Research Themes

Cytoskeleton dynamics during parasite motility and phagocytosis.

Nancy Guillén, Elisabeth Labruyère

Motility is based on physical and cellular mechanisms leading to both cell polarization and oriented displacement toward attraction sources such as inflammatory molecules. The distribution of adherence or cytoskeleton protein complexes induces a cell antero-posterior axe, which is visualized by a frontal cellular pseudopodial extension. Pseudopods also form during the phagocytosis of human cells. The objective is to understand the physical and cellular rules of pseudopod formation; as well as, the regulation of pseudopod persistence guiding random and tissue directed motility of E. histolytica. Little is known on E. histolytica signaling pathways controlling the activation of its cytoskeleton for cell polarization. Nevertheless, it is increasingly clear that the efficiency of migration requires the coordination with other cell activities such as the coordinate extrusion of membrane components (receptor capping and shedding of caps), the dynamics of microtubules, the renewal of amoeba-extracellular matrix interactions and the activation of specific genes required for parasite adhesion to human cells. This project take into account these different factors with the aim of identify the key factors of pseudopod extension and the signal pathway sustaining directional motility.

Imaging a hamster liver infected by E.histolytica.
Two-photon imaging was used for the movement analysis of wild type E. histolytica inside the liver parenchyma. The adhesion molecules are necessaries for parasite polarization (seen here by extension of a frontal pseudopod) and for the adhesive forces allowing to propel the cells forward. The lectin Gal/GalNAc is a protein complex important for parasite adhesion. Le PAK kinase, the myosins IB and II sustain pseudopod extension. In the video, 45 frames are shown at 0.25 s interval. These have been selected from stacks of 150 frames of one optical plane acquired at 2.5 s interval. (X 430 mm, Y 430.1 mm and Zoom = 1.1)

Experimental approaches: imaging in real time, two photons microscopy, image analysis, scanning microscopy, cryomicroscopy, physical and mathematical modelling, proteomics.

Collaborations :
Jean Christophe Olivo. Quantitative Image Analysis Unit. Pasteur Institute.
Spencer Shorte, Martin Sache. ImagoPole. Pasteur Institute.
François Amblard. Laboratory of Physics of the Cytoskeleton and Membrane Functions. CNRS. UMR 168. Curie Institute.
Alok Bhattacharya (J. Nehru University, India)

Molecular activation and signal transduction through receptors undergoing capping at the surface of E. histolytica

Nancy Guillén, Sylvie Syan, Jacques Marquay

Entamoeba histolytica displacements are intimately coupled to the flow of cell membranes and the cortical cytoskeleton toward the rear of polarized cells. As amoebae progress, an appendage called the uropod is formed at the rear edge, it concentrates the cluster of receptors that undergoes capping at the amoebic surface. The capping of receptors is characterized by recruitment by the cytoskeleton of large aggregates “caps“ of receptor-ligands complexes at the rear end of migrating cells. In Entamoeba histolytica the formation and expulsion in the medium of these aggregates enables the amoebas to eliminate the complement and the anti-amebic antibodies raised during the infection. This is a very efficient mechanism of resistance to the host’s immune response. We have shown the predominant role of the myosin II in the capping of the Gal-GalNAc receptor and the colocalization of proteins homologue to alpha-actinin. Proteomic analysis of caps and functional analysis of components involved in adhesion and cytotoxicity will provide insight in their role in virulence.

This movie shows the capping process induced by adding fluorescent Concanavalin A to Entamoeba histolytica. Concanavalin A is a ligand for numerous receptors on the amoeba’s surface including the Gal-GalNAc receptor. Binding to all receptors induces their capping with the formation of a large aggregate called uroïd.

Experimental approaches: Proteomics by LC/MS-MS, confocal microscopy, analysis of cell survival.
Collaborations: Miguel Vargas. CINVESTAV, Mexico

<i> E. histolytica</i> mobility and chemotaxis during invasive amoebiasis. Inflammatory host response

Elisabeth Labruyère, Samantha Blazquez, Devendra Bansal

The motility of E. histolytica is essential during the invasive and destruction processes of the intestinal barrier. When the inflammation occurs cytokines and chemokines are released from the epithelial cells and one of them, the tumor necrosis factor (TNF), is a key modulator of the inflammatory human response. To aboard the study of the molecular mechanisms that control the intestinal invasion by E. histolytica, we have hypothesized that chemotaxis could play a role in directing E. histolytica attack upon host tissue. Recently, we have demonstrated, in vitro, that TNF is chemotaxic and chemokinetic for E. histolytica. TNF is not known as a chemoattractant, but it is known to have pleiotropic effects on eukaryotic cells, inducing expression of gene involved in immune cell activation, cell survival or cell death depending on the context. Therefore our aim is to decipher the signaling pathways activated by TNF, in the parasite, and to determine whether E. histolytica TNF chemotaxis play a role in the physiopathology of amoebiases.
To address the first point, we have chosen two different approaches: (i) transfected amoeba deficient for proteins that are potential receptor for TNF are tested in chemotaxis and/or viability assay and (ii) the modulation of amebic gene expression, in the presence of TNF, is determined using micro array.
To address the second point, we have developed an ex-vivo human colonic explants model that mimic the first steps of intestinal barrier invasion by E. histolytica. The interaction between host/pathogen and parasite motility will be studied using scanning electron microscopy, histology and real time imaging (3D plus time); the inflammatory tissue response determined by the cell viability, cytokines assay and immunohistochemistry.

