|Director : GUILLEN-AGHION, Nancy (firstname.lastname@example.org)|
Amoebiasis is a parasitic infection of the instestine prevalent in the developing world. The causative agent of amoebic dysentery and amoebic liver abscess is Entamoeba histolytica, a protozoan parasite that infects about 50 million people and causes 100,000 deaths yearly. E. histolytica colonizes the lumen of the large bowel. Invasion of the colonic mucosa, which is triggered by unknown stimuli, causes dysentery. Our research project aim to elucidate the molecular and cellular mechanisms of E. histolytica motility as well as those supporting the interaction of this parasite with cells and matrix components of the intestinal epithelial barrier.
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 amoeba progresses, 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 milieu that lead to the cytoskeletal changes necessary for motility as well as for the interaction with human cells and their eventual phagocytosis.
1. Cell polarization during the movement of E. histolytica, pseudopod formation.
E. Labruyère, 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 analyzed phenotypes of wild-type amoeba population as well as those from the different modified strains. In E. histolytica, PAK does not possess the regulatory regions encountered in PAK from other cells. However, we demonstrate that the NH2-terminal domain possesses the regulatory activity leading to binding of the small GTPase Rac and to the regulation of cytoskeleton dynamic, cell polarization, motility and the number of protruding pseudopods.
2. Cytoskeleton rearrangements during phagocytosis of human cells by E. histolytica.
S. Marion with C. Laurent-Winter (Proteome, IP), C. Whilem et F. Gazeau (Paris VI 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. In the MyoIB+ strain, myosin IB is located in patches and actin and the Arp2/3 complex form comet tails around the phagosomes. Bidimensional electrophoresis analysis showed relevant differences in the protein content of phagosomes isolated from the MyoIB+ strain when compared with the wild-type amoeba.
3. Motility and adhesion play a major role in the virulence of E. histolytica
P. Tavares with M.C. Rigothier (Pharmacy Faculty) and H. Kunt, A. Cardona, M. Huerre (Histotechnology and Pathology Expertise Unit, IP).
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 which 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 observed the dramatic incidence of both cytoskeleton and adhesive molecules in the regulation of tissue invasion by E. histolytica. We showed 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. Characterization of a dominant negative mutant for myosin II or for the Gal-GalNAc lectin heavy chain in this model showed contrasting phenotypes. 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.
4. Cellular analysis of E. histolytica enterocyte interaction during amoebiasis.
N. Guillén with E. Coudrier and F. Amblard, (Curie Institute), M.C. Rigothier (Pharmacy Faculty) and M.C. Prevost (Microscopy Platform IP). The PRFMMIP program of the French Ministry of Education supports this project.
Using wild-type and myosin II or Gal-GalNAc deficient amoebae, we described morphological changes in amoeba (fluorescent) and enterocytes (carrying GFP-actin) Adhesion to epithelial cells and motility was analyzed by using biphotonic microscopy with living cells, and by confocal and electron microscopy, with fixed cells. E. histolytica moves quickly (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. 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 poorly penetrates the monolayer despite its cytotoxic activity. These changes were not seen when the myosin II-inactivated amoebae were used. This non-motile strain displays a pendulum movement, has few cell adhesion areas and is non-virulent. In addition, biochemical analysis of the amoeba-enterocyte interaction leads to the purification of new amoebic proteins involved in cell adhesion. Two of these have been identified and their molecular characterization is in progress.
5. Exploring pathogenesis of amoebiasis with cDNA microarrays
C. 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 of a non-virulent E. histolytica strain HM-1 was prepared and cDNA inserts are being sequenced. Following the computer sequence analysis, these clones will be used for the preparation of microarrays. We also purified the mRNA from amoebae isolated from liver abscesses and from the isogenic strain that has lost its ability to induce liver abscesses. With these two mRNAs, a subtractive hybridization experiment is in progress, subtracted mRNA will be used as a probe for microarray screening.
Cellular distribution of PAK and F-actin in a motile E. histolytica
Amoeba 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 staining 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.
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|Office staff||Researchers||Scientific trainees||Other personnel|
Lambrecht, Marie-Régine, email@example.com
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.
Vogt, Guillaume, DEA student.
Weber, Christian, Technician, I.P.
Lambrecht, Marie-Régine, Secretary, I.P. firstname.lastname@example.org