Tracking motile unicellular parasites in real time A dynamic view of Entamoeba histolytica virulence Nancy Guillén Unité Biologie Cellulaire du Parasitisme

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)

QuickTime™ et undécompresseur TIFF (non compressé)

sont requis pour visionner cette image.







1 60





156 172

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





*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





*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

Acknowledgments….

Eveyne Coudrier and François Amblard Curie Institute

Marie-Christine RigothierPharmacy Faculty. Paris XI University

Christophe Zimmer and Jean-Christophe Olivo UAIQ, Pasteur Institute

Pascal Roux, Imagopole, Pasteur Institute

Documents

Tracking motile unicellular parasites in real time A dynamic view of Entamoeba histolytica virulence Nancy Guillén Unité Biologie Cellulaire du Parasitisme