Unit: Cell Biology of Parasitism - INSERM U 786
Director: Nancy Guillén-Aghion
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 causes 50 million clinical cases and 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.
In the invasion of human tissues by E. histolytica, two major steps are recognized: (i) amoebic motility supported by the interaction with the substratum (ex: 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 dynamic of both the amoeba and the host cell. Our major findings these last years, have pointed out the crucial role of motility and adhesion in the success of the amoebic infection.
The major objectives of our project are :
1. Elucidate the cytoskeletal changes necessary for motility as well as the signaling pathway triggered by the interactions between the parasite and the external medium.
2. Identify the surface molecules necessary for amoebic interaction with human cells.
3. Establishment of the role of these molecules in (i) the cross-talk between amoeba and human cells and, (ii) the phagocytosis phenomenon.
4. Analyze amoebiasis by pathophysiology and by biotechnology developments.
The most innovative techniques that we are currently using are genomics, proteomics and imaging. Thanks to the performance of these technologies we got insight on the infection process of E. histolytica and we have opened avenues for the understanding of amoebiasis. In the last year of research our major findings are the following:
1. Expression analysis of genes involved in Entamoeba histolytica pathogenic process
C. Weber, J. Santi-Rocca, L-B. Luong-Nguyen, Genopole® Ile-de-France (PF2, PF4 et PF8), D. Mirelman (Weizmann Institute, Rehovot, Israel) and M-C. Rigothier (Faculté de Pharmacie, Châtenay-Malabry). Project supported by Pasteur-Weizmann Research Council.
- Identification and expression analysis of genes regulated depending on culture conditions by microarrays
A specific biochip has been developed, using 1300 oligonucleotides from E. histolytica unique sequences. A heat shock experiment allowed the validation of this biochip. The results show: (i) specific regulation of genes induced during such a stress and (ii) the differential expression, for genes involved in virulence, of copies of a same gene, including the Gal/GalNAc lectin heavy subunit-encoding one. We compared the gene expression patterns of virulent parasites purified from hamster liver abscesses and amoebae from the same strain that have lost their pathogenicity. Thus, a new pathogenic factor family was identified. All of the results obtained from biochips analysis were pooled in a database developed at the Institut Pasteur and are being validated.
- Analysis of target gene expression kinetics during the pathogenic process
To confirm the data obtained from the microarray analysis, we have developed a method allowing us to analyze the gene expression during the infectious process. The quantification of mRNA weakly expressed during the hamster hepatic infection has been made possible by the real-time PCR technique (using "Molecular Beacons" as higher sensitivity and specificity probes). We have developed this method using kerp1, a gene involved in E. histolytica pathogenic process; thus determining its expression profile in the course of the infection. Moreover, we compared by immunohistochemistry KERP1 protein level during liver abscess development. Finally, an antisense RNA approach allowed the confirmation of kerp1 essential role in amoebic liver abscess formation.
2. Analysis of the effects of TNF on E. histolytica and on amoebiasis
E. Labruyère, S. Blazquez with G. Guigon (PF8), O. Sismeiro (PF2), P. Roux (Platform of dynamic imaging) and P. Ave (Unit of Research and Expertise in Histology and Pathology). Project supported by the PTR 178.
TNF could have a dual role during amoebiasis: on the inflammatory reaction developed by the host and on trophozoïte migration (we have shown that TNF is chemoattractant and chemokinetic for E. histolytica). During liver abscess formation, there is an influx of macrophages and the presence of TNF in the lumen of vessels and around the inflammatory foci has been determined. On the contrary, after an infestation with parasites deficient in adhesion, the inflammatory reaction is delayed and reduced, suggesting that the adhesion to the target cells and the induced signalling are necessary to develop an inflammatory response. The modulation of gene expression of E. histolytica, induced by TNF has been studied using DNA chips, constructed in the laboratory. The results underline the up-regulation of genes coding for cytoskeletal proteins, ribosomal proteins and a factor of transcription, we will analyse the role of certain proteins. In the aim to study the physiopathology of E. histolytica, we are currently developing an "ex-vivo" model of human colon explants. The first steps of the invasive process of the human colon explants, by the trophozoïtes, present the characteristics described for human intestinal amoebiasis. The autofluorescence of structures of the human colon, in the absence and in the presence of the trophozoïtes, have been determined. This model will allow us to analyse in detail the molecular mechanisms of the first steps of intestinal amoebiasis.
3 - Myosin IB from E. histolytica is a cytoskeleton regulatory factor invoved in pseudopod extension during phagocytosis
S. Marion in collaboration with C. Laurent, Genopole® Ile-de-France, IP, supported by the European Union, INCO-DEV- PHAGOAMEBA.
Remarkably E. histolytica has adapted its very simple cytoskeleton to (i) motility, (ii) infection and (iii) killing and (iiii) phagocytosis of human cells. We developed proteomics analysis of purified phagosomes. Bidimensional electrophoresis and LS - MS/MS of these phagosomes allowed identification of proteins involved in cytoskeleton activities and among them several candidates for binding myosin IB and/or to regulated the cytoskeleton dynamics such as CARMIL, α-actinin and formin.
Figure 1. Human colonic tissue. (A) Histological section (longitudinal) of the mucosa and submucosa of a dissected human colon explant stained with haematoxylin and eosin. (B) Autofluorescence signature of a human colonic mucosa in multiphotonic imaging. (Stack of transversal images). The Lieberkühn crypts, the epithelium and the chorion are well defined in both images.
Figure 2. The virtual phagosome described the proteins identified by proteomics on the early phagosomes isolated from E. histolytica (from the wild type strain and from transfectants overexpressing myosin IB) and potentially participating in the different steps of the internalisation process. Notice important signalization factors and cytoskeleton related components.
Keywords: Amoebiasis, Entamoeba, motility, phagocytosis, microarrays, proteomics