ABSTRACT
Oxygen or nitrogen oxidative species and chemical stress induce the programmed cell death (PCD) of Entamoeba histolytica trophozoites. PCD caused by the aminoglycoside G418 is reduced by incubation with the cysteine protease inhibitor E-64; however, no typical caspases or metacaspases have been detected in this parasite. Calpain, a cysteine protease activated by calcium, has been suggested to be part of a specific PCD pathway in this parasite because the specific calpain inhibitor Z-Leu-Leu-Leu-al diminishes the PCD of trophozoites. Here, we predicted the hypothetical 3D structure of a calpain-like protein of E. histolytica and produced specific antibodies against it. We detected the protein in the cytoplasm and near the nucleus. Its expression gradually increased during incubation with G418, with the highest level after 9 h of treatment. In addition, a specific calpain-like siRNA sequence reduced the cell death rate by 65%. All these results support the hypothesis that the calpain-like protein is one of the proteases involved in the execution phase of PCD in E. histolytica. The hypothetical interactome of the calpain-like protein suggests that it may activate or regulate other proteins that probably participate in PCD, including those with EF-hand domains or other calcium-binding sites.
Subject(s)
Apoptosis , Calpain/metabolism , Entamoeba histolytica/physiology , Calpain/antagonists & inhibitors , Computational Biology , Entamoeba histolytica/drug effects , Enzyme Activators/metabolism , Gentamicins/metabolism , Protein Interaction MapsABSTRACT
Infections with protozoa parasites are associated with high burdens of morbidity and mortality across the developing world. Despite extensive efforts to control the transmission of these parasites, the spread of populations resistant to drugs and the lack of effective vaccines against them contribute to their persistence as major public health problems. Parasites should perform a strict control on the expression of genes involved in their pathogenicity, differentiation, immune evasion, or drug resistance, and the comprehension of the mechanisms implicated in that control could help to develop novel therapeutic strategies. However, until now these mechanisms are poorly understood in protozoa. Recent investigations into gene expression in protozoa parasites suggest that they possess many of the canonical machineries employed by higher eukaryotes for the control of gene expression at transcriptional, posttranscriptional, and epigenetic levels, but they also contain exclusive mechanisms. Here, we review the current understanding about the regulation of gene expression in Plasmodium sp., Trypanosomatids, Entamoeba histolytica and Trichomonas vaginalis.
Subject(s)
Eukaryota/genetics , Gene Expression Regulation , Entamoeba histolytica/genetics , Entamoeba histolytica/metabolism , Gene Expression Profiling/methods , Genes, Protozoan , Genome, Protozoan , Plasmodium/genetics , Plasmodium/metabolism , Trichomonas vaginalis/genetics , Trichomonas vaginalis/metabolism , Trypanosoma/genetics , Trypanosoma/metabolismABSTRACT
This study presents morphological and biochemical evidence of programmed cell death (PCD) in Entamoeba histolytica induced by exposure of trophozoites to the aminoglycoside antibiotic G418. Morphological characteristics of PCD, including cell shrinkage, reduced cellular volume, nuclear condensation, DNA fragmentation and vacuolization were observed, with preservation of trophozoite membrane integrity. PCD is orchestrated biochemically by alterations in intracellular ion fluxes. In G418-treated trophozoites, overproduction of reactive oxygen species (ROS), decreased intracellular K+, increased cytosolic calcium, and decreased intracellular pH levels were observed. However, externalization of phosphatidylserine was not detected. These results suggest that amoebae can undergo PCD under stress conditions, and that this PCD shares several properties with PCD reported in mammals and in a variety of unicellular organisms.
Subject(s)
Amebicides/pharmacology , Apoptosis , Entamoeba histolytica/drug effects , Entamoeba histolytica/physiology , Gentamicins/pharmacology , Animals , Calcium/metabolism , DNA Fragmentation , Entamoeba histolytica/growth & development , Entamoeba histolytica/ultrastructure , Hydrogen-Ion Concentration , Oxidative Stress , Potassium/metabolism , Reactive Oxygen Species , Trophozoites/drug effects , Trophozoites/physiologyABSTRACT
We have studied the cellular location and the efflux pump function of the Entamoeba histolytica P-glycoproteins (EhPgps) in drug-sensitive and -resistant trophozoites. Polyclonal antibodies against the EhPgp384 polypeptide (375-759 amino acids) revealed a 147-kDa protein by Western blot. The band intensity correlated with the emetine-resistance of the trophozoites. Through the confocal microscope, using the anti-EhPgp384 and fluorescein secondary antibodies, the EhPgps were found in a complex vesicular network, in the plasma membrane and outside of the cells. Transmission electron microscopy assays confirmed that drug-resistant trophozoites presented four to five times more EhPgps than sensitive cells. Fluorescence co-localization experiments using rhodamine-123 (R123) and the anti-EhPgp384 antibodies suggested the interaction between EhPgps and the drug. R123 efflux kinetics evidenced that the emetine-resistant trophozoites displayed a drug efflux kinetic four times higher than the drug-sensitive trophozoites, which was reduced by verapamil in both cases. EhPgps may participate in avoiding drug accumulation in the trophozoites by two putative mechanisms: (1) the direct extrusion of the drug from the plasma membrane, and (2) an indirect transport mechanism in which the drug is trapped by EhPgps and concentrated within vesicles that drive the drug to the plasma membrane.
