Demonstration of acid phosphatase in Entamoeba histolytica and Entamoeba gingivalis

의학과/박사[한글] Demonstration of Acid Phosphatase in Entamoeba histolytica and Entamoeba gingivalis Gau Young Cha, D.D.S. Department of Medical Science, The Graduate School, Yonsei University (Director: Professor Chin Thack Soh, M.D.) Entamoeba histolytica (E.histolytica) is a parasitic protozoa of intestinal tract which was first described by Losch (1875). It occasionally spreads from its enteric habitat and established metastatic spheres in liver, lung and skin etc. Entamoeba gingivalis (E. gingivalis) is a parasite of the mouth of man, which was found by Gros(1949) from soft tartar of the teeth, and is generally considered as a harmless commensal living in both diseased and healthy tissues of the mouth. The morphology of trophozoite of E.histolytica markedly resembles that of E. gingivalis. Recently, a number of investigators have observed ultrastructure of E. histolytica using electron microscope, and reported on several new structures which have not been hitherto detected by optical microscopy (Eaton et al., 1969; El-Hashimi and Pittman, 1970; Lowe and Maegraith, 1970 a,b,c,; Cho et al., 1972; Proctor and Gregory, 1972; Westphal et al., 1972; Diamond et al., 1972; Mattern et al., 1972). Those are small cytoplasmic processes, surface-active lysosome, a number of vacuoles of different size, shape and contents, endoplasmic reticulum, electron-dense granules and pigment bodies, filamentous structures, crystalloid aggregates and hellical structures, cylindrical rods in rosette patterns, and various shaped nuclear inclusions. Keller et al. (1967) and Yang and Cho (1972) observed E.gingivalis by electron microcope, and reported uroid-like structure at the plasma membrane in addition to several fine structures in the cytoplasm which were reported in E. histolytica, though some of those features were different. Although a number of reports have been appeared on the fine structures of both amoebae, very little work has been done for those structures in relation to their functional natures. Carrera and Changus (1948) first demonstrated acid phosphatase activity in e. histolytica using Gomori's histochemical method by optical microscopy. Eaton et al. (1970) demonstrated an enzyme-containing organelle equipped with a thread-like trigger, to the level of ultrastructure, on the surface of E. histolytica by Gomori staining. They suggested that the lysosome was responsible for the cell damage observed and that this would represent a pathogenic modification of an organelle which was possessed by other members of the genus Entamoeba. Recently, Ohashi (1971, 1972) observed localization of acid phosphatase activities in Trichomonas tenax (T.tenax) and Trichmonas vaginalis (T.vaginalis) by optical and electron microscopy. In T.tenax, the activity was demonstrated on plasma membrane, in cytoplasmic vacuoles and Golgi apparatus. In T. vaginalis the precipitates were found in vacuoles, Golgi appartus and endoplasmic reticulum, but no reaction was recognized on the cell membrane. The findings indicated that the food vacuoles of T.vaginalis might be associated with autophagy, whereas those of T.tenax might be assoicated with heterophagy, and suggested marked differences in functional natures between two trichomonads. In recent years, histochemical and biochemical studies on protozoa became considerable attentions and a great volume of information has been accumulated from investigations of living and fixed organisms. The purpose of this study was to compare localization of acid phosphatase activities between two species of amoebae, E. histolytica and E. gingivalis, by means of electron microscopic cytochemistry, and to elucidate the relations between this enzyme and the digestion of food as well as newly found fine structures. In the present study, YS 27-strain E. histolytica and YG 215-strain E. gingivalis were used throughout the investigation. YS 27-strain E. histolytica was isolated in liver abscess of a 72-year-old man at Severance Hospital in Septemer 1969, and maintained by subculture on a diphasic medium with calf serum and penicillin G. added (Cho, 1968), assoicated with unidentified intestinal bacterial flora. YG 215-strain E. gingivalis was collected from