J . Euk. M i r m b d . 4 3 3 ) . 199R pp. 265-272 0 1998 by the Society of Prorozoo1ogislr
Entarnoeba dispar: Ultrastructure, Surface Properties and Cytopathic Effect MARTHA ESPINOSA-CANTELLANO,' ARTURO G,ONZALEZ-ROBLES, BIBIANA CHAVEZ, GUADALUPE CASTAKON, CARLOS ARGUELLO, AMPARO LAZARO-HALLER and ADOLFO MARTINEZ-PALOMO Departamento de Patologia Experimental, Centro de Investigacidn y de Estudios Avanzados del IPN. Apartado Postal 14-740, Mexico, D. F., Mexico ABSTRACT. The cytological features of Entarnoeba dispar, recently recognized by biochemical and molecular biology criteria as a distinct species, were compared to those of Entamoebo histolytica. When cultured under axenic conditions. living trophozoites of E. d i s p r strain SAW 760RR clone A were more elongated in form, had a single frontal pseudopodium. and showed a noticeable uroid. In sections of E. dispar trophozoites stained with Toluidine blue, characteristic areas of cytoplasmic metachromasia were seen due to the presence of large deposits of glycogen, seldom found in E. histolytica strain HM1:IMSS. Under the light microscope the periphery of the nucleus in E. dispar was lined by finer, more regularly distributed dense granules. With transmission electron microscopy the surface coat of E. dispar was noticeable thinner. In addition, E. dispar had a lower sensitivity to agglutinate with concanavalin A and a higher negative surface charge, measured by cellular microelectrophoresis. The cytopathic effect of E. dispar was much slower, analyzed by the gradual loss of transmural electrical resistance of MDCK epithelial cell monolayers mounted in Ussing chambers. Whereas in E. histoIytica phagocytosis of epithelial cells plays an important role in its cytopathic effect, E. dispar trophozoites placed in contact with MDCK cells showed only rare evidence of phagocytosis. The results demonstrate that the morphology of E. dispar is different to that of E. histolytica, both at the light microscopical and the ultrastructural levels. In addition. they show that E. dispar in axenic culture has a moderate cytopathic effect on epithelial cell monolayers. However, when compared to E. Izistolyrica, the in vitro lytic capacity of E. dispar is much slower and less intense. Supplementary key words. Amebas, amebiasis, carrier, non-pathogenic, parasites, pathogenesis, virulence.
T
HE debate on the existence of two different species of Entamoeba histolytica, morphologically identical but biologically distinct, initially proposed by Brumpt in 1925, was solved at a satellite meeting of the XI11 Seminar on Amebiasis, held recently in Mexico City. E. dispar, formerly known as nonpathogenic E. histolytica, has now been classified as a different protozoan species of the human gut, not capable of causing invasive disease [ 191. The distinction between the two species, achieved through a combination of biochemical, immunological and genetic data will gradually change our understanding of the epidemiology, control, and treatment of amebiasis. At the present time, there is much need for simple and reliable tests to differentiate between both species of Entamoeba in clinical samples for use under the impoverished conditions prevalent in most countries where invasive amebiasis is a health problem. E. dispar is still considered as morphologically similar to E. histolytica but, until very recently, the comparative cell biological analysis of the two species of Entamoeba was hindered by the lack of axenic cultures of E. dispar. The successful axenization of the latter species [5] has allowed the present study of the morphology, ultrastructure, surface properties, and in vitro cytopathic effects of E. dispar. The results demonstrate the existence of morphological differences between the two species and reveal that E. dispar is endowed with a discrete in vitro lytic activity. MATERIALS AND METHODS
Cell cultures. E. dispar trophozoites strain SAW 760 RR clone A and E. histolytica trophozoites strain HML-IMSS were cultured in complete YI-S medium [6] in borosilicate glass culture tubes incubated at 36.5" C. YI-S medium is used to culture E. dispar, which does not grow in the widely used TYI-S-33 medium, and differs mainly from the latter in that it does not contain casein. Both species were periodically tested by PCR amplification with specific primers to confirm their identity [4]. Amebas were harvested after 72 h incubation by chilling the culture tubes in an ice bath for 5 min. After centrifugation at 160 g for 5 min the pellets were resuspended in fresh medium. Epithelial MDCK cells were grown on 13 mm round plastic tissue culture coverslips in Eagle's salts with 100 U/ml peni-
' To whom correspondence should be addressed. Fax: 525-747-7107; Email:
