Entamoeba histolytica: trophozoite, precyst and cyst studied by atomic force microscopy
J. Luis Menchaca Arredondo4,5, M. P. Barrón González1, A. León Coria1, J.E. Ortega2, J. Vargas Villarreal3, J.L. Hernández Piñero1,4 and M. R. Morales Vallarta1 1 Laboratorio de Biología Celular, Departamento de Biología Celular y Genética, Universidad Autónoma de Nuevo León,
C.P. 66451, San Nicolás de los Garza, Nuevo León, México 2 Department of Physics, Tecnológico de Monterrey, C., México 3 Biochemical and Cellular Physiology Laboratory, Centro de Investigaciones Biomédicas del Noreste, C.P. 64720,
México 4 Centro de Investigación en Innovación y Desarrollo en Ingeniería y Tecnología; PIIT/Universidad Autónoma de Nuevo
León, Apodaca, C.P. 66600, México 5 Facultad de Ciencias Físico Matemáticas, Universidad Autónoma de Nuevo León, C.P. 66451, México
Amoebiasis is a disease caused by the cosmopolite parasitic protozoa E. histolytica; it has been estimated that the 10% of the world population is infected by this protozoa, which represents approximately 50 million of invasive amoebiasis cases and as much as 100,000 deaths per year. E. histolytica have four stages: the trophozoite, precyst, cyst and metacyst; trophozoite is the invasive stage and the cyst is the infective stage. Trophozoite, or mobile form, is a uninucleated and pleomorphic structure, measures about 15-40 μm; the cyst measures 10-15 μm, and is a spherical tetranucleated structure covered by a chitin cell wall that confers its resistance to adverse conditions. Atomic force microscopy (AFM) has emerged in recent years as a powerful tool for the study of cellular structure at resolutions of a few nanometers. Morphological studies revealing details of living cells, which were previously impossible due to the resolution limits of the light microscope, are now possible using AFM. The AFM´s major contribution to biology has been its ability to study the dynamics of live cells in physiological medium at ultrahigh resolution and in real time. Despite the encysting process has been studied in vitro by different authors, many of the aspects about this process still remain unknown. In this work, we studied in situ by atomic force microscopy (AFM) the E. histolytica trophozoite, pre-cyst and cyst, and we made clear some of the differences between each one of the stages, making evident the differences in roughness, composition, elasticity and size. In situ characterization of the E. histolytica trophozoite, pre-cyst and cyst stages was performed. This is the first report of the pre-cyst stage observed on AFM. In addition, we performed a quantitative comparison of the elasticity of the outer surface of the cell, which shows that the trophozoite cell membrane is more elastic and less rigid than the cell wall of the cyst. Morphological differences between the different stages were also observed, both the morphology and measurements of all cell stages observed in this study are consistent with those reported in the literature. According to the data obtained, as cell goes through the encysting process it loses its elasticity, opposite occurs with the roughness and the adhesion work, which increase as the cell becomes encysted.
Keywords: amoebiasis; atomic force microscopy (AFM); cyst; Entamoeba histolytica; morphometric
1. Introduction
Entamoeba histolytica is a widely distributed parasitic protozoa and the major cause of morbidity and mortality in developing countries. Amoebiasis is a disease caused by the cosmopolite parasitic protozoa E. histolytica; it has been estimated that about 10% of the world population is infected by this protozoa, which represents approximately 50 million of invasive amoebiasis cases and as much as 100,000 deaths per year [1]. E. histolytica in its life cycle present four stages, trophozoite, pre-cyst, cyst and metacyst, however the trophozoite and cyst stages are the most studied. Inside humans, E. histolytica lives and multiplies as a trophozoite; which corresponds to the invasive and mobile form. This cell stage is a mononuclear and pleomorphic structure that measures about 15–40 µm and is wrapped by a cell membrane, inside abundant digestive vacuoles are present. In order to infect other humans they encyst and exit the body. The cyst corresponds to the infective and resistant stage, mature cyst is spherical and tetra-nucleated, its diameter is about 12-15 µm and the cell membrane is covered with a thick cell wall [2] formed by chitin (N-acetyl-glucosamine polymer), that confers its resistance to adverse conditions. Life cycle of E. histolytica does not require any intermediate host. Mature cysts are passed in the feces of an infected human. Another human can get infected by ingesting them in fecal contaminated water, food or hands. If the cysts survive the acidic stomach, they turn into trophozoites in the small intestine. Trophozoites migrate to the large intestine where they live and multiply by binary fission. Both cysts and trophozoites are sometimes present in the feces. Cysts are usually found in firm stool, whereas trophozoites are found in loose stool. Only cysts can survive longer periods (up to many weeks outside the host) and infect other humans. If trophozoites are ingested, they are killed by the gastric acid of the stomach. The E. histolytica cell differentiation takes place inside the colon, during this process the trophozoite stops its pseudopods formation, the nuclei starts division, the characteristically irregular shape is lost and the cell takes a
Microscopy: advances in scientific research and education (A. Méndez-Vilas, Ed.)