the gingival crevice of a periodontitis patient, a 41-year-old woman, in January 1972, and was cultured on a diphasic medium overlayed with egg-yolk fluid media (pH 7.5) associated with unidentified oral flora, and subcultured evey other day. For opitcal microscopy, several 48-hour cultures of the amoebae were spun at 500rpm for 5 minutes, and the supernate was discarded. The sediment was smeared on cover slips which were coated with egg-white glycerin previously. The cover slips were dried in air, but not dried completely before immersing in the fixative. The smears were fixed in chilled acetone for 30 seconds, rinsed in distilled water, left in 0.1M citrate buffer (pH 4.7) for 30 minutes, and then, rinsed again in distilled water. These were incubated at 37℃ for 45∼90 minutes in Gomori medium (Eranko et al., 1952). Composition of the incubation medium was as follows; 2% sodium glycerophosphate 6ml 0.1M acetate buffer (pH 4.7) 10ml 5% lead nitrate 4ml distilled water 80ml After the incubation the cover slips were washed in distilled water 5∼6 times at intervals of 5 minutes, and dried in air. These were immersed in 0.25% ammonium sulfide for 2 minutes, washed in distilled water, and mounted in Canada balsam. Control preparations were made either by omitting 2% sodium glycerophosphate from the buffered reagent or by adding to the control substrate sodium fluoride (M/300), a known acid phosphatase inhibitor. For electron microscopy, the trophozoites of amoebae were collected and spun as before. The supernate was discarded, and 2ml of 3% glutaraldehyde 0.1M cacodylate buffer (pH 7.2) was added to the sediment. In order to increase the concentration of the fixative, 1ml portion of the mixture of fixative was repeatedly withdrawn and replaced with fresh fixative, and fixed for 2∼4 hours at 4℃. These were spun at 500 rpm for 5 minutes and the supernate was discarded. 1% agar (45℃) was added in the sediment and solidified, and then, cut into 1mm cubes. The cubes were washed in 7.5% sucrose 0.1M cacodylate buffer (pH 7.2), and left overnight at 4℃. These were incubated at 37℃ for 45∼90 minutes ingomori medium (Eranko et al., 1952). Controls were run as before. After the incubation, the specimens were washed in 7.5% sucrose cacodylate buffer, post fixed in 1% O^^sO^^4 0.1M cacodylate buffer (pH 7.4) at 4℃ for 30 minutes, dehydrated in ascending series of ethanol from 60% and prophylene oxide, and then embedded in Epon 812 (Luft, 1961). One micron thick sections were made on a Sorvall Porter Blum MT^^2 ultramicrotome, stained with basic fuchsin, and examined under light microscope. When organisms were found, thin sections were made for electron microscopy. The sections were placed in 150-mesh copper grids, and examined using Hitachi HU-11E-1type electron microscope. Under optical microscopy, E. histolytica demonstrated weak reactions of acid phosphatase around the cytoplasmic membrane. The coarse precipitates were localized to the central parts of the organism, but indistinguishable from other organelles. Those precipitates resembled the portions of cytoplasmic vacuoles. Controls werre negative for the reactions. By electron microscopy of E. histolytica, weak reaction products were evenly distributed around the plasma membrane. The reactive organelles in the cytoplasm were vacuole membrane and its contents, and endoplasmic reticulum. The control showed negative reaction, and vacuoles present in the cytoplasm varied greatly in size and shape, and usually contained some sort of particulate matters probably represented ingested bacteria and starch grains under various stages of digestion. On the portion of plasma-membrane of E. histolytica, coarse precipitiates were demonstrated at the cytoplasmic projection, which was reported by Cho et al. (1972). Acid phosphatase activities in vacuoles of E. histolytica differed significantly from negative to highly active ones, and were grouped into 5 phases; (a) lysosome-like structure showing strong enzymic reaction, (b) large reaction products in vaculole lumen with distinctively enzyme active membrane, (c) marked reaction on vacuolar wall, but not in lumen, (d) no reaction products either vaculoar wall or its contents, (e) reaction negative empty vacuole. In some amoeba, (a) was demonstrated