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In Vivo Nanoimaging and Ultrastructure of Entamoeba histolytica By Using Atomic Force Microscopy
Experimental Parasitology 93, 95–100 (1999) Article ID expr.1999.4439, available online at http://www.idealibrary.com on
RESEARCH BRIEF In Vivo Nanoimaging and Ultrastructure of Entamoeba histolytica By Using Atomic Force Microscopy
N.V. Joshi,* H. Medina,* H. Urdaneta,† and L. Berrueta† *Department of Physiology, Faculty of Medicine, and †Institute of Clinical Immunology, University of Los Andes. P.O. Box 566, Me´rida, Venezuela
Joshi, N. V., Medina, H., Urdaneta, H. and Berrueta, L. 1999. In vivo nanoimaging and ultrastructure of Entamoeba histolytica by using atomic force microscopy. Experimental Parasitology 93, 95–100. q 1999 Academic Press
We used E. histolytica (Diamond and Clark 1993) pathogenic strain IDICULA 0593:2, isolated and axenized by us as previously described (Urdaneta et al. 1995), from a Venezuelan patient who presented abdominal pain, diarrhea with mucus and blood, nausea, tenesmus, fever, and chills. This strain was classified as pathogenic by a standard isoenzymatic pattern, inoculation in hamsters and PCR-SHELA (Aguirre et al. 1997; Urdaneta et al. 1998). For axenization the strain was grown in TYI-S-33 medium (Diamond et al. 1978), supplemented with 15% bovine serum, and harvested in mid log phase by 10-min ice bath. Centrifugation was carried out at 48C. For this investigation, an AMF unit attached to a conventional Olympus microscope (BX 60) was used. The system is known as SIS-ultraobjective, obtained from Surface Imaging System (Germany). We used Silicon cantilevers with a length of 450 mm and a width 50mm. The spring constant of the cantilever was 1 Nm21. The unit was installed on a vibration-free table and the complete system guarantees reproducibility. Trophozoites of E. histolytica were placed on a conventional microscopic slide. The membrane was just strong and healthy enough to be examined by contact mode; therefore, the use of tapping mode was avoided. The force applied was 0.8 nN/m and it was found to be an optimum force for this membrane. In some cases, the force was just sufficient to carry out one or two scans. The third scan in the same region often broke the membrane. The ultrastructures of three zones of E. histolytica, the nucleus, endoplasm, and ectoplasm, were investigated separately. A very organized structure was observed in the endoplasm, resembling aggregates of tubular parallel structures known as chromatoid bodies (Diamond et al. 1993; Chaves et al. 1997; Martinez-Palomo and Espinosa 1998), which are arranged in helices and aggregate to form characteristically shaped elongated bars with rounded ends as examined by electron microscopy. These structures are observed in the immature cyst and trophozoites, but frequently disappear as the cyst matures (Diamond
Atomic force microscopy (AFM) is a powerful technique for investigating the nanostructure of biological materials. The major advantage of this technique over electron microscopy is that it allows us to measure structural details at the resolution of a few nanometers in living cells, tissues, etc., as it does not require high vacuum and does not need surface preparation which damages or alters the sample. Unlike electron microscopy, AFM does not require fixation techniques, which frequently damage the sample. Thus, living cells can be imaged in their natural environmental provided that the temperature is adequately maintained. Moreover, since it is a scanning probe microscope, the technique endures 3-D image processing, enabling us to achieve resolution on the order of a few nanometers, which is an essential aspect for biological materials like living cells, parasites, red blood cells, etc. These species are ideal for examination with AFM as they have very slow or negligible movement and very frequently remain still for quite a long time, say 200 to 300 s, time enough to scan the image completely. In this way, the fixing technique can be avoided, as it perturbs the environment of the species to a certain extent. Moreover, it has an adhesive force to glass plates and sticks to the microscopic slides easily, reducing lateral movement. In addition to this, Entamoeba histolytica is known for its organized structure in the endoplasm. Although its ultrastructure has been studied by electron microscopy, this is the first time that a resolution on the order of nanometers has been shown in living E. histolytica. The purpose of the present investigation, therefore, was to perform ultrastructural and morphologic studies of E. histolytica using AFM.
0014-4894/99 $30.00 Copyright q 1999 by Academic Press All rights of reproduction in any form reserved.
