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It has been described that the pathogenicity of Entamoeba histolytica is influenced by environmental conditions and that transcription profile changes occur during invasion, suggesting that gene expression may be involved in the virulence of this parasite. However, the molecular mechanisms that are implicated in the control of gene expression in this microorganism are poorly understood. Here, we showed that the expression of the EhRabB protein, a small GTPase involved in phagocytosis, is modified through the interaction with red blood cells. By ELISA, Western blot, and immunofluorescence assays, we observed that the expression of EhRabB diminished after 5 min of the interaction of trophozoites with red blood cells, but protein level was recovered at subsequent times. In the EhRabB amino acid sequence, we found two lysine residues that could be target for ubiquitin modification and trigger the degradation of this GTPase at early times of phagocytosis. The analysis of the expression of the EhrabB mRNA showed that the interaction of trophozoites with red blood cells produces a drastic diminishing in its half-life. In addition, promoter assays using the chloramphenicol acetyltransferase reporter gene and electrophoretic mobility shift assays experiments showed that the URE1 motif located in the promoter region of EhrabB is involved in the expression regulation of this gene during phagocytosis. Moreover, the immunolocalization of the URE1-binding protein during phagocytosis indicated that the transcription of the EhrabB gene is determined, at least in part, by the translocation of this transcription factor to nuclei. These results suggested that the expression of particular genes of this parasite is controlled at several stages.

Calcium regulates many cellular processes in protozoa, including growth, differentiation, programmed cell death, exocytosis, endocytosis, phagocytosis, fusion of the endosomes of distinct stages with phagosomes, fusion of phagosomes with lysosomes, and recycling the membrane. In Entamoeba histolytica, the protozoa responsible for human amoebiasis, calcium ions are essential for signaling pathways that lead to growth and development. In addition, calcium is crucial in the modulation of gene expression in this microorganism. However, there is scant information about the proteins responsible for regulating calcium levels in this parasite. In this work, we characterized a protein of E. histolytica that shows a close phylogenetic relationship with Ca pumps that belong to the family of secretory pathway calcium ATPases (SPCA), which for several organisms are located in the Golgi apparatus. The amoeba protein analyzed herein has several amino acid residues that are characteristic of SPCA members. By an immunofluorescent technique using specific antibodies and immunoelectron microscopy, the protein was detected on the membrane of some cytoplasmic vacuoles. Moreover, this putative calcium-ATPase was located in vacuoles stained with NBD C6-ceramide, a Golgi marker. Overall, the current findings support the hypothesis that the presently analyzed protein corresponds to the SPCA of E. histolytica.

SummaryClC chloride channels perform a wide variety of physiological functions and they had been characterized in animals, yeast, plants and bacteria but not in protozoa. By BLAST search we found in Entamoeba histolytica , the protozoan responsible for human amoebiasis, two genes ( Ehclc-A and Ehclc-B ) encoding for putative polypeptides with 25-30% identity to ClC chloride channels of several organisms. Reverse transcription polymerase chain reaction (RT-PCR) experiments showed that both genes are transcribed in trophozoites. Phylogenetic analysis revealed that EhClC-A and EhClC-B polypeptides belong to the eukaryotic branch of plasma membrane ClCs. Specific antibodies against EhClC-A confirmed that it is located at the trophozoite plasma membrane. Xenopus laevis oocytes microinjected with Ehclc-A cRNA elicited anion currents not detected in oocytes microinjected with water. Induced currents were inwardly rectifying and had a permeability sequence of Cl -> Br -> I -> F ->> NO 3 -. The chloride channel blocker 4-acetamido-4 ¢ isothiocyanostilbene-2, 2 ¢ -disulphonic acid (SITS) strongly inhibited the oocytes anion currents and trophozoites growth. Experiments at diverse pHs suggested that EhClC-A is not a Cl -/H + exchanger, but it is an ion channel that could be involved in pH regulation. EhClC-A may also participate in cell volume regulation. As far as we know, EhClC-A is the first chloride channel characterized in protozoa.