closely to the limiting membrane of (b) or (c) vacuoles, and some of (a) appeared to be fusing with (b) or (c). Some of the endoplasmic reticulum with the attendant enzyme activity occasionally extended to the (b) or (c) vacuoles which showed positive reaction Vacuole (e) was usually found at the periphery of cytoplasm. In some vacuoles, which retained weak deposits of lead phosphates, a portion of the limiting membrane was opened and fused onto the plasma membrane. Nucleus showed negative reaction for the enzyme activity. Under optical microscopy of E. gingivalis, acid phosphatase activity was closely resembled to that showed in E. histolytica. By electron microscopy of E. gingivalis, plasma membrane showed generally negative reaction for acid phosphatase. But in some amoeba, dense deposits of lead phosphate were appeared at the limited portion of plasma membrane, which was identified as uroid-like structure reported by Yang and Cho (1972). In the cytoplasm of E. gingivalis, (a) (b) (c) (d) and (e) phase of vacuoles, shown in E. histolytica were almost presented. Some vacuoles with bacteria, which appeared as recently ingested, were devoid of enzyme activity except in the bacteria. Coarse lead phosphate deposits were located in the bacteria. Another sort of vacuoles contained strong enzyme active precipitates, whereas vacuole membrane showed negative or weak reaction of the activity. Enzyme reaction positive small granules, 0.2μ in diameter, were occasionally observed at near nucleus. In the nucleus of E. gingivalis, highly enzyme active small granules, 0.05∼0.1μ in diameter, were scattered or assembled in the nucleoplasm. Control showed negative reaction. The results above are summarized as follows; 1. In E. histolytica, the reaction product was evenly distributed over the entire plasma membrane, whereas, E. gingivalis presented no activity of acid phosphatase on the plasma membrane, except the portion of uroid-like structure. 2. In the cytpolasm, various precipitations of the reaction were observed in the vacuoles of both amoebae; vacuole limiting membrane, vacuole membrane and it scontents, or contents of vacuole only. Endoplasmic reticulum demonstrated moderate activity. In E. gingivalis vacuoles with bacteria were devoid of enzyme activity except in the bacteria. 3. Granule-like acid phosphatase reaction product was demonstrated in the nucleoplasm of E. gingivalis. However, the activity was negative in the nucleus of E. histolytica. [영문] Entamoeba histolytica (E.histolytica) is a parasitic protozoa of intestinal tract which was first described by Losch (1875). It occasionally spreads from its enteric habitat and established metastatic spheres in liver, lung and skin etc. Entamoeba gingivalis (E. gingivalis) is a parasite of the mouth of man, which was found by Gros(1949) from soft tartar of the teeth, and is generally considered as a harmless commensal living in both diseased and healthy tissues of the mouth. The morphology of trophozoite of E.histolytica markedly resembles that of E. gingivalis. Recently, a number of investigators have observed ultrastructure of E. histolytica using electron microscope, and reported on several new structures which have not been hitherto detected by optical microscopy (Eaton et al., 1969; El-Hashimi and Pittman, 1970; Lowe and Maegraith, 1970 a,b,c,; Cho et al., 1972; Proctor and Gregory, 1972; Westphal et al., 1972; Diamond et al., 1972; Mattern et al., 1972). Those are small cytoplasmic processes, surface-active lysosome, a number of vacuoles of different size, shape and contents, endoplasmic reticulum, electron-dense granules and pigment bodies, filamentous structures, crystalloid aggregates and hellical structures, cylindrical rods in rosette patterns, and various shaped nuclear inclusions. Keller et al. (1967) and Yang and Cho (1972) observed E.gingivalis by electron microcope, and reported uroid-like structure at the plasma membrane in addition to several fine structures in the cytoplasm which were reported in E. histolytica, though some of those features were different. Although a number of reports have been appeared on the fine structures of both amoebae, very little work has been done for those structures in relation to their functional natures. Carrera and Changus (1948) first