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Real-Time Tracking of Motile Unicellular Parasites: A Dynamic View of Entamoeba histolytica Virulence

This study focuses on the tracking and imaging of Entamoeba histolytica, a parasitic unicellular organism, in real-time. The research aims to understand the motility and virulence of the parasite, including its adhesion properties and ability to invade host cells and cause disease. The findings have important implications for public health and the development of interventions against amoebiasis.

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Real-Time Tracking of Motile Unicellular Parasites: A Dynamic View of Entamoeba histolytica Virulence

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  1. 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

  2. Cyst A public health problem DYSENTERY ABCESS 50 millions cases 100.000 death Trophozoite Parasite life cycle and infection Entamoeba histolytica In rural areas, 2 to 20% of humans carry E. histolytica. These reject daily millions of cysts into the environment Amoebiasis ASYMPTOMATIC SYMPTOMATIC

  3. Intestine invasion requires specific parasite adhesion and toxic factors Invasion of colon Surface molecules mucin Invasion of mucosa and submucosa Dissemination Inflammation N 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

  4. Eh Human enterocytes The Gal-GalNAc inhibitable lectin Gal/GalNAc lectin immunolabeling 170 kDa C-rich COOH 31-35 kDa CRD GPI TM cytoplasm Tavares et al., 2005, Infection and Immunity

  5. Signalling through the heavy chain is involved in parasite adhesive properties HgL PM -actinin Small GTPAses, PAK Cytoskeleton remodelling Dominant negative strategy for phenotype analysis LOST OF PARASITEADHESION TO CELLS HGL-negative parasite Vines 1998, MCB. Tavares , 2005, I and I

  6. Does changes in the signalling through theheavy 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

  7. 1 60 156 172 E. histolytica is a highly motile cell Computing based on active contours analysis Live cell imaging Parametric curve is deformed under internal forces (geometry) and external forces (image) Image frames Moves at ~ 1mm/sec C. Zimmer, J.C. Olivo - UAQI, IP

  8. MSD: mean square displacement MSD (m2) = <x2(t)> lm2  = 1, Brownian log time (sec) . . . . 1 2 3 Mathematical analysis of amoeba motility 1. Directionality and speed 2. Cell morphology changes Image frame WT Amoebic motility obey to a power law (> 1) indicating important regulations based on acto-myosin cytoskeleton activities

  9. Fluorescence emission from two-photons laser pulsations Continue Laser irradiation One photon Pulsed Laser irradiation Two photons Two-laser microscopy Weak energy Preserved samples Small volume of irradiation safranine

  10. Imaging E.histolytica in culture conditions trajectories morphology Wild type HGL Two-photons laser microscopy on living parasites

  11. Two-photons microscopy during liver infection by E. histolytica 50 m 20 m High laser power Weak laser power *Liver is “viewed” by its natural fluorescence *Parasites were labeled with a fluorescent cell tracker

  12. Imaging E.histolytica in a living liver trajectories morphology Wild type HGL HGL parasites do not deform and are immobile in living liver

  13. MSD: mean square displacement MSD (m2) = <x2(t)> lm2 On 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  = 1, Brownian log time (sec) Summary of motility mathematical analysis *Calculations were made on at least 50 individual cells from each experimental condition • Speed of displacements of WT parasites are reduced in living tissuecompared to culture conditions • Adhesion through the Gal/GalNAc lectin is essential to amoebic motility in the tissue (3D)

  14. Experimental infection with WT and HGL strains Wild type HGL ABCESSES AT 7 DAYS Adhesive properties of E. histolytica are essential to pathogenesis

  15. 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

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