Archives of Medical Research 31 (2000) S273–S274
Entamoeba histolytica Hybrids E. Orozco,*,** C. Gómez,** D.G. Pérez,** G. García-Rivera,*,** T. Sánchez,* C. Bañuelos,* L. Mendoza,*,** F. Solís,*** E. Gómez,**** R. Ramos* and E. Flores** *Departamento de Patología Experimental, Centro de Investigación y de Estudios Avanzados del I.P.N. (Cinvestav), Mexico City, Mexico **Programa de Biomedicina Molecular, Centro de Investigación en Ciencia Aplicada y Tecnología Avanzada del I.P.N. (CICATA-IPN), Mexico City, Mexico ***Facultad de Medicina, Universidad Autónoma de Chihuahua (UACH), Chihuahua, Mexico ****Departamento de Biomedicina Molecular, Cinvestav, Mexico City, Mexico
Key Words: Entamoeba histolytica hybrids, DNA horizontal transference, Ehpili, Variability, Drug resistance.
Introduction
Results and Discussion
Entamoeba histolytica is the protozoan responsible for human amebiasis. It provokes 100,000 deaths per annum around the world, placing it second only to malaria in mortality due to protozoan. No sexual stage has been described in this parasite. Trophozoites have a complex genome with at least eight lineal chromosomes of unknown ploidy (1,2), a huge amount of circular DNA (2,3), and extranuclear DNA in the EhkO organelles (4). Earlier experiments suggested the generation of E. histolytica hybrids. The appearance of trophozoites with double drug resistance or a new zymodeme after the coincubation of trophozoites from two different strains led to the conjecture that E. histolytica may be sexual, but no further data proved that the populations obtained were bona fide hybrids.
Taking advantage of the different phenotypes displayed by clones Aneo and C2, we first obtained E. histolytica hybrids using these clones. Trophozoites were mixed in 2 mL of medium at 37⬚C for 2 h. Putative hybrids were incubated for an additional 12 h, to allow them to divide and fix their double drug resistance. Subsequently, we added to the cultures 14 M neomycin and 40 M emetine, lethal doses for clones Aneo and C2, respectively. Hybrid frequency formation for the mentioned markers, was 0.3 ⫻ 10⫺6, calculated by the number of surviving trophozoites after 120 h of incubation in the presence of both antibiotics, divided by the initial number of trophozoites. This means that a single double-resistant trophozoite survived out of 0.3 ⫻ 105 trophozoites from parental clones. A double-resistant population (RR) was obtained and cloned by limited dilution. Another three hybrid clones (AneoC21, AneoC22, AneoC23) were isolated as colonies in semisolid agar. Hybrids grow well in 20 M neomycin and 50 M emetine, but died in 50 M neomycin and 80 M emetine. Their double drug resistance suggested that they carry and express the EhPgp5 and neo genes. As a part of the hybrids’ phenotypic characterization, we obtained their zymodemes. Patterns shown by hybrids and parental clones were identical for all enzymes, except glucophosphoisomerase (GPI). A single  band was detected in clones Aneo and hybrids RR, AneoC21, AneoC22, and AneoC23, whereas clone C2 displayed ␣ and  GPI bands. Clones A and C20, grown without drugs, presented the same GPI bands as clone Aneo and C2 grown in the presence of drugs. These results could suggest that hybrids were generated from trophozoites of clone Aneo, which received the EhPgp5 gene from clone C2. However, other markers, studied subsequently, demonstrated that it is necessary to perform a finer characterization of hybrids to define the direction of DNA transference. We detected both gene markers in all hybrid clones obtained.
Materials and Methods To obtain hybrid cells, we first used the clone C2, which is emetine resistant, and the clone Aneo, which is neomycin resistant. We also used the cloned strain MAVhyg, which is resistant to hygromycin and sensitive to emetine and neomycin, and the clones Aneo or C2. DNA exchange between trophozoites of a cloned culture was demonstrated by labeling the trophozoites with 5-bromo-2⬘ deoxyuridine (BdUr). Then, DNA was detected by immunofluorescence, using specific anti-BdUr antibodies.
Address reprint requests to: Esther Orozco, Departamento de Patología Experimental, Cinvestav, Apdo. Postal 14-740, 07000 México, D.F., México. Tel.: (⫹525) 747-3800, ext. 5650; FAX: (⫹525) 747-7108; E-mail:
[email protected] Presenting author: Esther Orozco.