demonstrated acid phosphatase activity in e. histolytica using Gomori's histochemical method by optical microscopy. Eaton et al. (1970) demonstrated an enzyme-containing organelle equipped with a thread-like trigger, to the level of ultrastructure, on the surface of E. histolytica by Gomori staining. They suggested that the lysosome was responsible for the cell damage observed and that this would represent a pathogenic modification of an organelle which was possessed by other members of the genus Entamoeba. Recently, Ohashi (1971, 1972) observed localization of acid phosphatase activities in Trichomonas tenax (T.tenax) and Trichmonas vaginalis (T.vaginalis) by optical and electron microscopy. In T.tenax, the activity was demonstrated on plasma membrane, in cytoplasmic vacuoles and Golgi apparatus. In T. vaginalis the precipitates were found in vacuoles, Golgi appartus and endoplasmic reticulum, but no reaction was recognized on the cell membrane. The findings indicated that the food vacuoles of T.vaginalis might be associated with autophagy, whereas those of T.tenax might be assoicated with heterophagy, and suggested marked differences in functional natures between two trichomonads. In recent years, histochemical and biochemical studies on protozoa became considerable attentions and a great volume of information has been accumulated from investigations of living and fixed organisms. The purpose of this study was to compare localization of acid phosphatase activities between two species of amoebae, E. histolytica and E. gingivalis, by means of electron microscopic cytochemistry, and to elucidate the relations between this enzyme and the digestion of food as well as newly found fine structures. In the present study, YS 27-strain E. histolytica and YG 215-strain E. gingivalis were used throughout the investigation. YS 27-strain E. histolytica was isolated in liver abscess of a 72-year-old man at Severance Hospital in Septemer 1969, and maintained by subculture on a diphasic medium with calf serum and penicillin G. added (Cho, 1968), assoicated with unidentified intestinal bacterial flora. YG 215-strain E. gingivalis was collected from the gingival crevice of a periodontitis patient, a 41-year-old woman, in January 1972, and was cultured on a diphasic medium overlayed with egg-yolk fluid media (pH 7.5) associated with unidentified oral flora, and subcultured evey other day. For opitcal microscopy, several 48-hour cultures of the amoebae were spun at 500rpm for 5 minutes, and the supernate was discarded. The sediment was smeared on cover slips which were coated with egg-white glycerin previously. The cover slips were dried in air, but not dried completely before immersing in the fixative. The smears were fixed in chilled acetone for 30 seconds, rinsed in distilled water, left in 0.1M citrate buffer (pH 4.7) for 30 minutes, and then, rinsed again in distilled water. These were incubated at 37℃ for 45∼90 minutes in Gomori medium (Eranko et al., 1952). Composition of the incubation medium was as follows; 2% sodium glycerophosphate 6ml 0.1M acetate buffer (pH 4.7) 10ml 5% lead nitrate 4ml distilled water 80ml After the incubation the cover slips were washed in distilled water 5∼6 times at intervals of 5 minutes, and dried in air. These were immersed in 0.25% ammonium sulfide for 2 minutes, washed in distilled water, and mounted in Canada balsam. Control preparations were made either by omitting 2% sodium glycerophosphate from the buffered reagent or by adding to the control substrate sodium fluoride (M/300), a known acid phosphatase inhibitor. For electron microscopy, the trophozoites of amoebae were collected and spun as before. The supernate was discarded, and 2ml of 3% glutaraldehyde 0.1M cacodylate buffer (pH 7.2) was added to the sediment. In order to increase the concentration of the fixative, 1ml portion of the mixture of fixative was repeatedly withdrawn and replaced with fresh fixative, and fixed for 2∼4 hours at 4℃. These were spun at 500 rpm for 5 minutes and the supernate was discarded. 