95
96 and Clark 1993). We investigated more ultrastructural details in trophozoites and carried out 3-D analysis by using AFM. All images presented in this work are unfiltered. In some cases contrast optimization was carried out to enhance some details and make them clearly visible. Figures 1a–1c show the central part of E. histolytica, which includes the nucleus. All the details of the nucleus are not clearly visible because of the presence of a double membrane, which covers the inner structure. The contact with the upper membrane is sufficient not to get information hidden under the second membrane; still, some aspects were revealed. Some roughness was visible due to the inner structural details of the nucleus. The dimensions and height can be appreciated with the help of
JOSHI ET AL.
the table given below the figure. Distance was measured in a horizontal column, while height in a vertical file between required points. In the present sample, it was found that the diameter of the nucleus was approximately 10 mm, which is slightly higher compared with the earlier reported value of 4–7 mm (Martinez-Palomo and Espinosa 1998). This might be due to the electron microscopy processing, which causes dehydration of the sample. Moreover, in the present study, we considered not only the top but also the points from where the elevation starts. A small spherical body, probably representing the karyosome (2.93 mm in diameter), was observed (Fig. 1c between Nos. 3 and 4). The three-dimensional view (see Fig. 1b) shows that the nuclear
FIG. 1. (a) Entamoeba histolytica image obtained by AFM. The nucleus (circular elevation at the lefthand side) is clearly visible. The intensity bar shown on the righthand side shows height with respect to the substrate. Relative heights can be estimated with the help of table given below. (b) 3-D view of Fig. 1a rotated through 1808; karyosome is just visible. (c) Nucleus with its surface profiles. Note that the height of the nucleus is measured with respect to its surroundings and is approximately 700 nm. Thickness and other dimensions can be appreciated with the help of the table given below.
97
ULTRASTRUCTURE OF Entamoeba histolytica
FIG. 1.—Continued
membrane has practically no folding and is rather smooth. Very close to it there is a small depression of about 3 mm. The topographical study (see Fig. 1c) shows that the nucleus has a slight elevation on the order of 238 nm compared to its surrounding components; meanwhile, the nuclear body is elevated about 80 nm with respect to the other components of the nucleus. Figures 2a–2d show the ultrastructure of a cromatoid body, in vivo, located within the endoplasm of E. histolytica. The structure of the
cromatoid bodies was observed earlier by Feria-Velasco and Trevin˜o (1992), who described a conglomeration of ribosomes (or ribosomal precursors) and polysomes forming helical bodies, organized in the cytoplasm, which aggregate as crystalloid bodies. The presence and the structure of these bodies were further extensively investigated and their organized and repetitive pattern was reported (Diamond and Clark 1993; Benkert et al. 1997; Martinez-Palomo 1998; Kusamrarn et al. 1995). The present investigation confirms the presence of chromatoid
98
JOSHI ET AL.
FIG. 2. (a) Organized structure of cromatoid body. (b) 3-D image of a. (c) An enlarged portion of a which clearly shows a repetitive pattern. (d) Topography of c showing periodicity of the structure.
ULTRASTRUCTURE OF Entamoeba histolytica
99
FIG. 3. (a) Amplified view of a very small portion of the lefthand corner of Fig. 2a. A well-organized structure is imaged. (b) 3 - D image of a.
100 bodies in non-cyst E. histolytica, as previously reported (Carosi 1975), and further reveals three-dimensional details for the first time. The upper part of the figure shows an organized structure (Fig. 2a) along with the 3-D image (Fig. 2b). The organized part shows periodicity and therefore it was extracted and is shown in Fig. 2c, which reveals impressive organization and periodicity in the structure. In order to appreciate the ultrastructure, which shows excellent details of the periodic behavior, we have also shown the topography (Fig. 2d) to appreciate the dimension of the sequences and their perfect organization. These images clearly confirm that this structure described in trophozoites of E. histolytica consists of a very well organized system in the endoplasm. Figure 3a shows an amplified portion (365) of Fig. 2d (upper corner of the left side). Combinations of the repeated patterns are clearly visible. Figure 3b is a 3-D image of Fig. 3a, showing a repetitive conical configuration of different sizes. In short, the ultra structure of a living E. histolytica was examined for the first time with the help of AFM. In spite of the double membrane, the nucleus with its karyosome was visible. The organized, periodic structure of the chromatoid body was revealed and its 3-D imaging permits us to appreciate structural details at the level of nanometers. A comprehensive analysis of the ultrastructure of in vivo E. histolytica will contribute to understanding its dynamic behavior. This powerful technique, if extended to the investigation of living organisms, should provide insights about the pathogenic mechanisms of this and other parasites. (We are thankful to CONICIT (Consejo Nacional de Investigacio´n Cientifica y Tecno´logica de Venezuela “CONICIT”) for financing the research project (No. G-97000820) under which the present work was carried out.)
