Figure 5.3).
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Figure 5.3. qRT-PCR analysis of Caco-2 cells incubated with either probiotics or pathogens
alone, or pre-incubated with probiotics. Probiotics were prepared either industrially or
cultured in MRS broth, while E. coli enteropathogens were cultured in LB broth. Expression of
pro-inflammatory genes (Panels A to C), anti-inflammatory genes (Panels D and E), cellular
growth/protection genes (Panels F to H), Cell arrest/death genes (Panels I and J) and tight
junction gene (Panel K) were analyzed by qRT-PCR. Fold expression changes were normalized
to three reference genes: ACTB, B2M, and RPLP0. All analyses were performed using
independently collected samples (n=3-6, Two-way ANOVA,* p < 0.05 compared to EDL933, &
p <0.05 compared to LF82).
126
127
5.5 DISCUSSION
This is the first study, at least to our knowledge, that has analyzed using microarray and qRT-
PCR the human immune pathways affected by the pathogenic E. coli strain EDL933 and LF82 in
combination with the probiotics L. helveticus R0052 and L. rhamnosus GG. In the present study,
we show through a series of complementary microarray and qRT-PCR analyses that two enteric
bacterial pathogens (EHEC O157:H7, strain EDL933 and AIEC, strain LF82) alter gene
expression changes in Caco-2 cells in a distinct manner compared to the probiotic bacteria L.
helveticus R0052 and L. rhamnosus GG alone. Moreover, probiotics prepared in MRS broth
prevented pathogen-mediated changes in Caco-2 cell gene expression in a co-infection model.
EHEC and AIEC are both human pathogens that modulate immune pathways upon contact with
the host cell. Their pathogen-associated molecular patterns (PAMPs) are recognized by host
pathogen-recognition receptors (PRRs) leading to the activation of an inflammatory response.
For example, Toll-like receptor signaling in the intestine activates cytokine production (Dalpke
et al., 2003), chemokine-mediated recruitment of acute inflammatory cells and immunoglobulin
A (IgA) production (Shang et al., 2008), epithelial cell proliferation (Fukata et al., 2009),
maintenance of the integrity of intercellular tight junctions and antimicrobial peptide expression
(Hooper and Macpherson, 2010).
Probiotics are live, non-pathogenic microorganisms that confer health benefits to the host, and
are increasingly being employed to prevent and treat bacterial infection (Gareau et al., 2010). For
instance, probiotic bacteria have been shown to inhibit pathogen adhesion and invasion, and
changes in epithelial cell permeability induced by enteroinvasive E. coli (Resta-Lenert and
Barrett, 2006), Salmonella typhimurium (Gill et al., 2001) and Shigella flexneri (Tien et al.,
2006). Probiotics also mediate the activation, expression and secretion of pro-inflammatory
cytokines (TNFα) and chemokines (CXCL-8) caused by enteric pathogen infection (Gareau et
al., 2011).
In the present study, we have shown that pre-incubation of Caco-2 cells with probiotics L.
helveticus R0052 and L. rhamnosus GG grown in MRS broth were able to significantly
ameliorate E. coli EDL933 and LF82-induced modulation of genes involved in pro-
inflammatory, anti-inflammatory, cellular growth/protection and cellular arrest/death signaling
pathways (Figure 5.3).
128
The results of this study raise several issues with regards to the use of probiotics as a
management option for intestinal infections. In a previous study, we observed a more beneficial
effect from industrially versus MRS broth grown probiotics in modulating epithelial cell barrier
function in T84 cells (Sherman et al., 2005). In the present study, we confirm that the specific
methods used to prepare probiotics can dramatically affect their biological effects. However, in
this case MRS grown probiotics were more efficacious in mediating host immune pathways
compared to industrially prepared L. helveticus R0052. As reviewed by (Foster et al., 2011), it
has been observed that the probiotics Lactobacillus rhamnosus R0011 can reduce the
pathogenicity of Campylobacter jejuni in HT-29 cells (Alemka et al., 2010), but not when using
T84 cells (Wine et al., 2009). These findings suggest that there is specificity to the interactions
between probiotics and human cells. Furthermore, while it is not currently known precisely how
preparation conditions affect probiotic effectiveness, this area of research is currently under
active investigation (Grzeskowiak et al., 2011).
The results arising from this study also show the benefits of employing probiotics as a
prevention, strategy, rather than as an intervention option, as pre-incubation with probiotics
prepared in MRS medium prevented pathogen-induced pro-inflammatory gene expression.
