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 Collège de France

Parasite life cycle and infection ASYMPTOMATICSYMPTOMATIC DYSENTERYABCESS 50 millions cases 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 mucosa and submucosa Dissemination Surface molecules Adapted from Haque R et al., N Engl J Med Surface molecules: LPG, KERP1, Gal/GalNAc lectin Cytotoxic molecules: amoebapores Lytic factors: cysteine proteinases

COOH C-rich The Gal-GalNAc inhibitable lectin Eh Human enterocytes Tavares et al., 2005, Infection and Immunity 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 PARASITE ADHESION TO CELLS HGL-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 cell Computing based on active contours analysis Moves at ~ 1  m/sec C. Zimmer, J.C. Olivo - UAQI, IP Live cell imaging Parametric curve is deformed under internal forces (geometry) and external forces (image) Image frames

WT Mathematical analysis of amoeba motility MSD: mean square displacement MSD (  m 2 ) = log time (sec) l    m 2  = 1, Brownian 1. Directionality and speed Amoebic motility obey to a power law (  > 1) indicating important regulations based on acto-myosin cytoskeleton activities 2. Cell morphology changes Image frame

Two-laser microscopy Weak energy Preserved samples Small volume of irradiation safranine 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 liver infection by E. histolytica High laser powerWeak laser power 50  m 20  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 tissue compared to culture conditions 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 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 (  m 2 ) = log time (sec) l    m 2  = 1, Brownian On slides Inside the liver WTHGLWTHGL Prefactor (  m 2 sec-  ) 1-100, Noise slope  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 Rigothier Pharmacy Faculty. Paris XI University Christophe Zimmer and Jean-Christophe Olivo UAIQ, Pasteur Institute Pascal Roux, Imagopole, Pasteur Institute