Outcome of E. histolytica (Eh) infection in the gut
Asymptomatic infection
Trophozoites colonize
Acute inflammatory response
Trophozoites breach innate defences
Infection resolved
Amebic colitis/ liver abscess
Clear Eh Eh persist
? ?
99% 1%
MUC2 mucus layer
Eh colonizes in/on the outer mucus layer
Eh
Inner densemucus layer
Outer loosemucus layer
Depletion of the mucus layer and Eh adherence to epithelial cells
Eh
Cover Photo J. Innate Immunity (2009)
De
gre
e o
f In
flam
mat
ion
Live/Dead Ehwhole lysates
membrane componentscytosolic components
Live Eh
High level of danger -strong inflammatory
response
Low level of dangermild inflammatory
response
What host pathogenicity sensors respond to this
scenario?
NLRP3 inflammasome monitor the pathogenicity of Eh
Asymptomatic infection Invasive disease
Inflammasomes - Caspase-1 Activating Platforms
Intracellular multi-protein complexes that link specific pro-inflammatory stimuli to caspase-1 activation
Pro-caspase-1
ASC
NLRP3
Pro-caspase-1
ASC
AIM-2
Pro-caspase-1
ASC
NLRP1
Pro-caspase-1
ASC
NLRC4
Naips
Bacillus anthracislethal toxin
Cytosolic flagellin
Type III and IV secretion systems
Cytosolic dsDNAToxins (nigericin, maitotoxin)
Endogenous danger molecules (ATP, uric acid crystals)
Bacteria, Fungi, Viruses
Environmental particulates (silica, asbestos)
DNA
mRNA
mRNA
Nucleus
Cytosol
Inflammasomes - Caspase-1 Activating Platforms
Pro-IL-1β
Pro-IL-18Active Caspase-1
Cell surface
Pro-caspase-1
ASC
NLRP3 sensor
Pyroptosis
CARDCARD
PYDPYD
Stimulus LRR
NBC
Active MultimericComplex
Signal 1Signal 2
PrimingActivating
kDa
11
SN20
35
45
p20/22
proCaspase-1
p35
CARDCaspase-1
IL-1β
Only live Eh activates the inflammasome
Eh
Mortimer, Moreau, Cornick and Chadee (2013) Mucosal Immunology 7:829
10 20 40
SN
LYS
pro-IL-1β
45
Eh (min)
17 p17
kDa
p20
35
20
11
31
CARD
Caspase-1
IL-1β
GAPDH
pro-caspase-1
pro-IL-1β
p17
p20
CARD
Caspase-1
IL-1β
GAPDH
pro-caspase-1
31
45
17
kDa
35
20
11
SN
LYS
Eh
Eh
0
300
600
900
1200
IL-1β (pg
/mL)
-V Eh
ZVADYVAD
0
300
600
900
1200
IL-1
b pg
/mL
***
***
10 20 400
500
1000
1500
2000
IL-1β (pg
/mL)
Eh (min)
-V
10 min
20 min
40 min
0
500
1000
1500
2000
IL-1
b p
g/m
L
***
***
***
Eh activates the NLRP3 inflammasome by direct contact
Uns
timulate
dLPS
Nigericin
Eh
Active caspase-1 FLICA Mammalian nuclei
MΦ
MΦ
MΦ
Eh
zVAD – pan-caspase inhibitorYVAD – caspase-1 specific inhibitor
Eh
p20/22
CARD
Caspase-1Pro
p35
Caspase-1
kDa
45
35
20
11
26
SN
Eh
IL-1β (pg
/mL) 800
400
0
600
200
Surface-bound Eh Gal-lectin is required to activate the inflammasome
Eh
kDa
SN
45
35
20
11
17
p20/22
CARD
Caspase-1Pro
IL-1β
p35
Caspase-1
3F48C12
895-998
H851033-1082
Cysteine poor Pseudo-repeat Cysteine Rich
NH2
1 436 624 1053
COOH
Eh
IL-1β (pg
/mL)
Inhibitory
Enhancing900
300
600
0
Gal-lectin MAbs bind to and inhibit Eh-induced inflammasome activation
1G7596-818
Soluble Eh Gal-lectin primes but does not activate the inflammasome
SN
kDa
45
35
20
11
p20/22
CARD
Caspase-1
p35
pro
Caspase-1
IL-1β (pg
/mL)
1000
250
500
0
750
31 IL-1β
117 NLRP3
GAPDH
Caspase-1pro45
LYS
kDa
IL-1β
p17
Caspase-1p1010
SN
kDa
17
31The Gal-lectin up-regulates (primes) IL-1β and NLRP3
similar to LPS
Humanmacrophage
BMM
What host surface receptor is at the contact site?