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cillin, 10 pg/ml streptomycin, and 10% calf serum, at 36.5" C in an 8% CO, atmosphere. Light microscopy. Living trophozoites were studied with phase contrast and Nomarski optics. Trophozoites fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer were dehydrated in increasing concentrations of ethanol and embedded in epoxy resin. Sections stained with Toluidine blue were analyzed with a Zeiss Axiophot photomicroscope equipped with planapochromatic objectives. Transmission electron microscopy. Trophozoites were placed in a rubber ring and cryofixed at -196" C on contact with a copper mirror for 2 min. The samples were cryosubstituted by constant agitation in 4% Os0,in acetone for 50 h at -80" C. Once cryosubstituted they were placed in acetone: epoxic resin (1:l) for 18 h at room temperature and embedded in resin for 24 h at 60" C. Ultrathin sections contrasted with uranyl acetate and lead citrate were observed with a Zeiss EM 9 10 transmission electron microscope. Scanning electron microscopy. Fixed samples were dehydrated with increasing concentrations of ethanol, critically point dried in a Samdri apparatus, gold coated with an ion sputtering device (Jeol-JFC- 1loo), and examined with a Zeiss 982 field emission scanning electron microscope. Surface properties. An agglutination reaction was carried out in 16-well tissue culture microtiter plates. Trophozoites (5 X 105/ml, 0.4 ml) were incubated at room temperature with concanavalin A for 10 min, with intermittent shaking. Concanavalin A was used at final concentrations of 1, 50, or 100 (*g/ ml. The specificity of the agglutination was tested by abolition of the reaction by previous incubation of the trophozoites with 0.1 M a-methylmannoside. All concanavalin A solutions were freshly prepared before each experiment. The surface charge of trophozoites in PBS was studied by measuring the electrophoretic mobilities in a cylindrical cell electrophoresis apparatus (Rank Brothers, Cambridge, England) using the 4-ml vol c h m ber fitted with platinum electrodes and immersed in water at 25 2 1" C. Migration rates were measured by timing the passage of trophozoites through a calibrated graticule when a current of 2.5 mA and a gradient of 4 V/cm were applied to the electrophoresis chambers. Trophozoites were timed in alternate directions to minimize electrode polarization. Calibration was controlled before each experiment by measuring the electrophoretic mobility of human red blood cells. Interaction of amebas with MDCK monolayers. When
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MDCK cell monolayers grew to confluence the medium was aspirated and E. dispar trophozoites in YI-S medium were added at a ratio of 1:l. Incubation at 36.5"C was allowed to proceed for different timepoints ( 5 . 30, 60. and 90 min), after which samples were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2. Electrophysiology. Measurements of transepithelial electrical resistance were carried out essentially as reported [7]. Briefly. Nylon disks with confluent MDCK monolayers were mounted between two Lucite chambers filled with Eagle's medium. The difference of potential between the two chambers was measured with Ag/AgCl electrodes. The potential difference across the monolayer was recorded and short ( 2 s) and low intensity (100 pA/cm') current pulses were delivered. The current deflection elicited was measured with a second electrode set placed 2 mm apart from the membrane. The contribution of the collagen support and the bathing solution were taken into account. All values correspond exclusively to the epithelial monolayer. The transepithelial electrical resistance was calculated following Ohm's law, and the normalized values reported represent means 2 SEM at each timepoint measured for E. dispur (n = 17) and E. tiisto1ytic.a (n = 6 ) . RESULTS Light microscopical differences between E. dispar and E. histolyrica live trophozoites cultured under similar conditions were evident with Nomarslu optics (Fig. l A , B). The overall shape of living trophozoites of E. dispar was elongated, with a prominent anterior and broad pseudopod and a noticeable posterior uroid. In contrast. E. tzisto/yrica trophozoites tended to be round in shape, had several small pseudopodia and the uroid was less commonly seen. In E. dispar the thin peripheral rim of the nucleus was made up of regularly disposed dense granules of circular profile, whereas in E. histolvtica the nuclear periphery was coarse and thick (Fig. lC, D). In sections of fixed trophozoites, the above mentioned differences were more evident (Fig. 1E. F). The cytoplasm of E. dispar appeared patchy: vacuoles were concentrated in some regions, while other areas devoid of vacuoles appeared metachromatic when stained with Toluidine blue. In contrast, vacuoles in E. histo/ytica were larger and had a uniform distribution throughout the cytoplasm. The density and thickness of the nuclear rim in sections of E. histolytica was clearly distinct from the thinner and uniform peripheral chromatin of E. dispar. The dividing nuclei of both species showed five to six central condensations. possibly corresponding to an equal number of metaphase chromosomes (Fig. 1E. E inserts). Under transmission electron microscopy both nuclear and cytoplasmic components of the two species of Eiirarnoeba were similar, the difference being the distribution and relative quantities of various organelles and particulate cytoplasmic components. A prominent feature of most E. dispar trophozoites was the existence of cytoplasmic areas containing large deposits of glycogen granules, with ribosomal bundles interspersed (Fig. 2A). E. tiistolytica showed the characteristic cytoplasm occupied by a heterogeneous population of vacuoles of different sizes (Fig. 2B). However, there was no single ultrastructural feature present in all amebas that could be used to distinguish one species from another, except for the plasma membrane of E. histolytica, which had a thicker surface coat when compared to that of E. dispur. In cryofixed and cryosubstituted trophozoites from E. histolytica, a thick uniform surface coat measuring approximately 16 nm can be observed at the outer surface of the plasma membrane (Fig. 3B). while E. dispar displays a loose surface coat, sometimes interrupted in vast areas of the plasma membrane (Fig. 3A).
