J. EUKARYOT. MICROBIOL., 2003 Purification of Entamoeba histolytica DNA Containing Organelles (EkhOs): A Further Characterization JUAN PEDRO LUNA-ARIAS, " TOMAS SANCHEZ,~ MARIA ESTHER HERRERA-AGUIRRE,' PEDRO CHAVEZ,~ EFRAIN GARRIUO" and ESTHER OROZCO~ De~.~'~rfm~wt's of 'Molecular Biomedicine, h~.~prrime~~tal Pathology & "Genetic.r arld Molecular Biologv, CINVESTAV-IPN, Av. IPN 2508. Col. Sat1 Pedro Zucutmco, CP 07.760 Mexico. D.E & 'Progrurrr of' Molacrdar Biomedicine, ENMYH-IPN, G~~illermo Mu.s.sirw Holguern 239 Fracc. La Escaleru, CP 07.720 Mexico, D.E Entamoebu histolytic~cr is the protozoan parasite responbibk for human amoebiasis [lo]. Its life cycle is comprised of two dominant phases: the cysts that are the infective forms, and the trophozoites, the invasive forms. After ingestion, the cysts in the intestine can differentiate into trophozoites. Trophozoites can invade the epithelium and go through the portal vein to the liver, where they produce abscesses. Thus, the trophozoites are able to live in poor and rich oxygen milieu, and therefore, E. histolyticu is considered as a facultative microaerophilic protozoan. Genes and proteins partici- pating in these changes are unknown. E. histolyticu trophozoites posses cytoplasmic DNA-containing organelles, named EhkOs [ 1,151. Crypton structures also contain DNA [4]. The pyruvate:ferredoxin oxidoreductase enzyme (EhPFO) is found in the EhkOs 1171. PFO is an enzyme in anaerobic metabolism that in Trichomo~zas vagirzulis is located in hydrogenosomes [19]. Additionally, ribosomal gene probes hybridize with EhkO's DNA [IS]. In this paper we report a method to purify EhkOs. Purified EhkOs appeared composed of small structures that associate them- selves together to form EhkOs of different sizes. The presence of DNA in these organelles is demonstrated here by different techniques including propidium iodide stain, DNase-sensitivity, ['H]thymidine labeling and autoradiography, and carbon-platinum shadowed pure EhkOs. MATERIALS AND METHODS Trophozoites of E. histolytica clone A (strain HM I :IMSS) [13] were cultured in TYI - S- 33 medium at 37°C and harvested during exponential growth phase [3]. For some experiments, the medium was supplemented with 2 pCi/ml [' HI-thymidine (Amersham). ['H]thymidine-labeled trophozoites (2 X 10" were washed with PBS and resuspended in eight vol of a solution containing 10 mM Hepes pH 7.9, 10 mM EDTA, 10 mM DTT, 1 mM each of aprotinin, E-64. leupeptin, pepstatin A, and phenyl-methyl-sulfonyl fluoride, and also included 0.1 mM each of benzamidine and N-ethylmaleimide (buffer A), supplemented with 250 mM sucrose. Trophozoites were disrupted in a Potter homogenizer at 4°C and centrifuged at 160X g for 10 rnin. The pellet (nuclei) was resuspended in 4 ml of buffer A containing 250 mM sucrose and then centrifuged through 2 ml of 30% Ficoll 400 in buffer A at 17,000X g for 45 min at 4OC in a SW40Ti rotor (Beckman). The pellet (nuclei) was resuspended in 2 ml of 250 mM sucrose and 15% Nycodenz (Nycomed) in buffer A and loaded on a Nycodenz discontinuous gradient (30, 40 and 50% in buffer A) and centrifuged at 13,000X g for I, 24 or 48 h at 4°C in a SW40Ti rotor to measure its buoyant density. An aliquot of the 160X g nuclei-depleted supernatant was fixed with 4% paraformaldehyde for 15 min at 37T. Unfixed aliquots were centrifuged at 12,000X g for 10 min at 4°C in a JA- 20 rotor (Beckman) and the pellets (containing the EhkOs) were resuspended in 4 ml of 158 Nycodenz in buffer A and ultracentrifuged as described above. After ultracentrifugation, the gradients were fractionated (DensiFlow I1 C, Buchler Instruments), fractions were TCA precipitated and radioactivity was determined in a scintillation counter (Beckman LS6500). For transmission electron microscopy (TEM) assays, the 1~~1th~- midine-labeled paraformaldehyde-treated or non-treated EhkOs were Corresponding author: E. Orozco. Telephone: (52)-(5)-50-61-38-00 ext. 5642; Fax: (52)-(5)-50-61-71-08; Email: esthe@n~ail.cinvestav.mx fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, and stained with 0.2% uranyl acetate (negative stain) [2] or carbon- platinum shadowed [2]. Samples were analyzed in a JEOL-100-SX microscope. Other aliquots were placed on slides, covered with Ilford emulsion in dark for five days and after developing, autoradiographies of the thymidine dine-labeled EhkO preparations were observed and photographed by light microscopy [14]. RESULTS AND DISCUSSION The trophozoites were stained by propidium iodide and examined by confocal microscopy. They exhibited red fluorescence in the nuclei and in EhkOs, which varied in number and size in each trophozoite (Fig. 1A). When the cells were DNase-treated before being stained with propidium iodide. fluorescence was not detected in nuclei and EhkOs (Fig. 1B). indicating that DNA is present in both organelles. The buoyant density of the (H]thymidine-labeled nuclei and EhkOs was determined after centrifugation at 1, 24 and 38 h through Nycodenz gradients. Both organelles showed 1.140 g/ml of density after 1 h centrifugation (Fig. 2A, 2B), which was the same after 24 and 48 h centrifugation, indicating that buoyant density equilibrium was reached at 1 h. The purified organelles were stained by propidium iodide. Stained nuclei appeared as 4- 7 pm diameter organelles (Fig. lC), whereas the purified EhkOs fraction presented 1 - 5 pm structures (Fig. ID). By transmission electron microscopy (TEM), the negatively stained radioactive EhkOs obtained from the Nycodenz gradient appeared composed of structures of approximately 0.1-1 pm in diameter (Fig. 3A, 3B, 3D, 3E). They had an electron-dense envelope with discontinuities (Fig. 3D, arrowhead) and strands in the exterior and inside the structures, which could be DNA (Fig. 3D, 3E). In DNase- treated preparations, before the negative staining, the structures became white and the strands disappeared (Fig. 3C). These small structures could associate with each other and with cytoplasmic Fig. 1. E. histol.~tictr trophozoite DNA is in the nucleus and EhkO indi- cated by propidium iodide staining. A. Confocal microscopy of trophozoites. Arrows indicate nuclei; arrowheads indicate the smaller EhkOs. B. Control. Trophozoites pre-treated with DNAse indicate the absence of stained structures. C. Nycodenz gradient-puritied nuclear fraction. D. Nycodenz gradient-purified EhkO fraction.