Recent findings presented from our laboratory demonstrate that probiotics can be effective in
both preventing and treating bacterial intestinal infections in vivo, but efficacy in the rodent
model of bacterial-induced colitis diminished the longer the infection goes unchecked
(Rodrigues et al., 2012).
Future work focusing on the preparation conditions mediating probiotic effectiveness, as well
experiments to determine the temporal restrictions involved in using probiotics as a therapeutic
option should now be undertaken. Such information could be used to elucidate the mechanisms
by which probiotics achieve their health benefits, and direct use as a management option for
bacterially-induced enterocolitis.
5.6 ACKNOWLEDGEMENTS:
This work was supported by an operating grant from the Canadian Institutes of Health Research
(MOP-89894). NH was supported by a doctoral research award from the Canadian Institutes of
Health Research (JDD-95413). PMS was supported by a Canada Research Chair in
Gastrointestinal Disease.
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Chapter 6
Discussion, Future Directions, and Significance
130
6.1 DISCUSSION
The studies presented in this thesis evaluated the mechanisms by which E. coli O157:H7 is able
to subvert host IFNγ/Jak1,2/Stat-1 signaling. Through a series of complementary biochemical
and genetic techniques, I have demonstrated the ability of EHEC to subvert the IFNγ pathway by
ways that are independent of direct bacterial contact with epithelial cells, and through the use of
secreted proteins (Chapter 3). I then showed that EHEC secreted Shiga toxins play a role in the
subversion of IFNγ signaling when testing several human epithelial cell lines (Chapter 4); and
that probiotics could ameliorate host immune activation responses induced following EHEC
exposure in a manner that is dependent on both the preparation employed and the conditions of
exposure (Chapter 5).
Chapters 3 and 4 address one of the central themes of this thesis as the concept of pathogen
mediated host immune activation, and the subversion by EHEC to evade the host immune system
and thereby potentially promote pathogenesis. It is not surprising to discover that EHEC has
evolved a way to subvert the host IFNγ pathway, as subversion of the host immune system is
increasingly recognized as a common theme as an underlying strategy employed among many
human pathogens (Jones and Neish, 2011). For example, viruses of the Paramyxoviridae family
subvert the IFNγ pathway by degrading intracellular Stat-1 (Didcock et al., 1999), while the
parasite Leishmania donovani prevents tyrosine-phosphorylation and activation of Stat-1
(Nandan and Reiner, 1995). The ability of EHEC O157:H7 to suppress the IFNγ pathway could
arguably promote its ability to colonize the gut of the infected host by reducing immune
surveillance (Jones and Neish, 2011).
As EHEC contains an array of virulence factors that aid in infection pathogenicity and
subversion of host immune signaling, such as the Locus of Enterocyte Effacement (LEE)
pathogenicity island encoded type three secretion system (T3SS), its pO157 plasmid, and phage
encoded Shiga toxins, I focused on each of these virulence factors to identify potential
mechanisms by which this pathogen can subvert the host IFNγ/Jak/Stat signal transduction
pathway.
One virulence factor of primary interest was the LEE pathogenicity island and the encoded
T3SS, which allows the bacterium to inject bacterial effector proteins directly into the host cell to
subvert host cytoskeleton processes, destroy brush border microvilli, and cause rearrangements
131
of F-actin resulting in attaching and effacing (A/E) lesions on epithelial cell apical membrane
surfaces (Frankel and Phillips, 2008; Garmendia et al., 2005; Kaper et al., 2004). Studies on the
T3SS and its effectors have found that many of these bacterial-derived proteins have redundant
and overlapping functions, each of which appears to have multiple roles in subverting eukaryotic
cellular processes, thereby aiding in EHEC-mediated disease pathogenesis (Dean and Kenny,
2009). For example, the EspF protein appears to play several roles in the subversion of host
cellular signaling pathways, such as effacing the microvilli of infected cells, disrupting the
nucleolus, and preventing phagocytosis (Holmes et al., 2010). Furthermore, EspF, along with Tir
and Map, all contribute to SGLT-1 transporter inactivation (Wong et al., 2011). As a whole, it
appears that many of the virulence proteins expressed by EHEC are multifunctional, cooperative,
and redundant (Dean and Kenny, 2009). However, experiments utilizing isogenic mutants in the
T3SS system demonstrated that the LEE PAI in E. coli O157:H7 was not responsible for EHEC
mediated subversion of the IFNγ signaling pathway (Chapter 3).
