Download E. histolytica.

Tracking motile unicellular parasites
in real time
A dynamic view of
Entamoeba histolytica virulence
Nancy Guillén
Unité Biologie Cellulaire du Parasitisme
Institut Pasteur, Inserm U786
Mini-symposium
Seeing is beleiving : imaging the infectious processes in vitro and in vivo
27 avril 2009. Collège de France
Parasite life cycle and infection
Entamoeba histolytica
Cyst
In rural areas, 2 to 20% of
humans carry E. histolytica.
These reject daily millions of
cysts into the environment
Amoebiasis
A public health problem
DYSENTERY ABCESS
Trophozoite
ASYMPTOMATIC
50 millions cases 100.000 death
SYMPTOMATIC
Intestine invasion requires specific
parasite adhesion and toxic factors
Invasion of colon
Surface molecules
mucin
Invasion of mucosa
and submucosa
N
Dissemination
Inflammation
Colitis
•Surface molecules: LPG, KERP1, Gal/GalNAc lectin
•Cytotoxic molecules: amoebapores
•Lytic factors: cysteine proteinases
Adapted from Haque R et al., N Engl J Med. 2003
The Gal-GalNAc inhibitable lectin
Gal/GalNAc lectin immunolabeling
170 kDa
C-rich
Eh
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COOH
décompresseur TIFF
(non compressé)
QuickTime™ et un
31-35
kDa
Human enterocytes
CRD
GPI
TM
cytoplasm
Tavares et al., 2005, Infection and Immunity
Signalling through the heavy chain is
involved in parasite adhesive properties
HgL
Dominant negative strategy
for phenotype analysis
PM
-actinin
LOST OF PARASITE
ADHESION TO CELLS
HGL-negative parasite
Small GTPAses, PAK
Cytoskeleton remodelling
Vines 1998, MCB. Tavares , 2005, I and I
Does changes in the signalling through the
heavy chain of the Gal/GalNac lectin affect the
infectious process of Entamoeba histolytica?
Tools….
the HGL strain reduced in adhesion capacities,
molecular strategies, pathophysiology
and living imaging
E. histolytica is a highly motile cell
Live cell imaging
1
Computing based on
active contours analysis
60
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Parametric curve
is deformed under
internal forces
(geometry) and
external forces
(image)
172
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Image frames
Moves at ~ 1mm/sec
C. Zimmer, J.C. Olivo - UAQI, IP
Mathematical analysis of amoeba motility
1. Directionality and speed
MSD: mean square displacement
MSD (mm2) = <x2(t)>
 = 1, Brownian
log time (sec)
2. Cell morphology changes
.
1
.
.
2
3
Image frame
.
WT important
Amoebic motility obey to a power law (> 1) indicating
regulations based on acto-myosin cytoskeleton activities
Fluorescence emission from two-photons
laser pulsations
Continue Laser irradiation
One photon
Pulsed Laser irradiation
Two photons
safranine
Two-laser microscopy
Weak energy
Preserved samples
Small volume of irradiation
Imaging E.histolytica in culture conditions
trajectories
Wild type
HGL
morphology
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Two-photons laser microscopy on living parasites
Two-photons microscopy during liver
infection by E. histolytica
50 mm
20 mm
High laser power
Weak laser power
*Liver is “viewed” by its natural fluorescence
*Parasites were labeled with a fluorescent cell tracker
Imaging E.histolytica in a living liver
trajectories
Wild type
HGL
morphology
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HGL parasites do not deform and are immobile in living liver
Summary of motility mathematical analysis
MSD: mean square displacement
MSD (mm2) = <x2(t)>
 = 1, Brownian
On slides
Inside the liver
WT
HGL
WT
HGL
Prefactor (mm2 sec-)
1-10
0,5-2
0.2
Noise
slope
1.25
1.13
1.6
NON applicable
log time (sec)
*Calculations were made on at least 50 individual cells from each experimental condition
•Speed of displacements of WT parasites are reduced in living tissue
compared to culture conditions
•Adhesion through the Gal/GalNAc lectin is essential to amoebic
motility in the tissue (3D)
Experimental infection with WT and HGL strains
Wild type
HGL
ABCESSES AT 7 DAYS
Adhesive properties of E. histolytica are essential to pathogenesis
Acknowledgments….
Eveyne Coudrier and François Amblard
Curie Institute
Marie-Christine Rigothier
Pharmacy Faculty. Paris XI University
Christophe Zimmer and Jean-Christophe Olivo
UAIQ, Pasteur Institute
Pascal Roux, Imagopole, Pasteur Institute