Expression of EhRAD54, EhRAD51, and EhBLM proteins during DNA repair by homologous recombination in Entamoeba histolytica.

Entamoeba histolytica, the protozoan responsible for human amoebiasis, exhibits a great genome plasticity that is probably related to homologous recombination events. It contains the RAD52 epistasis group genes, including Ehrad51 and Ehrad54, and the Ehblm gene, which are key homologous recombination factors in other organisms. Ehrad51 and Ehrad54 genes are differentially transcribed in trophozoites when DNA double-strand breaks are induced by ultraviolet-C irradiation. Moreover, the EhRAD51 recombinase is overexpressed at 30 min in the nucleus. Here, we extend our analysis of the homologous recombination mechanism in E. histolytica by studying EhRAD51, EhRAD54, and EhBLM expression in response to DNA damage. Bioinformatic analyses show that EhRAD54 has the molecular features of homologous proteins, indicating that it may have similar functions. Western blot assays evidence the differential expression of EhRAD51, EhRAD54, and EhBLM at different times after DNA damage, suggesting their potential roles in the different steps of homologous recombination in this protozoan.

Entamoeba histolytica and Trichomonas vaginalis: trophozoite growth inhibition by metronidazole electro-transferred water.

The influence of low-frequency electromagnetic (LF-EM) waves on microorganisms has been a subject of experimental investigations for more than two decades and the results are promising. In parallel, an interesting procedure known as biophysical-information-therapy or bioresonance therapy (BRT) which in principle is based on LF-EM stimulation, has emerged. BRT was discovered in the late 1980's but it is still poorly studied. This paper demonstrates that by transferring metronidazole information to water samples by an electronic amplifier (BRT device), the growth of axenically cultured trophozoites of Entamoeba histolytica and Trichomonasvaginalis is significantly inhibited, compared with those cultures treated with non and sham electro-transferred water samples. A positive control of metronidazole, a well-known cytotoxic drug against parasites, was used as a reference.

Impact of solar radiation in disinfecting drinking water contaminated with Giardia duodenalis and Entamoeba histolytica/dispar at a point-of-use water treatment.

AIMS: To determine the impact of natural sunlight in disinfecting water contaminated with cysts of Giardia duodenalis and Entamoeba histolytica/dispar using plastic containers. METHODS AND RESULTS: Known quantities of Giardia duodenalis and Entamoeba histolytica/dispar cysts in sterile water were exposed to the sun. Containers were made of polyethylene terephthalate, eight painted black on one side, one not painted and another cut open at the top and the last was a high density polypropylene container. Viability testing was performed using vital and fluorescent dyes. The same assays were conducted under cloudy conditions. Thermal control tests were also performed using heat without ultra violet light from the sun. Results show that 99.9% of parasites was inactivated when water temperatures reached 56 degrees C after sunlight exposure. CONCLUSION: Both solar radiation and heat produced by the sun have a synergistic effect in killing cysts of Giardia duodenalis and Entamoeba histolytica/dispar when temperatures rise above 50 degrees C, with complete death at 56 degrees C, using painted 2-l PET containers. SIGNIFICANCE AND IMPACT OF THE STUDY: Solar disinfection system using PET containers painted black on one side can be used to disinfect water against Giardia duodenalis and Entamoeba histolytica/dispar using natural sunlight.

Effects of DNA damage induced by UV irradiation on gene expression in the protozoan parasite Entamoeba histolytica.

Previously, we provided evidence for the role of E. histolytica RAD52 epistasis group genes and the EhRAD51 recombinase in DNA damage response. To identify other genes participating in DNA repair in this protozoan parasite, here we analyzed the transcriptional response to genetic damage induced by ultraviolet light (UV) using cDNA microarrays. We found that 11.6% (350 ORFs) and 17.2% (522 ORFs) of genes were modulated at 5 min and 3h after UV irradiation, respectively. Most genes were less than 2-fold changed evidencing a weak transcriptional activation. The genes encoding so-called "classical" DNA repair proteins were slightly regulated in trophozoites submitted to UV irradiation. We also observed the over-expression of genes encoding for Fe-S clusters-containing proteins, potentially involved in the stress adaptation in response to DNA damage. Several genes encoding cytoskeleton proteins were repressed suggesting that actin dynamics was impaired after UV irradiation. Our analysis highlights novel genes potentially involved in DNA damage response, and these data will contribute to further elucidation of mechanisms regulating genome integrity in this early branch protozoan.

The effect of electromagnetic waves on the growth of Entamoeba histolytica and Entamoeba dispar.

