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Cytoskeleton Activities During the Interaction of Entamoeba histolytica with Epithelial Cells
Archives of Medical Research 31 (2000) S134–S136
Cytoskeleton Activities During the Interaction of Entamoeba histolytica with Epithelial Cells Joelle Mounier,* Marie-Christine Prevost,** Evelyne Coudrier*** and Nancy Guillén* *Unité de Pathogénie Microbienne Moléculaire, INSERM U38, ** Unité d’Oncologie Virale, Pasteur Institute, Paris, France *** Unité de Morphogenèse et Signalisation Cellulaires, CNRS UMR 144, Curie Institute, Paris, France
Key Words: Entamoeba histolytica, Cytoskeleton, Motility, Myosin II, Microvilli, Actin.
Introduction Enterocytes that comprise the intestinal tract of humans are polarized epithelial cells that mediate specialized functions, such as transport of molecules, secretion, endocytosis, and transcytosis. The apex of these cells, oriented to the external milieu, forms a unique organelle termed the brush border (BB), which consists of microvilli providing an expanded surface that facilitates absorption of digested products in the intestine. Microvilli are maintained by actin microfilaments organized by the activity of actin-binding proteins. Among these, villin and fimbrin modulate microfilament assembly. The brush border myosin I (BBMI) and ezrin link microfilaments to the microvillus membrane (1). Although adherence to enterocytes is important for intestinal infection leading to diarrhea, very little is known about the interaction of pathogens with the BB. It has been shown that enteropathogenic Escherichia coli infects enterocytes, resulting in lesions on the cell surface. This is further characterized by localized effacement of microvilli and intimate attachment of the bacteria to the surface of the cell (2). The underlying cytoskeleton is modified, resulting in the formation of so-called pedestal-like structures upon which the bacteria reside for the duration of the infection (2). Thus, it appears that pathogens may contact proteins at the BB surface to induce changes in the cytoskeleton of the target cells. Adhesion to enterocytes is an early step of the intestinal invasive process of Entamoeba histolytica. Amebas contact the epithelial cells through their surface molecules, followed by killing the enterocytes and phagocytosis. The massive cytolysis combined with degradation of the extracellular matrix accounts for tissue destruction during progression of the parasite into the intestinal epithelium (3). During amebiasis, E. histolytica directly contacts the BB,
Address reprint requests to: Nancy Guillén, Unité de Pathogénie Microbienne Moléculaire, INSERM U38, Pasteur Institute, 28 Rue du Dr. Roux, 75724 Paris, Cedex 15, France; E-mail: [email protected] Presenting author: Nancy Guillén.
which is degraded as soon as the amebas arrive at the apex of the enterocytes. The parasites attach to the cells in areas in which microvilli have been eliminated. To date, information concerning the specificity of ameba-BB interaction does not exist, except that adherence of the parasite is diminished when the activity of the Gal-GalNAc lectin, a receptor involved in the mechanism of amebic adhesion, is inhibited. Interestingly, examination of the ameba–enterocyte interaction has demonstrated that the Gal-GalNAc lectin is transferred to the lateral side of epithelial cells (4). In this work, the cellular analysis of the ameba–enterocyte interactive process was performed by using a combination of confocal and electron microscopy. The interactions of E. histolytica with enterocytes are complex, resulting in dramatic cytoskeletal rearrangements of both cells. These changes certainly contribute to the loss of absorptive surface of microvilli to establish dysentery.
Materials and Methods Strains and culture conditions. The pathogenic E. histolytica strain HM-1:IMSS and the genetically engineered LLM30 strain (5) were cultivated axenically in plastic flasks containing TYI-S-33 medium at 36⬚C for 48 h prior to each experiment. The human carcinoma Caco-2 cell line was grown for 14 days to confluence in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal serum at 37⬚C in a 10% CO2 incubator. For electron microscopy analysis, epithelial cells were grown until polarization occurred on polycarbonate filters. For amebae–Caco-2 cell interactions, trophozoites and cells were washed twice with prewarmed, serum-free DMEM medium. Amebas were added to the cell monolayer at a ratio of 1 ameba:20 cells in DMEM serumfree medium. The coculture was incubated at 37⬚C in 10% CO2 atmosphere for 30 min. Confocal scanning laser microscopy (CSLM). For confocal analysis, the coculture was fixed in 3.5% paraformaldehyde (PFA) and treated with phalloidin-TRITC to label actin or with specific antibodies (1:100) raised against ezrin or vil-
0188-4409/00 $–see front matter. Copyright © 2000 IMSS. Published by Elsevier Science Inc. PII S0188-4409(00)00 1 3 4 - X
XIV Seminar on Amebiasis, Mexico City / Archives of Medical Research 31 (2000) S134–S136
S135
lin. In particular experiments, the Caco-2 cell line expressing the hybrid protein BBMI-GFP was used. Fluorescent samples were examined on a Zeiss LSM510 confocal laser scanning microscope. Observations were performed on 30 optical planes of 1 m. Images were further analyzed using LSM510 software from Zeiss.