spherical form at the time that a thick cystic wall appears. The trophozoite differentiation concludes with the tetranucleated cyst formation [3]. Most studies are focused mainly on aspects about the trophozoite, but recently the cyst has taken importance, for the last 10 years, these structures have been observed and studied using transmission electron microscopy (TEM) and scanning electron microscopy (SEM), these studies have provided information about some morphological characteristics of both the trophozoite and cyst stages of E. histolytica. The fine structure of parasitic protozoa has been the subject of intense investigation using TEM and SEM. The recent development of atomic force microscopy (AFM) and all of the techniques associated with AFM has created new ways to further analyze the structure of cells [4]. Atomic force microscopy (AFM) has emerged in recent years as a powerful tool for the study of cellular structure at resolutions of a few nanometers. Morphological studies revealing details of living cells, which were previously impossible due to the resolution limits of the light microscope, are now possible using AFM. The AFM´s major contribution to biology has been its ability to study the dynamics of live cells in physiological medium at ultrahigh resolution and in real time [5]. In contact mode AFM, the probe tip is mounted on a cantilever and scans over the surface of a sample while maintaining a contact with the surface. However, for soft samples, the tip-force may induce an irreversible damage of the surface [6]. To overcome this problem tapping mode AFM is used. In this method the cantilever oscillates vertically near its resonance frequency, so that the tip makes contact with the sample surface only briefly in each cycle of oscillation [7]. As the tip is brought close to the sample surface, the characteristics of the cantilever vibration (e.g., the amplitude, resonance frequency, and phase angle of vibration) change due to the tip-sample interaction. The detection of phase angle shifts provides enhanced image contrasts, especially for heterogeneous surfaces [8]. During this study we use AFM to describe the changes the cell goes throughout its encystment. The results here obtained show a clear difference between topology, elasticity, structure, morphology, roughness and size of E. histolytica stages during cell differentiation.
2. Materials and methods
2.1 E. histolytica
Culture: E. histolytica HM1-IMSS strain was axenically cultivated in PEHPS medium [9], distributed in 5 mL aliquots in 13x100 mm borosilicate screw-capped tubes. Cells were harvested by chilling of the tubes on ice water for 10 min and then inoculated with 5x103 trophozoites/mL and incubated for 72 h at 37°C. Trophozoites samples: E. histolytica trophozoites were grown in 16x125 mm borosilicate screw-capped Leighton tubes with a flat thin glass cover slip inside, and inoculated with 5x103 trofozoites/mL. After 72 hours of incubation, the cover slips, where the trophozoites grew adhered to the surface, from each tube were retired and immediately fixed with 2.5% glutaraldehyde for its observation at the AFM. Encystation process: To induce the encysting process, the high CO2 tension method was used [10], where tubes containing the trophozoites were gassed with CO2 (99.9% purity) with a flux of 0.4 L/min for 3 minutes. The tubes were incubated at 37°C, for 5 days. Every 24 hours cells samples were taken by chilling of the tubes on ice water for 10 min followed by centrifugation at 1,500 rpm/10 min, the cell pellet were collected and immediately fixed with glutaraldehyde 2.5% for its observation at the AFM. On day 4 (96 h) cells were tested for resistance to SDS 1% for 10 min, the resistant cells were fixed with 2.5% glutaraldehyde. Cysts isolated from patients: Pasty stool sample with positive diagnosis for amoebiasis was obtained from a clinical laboratory, samples were taken from the periphery of tarry stools and cysts were separated using the Faust technique [11], then the material was collected and separated using the Percoll gradient [12], finally the cysts were fixed with glutaraldehyde 2.5% and analyzed by AFM.
2.2 AFM and cantilevers
All samples were observed on a Multimode NanoScope IIIa from Digital Instruments Veeco Metrology Group attached to an optical video system XC-555 (Sony) equipped with 10× and 40× lenses (Nikon) (used to locate the biological samples). The system was sitting on an active damping table (Nano-K) to suppress mechanical noise. Images were presented using SPM software package WSxM [13]. All force values were calculated using non-conductive silicon nitride (Si3N4) triangular cantilevers (Veeco NP-20, tip radius ∼20 nm) with a length of 120/205 μm and nominal spring constants varying from 0.06 to 0.32 N/m which specific values were calculated with the thermal tune method[14]. The roughness analysis was carried out by comparing the images obtained in TappingModeTM using non-conductive silicon nitride rectangular cantilevers (Bruker MPP-11120, tip radius ∼8 nm) with a length of 125 μm and nominal frequency of 300 KHz. For the in situ measurements on water an electrochemistry TappingModeTM fluid cell (Veeco MMTMEC) was used together with the cantilevers mentioned above.