OBJECTIVE: The aim of this study was to investigate the influence of electromagnetic radiation of a digital Global System for Mobile Communication mobile telephone (900 MHz) on Entamoeba histolytica (E. histolytica) and Entamoeba dispar (E. dispar) (cysts or trophozoites, or both) in a 24-hour period. METHODS: This study was carried out from April 2004 to May 2004 at the Department of Parasitology, Medical Faculty of Dokuz Eylul University in Izmir, Turkey. The cultivated isolate tubes, which were exposed to electromagnetic field at 37 degrees C, were evaluated as study group, whereas the tubes without exposure were assessed as control group. Finally, only living parasites in all tubes were counted using a hemacytometer. The effect of the temperature was evaluated for both control and study groups. RESULTS: The influence of electromagnetic field and temperature was assessed separately for the study group. The parasite number of E. histolytica decreased after exposure at 37 degrees C and room temperature (p=0.009) compared to the decrease in the control group (p=0.009). The parasite number of E. dispar also decreased after exposure at 37 degrees C and room temperature (p=0.009). In comparison to control tubes, this was a significant decrease (p=0.008). In the case of exposure of E. histolytica the results did not reveal any significant difference between temperature degrees to magnetic field (p=0.459) and E. dispar (p=0.172). CONCLUSION: Our findings show that exposure to electromagnetic field for a certain period of time may cause damage that can lead to death in single-cell organisms.

Inflammation, complement, ischemia and amoebic survival in acute experimental amoebic liver abscesses in hamsters.

We have examined the role of inflammatory cells, ischemia and serum complement on the development of acute experimental amoebic liver abscess in hamsters (AEALAH). In hamsters made leukopenic by whole body radiation (800 rad) and daily intraperitoneal glycogen injections, the absence of inflammatory cells and liver tissue damage surrounding the parasites resulted in their rapid (24 h) disappearance from the liver, which showed no lesions. Focal liver ischemia, always present in control AEALAH with inflammation and tissue destruction, was reproduced in radiated hamsters by injection of amoebae mixed with Superdex microspheres, but again in the absence of inflammation, amoebae caused no liver damage and disappeared in 24 h. In hamsters made hypocomplementemic by injection of purified cobra venom factor (CVF), amoebae caused AEALA indistinguishable from controls, but in leukopenic + hypocomplementemic hamsters, amoebae were unable to produce lesions and disappeared from the liver in 48 h. We conclude that inflammation and tissue damage are required for the survival of amoebae in AEALAH and for the progression of the experimental disease.

Ehp53, an Entamoeba histolytica protein, ancestor of the mammalian tumour suppressor p53.

This paper reports the identification of Ehp53, a p53-like Entamoeba histolytica protein, which binds to the human p53 DNA consensus sequence (oli-p53). Monoclonal antibodies against p53 (Ab-1 and Ab-2) recognized a single 53 kDa spot in two-dimensional gels and inhibited the formation of complexes produced by E. histolytica nuclear extracts and oli-p53. Additionally, E. histolytica gene promoter sequences with high homology to oli-p53 formed complexes with nuclear proteins that were abolished by oli-p53. Ehp53 protein levels increased in UV-irradiated trophozoites. This protein was also detected in Entamoeba moshkovskii and Entamoeba invadens. By confocal microscopy, Ehp53 was located in the nuclei, EhkO organelles and cytoplasm. The Ehp53-encoding gene was cloned and its predicted amino acid sequence showed 30-54 % and 50-57 % homology with important domains of the human and the Drosophila melanogaster p53 proteins, respectively. This homology included the tetramerization domain, the nuclear export signal and a nuclear localization signal. Ehp53 also contains seven of the eight DNA-binding residues and two of the four Zn(2+)-binding sites described for p53. A recombinant Ehp53 was recognized by Ab-2. Ehp53 is believed to be the first p53-like protein found in protozoa and may be the evolutionary ancestor of the mammalian p53.

Entamoeba histolytica. Phagocytosis as a virulence factor.

In this paper, we attempted to define the role of phagocytosis in the virulence of Entamoeba histolytica. We have isolated, from a highly phagocytic and virulent strain, a clone deficient in phagocytosis. Trophozoites of wild-type strain HM1:IMSS were fed with Escherichia coli strain CR34-Thy- grown on 5-bromo,2'-deoxyuridine. The trophozoites that had incorporated the base analog through phagocytosis of the bacteria were killed by irradiation with 310 nm light. The survivors, presumably trophozoites defective in phagocytosis, were grown until log phase and submitted two more times to the selection procedure. Clone L-6, isolated from a subpopulation resulting from this selection procedure, showed 75-85% less erythrophagocytic activity than the wild-type strain. The virulence of clone L-6 and strain HM1:IMSS was measured. The inoculum required to induce liver abscesses in 50% of the newborn hamsters inoculated (AD50) of HM1:IMSS was 1.5 X 10(4) trophozoites. Clone L-6 trophozoites failed to induce liver abscesses in newborn hamsters even with inocula of 5 X 10(5) trophozoites. Virulence revertants were obtained by successive passage of L-6 trophozoites through the liver of young hamsters. The trophozoites that recovered the ability to produce liver abscesses simultaneously recuperate high erythrophagocytic rates. These results show that phagocytosis is involved in the aggressive mechanisms of E. histolytica.