Scanning electron microscopy of E. dispar trophozoites (Fig. 4A) revealed a smoother surface and a polarized shape, with a single prominent pseudopodium at the anterior end. E. histo/yticu trophozoites (Fig. 4B) had a rougher cell surface, were more pleomorphic, and showed several small pseudopodia. However, it was not possible to differentiate with certainty between single trophozoites of the two species of amebas. The surface properties studied were sensitivity to agglutinate with concanavalin A and microelectrophoretic mobility. In both species of Enramoeba agglutination inhibited by a-methylmannoside was found in the presence of concanavalin A, at concentrations ranging from 1 to 100 pg/ml. However, in all agglutination experiments E. histolytica cultures formed clumps two to four times larger than those of E. dispar. We confirmed our early observation [17] that the surface charge of E. histo/yica is so low that i t cannot be measured with the technique of cell electrophoresis under the conditions of pH and ionic strength used. In contrast, under the same conditions E. dispur displayed a large value of electrophoretic mobility of 1.27 ( * m - ' V 'cm ~ (SD = 0.26). The cytopathic activity of E. dispar, measured as the drop in transepithelial resistance (TER) of MDCK monolayers in Ussing chambers placed in contact with trophozoites showed important differences when cornpared to E. histolvtica (Fig. 5 ) . While E. dispar required 30 min incubation with the monolayer to drop the electric transepithelial resistance to around 30% of control, E. hisrolvtica showed a similar effect after less than 5 min incubation. In fact, transmural resistance was practically abolished after 15 min incubation with E. histolytica trophozoites, the values obtained mostly corresponding to background registration. E. dispar was able to drop the transmural resistance only after 60 min incubation. The differences were more striking when MDCK monolayers were analyzed with scanning electron microscopy after interaction with trophozoites of either strain. After one hour E. histolTtica had produced an almost complete disappearance of the epithelial cell cultures as a result of the lysis, detachment and phagocytosis of target cells. In contrast, trophozoites of E. disp a r displayed a more discrete cytopathic effect, seen first as disappearance of microvilli at the luminal surface of MDCK cells (Fig. 6B), progressing slowly to separation of neighboring cells and gradual detachment of MDCK from the substrate (Fig. 6C). Phagocytosis was only seldom seen, in contrast to the frequent internalization of target cells displayed by E. histolytica amebas.
DISCUSSION Until recently, two of the most puzzling aspects of the biology of E. histolvtica were the unexplained variability of its pathogenic potential and the restriction of human invasive amebiasis to certain geographical areas despite the worldwide distribution of the parasite. The main debate centered on the question of whether there are one or two species of E. histolytica. In 1925 the French parasitologist Emile Brumpt proposed that invasive amebiasis is produced by a species of ameba of restricted distribution, biologically distinct, but morphologically similar to nonpathogcnic amebas having a worldwide distribution [ 2 ] . Except for some confirmatory studies carried out by Simic, nothing refuted or confirmed this hypothesis for almost 50 years 1141. However, in the 1970s, differences in surface properties were found between strains of E. histolytica isolated from carriers and those obtained from patients with invasive amebiasis [9, 17, 181. Subsequently, the isoenzyme technique was applied to thousands of isolates of amebas isolated in several continents (131. This technique, based on the analysis of band patterns
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Fig. 1. Light micrographs of E. histo!\.riccz (A, C, E) and E. dispar (B. D, F) trophozoites. Nomarski optics (A-D) show a round shape with several small pseudopodia and a coarse nuclear periphery in E. hisfolyfica(A, C), and a more elongated form with a prominent anterior pseudopod, a noticeable posterior uroid and a thin peripheral rim of the nucleus in E. dispar (B, D). Sections of fixed trophozoites stained with Toluidine blue (E, F) show the characteristic uniform distribution of large vacuoles in the cytoplasm of E. hisfolytica (E) and a thick. dense nuclear rim containing six central condensations in the dividing nuclei (E, insert). In contrast. the cytoplasm of E. dispar appears patchy. with vacuoles concentrated in some regions and large glycogen deposits giving a metachromatic appearance (F). Details of the thin peripheral chromatin with five condensations in a dividing nucleus are shown (E insert). Bar = 10 pm.