actin. Microfilaments delocalize from cell junctions and then aggregate in areas where ameba contacts the cell. Following these morphological changes, the BB is degraded, and the ameba phagocytoses fragments containing actin. Cell death occurs in the interacting cell and adjacent cells exhibit, in turn, remodeling of microfilaments.
Transmission electron microscopy (TEM) and immunolabeling. After interaction of amebas with Caco-2 cells, the medium was removed and fixation buffer containing 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M Sörensen buffer (pH 7.2) was added to the samples for 1 h at 4⬚C. The filters were washed in 0.1 M Sörensen, and then briefly washed three times with distilled water. The samples were stained for 30 min with 0.5% uranyl acetate at 4⬚C, diluted in Michaëlis buffer, dehydrated with increasing concentrations of alcohol at decreasing temperatures, and embedded in Lowicryl K4M. After polymerization under ultraviolet (UV) light, thin sections were cut, collected on Formvar carbon-coated nickel grids, and immunolabeled. The grids were floated for 1 h on PBS containing 0.5% BSA and 5% normal goat serum, and subsequently incubated for 1 h at room temperature with monoclonal CD6 antibody against the Gal-GalNAc lectin (4) or a monoclonal antibody against actin (Amersham Life Science N350) (1/10). The grids were then washed with PBS containing 0.5% BSA, 0.1% cold water skin fish (PBG), and floated on PBG containing goat antimouse IgG⫹IgM (H⫹L) diluted at 1/25 (Amersham Life Science) antibodies coupled to 10-nm colloidal gold particles. After 1–2 h of incubation, the grids were washed, postfixed with 1% glutaraldehyde diluted in PBS, washed with water, stained with 2% uranyl acetate, and observed under a JEOL 1200EX microscope at 80 kV.
Amebic actomyosin cytoskeleton is essential for interaction with the enterocytes and their cytolysis. Cellular analysis of ameba–enterocytes interactions also suggests a role for the amebic cytoskeleton in the cytolysis of enterocytes. Using molecular approaches, we have constructed an ameba strain deficient in myosin II activity (4). This strain shows a significant reduction in movement, loss of cell polarity, and is inhibited for capping of receptors at the amebic surface. A dramatic change was also observed when cytolytic activity of the myosin II-deficient strain was assessed on human enterocytes. Whereas the amebic wild-type strain destroyed an enterocyte monolayer in 3 h, the myosin II-deficient strain was less virulent. Interestingly, cell microfilament delocalization was not seen in enterocytes contacting the myosin II-inactivated amebas. After a 30-min incubation, the confocal analysis of both the ameba (LMM30 strain) and the enterocytes showed that the BB is very lightly modified in the presence of these amebas. There is no degradation of microvilli, and microfilaments are still organized normally. Biochemical analysis of the myosin II-inactivated strain indicated that there is no modification of the amebic surface.
Results and Discussion E. histolytica contact promotes dramatic changes on the enterocyte apical surface. Monolayers of Caco-2 cell line were incubated in the presence of the E. histolytica strain HM-1:IMSS. Following interaction, amebas were fixed and treated for analysis with both confocal and electron microscopy. These microscope analyses led to the following conclusions: (i) there is a particular tropism of amebas for the BB, suggesting that BB components may function as a signal for tissue invasion; (ii) the BB undergoes dramatic changes following the ameba–enterocyte interaction, and (iii) membrane projections are formed and these projections attempt to surround the ameba (Figure 1). Labeling of cells with antibodies against actin, ezrin, or villin, as well as the utilization of Caco-2 cells expressing a GFP-BBMI hybrid protein, indicated that these projections are enriched in BB compounds. Concurrent with the BB deformation, there is a modification of the cellular actin-rich cytoskeleton. Major changes were visualized in the localization of filamentous
Figure 1. Electron microscopic analysis of E. histolytica interacting with enterocytes. The micrographs show ultrathin sections that were labeled with specific antibodies and with IgG coupled to gold as secondary antibodies. (A) The regular structure of the brush border demarcated by actin labeling of microvilli (1–2 m). (B,C) A view of the area in which ameba interacts with the cell. The disruption of the regular array of the BB is observed, as well as the elongation of some microvilli that expanded until 7 m. These elongated fragments are specific to the BB, because they are not labeled with an anti-Gal-GalNAc lectin antibody (B), as is the amebic surface, but are labeled by an antiactin antibody (C). Bar ⫽ 1 m.
