Cell Biology of Parasitism - INSERM – U786  


  HEADDr. GUILLEN-AGHION, Nancy, DR1 – CNRS / nguillen@pasteur.fr
  MEMBERSDr. FAUST, Daniela, CR-IP / Dr. GIRARD-MISGUICH, Fabienne, MCU / Dr. LABRUYERE, Elisabeth, CR-IP / MARQUAY MARKIEWICZ, Jacques, PhD student
SANTI-ROCCA, Julien, PhD student / Dr. SOLIS, Carlos, Post-doctorant IP / WEBER, Christian, Engineer-II-IP / SYAN Sylvie, Technician IP / LAMBRECHT, Régine, secretary


  Annual Report

Amoebiasis, a human infection developed by 50 million persons every year, is caused by the protozoan parasite, Entamoeba histolytica. The parasite invades the intestinal mucosa where it causes dysentery.In some cases liver abscesses are formed as well. Major processes sustain amoebiasis: (i) amoebic motility, supported by the interaction with the extracellular matrix and the reorganization of the amoebic cytoskeleton, (ii) adhesion to human cells that is a consequence of the activity of surface receptors and cytoskeleton dynamics of both the amoeba and the host cell and (iii) host responses leading to inflammation and cell death. Our project aims to:

  1. Elucidate the cytoskeletal changes necessary for motility as well as the signalling pathways triggered by the interactions between the parasite and the external environment.

  2. Identify the molecules necessary for amoebic interactions with human cells and their role in the cross-talk between amoeba and human cells, leading to cell death and inflammation.

  3. Analyze amoebiasis by pathophysiology and by biotechnology developments.

Cellular analysis of cytoskeleton activities during chemotaxis.

S. Blazquez, C. Weber, E. Labruyère and S. Syan, with the Pasteur ImagoPole. Granted by EU- INCO-DEV.

The analysis of parasite signalling and cytoskeleton changes leading to directional motilitywas conducted by cell biology and microarrays. TNF-induced signalling during chemotaxis was PI3K-dependent and could lead to modifications in the polarisation of certain cytoskeleton-related proteins. Transcriptome analysis of TNF-chemoattracted parasites shown that genes encoding proteins involved in cytoskeleton dynamics along with the galactose/N-acetylgalactosamine lectin were up regulated during chemotaxis . Parasites blocked for Gal/GalNAc lectin signal were no longer able to chemotax towards TNF. The α actinin protein is the candidate to link the Gal/GalNAc lectin to the cytoskeleton. These results have given us an insight on how E. histolytica changes its cytoskeleton dynamics during chemotaxis and revealed the capital role of PI3K and Gal/GalNAc lectin signalling in chemotaxis.

Pathophysiology of amoebiasis.

E. Labruyère, D. Faust, D. Bansal, J. Marquay Markiewicz and S. Syan with the ImagoPole and Genopole (PF2).Granted by PTR-178 and Pasteur-Weizmann Reserach Council

We have set up an ex vivohuman colon model that mimics the early steps of amoebiasis. For imaging the infectious process we used two photons laser microscopy, scanning electron microscopy and histology. To characterise the tissue responses, human cells lyses and the pattern of secreted cytokines were quantified. The behaviour of trophozoites silenced in genes coding for known virulent factors such amoebapores, the Gal/GalNAc lectin and cysteine protease 5, which have major roles in cellular death, adhesion and degradation of the mucus, respectively, were examined. Our data revealed the essential role of the cysteine protease 5 in the parasite’s penetration of the colonic mucosa and in the induction of the host inflammatory response.

After disruption of the intestinal barrier, liver infection starts with the destruction of sinusoidal endothelial cells. Using a transcriptome approach, we have determined gene expression changes occurring in these cells upon contact with the parasite, allowing the identification of regulatory and structural components as well as signalling cascades modified by the interaction. We are characterizing the role of amoebic candidate proteins in this signalling process.

Expression analysis of genes involved in the Entamoeba histolytica pathogenic process.

J. Santi-Rocca, F. Misguich-Girard, C. Weber and C. Solis with the Genopole (PF1, PF2). Granted by the Pasteur-Weizmann Research Council.

To study identified pathogenic factors we have engineered bacteria to express a double-stranded RNA (dsRNA) corresponding to amoeba sequences. Taking advantage of the "professional" phagocytic capacities of Entamoeba we have fed parasite cultures with the dsRNA-expressing bacteria. RNA interference by dsRNA enabling down-regulation of E. histolytica gene expression has been used with large success for the analysis of newly identified pathogenic factors including KERP1, KRiP3 and ARIEL. These factors are involved in amoebic virulence and play a role during the onset of inflammation following parasite liver infection. Amoebic responses against the major markers of inflammation have been investigated on microarrays.

During infection, macrophages produce TNF, which promotes production of reactive oxidative species (ROS) and, among them, nitric oxide (NO). These are detoxified by reducing enzymes (purple ellipses): and by an arginase. Amoebae can also synthesize PGE-2, which reduces TNF and class II MHC production. This mechanisms used by E. histolytica to subvert the inflammatory response is essential for abscesses development, along with other amoebic features, supported by the Gal/GalNAc lectin (blue ellipse), by lytic proteins (white ellipse), like amoebapores and cysteine proteases, and by molecules of unknown function (black ellipse), such as the virulence factor KERP1.

Keywords: Amoebiasis, Entamoeba, motility, chemotaxis, microarrays, inflammation

Bcp.jpg

During infection, macrophages produce TNF, which promotes production of reactive oxidative species (ROS) and, among them, nitric oxide (NO). These are detoxified by reducing enzymes (purple ellipses): and by an arginase. Amoebae can also synthesize PGE-2, which reduces TNF and class II MHC production. This mechanisms used by E. histolytica to subvert the inflammatory response is essential for abscesses development, along with other amoebic features, supported by the Gal/GalNAc lectin (blue ellipse), by lytic proteins (white ellipse), like amoebapores and cysteine proteases, and by molecules of unknown function (black ellipse), such as the virulence factor KERP1.



  Publications

Girard-Misguich F, Sachse M, Santi-Rocca J, Guillén N. 2008. The endoplasmic reticulum chaperone calreticulin is recruited to the uropod during capping of surface receptors in Entamoeba histolytica. Mol Biochem Parasitol. 157:236-40.

Santi-Rocca J, Weber C, Guigon G, Sismeiro O, Coppee JY, Guillen N. (2008). The lysine- and glutamic acid-rich protein KERP1 plays a role in Entamoeba histolytica liver abscess pathogenesis. Cell Microb. 10:202-17.

Blazquez S, Guigon G, Weber C, Syan S, Sismeiro O, Coppée JY, Labruyère E, Guillén N. (2008). Chemotaxis of Entamoeba histolytica towards the pro-inflammatory cytokine TNF is based on PI3K signalling, cytoskeleton reorganization and the Galactose/N- acetylgalactosamine lectin activity. Cell Microbiol. 10:1676-1686

Solis CF, Guillén N.Silencing genes by RNA interference in the protozoan parasite Entamoeba histolytica. (2008). Methods Mol Biol.;442:113-28.

Weber C, Blazquez S, Marion S, Ausseur C, Vats D, Krzeminski M, Rigothier MC, Maroun RC, Bhattacharya A, Guillén N.(2008). Bioinformatics and Functional Analysis of an Entamoeba histolytica Mannosyltransferase Necessary for Parasite Complement Resistance and Hepatical Infection. PLoS Negl Trop Dis..13:e165.



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