REFERENCES Aguirre, A., Molina, S., Urdaneta, H., Cova, J. A., and Guhl, F. 1997. Characterization of two Venezuela E. histolytica strains using electrophoretic isoenzyme patterns and PCR SHELA. Archives of Medical Research 28, 285–287.
JOSHI ET AL.
Benkert, C., Jacobs, T., Berninghausen, A. J., and Leippe, M. 1997. Molecular basis of aggresive and defensive function of E. Histolytica. Archives of Medical Research 8(Suppl.), S152–153. Carosi, C. 1975. “Concepts of Structure-Function Relationship in Amoebic Organelles,” pp. 289–299. Proceedings of the International Conference on Amebiasis. Chaves, M. B., Gonzales, R. A., Espinoza, C. M., Cristobal, R. A. R., and Martinez, P. A. 1997. Entamoeba dispar: Ultrstructure and cytopatic effect. Archives of Medical Research 28, S116–S118. Diamond, L. S., and Clark, G. 1993. A re-description of Entamoeba histolytica Shaudinn, 1903 (Emeded Walker, 1911). Separating If from Entamoeba dispar Brumpt, 1925.” J. Eukaroyte Microbiology 40, 340–344. Diamond, L. S., Harlow, D. R., and Cunnick, C. C. 1978. A new medium for the axenic cultivation of Entamoeba histolytica and others Entamoeba. Transations of the Royal Society Tropical Medicine and Hygiene 72, 131. Feria-Velasco, A., and Trevin˜o, N. 1992. The ultrastructure of trophosoites of E. histolytica with particular reference to spherical arrangements of osmiophilic cylindrical bodies. Journal of Protozoology 19(10), 200–211. Kusamrarn, T., Sobhon. P., and Bailey, G. B. 1995. The mechanism of formation of inhibitor- induced ribosome helices in Entamboeba Invadens. Journal of Cell Biology 65, 529–539. Martinez-Palomo, A., and Espinosa, C. M. 1998. “Intestinal Amoebace”. International Amoeba Microbiology and Microbial Infections 5, 157–177. Urdaneta, H., Cova, J. A., Molina, S., Aguirre, A., and Hernandez, M. 1998. “Evaluacio´n Inmunoquı´mica de cepas de Entamoeba histolytica Venezolanas.” Revista Kasmera 26. Urdaneta, H., Rondon, M., Mun˜oz, M., and Hernandez, M. 1995. “Isolation and axenization of two E. histolytica strains”. Gen 49, 23–28. Received 27 January 1999; accepted with revision 14 June 1999
RESEARCH BRIEF In Vivo Nanoimaging and Ultrastructure of Entamoeba histolytica By Using Atomic Force Microscopy
N.V. Joshi,* H. Medina,* H. Urdaneta,† and L. Berrueta† *Department of Physiology, Faculty of Medicine, and †Institute of Clinical Immunology, University of Los Andes. P.O. Box 566, Me´rida, Venezuela
Joshi, N. V., Medina, H., Urdaneta, H. and Berrueta, L. 1999. In vivo nanoimaging and ultrastructure of Entamoeba histolytica by using atomic force microscopy. Experimental Parasitology 93, 95–100. q 1999 Academic Press
We used E. histolytica (Diamond and Clark 1993) pathogenic strain IDICULA 0593:2, isolated and axenized by us as previously described (Urdaneta et al. 1995), from a Venezuelan patient who presented abdominal pain, diarrhea with mucus and blood, nausea, tenesmus, fever, and chills. This strain was classified as pathogenic by a standard isoenzymatic pattern, inoculation in hamsters and PCR-SHELA (Aguirre et al. 1997; Urdaneta et al. 1998). For axenization the strain was grown in TYI-S-33 medium (Diamond et al. 1978), supplemented with 15% bovine serum, and harvested in mid log phase by 10-min ice bath. Centrifugation was carried out at 48C. For this investigation, an AMF unit attached to a conventional Olympus microscope (BX 60) was used. The system is known as SIS-ultraobjective, obtained from Surface Imaging System (Germany). We used Silicon cantilevers with a length of 450 mm and a width 50mm. The spring constant of the cantilever was 1 Nm21. The unit was installed on a vibration-free table and the complete system guarantees reproducibility. Trophozoites of E. histolytica were placed on a conventional microscopic slide. The membrane was just strong and healthy enough to be examined by contact mode; therefore, the use of tapping mode was avoided. The force applied was 0.8 nN/m and it was found to be an optimum force for this membrane. In some cases, the force was just sufficient to carry out one or two scans. The third scan in the same region often broke the membrane. The ultrastructures of three zones of E. histolytica, the nucleus, endoplasm, and ectoplasm, were investigated separately. A very organized structure was observed in the endoplasm, resembling aggregates of tubular parallel structures known as chromatoid bodies (Diamond et al. 1993; Chaves et al. 1997; Martinez-Palomo and Espinosa 1998), which are arranged in helices and aggregate to form characteristically shaped elongated bars with rounded ends as examined by electron microscopy. These structures are observed in the immature cyst and trophozoites, but frequently disappear as the cyst matures (Diamond