EHEC O157:H7 also harbors a pO157 virulence plasmid that encodes several putative virulence
factors, such as: a metalloprotease (stcE) (Lathem et al., 2002), a serine protease (espP)
(Brockmeyer et al., 2007), a hemolysin (ehxA) (Schmidt et al., 1994), a catalase-peroxidase
(katP) (Brunder et al., 1996), and a putative adhesin (toxB) (Tatsuno et al., 2001). However,
experiments using pO157 plasmid cured strains continued to subvert the host IFNγ pathway as
well as wild-type strains (Chapter 3), indicating that this plasmid does not contain a factor
related to subverting of this signal transduction cascade.
Shiga toxins serve as a distinct marker for EHEC infections, and aid the pathogen in causing
systemic complications, including hemorrhagic colitis and the hemolytic-uremic syndrome
(HUS) (Obrig, 2010). In North America, approximately 75,000 cases of EHEC infections are
reported annually. Of these, 10-15% of cases develop HUS (Panos et al., 2006). In Chapter 5, I
demonstrate through the use of knockout mutagenesis, genetic complementation in trans, as well
as use of purified Stx that these toxins are involved in the suppression of the IFNγ/Jak 1,2/Stat-1
pathway. To our knowledge, this is the first study to show that Shiga toxins modulate IFNγ
signaling, which supports recent findings that many EHEC effectors have multiple and
overlapping roles in subverting host cell processes (Dean and Kenny, 2009; Hamada et al.,
2010).
132
The second theme of this thesis is that probiotics are a potential therapeutic option for use in the
management of EHEC infections. Studies of these beneficial microbes over recent years
demonstrate that probiotics can prevent and perhaps be used to treat enteric infections
(Rodrigues et al., 2012) through a multitude of mechanisms ranging from secreting antimicrobial
peptides (Corr et al., 2007), physical displacement of pathogenic bacteria (Johnson-Henry et al.,
2007), to modulation of host immune pathways (Seth et al., 2008). In Chapter 6, I evaluated two
such strains of probiotics, Lactobacillus helveticus R0052 and Lactobacillus rhamnosus GG, and
showed that pre-treatment with these probiotics can ameliorate host immune activation responses
to challenge of epithelial cells with two pathogenic bacteria, E. coli O157:H7, strain EDL933
and E. coli O83:H1, strain LF82, in a preparation dependent manner. The findings of this PhD
thesis can be used to form a model of EHEC O157:H7 pathogenesis, and is illustrated in Figure
6.1.1.
133
Figure 6.1.1. The results of this thesis presented as a model of EHEC O157:H7
pathogenesis. EHEC has evolved a method to subvert the IFNγ signaling pathway. The results
of Chapter 4 show that EHEC can mediate this effect through the secretion of its proteins, but
YodA does not appear to be involved. The results of Chapter 5 demonstrate that the secreted
EHEC Shiga toxins are involved, but also indicate that non-secreted proteins can play a
significant part in subverting the IFNγ signaling pathway. The results of Chapter 6 demonstrate
that probiotics could help prevent pathogen gene mediated effects on human intestinal epithelial
cells.
134
135
6.2 FUTURE DIRECTIONS
There are several areas presented in this thesis which could direct future research in the field. For
instance, YodA was identified as a candidate protein mediating the inhibition of IFNγ induced
Stat-1 phosphorylation in Chapter 3. While I demonstrated that a YodA knockout in EHEC, as
well as a purified YodA product were still able to subvert Stat-1 phosphorylation, further work
could be done to verify these results. For example, column chromatography could be repeated
using YodA CS to determine if the inhibitory protein is again observed, and subsequent isolation
experiments, using the methodologies described in Chapter 3 could then be undertaken to
identify the secreted factor.
Furthermore, the complete mechanisms by which EHEC O157:H7 subverts the IFNγ pathway
remains to be elucidated. I have demonstrated that Stx are involved in mediating this ability
(Chapter 5), but it is also clear that other non-secreted factors are likely to be involved, since
direct infection with StxDKO mutants retain the ability to completely suppress IFNγ mediated
Stat-1 tyrosine phosphorylation.