Microscopy: advances in scientific research and education (A. Méndez-Vilas, Ed.)
AFM images were obtained under ambient conditions or liquid environment while operating the instrument in tapping mode and contact mode. Height, amplitude, and phase images were recorded simultaneously using tapping mode AFM. Images were taken at the fundamental resonance frequency of the Si cantilevers. Images were recorded with typical scan speeds of ½ - 1 line/s using a scan head with a maximum range of 148 ˟ 148 µm2. Roughness and size: The root-mean-square average of the surface roughness (Rm) was measured with the following expression: = ∑( )
(1)
where Zi is the current Z value, Zavg is the average of the Z values within the given area, and Np is the number of point within the given area. Elasticity: Measurements were obtained from force plots acquired (120 per zone with an average count of three) at different points of the samples (at least 3). The force plots are a series of graphs that display the cantilever-deflection signal against the Z-axis piezo voltage (sample height). As the tip is pressed deeper and deeper into the material, the probe's cantilever flexes. The amount of cantilever flexion for a given amount of downward tip movement gives information about the material's elasticity. Then, relating the experimental graphs obtained with the standard deflection of the cantilever on a rigid substrate (calibration curve) the tensile modulus in each plot was obtained using a customized Igor Pro v6 program with a modified Hertz model [15] that correlate the data of the applied force ‘F’ and the indentation depth ‘δ’ of the tip into the membrane:
= tan (2)
where α is the half-opening angle of the tip (35° for the tip used) and ν is Poisson’s ratio of a sample (assumed to be 0.5 because the cell was considered incompressible [16]).
3. Results
3.1 Cell morphology
Trophozoite observation and measurement: E. histolytica trophozoites were observed in situ using tapping and contact mode. Groups of cells, cell morphology as well as characteristics of the cell membrane were observed (Figure 1A, Figure 1B). Trophozoites were observed as pleomorphic cells, measuring about 40 μm, as reported on literature [1,6,7]. Pre-cyst observation and measurement: Pre-cyst stages samples correspond to 24 h (CS1), 48 h (CS2), and 72 h (CS3) of incubation in encystment medium. Samples were observed using tapping mode. As the days passed, cell morphology was changing from trophozoite to cyst; the cell becomes smaller and takes round shape, in the final stages groups of cells begin to clump together (Figure 1B, Figure 2B). Cyst observation and measurement: The cyst stage (CS4) correspond to samples with 96 h of incubation in encystment medium and resistant to SDS 1% for 10 min. Cyst obtained had rounded morphology, with a rough outer surface (cyst wall), measured between 12-15 μm (Figure 1C, Figure 2C), and cyst isolated from patients (Figure 1D, Figure 2D).
Microscopy: advances in scientific research and education (A. Méndez-Vilas, Ed.)
Fig. 2 (A) Zoom of E. histolytica trophozoite membrane observed in situ in tapping mode. Scanned area: 4x4μm2. Zscale: H= 400nm, A=1V, P=120°. (B) Zoom of the pre-cyst (CS2) surface observed in tapping mode. The difference in the composition and topography on the same cell surface of pre-cyst is evident. Scanned area: 4x4μm2. Zscale: H= 300nm, A= 600mV, P=70°. (C) Cyst in vitro membrane observed in tapping mode. Scanned area: 4x4μm2. Zscale: H= 500nm, A= 800mV, P=120°. (D) Cyst in-vivo membrane observed in tapping mode. Scanned area: 4x4μm2. Zscale: H=600nm, A=0.7V, P=75°.
Cell roughness: The images obtained in the AFM were analyzed using the MultiMode SPM 4.31ce software for cell roughness. Trophozoites were analyzed in tapping and contact mode using both liquid and dry samples. Only tapping mode was used for pre-cyst and cyst in vitro and in vivo samples. The results presented in Table 1 and Figure 4 derived from statistical analysis of the several images acquired. Cell elasticity: Quantitative measures of the elasticity of the cellular external surface come from the adjustment of the force-distance curves to the Hertz model (Figure 3). Table 1 shows the values obtained from the elasticity curves for the trophozoite cell membrane as well as the cyst cell wall, both in vitro and in vivo samples.