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Fig. 2. Transmission electron micrograph of cryofixed and cryosubstituted E. dispar and E. histdytica trophozoites. In E. dispar ( A ) the cytoplasmic vacuoles are concentrated in some regions of the cytoplasm. leaving extensive areas devoid of vacuoles, which are occupied by large deposits of glycogen granules with ribosomal bundles interspersed. E. hisrolyrica (B) shows a heterogeneous population of vacuoles uniformly distributed throughout the cytoplasm. Bar = I km. N = nucleus, G = glycogen granules. R = ribosomal bundles, V = vacuoles.
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Fig. 3. Transmission electron micrograph showing a magnification of E. dispar and E. histolvrica plasma membranes. E. dispur (A) displays a loose surface coat which sometimes appears interrupted, while E. histolytica (B) shows a uniformly thick surface coat. Bar = 100 nm.
obtained after gel electrophoresis of the enzymes hexokinase and phosphoglucomutase, revealed that invasive amebiasis is produced by strains that have characteristic isoenzyme patterns (zymodemes) distinct from those obtained from amebas har-
bored by most carriers. Moreover, monoclonal antibodies specifically recognized amebas belonging to pathogenic zymodemes [ 11, 15, 161. However, the results obtained on the alleged conversion of isolates with nonpathogenic zymodemes to patho-
Fig. 4. Scanning electron micrograph of E. &par and E. hisfolytica trophozoites in axenic culture. The elongated form with a single prominent pseudopodium and a smooth surface found in most E. dispar (A) trophozoites is compared to the pleomorphic shape with several small pseudopodia and a rough cell surface of E. hisrolytica (B). Bar = 10 p m .
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Time (min) Fig. 5 . Effect of E. d / s p u r and E. h i . s t o / x f i w trophozoites on the transepithelial electrical resistance ( T E R ) of MDCK cell monolayers. Trophoroitcs of each species were added at a ratio 2: I parasite:epithelial cell. Incubation in U\sinp chambers was allowed for 5. 15, 30, and 60 min. Values have been normalized and expre\s incans ? SEM for E. d i s p f - ( n = 17) and E. / i i . s t d y f i r u (n = 6). An impaired two tailed Student t test \vith il 9 5 8 confidence interval reveals significant differences between the values obtained for E. d i . c p r ( p = 0.0469) and €. Izi.sto1yfir.a ( p = 0.0015) at 5 rnin.
genic ones during the process of axenization reopened the debate as to whether there are one or two species, the former living usually as a commensal in the human intestine and, under conditions not understood, becoming pathogenic to the host [ 1, 101. More recently the application of molecular biology has gradually accumulated evidence that confirms the existence of distinct variations between the genomes of both species. In fact. the debate on the existence of two different species of Entamoeba was solved at a satellite meeting of the XIXI Seminar on Amebiasis, held in Mexico City. E. dispur. formerly known as non-pathogenic E. histolytica, has now been classified as a separate species, not capable of causing clinical disease [ 191. This distinction will change our understanding of the epidemiology, control, and treatment of amebiasis. There are still both conceptual and practical issues to address in order to fully clarify our understanding of the human infections produced by the two species of protozoa. For example, E. dispar is still considered to be morphologically similar to E. hisrolvrica. This issue is of considerable practical importance because by far the technique most employed in developing countries to identify Entanzoeba as the causative agent of a case of diarrhea or dysentery is the microscopic identification of the parasite in stool samples. On the other hand, understanding the immunological reactions elicited by infection with E. dispur requires knowledge of the lytic capacity of E. dispar, a subject that still remains controversial. The recent successful axenization of E. dispar [S] has allowed us to tackle these problems by comparing the morphology, surface properties and lytic properties of both amebas cultured under comparable conditions. In this first approach we have used only one isolate of each species: E. hisrolvrica HM1:IMSS and E. dispar SAW 760RR clone A. Both strains have been cultured in the laboratory for many years; however, E. dispar was only recently axenized [S]. This study will be extended to include more isolates of each species to assure that the differences observed are characteristic of the two species. and not isolate specificities or changes induced by prolonged in vitro cultivation. Since cross contamination between cultures may occur, we verified periodically the identity of the two species here studied by ampli-