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Mounier et al./ Archives of Medical Research 31 (2000) S134–S136
Thus, we attribute a major role in cell adhesion and the invasive process of ameba to the activity of myosin II.
Acknowledgments Many thanks are due to M. Mavris for critical reading the manuscript. Special thanks go to P. Sansonetti for his support of our research project, and to P. Roux for his advice concerning confocal microscopy analysis. This work was supported by grants from the French Ministère de l’Education Nationale, de l’Enseignement Supérieur, de la Recherche et de l’Insertion Professionnelle, and from the NORD-SUD INSERM program (Grant No. 4N0016). The confocal microscope used in this work is a gift of M. and L. Pollack.
References 1. Louvard D, Kedinger M, Hauri HP. The differentiating intestinal epithelial cell: establishment and maintenance of functions through interactions between cellular structures. Annu Rev Cell Biol 1992;8:157. 2. Goosney DL, de Grado M, Finlay BB. Putting E. coli on a pedestal: a unique system to study signal transduction and the actin cytoskelton. Trends Cell Biol 1999;9:11. 3. Guillén N. Role of signalling and cytoskeletal rearrangements in the pathogenesis of Entamoeba histolytica. Trends Microbiol 1996;4:191. 4. Leroy A, De Bruyne G, Mareel M, Nokkaew C, Bailey GB, Nelis H. Contact-dependent transfer of the Galactose-specific lectin of Entamoeba histolytica to the lateral surface of enterocytes in culture. Infect Immun 1995;63:4253. 5. Arhets P, Olivo JC, Gounon P, Sansonetti P, Guillén N. Virulence and functions of myosin II are inhibited by overexpression of light meromyosin in Entamoeba histolytica. Mol Biol Cell 1998;8:1537.
Cytoskeleton Activities During the Interaction of Entamoeba histolytica with Epithelial Cells Joelle Mounier,* Marie-Christine Prevost,** Evelyne Coudrier*** and Nancy Guillén* *Unité de Pathogénie Microbienne Moléculaire, INSERM U38, ** Unité d’Oncologie Virale, Pasteur Institute, Paris, France *** Unité de Morphogenèse et Signalisation Cellulaires, CNRS UMR 144, Curie Institute, Paris, France
Key Words: Entamoeba histolytica, Cytoskeleton, Motility, Myosin II, Microvilli, Actin.
Introduction Enterocytes that comprise the intestinal tract of humans are polarized epithelial cells that mediate specialized functions, such as transport of molecules, secretion, endocytosis, and transcytosis. The apex of these cells, oriented to the external milieu, forms a unique organelle termed the brush border (BB), which consists of microvilli providing an expanded surface that facilitates absorption of digested products in the intestine. Microvilli are maintained by actin microfilaments organized by the activity of actin-binding proteins. Among these, villin and fimbrin modulate microfilament assembly. The brush border myosin I (BBMI) and ezrin link microfilaments to the microvillus membrane (1). Although adherence to enterocytes is important for intestinal infection leading to diarrhea, very little is known about the interaction of pathogens with the BB. It has been shown that enteropathogenic Escherichia coli infects enterocytes, resulting in lesions on the cell surface. This is further characterized by localized effacement of microvilli and intimate attachment of the bacteria to the surface of the cell (2). The underlying cytoskeleton is modified, resulting in the formation of so-called pedestal-like structures upon which the bacteria reside for the duration of the infection (2). Thus, it appears that pathogens may contact proteins at the BB surface to induce changes in the cytoskeleton of the target cells. Adhesion to enterocytes is an early step of the intestinal invasive process of Entamoeba histolytica. Amebas contact the epithelial cells through their surface molecules, followed by killing the enterocytes and phagocytosis. The massive cytolysis combined with degradation of the extracellular matrix accounts for tissue destruction during progression of the parasite into the intestinal epithelium (3). During amebiasis, E. histolytica directly contacts the BB,
Address reprint requests to: Nancy Guillén, Unité de Pathogénie Microbienne Moléculaire, INSERM U38, Pasteur Institute, 28 Rue du Dr. Roux, 75724 Paris, Cedex 15, France; E-mail: [email protected] Presenting author: Nancy Guillén.