Collaborations: P. Roux and A; Danckaert (Imagerie Dynamique-IP);. S. Kerneis: (Microscopie Electronique-IP); A. S. Leguern (Centre Médical-I.P); P.Ave: Unité Histotechnologie-IP); C. Zimmer (Unité AI Q-IP). Supported by the PTR 178 : “set up a human colon model to analyze tissue invasion by pathogens and for cell motility studies” and D. Bansal by the Cantarini fellowship from I.P.
Destruction of the colonic mucosa by E. histolytica. The trophozoite (e) revealed by an antibody against a surface protein, invade the mucosa of a colonic explant (hématoxylin staining) by degrading the mucus, (m), then detaching the epithelium at the surface (ep) and which decorating the cryptes of Lieberkün (cl).

Liver tissue response to invasion by <i>Entamoeba histolytica</i> - characterization of gene expression changes of human sinusoidal endothelial cells in response to contact with the parasite.

Daniela M. Faust, Sylvie Syan

Liver is composed of a parenchymal (hepatocytes, cholangiocytes, stellate cells) and a non-parenchymal compartment (Kupffer cells, endothelial cells) and is irrigated through the liver sinusoids, in which the blood flow is slow, allowing efficient removal of macromolecules and contact of sinusoidal cell populations with passenger leukocytes.
In amoebiasis, liver sinusoidal endothelial cells (LSEC) present one of the first cell types of the liver in contact with the parasite upon its arrival via the portal venous system, after rupture of the intestinal barrier. Crossing of the sinusoidal barrier is considered a pre-requisite for the invasion of the parenchyma. During the slow process of invasion, human cells die by apoptosis and by necrosis. The hepatic abscess is formed after massive parasite death and adaptation of the surviving trophozoites, and characterized by extensive necrosis of the tissue. Infected regions contain areas of dead hepatocytes and debris, surrounded by a relatively low number of parasites and infiltrated inflammatory cells (polynuclear neutrophils and macrophages).
Using transcriptome analysis, our aim is to identify key regulators modified by the interaction of hepatic cells and E. histolytica, in particular those involved in hepatic abscess formation.

Collaborations : A. Cardona et M. Huerre (Unité Histotechnologie-IP) ; B. Regnault, G. Soubigou et J.-Y. Coppée (PF2, Génopole-IP) ; M.-C. Rigothier (Faculté de Pharmacie, Chatenay-Malabry) Soutenu par un financement DARRI (IP).
Radio-imaging of abscess formation. The micrograph shows a section of liver infected for 24 hours with 35S-E. histolytica (immunodecteted), radioactive proteins are precipitated (silver dots) and the host cells visualized by sucleus staining (X1000)

Analysis of pathogenesis functions by microarrays: <i>Entamoeba histolytica</i> response during liver abscess formation.

Christian Weber, Julien Santi-Rocca, Fabienne Girard-Misguich

Liver abscess is the most common complication of amebiasis. Using an experimental model, we determined that a critical step for Entamoeba histolytica survival occurs around 12 hours post-infection. The factors modulated during this stage of the invasive process allow the amebae to adapt to their environment and to survive; the study of the regulation of these factors’ gene expression is crucial to comprehend the pathogenic mechanism and to propose alternative therapies.
By using DNA biochips, we characterize the expression profile, during liver invasion, of genes potentially involved in pathogenesis. Several thus-identified genes that are modulated in virulent parasites are studied, and their protein products analyzed by imaging and biochemistry techniques. The expression of one of these factors, KERP1 (lysine- and glutamic acid-rich protein) was followed during liver abscess development, confirming its upregulation. We proved that KERP1 is essential for the formation of liver abscess by using a strain in which its production was hindered by an antisense RNA strategy. The precise role of KERP1 during hepatic infection is studied by analyzing the stimuli that trigger its synthesis and the mechanisms involved in its intracellular trafficking. This project also involves the transcriptome analysis of stool-isolated parasites from patients suffering from amebiasis; this will allow the discovery of potential targets for new therapies.

Experimental approaches: Biochips, quantitative real-time PCR using molecular beacons, immunocytochemistry, immuno-precipitation, western blot, real-time imaging by videomicroscopy with a KERP1-Green Fluorescent Protein hybrid, antisense RNA.

Collaborations : J.-Y. Coppée, G. Guigon et O. Sismeiro (PF2, Génopole-IP) ; S. Shorte (Imagopole-IP). Supported by the Pasteur-Weizmann Council.
Analysis by histoimmunochemistry of inflammatory foci sections from hamster livers (a) 1 day, (b) 2 days, (c) 3 days and (d) 5 days after infection. Host cells’ nuclei are revealed in purple-blue. KERP1 protein and, thus, amebae are stained in red. KERP1 undergoes a relocalization to the trophozoites’ cortical regions and its abundance increases during the course of liver abscess formation in our model. (Bars = 20µm)

Biochemical characterization of virulent factors by proteomics approaches.

