tracking motile unicellular parasites in real time a dynamic view of entamoeba histolytica virulence...
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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-symposiumSeeing is beleiving : imaging the infectious processes in vitro
and in vivo27 avril 2009. Collège de France
Parasite life cycle and infection
ASYMPTOMATIC SYMPTOMATIC
DYSENTERY ABCESS
50 millions cases 100.000 death
A public health problem
Cyst
Trophozoite
Entamoeba histolytica
Amoebiasis
In rural areas, 2 to 20% of
humans carry E. histolytica.
These reject daily millions of
cysts into the environment
Intestine invasion requires specific parasite adhesion and toxic factors
N
mucin
Colitis
Invasion of colon
Inflammation
Invasion of mucosaand submucosa
Dissemination
Surface molecules
Adapted from Haque R et al., N Engl J Med. 2003
•Surface molecules: LPG, KERP1, Gal/GalNAc lectin
•Cytotoxic molecules: amoebapores
•Lytic factors: cysteine proteinases
COOH
C-rich
The The Gal-GalNAc inhibitable lectinGal-GalNAc inhibitable lectin
Eh
Human enterocytes
Tavares et al., 2005, Infection and Immunity
31-35 kDa
170 kDa
GPI
TM
cytoplasm
CRD
Gal/GalNAc lectin immunolabeling
Signalling through the heavy chain is involved in parasite adhesive properties
-actinin
Small GTPAses, PAK
Cytoskeleton remodelling
HgL
PM
LOST OF PARASITEADHESION TO CELLSHGL-negative parasite
Vines 1998, MCB. Tavares , 2005, I and I
Dominant negative strategy for phenotype analysis
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 cellComputing based on Computing based on
active contours analysisactive contours analysis
Moves at ~ 1m/sec C. Zimmer, J.C. Olivo - UAQI, IPC. Zimmer, J.C. Olivo - UAQI, IP
Live cell imaging
Parametric curve is deformed underinternal forces (geometry) and external forces (image)
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1 60
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Image frames
WT
Mathematical analysis of amoeba Mathematical analysis of amoeba motilitymotility
MSD: mean square displacement
MSD (m2) = <x2(t)>
log time (sec)
l
m
2
= 1, Brownian
1. Directionality and speed
1 2 3
. . ..
Amoebic motility obey to a power law (Amoebic motility obey to a power law (> 1) indicating important > 1) indicating important regulations based on acto-myosin cytoskeleton activitiesregulations based on acto-myosin cytoskeleton activities
2. Cell morphology changes
Image frame
Two-laser microscopy
Weak energy
Preserved samples
Small volume of irradiationsafranine
Continue Laser irradiation
Pulsed Laser irradiation
One photon
Two photons
Fluorescence emission from two-photons laser pulsations
Wild type
HGL
Imaging E.histolytica in culture conditions
Two-photons laser microscopy on living parasites
trajectories morphology
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*Liver is “viewed” by its natural fluorescence
*Parasites were labeled with a fluorescent cell tracker
Two-photons microscopy during liverTwo-photons microscopy during liver infection by infection by E. histolyticaE. histolytica
High laser power Weak laser power
50 m20 m
Wild type
HGL
Imaging E.histolytica in a living liver
HGL parasites do not deform and are immobile in living liver
trajectories morphology
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*Calculations were made on at least 50 individual cells from each experimental condition
•Speed of displacements of WT parasites are reduced in living tissueSpeed of displacements of WT parasites are reduced in living tissuecompared to culture conditions compared to culture conditions
•Adhesion through the Gal/GalNAc lectin is essential to amoebic Adhesion through the Gal/GalNAc lectin is essential to amoebic motility in the tissue (3D)motility in the tissue (3D)
Summary of motility mathematical analysis
MSD: mean square displacement
MSD (m2) = <x2(t)>
log time (sec)
l
m
2
= 1, BrownianOn slides Inside the liver
WT HGL WT HGL
Prefactor (m2 sec-) 1-10 0,5-2 0.2 Noise
slope 1.25 1.13 1.6 NON applicable
Experimental infection with WT and HGL strains
ABCESSES AT 7 DAYS
Wild type HGL
Adhesive properties of E. histolytica are essential to pathogenesis