Cell Biology of Parasitism - INSERM – U786  


  HEADDr. GUILLEN-AGHION, Nancy / nguillen@pasteur.fr
  MEMBERSDr. FAUST, Daniela / Dr. GIRARD-MISGUICH, Fabienne / Dr. LABRUYERE, Elisabeth / WEBER, Christian / SYAN Sylvie / LAMBRECHT, Régine / Dr. HERNANDEZ, Nora / Dr. HON, Chung / Dr. SMITH, Sherry / MARQUAY MARKIEWICZ, Jacques / PERDOMO, Doranda / THIBEAUX, Roman


  Annual Report

Our project aims to understand the cellular and molecular mechanisms responsible for amoebiasis, an infectious disease caused by an amoeba parasite named Entamoeba histolytica. The parasite invades the intestinal mucosa and causes dysentery.In some cases, the infection may also cause liver abscesses. Successful infection of the parasite mainly relies on: (i) amoebic motility, which is supported by the interaction with the extracellular matrix and the reorganization of the cytoskeleton, (ii) adhesion to human cells, which 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.

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

To shed light on the molecular mechanisms of the parasite’s motility and its phagocytosis-driven tissue invasion, we have re-annotated parasite cytoskeleton related genes and investigated their phylogenetic origins. These genes include the structural and signaling components of the actin cytoskeleton from available genomes of three Entamoebaspecies, includingE. histolytica, E.disparandE. invadens. Emphasis has been accorded to actin-related components (ARPs), which has been characterized by cellular and molecular approaches. Analysis of actin ortologues genes expression combined with living imaging of actin (during amoebic displacements and interaction with human cells) as well as biochemical studies on F-actin have been performed. Genotoxic responses of Entamoebahave been analyzed using a whole genome microarray. GenoScript (our open access database) has been improved and a Cytoscape plug-in associating transcriptome data with KEGG database implemented.

Aim 2: Identification the molecules necessary for amoebic interactions with human tissues and cells and their role in cell death and inflammation.

Our multidisciplinary approaches covering genomics, in vivoimaging and relevant animal models (e.g. pigs and hamsters), aiming to identify important host and parasitic components sustaining infection, which may be used to develop tools diagnosis and therapies of amoebiaisis.

We have also established an ex vivohuman intestinal colon infection model, and demonstrated E. histolyticastrain with cysteine protease 5 (CP5) silenced is unable to induce ahost inflammatory responseand to penetrate the colonic mucosa in the ex vivo model. The lack of TNF secretion could affect the process, as we had shown that TNF, in vitro, induces chemotaxis of E. histolytica. To decipher whether CP5 has a role in colonic invasion by degrading the extracellular matrix fibres and/or by inducingan inflammatory response, we are in the process to characterize (1) the mode of migration of E. histolyticawithin a three dimensional matrix, and (2) the primary cell source of TNF that is stimulated in the early phase of intestinal amoebiasis.

Upon intestinal disemination, hepatic sinusoidal endothelial cells are damaged during early stages of amoebic liver infection. Characterization of gene expression changes occurring in cultures of these cells in contact with E. histolyticaallowed us to identify cellular processes and signalling cascades modified by the interaction and to propose a mechanism participating in the induction of morphological changes and death of the human cells.

Amoebic responses against the major markers of inflammation have been investigated on microarrays. RNA interference by dsRNA enabling down-regulation of E. histolyticagene expression has been used with large success for the analysis of newly identified pathogenic factors including KERP1 and KRiP3. These factors are involved in amoebic virulence and play a capital role during liver infection. Their potential roles in the parasite’s interaction with human or mouse macrophages are currently under investigation.

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

guillen.jpg

Scanning electron micrograph of Entamoeba histolytica trophozoites adhering to the human mucus covering the intestinal epithelium. (T: trophozoite; M: mucus; P: pseudopode)



  Publications

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 histolyticaliver abscess pathogenesis.Cell Microb. 10:202-17. PMID: 18341598

Meurens F, Girard-Misguich F, Melo S, Grave A, Salmon H, GuillEn N. (2009). Broad early immune response of porcine epithelial jejunal IPI-2I cells to Entamoeba histolytica.Mol Immunol., 46(5):927-36. PMID: 19110312

Weber C, Marchat LA, Guillen N, LOpez-Camarillo C.( 2009). Effects of DNA damage induced by UV irradiation on gene expression in the protozoan parasite Entamoeba histolytica. Mol Biochem Parasitol.164(2):165-9. PMID: 19138709

Solis CF, Santi-Rocca J, Perdomo D, Weber C, GuillEn N. (2009). Use of bacterially expressed dsRNA to downregulate Entamoeba histolyticagene expression. PLoS One, 4(12):e8424. PMID: 20037645

Bansal D, Ave P, Kerneis S, Frileux P, BochE O, Baglin AC, Dubost G, Leguern AS, Prevost MC, Bracha R, Mirelman D, GuillEn N, LabruyEre E. (2009). An ex-vivo human intestinal model to study Entamoeba histolyticapathogenesis. PLoS Negl Trop Dis. 3(11):e551.PMID: 19936071



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Activity Reports 2009 - Institut Pasteur
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