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 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.
Molecular and Cellular analysis of Entamoeba histolytica pathogenesis
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 years of research our major findings are the following:
1 - Cell polarization during E. histolytica chemotaxis towards inflammatory molecules
E. Labruyère, S. Blazquez in collaboration with C. Zimmer (AIQ Unit, IP)
Amoebiasis trigger an acute inflammation in both the intestine and the liver. We have observed that mobility of E. histolytica is enhanced by pro-inflammatory molecules such as TNF suggesting that early inflammation enhances capacities of the parasite to penetrate human tissues. We have described by cell biology and video-microscopy the physical parameters of E. histolytica chemotaxis and chemokinesis towards TNF. Motile amoeba display a frontal pseudopod upon polarization. Changes triggered by TNF in pseudopod components and in parasite morphology lead us to propose the existence of a specific TNF amoebic receptor under description.
2 Role of tubulins in E. histolytica motility
L. Vayssié, C. Weber in collaboration with M. Vargas (CINVESTAV, Mexico) supported by the European Union, INCO-DEV- PHAGOAMEBA.
In mammalian cells, microtubules (MTs) in conjunction with microfilaments participate in cell motility. E. histolytica is a highly motile cell displaying a very versatile cytoskeleton, we hypothetised on the existence of dynamic parasite MTs contributing to motility. To asses the role of MTs in parasite motility, we first labeled tubulins and discovered that γ -tubulin has an intra-nuclear localization in an unknown structure that supports MTs arrays. siRNA treatment against γ -tubulin mRNA disrupts the nuclear structure enriched in γ -tubulin and disorganizes MTs scaffold thus inhibiting amoeba growth. Cytoplasmic MTs were not identified in this approach. A deeper analysis of MTs structure by EM is underway and the consequences of MTs disruption on parasite motility still needs to be described.
3 - Myosin IB from E. histolytica is a cytoskeleton regulatory factor invoved in pseudopod extension
S. Marion in collaboration with C. Wilhem and J-C. Bacri (PVII University-CNRS), and with C. Laurent, Genopole® Ile-de-France, IP and V Meas-Yedid (AIQ Unit, 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 have identified principal actors of cytoskeleton dynamics, cloned the encoding genes and further analyzed their functions. Example of these is the unconventional myosin IB that localizes in the pseudopod during motility and during phagocytosis of human cells. Cellular and biophysical analyses showed that myosin IB has a dual activity in E. histolytica. In resting cells, myosin IB crosslinks actin filaments throught its two actin binding sites and then regulates the mechanical properties of the cytoskeleton. Upon activation of the phagocytic process, myosin IB nucleates actin filament trought its SH3 domain. 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.
4 - Identification of new molecules involved in parasite-human cells interplay
M. Seigneur, J. Santi-Rocca in collaboration with M-C. Rigothier (Pharmacy Faculty). supported by the European Union, INCO-DEV- PHAGOAMEBA and by The PRFMMIP program of the French Ministry of Education.
We identify several proteins in an affinity chromatography approach using parasite surface molecules and the brush border of enterocytes. Among them KERP1, a lysine and glutamic acid rich protein. Cloning of the gene encoding KERP1, production of a recombinant protein in E. coli, production of specific antibodies and use of animal models of infection, allowed us to conclude that (i) KERP1 is located on the plasma membrane and in vesicles of the amoeba, (ii) KERP 1 is delivered in the interstitial area between the cells and the parasite, iii) purified KERP1 binds to epithelial cell surfaces. We observed that KERP1 concentration is increased after three days of liver abscess formation in hamsters along with a relocalisation to the amoebae membrane and an amoeba strain expressing kerp-1 anti-sense mRNA is unable to form liver abscesses. We have recently demonstrated that sera from patients suffering from amoebic liver abscesses recognized KERP1. The secretory pathway of KERP1 and the regulation of kerp1 gene expression are under study.
5 - Performing microarrays for the analysis of parasite gene expression during liver infection
C. Weber, in collaboration with Genopole® Ile-de-France (J-Y.Coppee, G. Guigon, O. Sismeiro, C. Bouchier, L. Frangeul, C. Gouyettte) and with D. Mirelman, The Weizmann Institute. Rehovot. Israel. ). Supported by the Pasteur-Weizmann Research Council.
The presence of E. histolytica in the liver environment probably requires expression of a repertoire of genes necessary for survival and for escape from the immune system. Understanding this genetic expression program should enable the identification of major parasite factors accounting for the aggressive behavior of E. histolytica at the different stages of abscess. The first set of microarrays using transcripts from E. histolytica has been built in this project. The gene sequence information was obtained from: i) a cDNA library of the HM1 :IMSS virulent strain, ii) clones from a library generated following a cDNA subtraction between HM-1:IMSS isolated from liver lesions in hamsters and cultured HM-1:IMSS that have lost this capability due to a long term axenic cultivation, iii) the genome information taking into account genes related to cytoskeleton and signaling functions. Our bioinformatic analysis has allowed us to define 1300 bonafide transcripts from E. histolytica. To investigate whether this array was sufficiently sensitive to detect gene expression changes in E. histolytica we first performed a heat shock experiment. The results were positive since genes related to heat shock response were overexpressed. Screening of transcripts modified during infection is underway.
Our future plans are to develop a deeper analysis of cytoskeleton related activities and of molecular and cellular analysis of the new pathogenic factors identified during these last years. Our ultimate aim is to use modern biotechnology approaches to progress in the understanding of the amoebic invasive process. This will enable the formulation of innovative strategies for the diagnosis and treatment of amoebiasis.
The confocal microscopy micrographe shows nuclear division in E. histolytica. The intranuclear microtubules are labelled by an anti-β-tubulin (green) and the DNA by an intercalant compound (red).
Keywords: Amoebiasis, Entamoeba, motility, chemotaxis, myosin IB, microarrays