An interesting direction for future research would be to identify and characterize differences
between proteins expressed and localized in the outer membranes of EPEC and EHEC. As
illustrated by Molley et al. (2001), this could be accomplished by collecting total bacterial
proteins, and then use an alkaline pH wash to remove non-membrane bound proteins. The
remaining membrane bound proteins could then be solubilized using strong denaturing
conditions and analyzed using 2D Difference Gel Electrophoresis, as was previously done in
Chapter 5. Differing proteins can then be verified for their role in subverting the IFNγ pathway
by E. coli O157:H7 using knockout mutagenesis and complementation in trans.
Another prime area for future study would be to further elucidate mechanisms by which Stx
suppress IFNγ mediated Stat-1 tyrosine phosphorylation. It can be presumed that the toxin’s
effects are mediated by direct protein interaction with at least one part of the IFNγ pathway.
Hence, experiments using co-immunoprecipitation (co-IP) techniques could be employed to
determine any direct binding of Stx’s to either the IFNγ receptors (IFNGR1, IFNGR2) the Jak’s
(Jak1, Jak2), or perhaps Stat-1 itself. The scope of such experiments then could be expanded to
include other EHEC proteins that might interact with Stx, as it was observed that purified Stx
136
alone did not affect IFNγ signaling, and that culture supernatants were required for IFNγ
suppression (Chapter 5).
In parallel with these proposed studies, alternative experiments employing modified Stx could be
utilized to elucidate the active sites required for mediating IFNγ subversion. Since nothing is
known about the mechanisms by which Stx mediates effects on the IFNγ pathway, either the use
of inactive Stx toxins (Glu167 and Arg176 double mutations in their active site) (Di et al., 2011),
or employing purified Stx A and B subunits could be tested to infer the minimal requirements for
Stx mediated IFNγ subversion. Results from these experiments could potentially be applied to
developing therapeutic options for use in the treatment of chronic diseases which have elevated
IFNγ levels, such as Crohn’s disease (Pak et al., 2012).
An interesting corollary of the Stx mutations is that, despite knockouts in stx1A, stx2A or both,
there remains some residual expression of toxin activity in the isogenic mutants (Table 4.3).
Theoretically, there shouldn’t be any detected at all since 1) the genes were ablated entirely, 2)
the antibody used in the ELISA are monoclonal towards the toxins, and 3) the technique used is
sensitive to pg/ml levels of the toxin. It is likely that this is an experimental artifact. Further
inquiry to define the exact specificity of the antibodies used in the immunoassay would be of
interest in determining the robustness of the knockout mutation studies.
Future work with probiotics would also likely prove to be meaningful. Additional microarray
experiments using MRS grown probiotics would complete the data currently present in Chapter
5. It would also be of interest to determine the ineffectiveness of the industrially prepared
probiotics compared to the MRS grown variants. One hypothesis is that since the industrially
prepared R0052 is packaged for human consumption and protection from the stomach acids in
vivo, it may require more time to activate the bacteria, a possibility which was not considered
during our experiments in vitro. One way to test this hypothesis would be to perform a growth
curve experiment on both the industrially and MRS prepared probiotics. Should a significant
delay in growth be seen in the industrially prepared probiotics, this could indicate a fault with the
in vitro experimentation design, and explain why the industrially prepared R0052 was ineffective
in modulating pathogen mediate host immune responses compared to MRS prepared probiotics.
137
It would be of great interest to assess the effects of probiotic administration during and post-
infection so as to determine if these beneficial microbes could be extended to use beyond
preventative strategies, such as in the setting of an outbreak of infection or to prevent intra-
familial or nosocomial spread of the illness. The inclusion of different preparation strategies,
such as collection during different growth phases (exponential vs. stationary), as well as
experiments with varying initial dosages would also be of value in determining the optimal
preparation conditions required for creating an effective probiotic for use in a therapeutic setting.
6.3 SIGNIFICANCE
EHEC is responsible for 75,000 infections annually in North America. The hemolytic-uremic
syndrome is the most severe clinical manifestation of EHEC infections, which accounts for >200
deaths every year. Antibiotics exacerbate EHEC infection as they increase the potential for
developing systemic disease. Characterizing novel EHEC virulence factors and the mechanisms
of bacterial disruption of host cellular responses will, therefore, advance current knowledge of
EHEC immune evasion strategies. Although the results of my thesis are reductionist in nature,
the finding that EHEC can subvert the IFNγ pathway is significant and should be verified in
relevant animal models (Higgins et al., 1999) before extended to human intervention studies.
Such information could lead to the development of novel alternative strategies to prevent and
treat EHEC infections in humans.
138
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