Atomic force microscopy (AFM) is a powerful technique for investigating the nanostructure of biological materials. The major advantage of this technique over electron microscopy is that it allows us to measure structural details at the resolution of a few nanometers in living cells, tissues, etc., as it does not require high vacuum and does not need surface preparation which damages or alters the sample. Unlike electron microscopy, AFM does not require fixation techniques, which frequently damage the sample. Thus, living cells can be imaged in their natural environmental provided that the temperature is adequately maintained. Moreover, since it is a scanning probe microscope, the technique endures 3-D image processing, enabling us to achieve resolution on the order of a few nanometers, which is an essential aspect for biological materials like living cells, parasites, red blood cells, etc. These species are ideal for examination with AFM as they have very slow or negligible movement and very frequently remain still for quite a long time, say 200 to 300 s, time enough to scan the image completely. In this way, the fixing technique can be avoided, as it perturbs the environment of the species to a certain extent. Moreover, it has an adhesive force to glass plates and sticks to the microscopic slides easily, reducing lateral movement. In addition to this, Entamoeba histolytica is known for its organized structure in the endoplasm. Although its ultrastructure has been studied by electron microscopy, this is the first time that a resolution on the order of nanometers has been shown in living E. histolytica. The purpose of the present investigation, therefore, was to perform ultrastructural and morphologic studies of E. histolytica using AFM.
0014-4894/99 $30.00 Copyright q 1999 by Academic Press All rights of reproduction in any form reserved.
95
96 and Clark 1993). We investigated more ultrastructural details in trophozoites and carried out 3-D analysis by using AFM. All images presented in this work are unfiltered. In some cases contrast optimization was carried out to enhance some details and make them clearly visible. Figures 1a–1c show the central part of E. histolytica, which includes the nucleus. All the details of the nucleus are not clearly visible because of the presence of a double membrane, which covers the inner structure. The contact with the upper membrane is sufficient not to get information hidden under the second membrane; still, some aspects were revealed. Some roughness was visible due to the inner structural details of the nucleus. The dimensions and height can be appreciated with the help of
JOSHI ET AL.
the table given below the figure. Distance was measured in a horizontal column, while height in a vertical file between required points. In the present sample, it was found that the diameter of the nucleus was approximately 10 mm, which is slightly higher compared with the earlier reported value of 4–7 mm (Martinez-Palomo and Espinosa 1998). This might be due to the electron microscopy processing, which causes dehydration of the sample. Moreover, in the present study, we considered not only the top but also the points from where the elevation starts. A small spherical body, probably representing the karyosome (2.93 mm in diameter), was observed (Fig. 1c between Nos. 3 and 4). The three-dimensional view (see Fig. 1b) shows that the nuclear
FIG. 1. (a) Entamoeba histolytica image obtained by AFM. The nucleus (circular elevation at the lefthand side) is clearly visible. The intensity bar shown on the righthand side shows height with respect to the substrate. Relative heights can be estimated with the help of table given below. (b) 3-D view of Fig. 1a rotated through 1808; karyosome is just visible. (c) Nucleus with its surface profiles. Note that the height of the nucleus is measured with respect to its surroundings and is approximately 700 nm. Thickness and other dimensions can be appreciated with the help of the table given below.
97
ULTRASTRUCTURE OF Entamoeba histolytica
FIG. 1.—Continued
membrane has practically no folding and is rather smooth. Very close to it there is a small depression of about 3 mm. The topographical study (see Fig. 1c) shows that the nucleus has a slight elevation on the order of 238 nm compared to its surrounding components; meanwhile, the nuclear body is elevated about 80 nm with respect to the other components of the nucleus. Figures 2a–2d show the ultrastructure of a cromatoid body, in vivo, located within the endoplasm of E. histolytica. The structure of the
cromatoid bodies was observed earlier by Feria-Velasco and Trevin˜o (1992), who described a conglomeration of ribosomes (or ribosomal precursors) and polysomes forming helical bodies, organized in the cytoplasm, which aggregate as crystalloid bodies. The presence and the structure of these bodies were further extensively investigated and their organized and repetitive pattern was reported (Diamond and Clark 1993; Benkert et al. 1997; Martinez-Palomo 1998; Kusamrarn et al. 1995). The present investigation confirms the presence of chromatoid
98
JOSHI ET AL.