which is degraded as soon as the amebas arrive at the apex of the enterocytes. The parasites attach to the cells in areas in which microvilli have been eliminated. To date, information concerning the specificity of ameba-BB interaction does not exist, except that adherence of the parasite is diminished when the activity of the Gal-GalNAc lectin, a receptor involved in the mechanism of amebic adhesion, is inhibited. Interestingly, examination of the ameba–enterocyte interaction has demonstrated that the Gal-GalNAc lectin is transferred to the lateral side of epithelial cells (4). In this work, the cellular analysis of the ameba–enterocyte interactive process was performed by using a combination of confocal and electron microscopy. The interactions of E. histolytica with enterocytes are complex, resulting in dramatic cytoskeletal rearrangements of both cells. These changes certainly contribute to the loss of absorptive surface of microvilli to establish dysentery.
Materials and Methods Strains and culture conditions. The pathogenic E. histolytica strain HM-1:IMSS and the genetically engineered LLM30 strain (5) were cultivated axenically in plastic flasks containing TYI-S-33 medium at 36⬚C for 48 h prior to each experiment. The human carcinoma Caco-2 cell line was grown for 14 days to confluence in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal serum at 37⬚C in a 10% CO2 incubator. For electron microscopy analysis, epithelial cells were grown until polarization occurred on polycarbonate filters. For amebae–Caco-2 cell interactions, trophozoites and cells were washed twice with prewarmed, serum-free DMEM medium. Amebas were added to the cell monolayer at a ratio of 1 ameba:20 cells in DMEM serumfree medium. The coculture was incubated at 37⬚C in 10% CO2 atmosphere for 30 min. Confocal scanning laser microscopy (CSLM). For confocal analysis, the coculture was fixed in 3.5% paraformaldehyde (PFA) and treated with phalloidin-TRITC to label actin or with specific antibodies (1:100) raised against ezrin or vil-
0188-4409/00 $–see front matter. Copyright © 2000 IMSS. Published by Elsevier Science Inc. PII S0188-4409(00)00 1 3 4 - X
XIV Seminar on Amebiasis, Mexico City / Archives of Medical Research 31 (2000) S134–S136
S135
lin. In particular experiments, the Caco-2 cell line expressing the hybrid protein BBMI-GFP was used. Fluorescent samples were examined on a Zeiss LSM510 confocal laser scanning microscope. Observations were performed on 30 optical planes of 1 m. Images were further analyzed using LSM510 software from Zeiss.
actin. Microfilaments delocalize from cell junctions and then aggregate in areas where ameba contacts the cell. Following these morphological changes, the BB is degraded, and the ameba phagocytoses fragments containing actin. Cell death occurs in the interacting cell and adjacent cells exhibit, in turn, remodeling of microfilaments.
Transmission electron microscopy (TEM) and immunolabeling. After interaction of amebas with Caco-2 cells, the medium was removed and fixation buffer containing 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M Sörensen buffer (pH 7.2) was added to the samples for 1 h at 4⬚C. The filters were washed in 0.1 M Sörensen, and then briefly washed three times with distilled water. The samples were stained for 30 min with 0.5% uranyl acetate at 4⬚C, diluted in Michaëlis buffer, dehydrated with increasing concentrations of alcohol at decreasing temperatures, and embedded in Lowicryl K4M. After polymerization under ultraviolet (UV) light, thin sections were cut, collected on Formvar carbon-coated nickel grids, and immunolabeled. The grids were floated for 1 h on PBS containing 0.5% BSA and 5% normal goat serum, and subsequently incubated for 1 h at room temperature with monoclonal CD6 antibody against the Gal-GalNAc lectin (4) or a monoclonal antibody against actin (Amersham Life Science N350) (1/10). The grids were then washed with PBS containing 0.5% BSA, 0.1% cold water skin fish (PBG), and floated on PBG containing goat antimouse IgG⫹IgM (H⫹L) diluted at 1/25 (Amersham Life Science) antibodies coupled to 10-nm colloidal gold particles. After 1–2 h of incubation, the grids were washed, postfixed with 1% glutaraldehyde diluted in PBS, washed with water, stained with 2% uranyl acetate, and observed under a JEOL 1200EX microscope at 80 kV.