Eh
MΦ
Con
trol
α5β1 integrin phospho-paxillin merge mammalian nuclei
MΦ
Eh
MΦ
Eh
α5β1 integrin and phosphorylated-paxillin are recruited to sites of contact where there is active integrin signaling
MΦ
MΦ
MΦ
Eh
Eh
MΦ
Hou, Mortimer and Chadee (2010)JBC 285:35497
pro-domain mature EhCP-A5
Cysteine protease
RGD(92-94)ERFNIN
RNS(266-268)
Q99 C105 H247 N267
Structural Organization of EhCP-A5
α5 β
1
rCP5 rCP5 RAD
EhCP-
A5
nuclei
MΦ MΦ
Surface-bound EhCP-A5 interacts with α5β1 integrin
IP: α5β1
IB: EhCP-A5
Input
Lysate
IB: α5β1
GAPDH
130
IB: α5β1
IP
Lysate
26
130
MΦ
MΦ
Eh
EhCP-A5 mammalian nuclei
Mortimer, Moreau, Cornick and Chadee (2015) PLoS Pathogens. doi: 10.1371
170
130
170
130
α5 integrin
β1 integrinclone LM 534
Phospho-Tyrβ1 integrin
β1 integrinClone LM 534
GAPDH
IP: α5β1 integrin
IP ly
sate
sinput ly
sate
sα5 integrin
Eh
170
130
170
130
170
130
α5 integrin
β1 integrinclone LM534
Phospho-Tyrβ1 integrin
IP ly
sate
s
α5 integrin
β1 integrinclone LM534
GAPDH
input lysa
tes
Eh
170
130
IP: α5β1 integrin
EhCP-A5 RGD-α5β1 integrin signaling upon contact
Specificity for EhCP-A5 RGD bindingwith RGDSP-inhibitory peptide but
not RADSP control
SFK phosphorylation (160/140/120kDa)inhibited by SFK inhibitor PP1 but not
inactive analog PP3
Is α5β1 integrin activation required for Eh to activate the NLRP3 inflammasome?
PP1 (μM)
0
200
400
600
1000
IL-1β (pg
/mL)
Eh
-V Eh
PP1 5 µM
PP1 10
µM
PP1 20
µM
PP30
200
400
600
800
1000
IL-1
b (
pg/m
L) ***
Eh
***
***
800
PP1 (μM)
11
Eh
CARD
kDa
SNCaspase-1
Eh
0
600
900
1200
IL-1β (pg
/mL)
300
-V Eh
RGDSP
RADSP0
300
600
900
1200
IL-1
b p
g/m
L
***
***
11
kDa
SN
Eh
CARDCaspase-1
RGDSP = inhibitory peptide PPI = SFK inhibitor
Is activation of α5β1 integrin required for Eh to activate the NLRP3 inflammasome?
CARD
Eh
Caspase-111SN
kDa
IL-1β (pg
/mL)
Eh
400
600
0
800
200
-V Eha5b1
isoty
pe0
200
400
600
800
IL-1
b p
g/m
L
***
Function blocking Abs to α5 and β1 subunits and α5β1 abrogated caspase-1 and IL-1β secretion
20
45
35
10
10 20 40 10 20 40
Eh EhCP-A5
pro-IL-1β
pro- caspase-1
GAPDH
IL-1β p17
(min)kDa
31
17
Caspase-1 CARD
Caspase-1 p20/22
pro-IL-1β
SN
LYS
***
Eh EhCP-A5
******
***
Inflammasome activation requires EhCP-A5
IL-1β
How does EhCP-A5-α5β1 integrin signaling regulate NLRP3 activation?
The kinetics of NLRP3 activation is very rapid
(can detect within 10 minutes)
This is similar to the rate of NLRP3 activation by ATP
(high extracellular ATP is an endogenous activator of NLRP3)
35
Eh
45
kDa
20
11
SN p20
CARD
Caspase-1
KN-62 (μM)
Eh
600
900
0
1200
IL-1β (pg
/mL)
300
-V Eh 5 100
300
600
900
1200
IL-b
(pg/
mL)
Eh + KN-62(mM)
***
***
Eh
600
900
0
1200
IL-1β (pg
/mL)
300
-V Eh
apyr
ase
0
300
600
900
1200
***
IL-b
(p
g/m
L)
Eh
oATP (μM)
1500
2000
0
2500
IL-1β (pg
/mL)
500
1000
-V Eh 50100
0
500
1000
1500
2000
2500
IL-1
b pg
/mL
***
***
Eh+ oATP (mM)
Is ATP required for Eh to activate the NLRP3 inflammasome?
ATP
P2X7
ATP
NLRP3
Cell surface
35
20
45
kDa
11
p20
CARD
Caspase-1
Eh
oATP (μM)
SN
35
20
45
kDa
11
p20
CARD
Caspase-1
Eh
KN-62 (μM)
SN
oATPKN-62
Apyrase (hydrolyses ATP to AMP)
Antagonist of P2X7
receptor signaling
Does EhCP-A5 trigger ATP release from macrophages?
