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  Director : GUILLEN-AGHION, Nancy (nguillen@pasteur.fr)


  abstract

 

Cell polarization necessary for motility of E. histolytica is dependent upon the formation at the leading edge of the cell, of an appendage called the pseudopod enriched in actin and its associated proteins. Pseudopods are also formed during early steps of phagocytosis. As amoebae progress, another appendage called the uropod is formed at the rear edge, it concentrates the cluster of receptors that undergoes capping at the amoebic surface. These appendages allow parasite motility and phagocytosis. We expect to elucidate the signaling pathway triggered by the interactions between the parasite and the external medium that lead to the cytoskeletal changes necessary for motility as well as for the interaction with human cells and their eventual phagocytosis.



  report

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1. Cell polarization during the movement of E. histolytica, pseudopod formation.E. Labruyère, S. Blazquez, G. Vogt with C. Zimmer and J.C Olivo in the PTR-IP " New approaches to study molecular polarization in motile cells by quantitative image analysis" conducted by A. Alcover.

Cellular and molecular analyses of amoeba polarization and the dissection of the signals involved in pseudopod formation allowed us to identify proteins able to regulate cytoskeleton dynamics. Among them, the PAK kinase involved in cytoskeleton regulation during morphological changes of eukaryotic cells. By using E. histolytica carrying different PAK domains combined with imaging analysis of motility, we showed that EhPAK is a key regulator of cell polarity, motility and phagocytosis in E. histolytica

Motility is stimulated during the invasive process of E. histolytica. Preliminary results showed that human cytokines trigger a chemotactic response in this parasite suggesting that these molecules are eventual regulators of both cell polarization and rate of movement. Cell surface proteins are under investigation according to their capacities to bind these cytokines.

3. Motility and adhesion play a major role in the virulence of E. histolyticaP. Tavares, N. Guillén with M.C. Rigothier (Pharmacy Faculty) with E. Coudrier and F. Amblard (Curie Institute), H. Kunt, A. Cardona, M. Huerre (Histotechnology and Pathology Expertise Unit, IP) and P. Roux (Centre for Dynamic Imaging).The PRFMMIP program of the French Ministry of Education supports this project.

The coordinated action of the cytoskeleton and surface molecules supports various processes essential for pathogenesis like adhesion to substrata, motility and capping of cell surface receptors. For the analysis of these functions we have selected a dominant antigen during the disease, the Gal-GalNAc lectin. We showed that this receptor is enriched in membrane detergent-resistant domains and undergoes capping in the presence of specific antibodies. Looking for capping regulation, we have identified, cloned and characterized the amoebic conventional myosin II that is essential for motility and for parasite adhesion to cells. Myosin II is enriched into the parasite cytoskeleton following the amoeba-enterocyte contact. By using an experimental model of amoebiasis we have shown that a critical point for success of infection is reached after 6 to 12 h when the parasite may acquire functions allowing its survival and development into the tissue. As soon as E. histolytica arrives in the liver, endothelial cells undergo apoptosis and hepatocytes, as well as inflammatory cells, are killed by necrosis. Using wild type and myosin II or Gal-GalNAc deficient amoebae, combined with confocal or two-photon microscopy, we analyzed the impact of adhesion and motility in the invasive process. Parasites were incubated on different surfaces: glass, the apex of enterocytes and the entire liver of an animal model. The major conclusion of these experiments is that adherence regulates the rate and the progression of parasites, supporting thus the pathogenic process. The Gal-Gal lectin deficient strain presents a reduced rate of adhesion (40% of the WT), moves faster, somersaults on the apex of cells and glides into the tissue. The myosin II-inactivated amoeba is non-motile, displays a pendulum movement and has few cell adhesion areas. During live tissue invasion, the Gal-GalNAc strain is deficient in adhesion and rapidly spread in to the tissues undergoing a metastasis-like process. The non-motile myosin II deficient strain is avirulent as it is incapable of forming liver abscess. Regulatory factors accounting for these phenotypes are under investigation.

3. Cellular and biochemical analysis of E. histolytica — enterocyte interaction.M. Seigneur with E. Coudrier (Curie Institute). The PRFMMIP program of the French Ministry of Education supports this project.

E. histolytica moves fast (1 m m/sec) and stops when it comes into contact with the apex of enterocytes. Adhesion triggers dramatic changes on the target cells: microfilaments are delocalized and membrane projections are formed, then parasites invade the cell monolayer. By using an affinity chromatography strategy with purified brush border as a ligand, we have isolated several amoebic proteins. Among them, one of 20 kDa is under characterization. The gene encoding this protein has been cloned, the protein was produced in E. coli and specific antibodies were raised. We have demonstrated that p20 is secreted by the parasites and that it has a remarkable adhesion capacity for differentiated human enterocytes. Further molecular characterization of p20 is in progress.