Amebic actomyosin cytoskeleton is essential for interaction with the enterocytes and their cytolysis. Cellular analysis of ameba–enterocytes interactions also suggests a role for the amebic cytoskeleton in the cytolysis of enterocytes. Using molecular approaches, we have constructed an ameba strain deficient in myosin II activity (4). This strain shows a significant reduction in movement, loss of cell polarity, and is inhibited for capping of receptors at the amebic surface. A dramatic change was also observed when cytolytic activity of the myosin II-deficient strain was assessed on human enterocytes. Whereas the amebic wild-type strain destroyed an enterocyte monolayer in 3 h, the myosin II-deficient strain was less virulent. Interestingly, cell microfilament delocalization was not seen in enterocytes contacting the myosin II-inactivated amebas. After a 30-min incubation, the confocal analysis of both the ameba (LMM30 strain) and the enterocytes showed that the BB is very lightly modified in the presence of these amebas. There is no degradation of microvilli, and microfilaments are still organized normally. Biochemical analysis of the myosin II-inactivated strain indicated that there is no modification of the amebic surface.
Results and Discussion E. histolytica contact promotes dramatic changes on the enterocyte apical surface. Monolayers of Caco-2 cell line were incubated in the presence of the E. histolytica strain HM-1:IMSS. Following interaction, amebas were fixed and treated for analysis with both confocal and electron microscopy. These microscope analyses led to the following conclusions: (i) there is a particular tropism of amebas for the BB, suggesting that BB components may function as a signal for tissue invasion; (ii) the BB undergoes dramatic changes following the ameba–enterocyte interaction, and (iii) membrane projections are formed and these projections attempt to surround the ameba (Figure 1). Labeling of cells with antibodies against actin, ezrin, or villin, as well as the utilization of Caco-2 cells expressing a GFP-BBMI hybrid protein, indicated that these projections are enriched in BB compounds. Concurrent with the BB deformation, there is a modification of the cellular actin-rich cytoskeleton. Major changes were visualized in the localization of filamentous
Figure 1. Electron microscopic analysis of E. histolytica interacting with enterocytes. The micrographs show ultrathin sections that were labeled with specific antibodies and with IgG coupled to gold as secondary antibodies. (A) The regular structure of the brush border demarcated by actin labeling of microvilli (1–2 m). (B,C) A view of the area in which ameba interacts with the cell. The disruption of the regular array of the BB is observed, as well as the elongation of some microvilli that expanded until 7 m. These elongated fragments are specific to the BB, because they are not labeled with an anti-Gal-GalNAc lectin antibody (B), as is the amebic surface, but are labeled by an antiactin antibody (C). Bar ⫽ 1 m.
S136
Mounier et al./ Archives of Medical Research 31 (2000) S134–S136
Thus, we attribute a major role in cell adhesion and the invasive process of ameba to the activity of myosin II.
Acknowledgments Many thanks are due to M. Mavris for critical reading the manuscript. Special thanks go to P. Sansonetti for his support of our research project, and to P. Roux for his advice concerning confocal microscopy analysis. This work was supported by grants from the French Ministère de l’Education Nationale, de l’Enseignement Supérieur, de la Recherche et de l’Insertion Professionnelle, and from the NORD-SUD INSERM program (Grant No. 4N0016). The confocal microscope used in this work is a gift of M. and L. Pollack.
References 1. Louvard D, Kedinger M, Hauri HP. The differentiating intestinal epithelial cell: establishment and maintenance of functions through interactions between cellular structures. Annu Rev Cell Biol 1992;8:157. 2. Goosney DL, de Grado M, Finlay BB. Putting E. coli on a pedestal: a unique system to study signal transduction and the actin cytoskelton. Trends Cell Biol 1999;9:11. 3. Guillén N. Role of signalling and cytoskeletal rearrangements in the pathogenesis of Entamoeba histolytica. Trends Microbiol 1996;4:191. 4. Leroy A, De Bruyne G, Mareel M, Nokkaew C, Bailey GB, Nelis H. Contact-dependent transfer of the Galactose-specific lectin of Entamoeba histolytica to the lateral surface of enterocytes in culture. Infect Immun 1995;63:4253. 5. Arhets P, Olivo JC, Gounon P, Sansonetti P, Guillén N. Virulence and functions of myosin II are inhibited by overexpression of light meromyosin in Entamoeba histolytica. Mol Biol Cell 1998;8:1537.