Unit: Cell Biology of Parasitism - INSERM U-389

Director: GUILLEN-AGHION, Nancy

Amoebiasis is a parasitic infection of the intestine 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 projects aim to elucidate the molecular and cellular mechanisms of E. histolytica motility as well as those involved in the interaction of this parasite with cells.

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.

1. Cell polarization during the movement of E. histolytica, pseudopod formation and chemotaxis.

E. Labruyère, S. Blazquez, 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.

During invasive amoebiasis, Entamoeba histolytica motility, a key factor to achieve its progression across tissues, could be directed by chemotaxis. We have hypothesized that inflammatory molecules could be chemoattractant for E. histolytica. Our results show that TNFα chemoattracts E. histolytica in an under agarose assay. To study the kinetic and morphological parameters during chemotactic migration, images obtained by video microscopy were analyzed with the help of a dedicated program. Under a gradient of TNFα, E. histolytica migrates with non-classical amoebic morphological changes. Quantitative analysis revealed that the fraction time of elongation and retraction during motility is 50% for classical amoebic movement and 80% and 20% respectively for chemotactic migration. The mean square displacement of each ameba indicates that in the presence of a gradient of TNFα, parasite motion is enhanced by a factor 1,5 (p<0,05). In conclusion, TNFα is chemotactic and chemokinetic for E. histolytica. These results suggest that TNFa induces through an amoebic receptor a signaling pathway that affects actin cytoskeleton dynamic and cell polarization. Search for an amoebic protein specifically binding TNFα and for the triggered signaling pathway is underway.

3. Motility and adhesion play a major role in the virulence of E. histolytica

N. Guillén with M.C. Rigothier (Pharmacy Faculty) with E. Coudrier and F. Amblard (Curie Institute), 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.

To investigate the molecular basis of in vivo motion, we performed a high resolution dynamic imaging analysis using two-photon laser scanning microscopy. We show that E. histolytica undergoes non-Brownian motion when it invades a monolayer of epithelial cells and hamster liver. We find that myosin II, a central component of the cytoskeleton, is essential for penetration of the cell mono-layer and liver invasion, and that the Gal-GalNAc lectin, exclusively triggers invasion of the liver. During live tissue invasion, the Gal-GalNAc strain is deficient in adhesion, it is rapidly propelled into the tissue through the blood circulation, and then small abscessess are created very close to the blood capillary. The non-motile myosin II deficient strain is avirulent as it is incapable of forming liver abscessess. Regulatory factors accounting for these phenotypes are under investigation.

3. Cellular and biochemical analysis of E. histolytica - enterocyte interaction.

M. Seigneur, J. Santi-Rocca. Supported by the European Union, INCO-DEV- PHAGOAMEBA

Invasion of tissues by E. histolytica starts by the development of an interaction between the amoeba and the host cell. Amoeba-enterocyte interaction leads to adhesion followed by host cells cytoskeletal and membrane modifications and in the end cell destruction. Analysis of this interaction showed that the Gal-GalNAc lectin is not the only amoebic protein that binds the enterocyte's brush border. Affinity chromatography on the enterocyte brush border revealed several amoebic proteins binding this brush border. Among these, two basic proteins were found, KERP-1 and KERP-2, rich in lysin (25% K) and in glutamic acid (19 and 14% E). KERP-1 (but not KERP-2) has no homologue in E. dispar, a non-pathogenic species closely related to E. histolytica, indicating a possible role in pathogenesis. Analysis of KERP-1showed us that (i) KERP-1is located on the plasma membrane and in vesicles of the amoeba, (ii) its concentration is increased during liver abscess formation in the hamster along with a relocalisation to the membrane, (iii) its concentration decreases with time when trophozoites from a liver abscess are axenically cultured. These results show that KERP-1 plays a role in E. histolytica's pathogenesis and we have now to determine this role in virulence or protection of amoebas facing host cells. KERP-1 is a good candidate for specific E. histolytica diagnosis and a patent has been taken out.

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 molecular mechanisms involved in the formation of the pseudopod allowed us to identify Rac1, PI3-K, 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+), deficient in the uptake step of the phagocytic process. Measurement of cytoplasm visco-elasticity showed a denser and less flexible cytoskeleton in MyoIB+ strain, a fact that correlates with the delay in initiating phagocytosis. We have recently constructed a set of amoeba strains carrying deletions of specific MyoIB functional domains. Cellular and biophysical analysis of these strains allowed the conclusion that the two actin-binding domains are simultaneously responsible for cytoskeleton rigidification. These results suggest that in resting cells, myosin IB acts as an F-actin cross-linker and that this activity is involved in regulating the mechanical properties of the cytoplasm and cortical gels.

By quantitative imaging, we also observed that myosin IB induces "de novo" polymerization of actin filaments, and that this activity may be mediated by the COOH-terminal SH3 domain. We are now focusing our interest in the identification of regulators of myosin IB activities during phagocytosis. We thus initiated the analysis, by bi-dimensional electrophoresis, of the amoebic proteins specifically recruited at the phagocytic cup step. The comparison with the next step of the process, the closed phagosome, will allow the identification of the proteins involved in the signaling pathway induced after the binding of the amoebic receptor to the human cells.

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 γ-tubulin encoding gene since the protein is responsible for essential functions in all eukaryotic cells. Double stranded RNA was produced in bacteria that were then phagocytozed by the amoeba. Simultaneously, small RNA (siRNA) specific and complementary of γ-tubulin nucleotide sequence were synthesized and tested. Both of these strategies allowed gene inactivation in E. histolytica. We were able to show that the microtubular network located inside the nucleus disappears in E. histolytica treated by the RNAi. Thus, we conclude that γ-tubulin is a regulator of microtubules dynamic in this parasite.

6. Exploring pathogenesis of amoebiasis with cDNA microarrays

C. Weber with M.-C. Rigothier (Pharmay Faculty), D. Mirelman (Weizmann Institute, Israel) and L. Frangeul, JY Coppée, C. Gouyette (Genopole, IP). Supported by the Pasteur-Weizmann Research Council.

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. These clones combined with already known genes of our interest constituted an 1500 gene computer file that will be used for the preparation of microarrays. A pilot experiment using 110 of these genes was conducted to determine experimental conditions for transcript microarrays analysis. The 1500 array is under preparation and it will be screened with mRNA from wild type strains of those modified in cytoskeleton or Gal-GalNAc receptor or amoebapore functions.

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

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