0188-4409/00 $–see front matter. Copyright © 2000 IMSS. Published by Elsevier Science Inc. PII S0188-4409(00)00 1 8 9 - 2
S274
Orozco et al./ Archives of Medical Research 31 (2000) S273–S274
Horizontal DNA transference among the trophozoites of a given culture will generate variability in strains and clones. We investigated whether this event was occurring among trophozoites of the same culture. However, no selectable markers are available to separate trophozoites of the same clone without altering them. Thus, trophozoites of clone A were grown on coverslips in medium supplemented with BrdU for 24 h and harvested in their late logarithmic growth phase. By laser confocal microscopy and specific antibodies, the BrdU incorporated in DNA was detected in disks, in nuclei, and EhkO organelles that have been defined as any extranuclear DNA-containing structures in the trophozoites (4). In all cells, the red propidium iodide stain overlapped with the green fluorescent antibodies. About 14% of the trophozoites appeared to be joined by tube-like structures carrying DNA. Due to their physical and functional analogy with bacteria pili, we called them Ehpili. In 2-m optical sections we observe a couple of trophozoites connected by a ⵑ40 m Ehpili carrying DNA. In the Ehpili extremes, EhkOs appeared, formed by grouped disks of ⵑ0.5 m in diameter, heavily loaded with DNA. Controls using only secondary antibodies did not show green fluorescence. In conclusion, we present here experimental evidence that DNA transference occurs among E. histolytica trophozoites. The EhkO organelle and the pili-like structures that we call Ehpilli participate in this process. The biological relevance of this mechanism lies in the fact that it causes phenotypic and genotypic variability in ameba strains. The generation of bona fide E. histolytica hybrids—RR, AneoC21, AneoC22, AneoC23, MAVhygC2, and MAVhygAneo— was possible only after the existence of genetically labeled clones, such as clone C2 (emetine-resistant) and clones Aneo and MAVhyg transfected with plasmids, to make them resistant to different drugs. Neomycin- and emetine-resistant hybrids were generated with a 0.3 ⫻ 10⫺6 frequency. The short times used for these experiments (2 h contact and 120 h incubation with the drugs) discard the generation of spontaneous emetine-resistant mutants of clone Aneo. Our previous results indicated that the rate of spontaneous mutation for emetine-resistance is very low. Consequently, the control tubes used for these experiments with trophozoites of clone Aneo incubated with distinct emetine concentrations did not produce emetine-resistant Aneo trophozoites. Currently we are working on the characterization of the MAVhygAneo and MAVhygC2 hybrids.
The expression of a GPI isoenzyme of identical migration in clones Aneo and AneoC2 hybrids could suggest that the EhPgp5 gene was passed from clone C2 to clone Aneo. However, the molecular karyotypes, TAFE, and in situ hybridization experiments showed that DNA transference between the trophozoites occurs in ways not yet clearly defined. We used two markers carried in plasmids to generate hybrid MAVhygAneo. Thus, not only ameba genes are being transferred, but also exogenous genes transfected into the trophozoites are shared during horizontal DNA transference. Mitosis occurs in E. histolytica without nuclear membrane rupture. After replicated chromosomal DNA is separated in two nuclei, cytokinesis gives rise to two daughter cells. No DNA was observed between the two new trophozoites before they come apart. These data and studies throughout the cell cycle shown here support that horizontal transference of DNA occurs before mitosis begins. However, we do not know whether all trophozoites receive DNA from other trophozoites before they divide, or whether this phenomenon occurs only in a percentage of the population. Thus, division of the new population, enriched by DNA horizontal transference, would compete in the culture with other populations, explaining variability of the strains.
Acknowledgments We are grateful for the excellent technical assistance of A. PadillaBarberi. E. Orozco is an International Fellow of Howard Hughes Medical Institute (USA). Conacyt (Mexico) also supported this work.
References 1. Gómez E, Hernández P, Argüello C. A morphodynamic study of the structural organization of Entamoeba histolytica chromatin during nuclear division. Arch Med Res 1997;28:124. 2. Riverón AM, Baez-Camargo M, López-Canovas L, Flores E, PérezPérez G, Luna-Arias JP, Orozco E. Circular and linear DNA molecules in the Entamoeba histolytica complex molecular karyotype. Eur J Biophys 2000 (In press). 3. Bhattacharya S, Som I, Bhattacharya A. The ribosomal DNA plasmids of Entamoeba. Parasitol Today 1998;14:181. 4. Orozco E, Gharaibeh R, Riverón AM, Delgadillo DM, Mercado M, Sánchez T, Gómez Conde E. A novel cytoplasmic structure containing DNA networks in Entamoeba histolytica trophozoites. Mol Gen Genet 1997;254:250.