FIG. 2. (a) Organized structure of cromatoid body. (b) 3-D image of a. (c) An enlarged portion of a which clearly shows a repetitive pattern. (d) Topography of c showing periodicity of the structure.
ULTRASTRUCTURE OF Entamoeba histolytica
99
FIG. 3. (a) Amplified view of a very small portion of the lefthand corner of Fig. 2a. A well-organized structure is imaged. (b) 3 - D image of a.
100 bodies in non-cyst E. histolytica, as previously reported (Carosi 1975), and further reveals three-dimensional details for the first time. The upper part of the figure shows an organized structure (Fig. 2a) along with the 3-D image (Fig. 2b). The organized part shows periodicity and therefore it was extracted and is shown in Fig. 2c, which reveals impressive organization and periodicity in the structure. In order to appreciate the ultrastructure, which shows excellent details of the periodic behavior, we have also shown the topography (Fig. 2d) to appreciate the dimension of the sequences and their perfect organization. These images clearly confirm that this structure described in trophozoites of E. histolytica consists of a very well organized system in the endoplasm. Figure 3a shows an amplified portion (365) of Fig. 2d (upper corner of the left side). Combinations of the repeated patterns are clearly visible. Figure 3b is a 3-D image of Fig. 3a, showing a repetitive conical configuration of different sizes. In short, the ultra structure of a living E. histolytica was examined for the first time with the help of AFM. In spite of the double membrane, the nucleus with its karyosome was visible. The organized, periodic structure of the chromatoid body was revealed and its 3-D imaging permits us to appreciate structural details at the level of nanometers. A comprehensive analysis of the ultrastructure of in vivo E. histolytica will contribute to understanding its dynamic behavior. This powerful technique, if extended to the investigation of living organisms, should provide insights about the pathogenic mechanisms of this and other parasites. (We are thankful to CONICIT (Consejo Nacional de Investigacio´n Cientifica y Tecno´logica de Venezuela “CONICIT”) for financing the research project (No. G-97000820) under which the present work was carried out.)
REFERENCES Aguirre, A., Molina, S., Urdaneta, H., Cova, J. A., and Guhl, F. 1997. Characterization of two Venezuela E. histolytica strains using electrophoretic isoenzyme patterns and PCR SHELA. Archives of Medical Research 28, 285–287.
JOSHI ET AL.
Benkert, C., Jacobs, T., Berninghausen, A. J., and Leippe, M. 1997. Molecular basis of aggresive and defensive function of E. Histolytica. Archives of Medical Research 8(Suppl.), S152–153. Carosi, C. 1975. “Concepts of Structure-Function Relationship in Amoebic Organelles,” pp. 289–299. Proceedings of the International Conference on Amebiasis. Chaves, M. B., Gonzales, R. A., Espinoza, C. M., Cristobal, R. A. R., and Martinez, P. A. 1997. Entamoeba dispar: Ultrstructure and cytopatic effect. Archives of Medical Research 28, S116–S118. Diamond, L. S., and Clark, G. 1993. A re-description of Entamoeba histolytica Shaudinn, 1903 (Emeded Walker, 1911). Separating If from Entamoeba dispar Brumpt, 1925.” J. Eukaroyte Microbiology 40, 340–344. Diamond, L. S., Harlow, D. R., and Cunnick, C. C. 1978. A new medium for the axenic cultivation of Entamoeba histolytica and others Entamoeba. Transations of the Royal Society Tropical Medicine and Hygiene 72, 131. Feria-Velasco, A., and Trevin˜o, N. 1992. The ultrastructure of trophosoites of E. histolytica with particular reference to spherical arrangements of osmiophilic cylindrical bodies. Journal of Protozoology 19(10), 200–211. Kusamrarn, T., Sobhon. P., and Bailey, G. B. 1995. The mechanism of formation of inhibitor- induced ribosome helices in Entamboeba Invadens. Journal of Cell Biology 65, 529–539. Martinez-Palomo, A., and Espinosa, C. M. 1998. “Intestinal Amoebace”. International Amoeba Microbiology and Microbial Infections 5, 157–177. Urdaneta, H., Cova, J. A., Molina, S., Aguirre, A., and Hernandez, M. 1998. “Evaluacio´n Inmunoquı´mica de cepas de Entamoeba histolytica Venezolanas.” Revista Kasmera 26. Urdaneta, H., Rondon, M., Mun˜oz, M., and Hernandez, M. 1995. “Isolation and axenization of two E. histolytica strains”. Gen 49, 23–28. Received 27 January 1999; accepted with revision 14 June 1999