-V1
wt
5 w
t
10 w
t
30 w
t
1 cp
5
5 cp
5
10 c
p5
30 c
p5W
t
EhC
P5-
0
100
200
300
400
500
AT
P (
nM
)
***
***
EhCP-A5
300
400
0
500
200
100
ATP
(nM
)
Wt Eh
(min)
EhCP-A5Wt Eh
50
75
0
100
25
rCP5 (μg mL-1)
ATP
(nM
)
-V 1.0
0.5
0.25
0.12
5
0.06
250
25
50
75
100
AT
P (
nM
)
***
**
*
rCP5
ATP
P2X7
ATP
NLRP3
Cell surface
Eh
MacrophageEhCP-A5
20
30
0
40
IL-1β (pg
/mL)
10
50
60
20
30
0
40
IL-1β (pg
/mL)
10
50
60
-V Wt
Nlrp3-/-
Asc-/-
0
10
20
30
40
50
60
IL-1
b (p
g/m
L)
**
Is NLRP3 inflammasome required for Eh-inducedpro-inflammatory responses in the colon?
Healthy human colon biopsy exposed to Eh
Mouse colonic loops inoculated with Eh
Glyburide – NLRP3 inhibitor
Take home messages
Eh primes the NLRP3 inflammasome upon formation of an immune synapse mediated by the Gal-lectin
Gal-lectin binding facilitates EhCP-A5 ligation and activation of α5β1 integrin and NLRP3 inflammasome
activation
NLRP3 inflammasome functions as a pathogenicity sensor for detecting invading Eh
CASP1CASP4
ASCNLRP3
Gal-lectin α5β1
EhCP-A5
Eh
MΦ
IL-1β
ROSK+
efflux
EhCP-A1
EhCP-A4
CASP6
Paxillin
Talin Pyk2
ATP
Pannexin 1
P2X7
Mortimer et al.
(2013) Mucosal Immunol
7:829-841
Mortimer et al. (2015)
PLoS Pathogen
11:e1004887
St-Pierre et al.
(2017) PLoS Pathogen
13:e1006592
Quach et al.
(2018) Mucosal Immunol
doi:10.1371/journal.ppat.
1007466
GSDMD
AcknowledgementsChadee Lab
France Moreau
Jeanie Quach
Sharmin Begum
Aralia León Coria
Dr. Manish Kumar
Dr. Sameer Tiwari
Dr. Preeti Shahi
Hayley Gorman
Past Chadee Lab Members
Dr. Steve Cornick
Dr. Joëlle St-Pierre
Dr. Leanne Mortimer
Dr. Adelaide Tawiah
Cells/Mice
Dr. Yan Shi (Casp-1/11 KO mice),
Dr. Yates (HEK-IL-1β cells, COS-7)
Dr. Dan Muruve (NLRP3 and ASC KO mice, NLRP3 CRISPR KO THP-1)
Dr. Bruce Vallance (Casp-11 KO mice)
Funding:
CIHR
NSERC
CCC
CFI
Trainee Scholarships
α5β1 integrin
Gal-lectin Gal-lectin + EhCP-A5 PGE2
TLR4 EP4
Eh virulent molecules
Host receptors
?
?
Mortimer . . Chadee (2016)Nature Immunology 10:1
The secretory response consist of water, mucus, serum albumin and pro-inflammatory cytokines
Muc2-/-
Wt6h Eh PI
3h Eh PI
Wt
Muc2-/-
Kissoon-Singh. . .Chadee (2013) Am J Pathology 182: 852
SENSOR
Pro-caspase-1
ASC
pro-IL-1β active
IL-1βpro-IL-18
active
IL-18
Sensor activation
Complexoligomerization
Parasite stimuli
Inflammasomes control the activation of caspase-1
Caspase-1
CARD
Active Multimeric Complex
NLRP3
CARD
PYD
PYD Active Caspase-1
Model of NLRP3 inflammasome activation by EhCP5
NLRP3 Inflammasome
ATP
P2X7
pannexin-1
ATP
pro-caspase-1
ASC
NLRP3
Eh
Macrophage
EhCP5
Src kinases
Actin cytoskeleton
α5β1
integrin
Goblet Cell
PKCδ activation
Green – mucus secretion
Live Eh
Live cell imaging showing PKC activation and mucus secretion
Global Prevalence and Incidence of Amebiasis
• About 10% of the world ’ s population is infectedwith Eh
• 1% get invasive disease (amebic colitis and/or liverabscess) causing @105 deaths/year
• Second leading cause of death by a protozoanparasite
MUC2 Mucus Barrier
Unresolved issues in the biology of Eh
Does the host sense/respond to
colonized Eh in the gut?
Why does Eh elicit a rapid pro-inflammatory response upon
contact/invasion?
Eh