Fabienne Girard- Misguich, Dorota Jeriorowska, Julien Santi-Rocca.

To identify news pathways and genes that are involved in liver abscess, we designed a gene expression array. A family of new virulence factors, characterized by abounding in Lysine (>25%) and glutamate, was highlight by this way.
These proteins rich in Lysine (Lysin Rich Protein, KRiP) were not associated with any known function. We have shown in a confocal microscopy analysis that they were located at the plasma membrane and in the phagosomes after a proteomic approach suggesting a role during adhesion and/or host cell lysis.
Biochemical and cellular approaches have been engaged to study the trafficking of these proteins in E. hystolytica. Because they have no transmembrane domains we supposed that they are associated with others proteins at the plasma membrane. A proteomic approach is developed to identify molecular partners for this association. Blue-Native PAGE (BN PAGE) was applied as a useful tool for analysis of KRIP-protein interactions. Several candidates are under analysis.

Experimental approache: Blue-Native (BN-PAGE) electrophoresis, MALDI, LC-MS/MS
BN-PAGE of E. histolytica membrane protein complexes.
First Dimension : After solubilization, the mixture of different protein complexes is separated by BN- PAGE. Following the run the lane is excised and subjected to a denaturation solution so that the native complexes dissociates. Second dimension : The subunits of the protein complexes are forced electrophoretically into the second dimension gel. Subunits of a protein complex form a vertical row on the second dimension gel. The proteins are then selected, eluted and identified by MS-MS technology.

RNA interference as a molecular tool for the study of <i>Entamoeba histolytica</i> virulence.

Carlos F. Solis, Christian Weber

Entamoeba histolytica is a protozoan parasite for which gene replacement techniques have not yet been developed. In our laboratory we have tested two different approaches based on RNA interference (RNAi): (i) “Soaking” approach which relies on the use of chemically synthesized small interfering RNAs (siRNAs) specific and complementary to target gene coding sequence and (ii) “Feeding” approach which benefits from the handily ability of E. histolytica to phagocyte bacteria genetically engineered for the expression of double-stranded RNA (dsRNA) specific for target gene. Based on these two RNAi approaches, our purpose is to establish this methodology as a standard molecular tool for E. histolytica functional genomics.
We recently showed that E. histolytica trophozoites fed with dsRNA-expressing bacteria results in a reduction in the amount of mRNA for beta-tubulin and KERP1. Reduction in the amount of transcript also correlates with a reduction in the amount of encoded product as shown through in situ immuno-localization studies, as well as western blotting analysis of trophozoite crude extracts using specific antibodies raised against cognate proteins. Initial results also revealed that silencing through RNAi of E. histolytica essential genes such as tubulins lowers amoeba growth. We are currently extending these observations to carry out a genome-wide high throughput screen using a bacterial library containing E. histolytica cDNA sequences for the expression of dsRNA. The aim of bacterial library screen through “feeding” of trophozoites cultured in microtiter plates is to identify genes, which are essential for parasite survival that could lead to new alternatives in the development of prophylactic and/or therapeutic treatment against amoebiasis.

Experimental approaches: RNA interference through “soaking” and “feeding” approaches, quantitative real-time PCR, western-blotting analysis, microarrays, confocal microscopy.

Collaborations: S. Shorte, M. Nguyen, and M.A. Nicola (ImagoPole IP); C. Bouchier, and E. Dessez (Genopole-IP, PF1).

Development of a new diagnosis test for amebiasis.

Julien Santi-Rocca, Nancy Guillén

Intestinal amebiasis is responsible for a very high morbidity, by handicapping 50 million people per annum. Indeed, symptoms are very close to other hemorrhagic diarrhoeas and do not allow a direct diagnosis by the physician, leading to inaccurate antibiotherapy prescription and to persistence of the dysenteric syndrome. Nonetheless, diagnosis tests are available but not adapted to affected people by either their cost or reliability. We develop a diagnosis test for intestinal amebiasis responding to four requirements:
- reliability: maximum sensitivity and specificity
- rapidity: result obtained in less than 10 minutes
- easiness: test can be carried out by a non-experimented user
- cost: adapted to population living in endemic areas

The test will be based on the immunochromatography technique, which is already used at the Pasteur Institute. In practice, the contact between a dipstick and the patient’s stools will reveal, by detection with the naked eye of a coloured band (or its absence), the infection (or not) by Entamoeba histolytica. The use of monoclonal antibodies specific for virulence factors identified in our unit will allow the exclusive detection of the pathogenic specie E. histolytica. The test will be validated by an epidemiologic study performed in the Pasteur Institutes International Network (Réseau International des Instituts Pasteur).

Collaborations : E. Fournié-Amazouz (Unité PMM-IP), F. Nato (PF5-IP) and Y. Germani (Unité PMM-IP). Supported by the Transversal Research Program 179 (Institut Pasteur).