fication of the small subunit rRNA genes of the organisms by PCR [4]. At first sight, under light microscopy, the morphology of E. dispar and E. histolytica may appear to be similar. However, closer examination reveal cytological differences, even in live cultures examined with an inverted optic microscope. Trophozoites of the former species are more elongated in form, their cytoplasmic vacuoles have a patchy distribution and they do not tend to form clumps, as E. histolytica does. With Nomarski optics these variations become more evident. In addition, the nuclei of E. dispar is lined by a thin layer of regularly distributed dense granules, as reported by Brumpt [ 3 ] . In plastic sections of cultured trophozoites, the nuclei of E. dispar appears again distinct from that of E. histolytica; the two species can be further differentiated by the patchy appearance of the cytoplasmic vacuoles, and also by the presence of large and irregular metachromatic areas apparent when stained with Toluidine blue. Transmission electron microscopy demonstrated that these metachromatic areas in E. dispar represent large cytoplasmic deposits of glycogen. Taken together, these results suggest the possibility of finding, with the use of simple staining techniques, light microscopical differences between the two species of Etitutnoeba in samples of fecal material. The main characteristic of E. histolytica in coprological samples of cases with invasive intestinal amebiasis studied by microscopy has been traditionally considered to be the existence of phagocytosis. Brumpt himself, however, stated that E. dispar could eventually phagocytize red blood cells [3]. In the present work the phagocytic activity of E. dispar on MDCK cells was less evident than with E. hisrolyticu, but the difference is not absolute. The same can be said with regard to our unpublished observations of E. dispar erythrophagocytosis. Both in vitro and in vivo erythrophagocytic E. dispar trophozoites may be found, but again much less frequently than in E. histolyticu. The difference in agglutinability with concanavalin A and surface charge between E. dispar and E. histolytica may be related to the clear-cut variations in the ultrastructure of the surface coat of both species. It is of interest that similar ultrastructural results pertaining to the surface coat of E. dispar were reported recently by another laboratory in preliminary form [ 121. The difference in sensitivity to agglutinate with the lectin is now of almost historical interest, since it was the first biological evidence that pointed out to differences between amebas isolated from asymptomatic carriers and those from invasive cases [ 9 ] . More than two decades latter the same results were obtained, now with axenic cultures of both species, periodically checked for their identity with the PCR technique. Similarly, E. histolytica trophozoites were confirmed to have no surface charge detectable by cell microelectrophoresis, as reported earlier [17]. As was the case with another nonpathogenic ameba, E. moshkovskii, E. dispar has now been shown to have a high negative surface charge. Since the potential lytic activity of E. dispar has been the subject of much recent discussion, the present results obtained by placing trophozoites of this species in contact with cell monolayers of epithelial MDCK cells are of interest. E. dispar under in vitro conditions does have a lytic effect, but this effect is much milder and slower than the one shown by E. histolyticu under the same conditions [8]. Our results tend to agree therefore with those of Brumpt who found that E. dispar produces only a discrete and very slow necrosis of the mucosal surface of the large intestine in susceptible animals 131. In conclusion. the present observations demonstrate the existence of morphological differences between E. histolytica and E. dispar and reveal that E. dispar is endowed with a moderate in vitro cytolytic activity.
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Fig. 6. Scanning electron micrograph of the in vitro cytopathic effect of E. dispar trophozoites on MDCK cell monolayers. At time 0 ( A ) . the characteristic architecture of MDCK monolayers with surface microvilli and closely apposed cells is observed. However. at 1S niin incubation with the parasite (B), the luminal surface of MDCK cells shows loss of microvilli; after 30 min incubation ( C ) a gradual detachment of the epithelial cells is observed. Bar = 10 p n
ACKNOWLEDGMENTS We gratefully acknowledge Dr. Cecilia Ximenez for carrying out periodic PCR amplifications to confirm the identity of E. histolvtica and E. dispar, and Dr. Lorenza Gonz6lez Mariscal for invaluable help in the statistical analysis. This work was partially supported by Conacyt grant No. 3694P-M9607, and special funding to MEC from the Fundacidn Mexicana para la Salud and from the Young Researchers Programme (JIRA) from Cinvestav, Mexico. The results reported here were partially pre-
sented at the XI11 Seminar on Amebiasis, held in Mexico City January 29-31, 1997.
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