4. Cytoskeleton rearrangements during phagocytosis of human cells by E. histolytica.S. Marion with C. Laurent-Winter (Proteome, IP), C. Whilem et F. Gazeau (Paris VII University), supported by the European Union, INCO-DEV- PHAGOAMEBA.

Killing and phagocytosis of human cells occurs after establishment of amoeba-cell contact though specific receptors. The signaling pathway, triggered by phagocytosis, activates parasite cytoskeleton rearrangements. Dissection of the signals and structural proteins involved in the formation of the pseudopod allowed us to identify ABP120 (the actin gelation factor), myosin IB, and Arp2/3 complex as its major components. We analyzed the role of myosin IB in phagocytosis by construction of a dominant negative strain (MyoIB+). These amoebae were deficient in phagocytosis, at the uptake step. Measurement of cytoplasm visco-elasticity showed a denser cytoskeleton of MyoIB+ strain, a fact that correlates with the inhibition of phagocytosis. We have recently constructed a set of amoeba strains carrying deletions of specific MyoIB domains. Cellular and biophysical analysis of these strains allowed the conclusion that the two actin-binding domains are simultaneously responsible for the encountered phenotypes. Bidimensional electrophoresis analysis showed relevant differences in the protein content of phagosomes isolated from the MyoIB+ strain when compared with the wild-type amoeba.

5. Genetic analysis of E. histolytica pathogenesis using the RNAi approach.L. Vayssié-Niger with M. Vargas (CINVESTAV, Mexico). supported by the European Union, INCO-DEV- PHAGOAMEBA.

The aim of this project is to analyze the pathogenic process of E. histolytica with a RNA interference strategy. To achieve this goal we have chosen the g-tubulin encoding gene since the protein is responsible for essential functions in all eukaryotic cells. RNA double strand were produced in bacteria that were then phagocytozed by the amoeba. Simultaneously, small RNA (siRNA) specific and complementary of g-tubulin nucleotide sequence were synthesized and tested. Both of these strategies allowed gene inactivation in E. histolytica and we were able to show that g-tubulin is a regulator of microtubules dynamic in this parasite.

6. Exploring pathogenesis of amoebiasis with cDNA microarraysC. Weber with C.Bouchier (Genomic-IP) and D. Mirelman (Weizmann Institute, Israel).

Our objective is the functional analysis of genes that are upregulated or exclusively expressed in highly virulent strains or induced following the contact of parasites with human cells. A complementary DNA (cDNA) library enriched in transcripts isolated from amoeba in a liver abscess, as well as a cDNA library from a virulent strain in culture, were constructed and sequenced. Following the computer sequence analysis we found that 30% of inserts encode proteins of unknown functions. These genes are therefore candidates for specifying new virulence factors. These clones will be used for the preparation of microarrays that will be screened with mRNA from wild type strains of those modified in cytoskeleton or Gal-GalNAc receptor.

Photo: Cellular distribution of PAK and F-actin in a motile E. histolyticaAmoeba moving from left to right displaying a prominent pseudopode that guides its locomotion. A confocal micrograph of the parasite showed that: F-actin (green) is lightly concentrated in the pseudopod, distributed in the cytoplasm and enriched at the posterior end of the parasite. PAK (red) is dispersed throughout the cytoplasm and is concentrated in pseudopodia extension. Superimposition of the two stainings indicates that F-actin and PAK are concentrated at specific opposite sites of the parasite. In moving parasites, the concentrated labeling, at opposite ends, of PAK versus F-actin reinforces the polarized state of migrating E. histolytica. The differential interference contrast (DIC) image shows E. histolytica with a pseudopod at the front of migration. Bar, 5 µm.

Keywords: amoebiasis, cytoskeleton, cell adhesion, Entamoeba, imaging



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  personnel

  Office staff Researchers Scientific trainees Other personnel
  Lambrecht, Marie-Régine,lambrech@pasteur.fr Guillén-Aghion, Nancy, CNRS. Research Director, CNRS.

Labruyère-Dadaglio, Elisabeth, Researcher, I.P.

Seigneur, Marie, Researcher, INRA.
Vayssié-Niger, Laurence, Postdoc EU.

Marion, Sabrina. PhD student.

Blazquez, Samantha. PhD student.

Ausseur, Christophe. DEA Student.
Weber, Christian, Technician, I.P.

Lambrecht, Marie-Régine, Secretary, I.P.lambrech@pasteur.fr

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