Is Entamoeba histolytica contact with host cells the key to disease

pathogenesis?

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

Mucus hypersecretion in response to Eh

Kissoon-Singh. . .Chadee (2013) Am J Pathology 182: 852

Depletion of the mucus layer and Eh adherence to epithelial cells

Eh

Cover Photo J. Innate Immunity (2009)

Eh invasion of the laminar propria and crypts

Lamina propria

Crypts

Invasive Amebiasis

2

1

Amebic ulcerativecolitis

Amebic liverabscess

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

Leanne

Leanne

France

Steve

Jeanie

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

What component on the surface of Ehactivates the inflammasome?

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

How does Eh Gal-lectin activate the inflammasome?

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

If the Gal-lectin primes the inflammasome - what other surface Eh

molecule activates it?

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

Parasite-induced inflammasome activation in disease pathogenesis

Pathogen driven NLRP3 inflammasome activation is a sensor for initiating gut

inflammation

What are the predominant responses in the gut in the absence of Eh-epithelial cell

contact?

Wt 3h PI Muc2-/- 3h PI

No Eh-epithelial cell damage in mucus-deficient animals

Mucus plug

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

SharminJeanie Aralia

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

α5β1 integrin

Gal-lectin Gal-lectin + EhCP5 PGE2

TLR4 EP4

Eh virulent molecules

Host receptors

?

?

Goblet Cell

PKCδ activation

Green – mucus secretion

Live Eh

Live cell imaging showing PKC activation and mucus secretion

Leanne

France

Steve

Life cycle of Entamoeba histolytica

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

Pathogenesis of Entamoeba histolytica colitis: a global health

problem in developing nations

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

CASP1CASP4

ASCNLRP3

Gal-lectin α5β1

EhCP5

Eh

MΦ

IL-1β

ROSK+

efflux

EhCP1

EhCP4

CASP6

Paxillin

Talin Pyk2

ATP

Pannexin 1

P2X7

1 2

3

4

4

5 5

6