Microscopy: advances in scientific research and education (A. Méndez-Vilas, Ed.)
Table 1 Topology and morphology of different E. histolytica life stages. Elastic modulus of the cells were obtained after the adjustement for 120 experimental curves (with an average count of 3)
ENVIRONMENT
PROPERTY
TROPHOZOITES PRE-CYST CYST (IN VITRO ) CYST (IN VIVO)
Fig. 4 Nanomecanic analyses of E. histolytica stages.
4. Discussion
In situ characterization of the E. histolytica trophozoite, pre-cyst and cyst stages was performed. This is the first report of the pre-cyst stage observed on AFM, also the first one that compares nanomechanical properties such as: elasticity and roughness of E. histolytica trophozoite and cyst stages (Table 1 and Figure 4). Morphological differences between the different stages were also observed, both the morphology and measurements of all cell stages observed in this study are consistent with those reported in the literature [9, 17]. Variation in size occurs because, once the conditions are unfavorable for trophozoite cell division, a process of cell outgrow begins causing loss of cell material through excretory vacuoles and a decrease in cell size [14]. We performed a quantitative comparison of the elasticity of the outer surface of the cell, which shows that trophozoite cell membrane is less rigid than the cell wall of the cyst (in vivo and in vitro samples). With the values obtained, we can see that in vitro cyst cell wall is about twice stiffer than trophozoite cell membrane. The difference in elasticity is bigger when comparing trophozoite and in vitro cyst, this may be due to the methodology used to produce the cystic structures (CS). These results could be explained due to the presence of N-acetyl-glucosamine (chitin) and probably other minor components as happens in Entamoeba invadens [18], which confers its resistance to adverse external agents that would easily kill the cell on trophozoite stage. When comparing E. histolytica elasticity to other biological samples, we have more flexible structures like the cartilage with 0.6 MPa [19] and clathrine with 10 MPa [20], and stiffer cell walls like the indiangrass (Sorghastrum mutans) with 60.1 MPa [13] and the one from bacteria Methanospirillum hungatei with 33 GPa [14]. Based on these results we can infer that the chitin present on the E. histolytica cell wall is softer than the components of some of the vegetal and bacterial wall, this could be as a result of the layer of β-(1,3)-N-acetylglucosamine and N-acetyltalosaminuronic acid of the pseudopeptidoglycan and the nonglycosylated polypeptides present on the cell wall of Archea M. hungatei [21]; and the presence of cellulose, hemicelluloses and lignin [22] of the indiangrass cell wall, which provide them a greater hardness. Regarding to the cell roughness, there is a notorious difference between the trophozoite and the cyst, the cyst cell wall is more than 4 times rougher than the trophozoite cell membrane (Fig. 4). This difference may be due to a great diversity of proteins, particularly cell surface glycoproteins such as lectins Jacob and Jessie and chitinase, which are present on the cyst stage [23]. The trophozoite topology certainly experience changes when encysting, due to the cell wall’s development which consist mainly in N-acetyl-glucosamine polymers, in addition to the ultrastructural changes the cell goes when encysting (Figure 1). Figure 2B shows an image of a portion of the outer surface of pre-cyst where, in the same image, different cellular composition and topography can be appreciated. The difference in color is given by the different hardness and chemical nature of the sample. This seems to be due to the formation of the cyst wall in the outer cell surface is not performed uniformly; as was reported previously on other parasitic protozoa, such as the “wattle and daub” model for Entamoeba cyst wall, where the chitin fibrils are cross-linked by Jacob lectins prior to the addition of the Jessie3 lectins [24], in addition, a similar process has been described on E. invadens [10], this report brings some evidences, based on SEM and TEM, of the differentiation process carried out on E. invadens. Based on these results we
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Microscopy: advances in scientific research and education (A. Méndez-Vilas, Ed.)
hypothesize that E. histolytica encysting shares certain features with the same process carried out in biological model E. invadens. The results here presented aid to the efforts for understanding the morphologic aspects that prevail in the E. histolytica cellular differentiation process. Taking into account that the mature cyst is the responsible for this disease dissemination, in the future, we consider necessary to continue the research about the E. histolytica cellular differentiation process, since this would bring important data that could be used to fight amoebiasis.
Acknowledgements The authors thank Consejo Nacional de Ciencia y Tecnologia (CONACYT) for the repatriation project #120431 and the grant # 44144. Also we thank the CAT-120 Research Program (Synthesis of nanostructured materials) by the Tecnologico de Monterrey for the support at this research.
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Microscopy: advances in scientific research and education (A. Méndez-Vilas, Ed.)