1% agar (45℃) was added in the sediment and solidified, and then, cut into 1mm cubes. The cubes were washed in 7.5% sucrose 0.1M cacodylate buffer (pH 7.2), and left overnight at 4℃. These were incubated at 37℃ for 45∼90 minutes ingomori medium (Eranko et al., 1952). Controls were run as before. After the incubation, the specimens were washed in 7.5% sucrose cacodylate buffer, post fixed in 1% O^^sO^^4 0.1M cacodylate buffer (pH 7.4) at 4℃ for 30 minutes, dehydrated in ascending series of ethanol from 60% and prophylene oxide, and then embedded in Epon 812 (Luft, 1961). One micron thick sections were made on a Sorvall Porter Blum MT^^2 ultramicrotome, stained with basic fuchsin, and examined under light microscope. When organisms were found, thin sections were made for electron microscopy. The sections were placed in 150-mesh copper grids, and examined using Hitachi HU-11E-1type electron microscope. Under optical microscopy, E. histolytica demonstrated weak reactions of acid phosphatase around the cytoplasmic membrane. The coarse precipitates were localized to the central parts of the organism, but indistinguishable from other organelles. Those precipitates resembled the portions of cytoplasmic vacuoles. Controls werre negative for the reactions. By electron microscopy of E. histolytica, weak reaction products were evenly distributed around the plasma membrane. The reactive organelles in the cytoplasm were vacuole membrane and its contents, and endoplasmic reticulum. The control showed negative reaction, and vacuoles present in the cytoplasm varied greatly in size and shape, and usually contained some sort of particulate matters probably represented ingested bacteria and starch grains under various stages of digestion. On the portion of plasma-membrane of E. histolytica, coarse precipitiates were demonstrated at the cytoplasmic projection, which was reported by Cho et al. (1972). Acid phosphatase activities in vacuoles of E. histolytica differed significantly from negative to highly active ones, and were grouped into 5 phases; (a) lysosome-like structure showing strong enzymic reaction, (b) large reaction products in vaculole lumen with distinctively enzyme active membrane, (c) marked reaction on vacuolar wall, but not in lumen, (d) no reaction products either vaculoar wall or its contents, (e) reaction negative empty vacuole. In some amoeba, (a) was demonstrated closely to the limiting membrane of (b) or (c) vacuoles, and some of (a) appeared to be fusing with (b) or (c). Some of the endoplasmic reticulum with the attendant enzyme activity occasionally extended to the (b) or (c) vacuoles which showed positive reaction Vacuole (e) was usually found at the periphery of cytoplasm. In some vacuoles, which retained weak deposits of lead phosphates, a portion of the limiting membrane was opened and fused onto the plasma membrane. Nucleus showed negative reaction for the enzyme activity. Under optical microscopy of E. gingivalis, acid phosphatase activity was closely resembled to that showed in E. histolytica. By electron microscopy of E. gingivalis, plasma membrane showed generally negative reaction for acid phosphatase. But in some amoeba, dense deposits of lead phosphate were appeared at the limited portion of plasma membrane, which was identified as uroid-like structure reported by Yang and Cho (1972). In the cytoplasm of E. gingivalis, (a) (b) (c) (d) and (e) phase of vacuoles, shown in E. histolytica were almost presented. Some vacuoles with bacteria, which appeared as recently ingested, were devoid of enzyme activity except in the bacteria. Coarse lead phosphate deposits were located in the bacteria. Another sort of vacuoles contained strong enzyme active precipitates, whereas vacuole membrane showed negative or weak reaction of the activity. Enzyme reaction positive small granules, 0.2μ in diameter, were occasionally observed at near nucleus. In the nucleus of E. gingivalis, highly enzyme active small granules, 0.05∼0.1μ in diameter, were scattered or assembled in the nucleoplasm. Control showed negative reaction. The results above are summarized as follows; 1. In E. histolytica, the reaction product was evenly distributed over the entire plasma membrane, whereas, E. gingivalis presented no activity of acid phosphatase on the plasma membrane, except the portion of uroid-like structure. 2. In the cytpolasm, various precipitations of the reaction were observed in the vacuoles of both amoebae; vacuole limiting membrane, vacuole membrane and it scontents