D.Guillermo Pérez

Two CCAAT/enhancer binding protein sites are cis‐activator elements of the Entamoeba histolytica EhPgp1 (mdr‐like) gene expression

Here, we show the relevance of promoter regions (−74 to +24, −167 to −75 and −259 to −168 bp) in the transcriptional activation of the multidrug resistance gene EhPgp1 in Entamoeba histolytica, using mutated plasmids and transfection assays. We also demonstrate that both CCAAT/enhancer binding protein sites (−54 to −43 bp and −198 to −186 bp) are cis‐activating elements of gene expression in the drug‐resistant (clone C2) and ‐sensitive (clone A) trophozoites. Nuclear proteins from trophozoites of both clones and C/EBP sequences of the core promoter formed specific complexes, which were abolished by anti‐human C/EBPβ antibodies. UV cross‐linking and Western blot assays revealed 25 and 65 kDa bands in urea treated and untreated proteins respectively. The nuclear factors that bind to C/EBP sites were semi‐purified by affinity chromatography. They were immunodetected by anti‐human C/EBPβ antibodies and formed a specific complex with the C/EBP probe. The antibodies recognized proteins in the cytoplasm, nucleus and EhkO organelles in immunofluorescence and confocal microscopy experiments. Based on our results, we propose that the C/EBP site at −54 bp stabilizes the transcription pre‐initiation complex, whereas the other site at −198 bp may be involved in the formation of a multiprotein complex, which provokes DNA folding and promotes the EhPgp1 gene transcription.

Cellular location and function of the P-glycoproteins (EhPgps) in Entamoeba histolytica multidrug-resistant trophozoites.

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.

The Multidrug-Resistance Phenotype of Entamoeba histolytica-Transfected Trophozoites with the EhPgp5 Gene

The ability of mammalian cells and microorganisms to develop drug resistance has become a world health problem of increasing proportions. Entamoeba histolytica presents the evolutionarily conserved multidrug-resistance phenotype (MDR) discovered in mammalian cells (1). The P-glycoprotein acts provoking a reduction in the intracellular cytotoxic drug concentrations via an active transport against concentration gradients at the plasma membrane. This results in a reduction of the number of effective drug interactions with biological targets, and hence a reduction in toxicity in PGPexpressing cells. In E. histolytica , very little is known concerning the biochemical and physiological properties of the P-glycoproteins encoded by the EhPgp genes (mdr-like). In 1996, Gosh and coworkers demonstrated that the EhPgp1 gene confers resistance to 40 m M emetine to the sensitive clone A (2), supporting the hypothesis that the EhPgp1 gene participates in emetine resistance at low drug concentrations. It has been suggested that EhPgp5 gene expression may be necessary for trophozoite resistance at high drug concentrations. To begin the study of P-glycoprotein function encoded by the EhPgp5 gene, we cloned and transfected the EhPgp5 gene into sensitive trophozoites.

Possible Role of the CCAAT/Enhancer Binding Protein in the Expression Regulation of the EhPgp1 Multidrug Resistance Gene in Entamoeba histolytica

Several potential binding sites for transcription factors participating in gene expression regulation have been identified in the 5 9 -flanking region of the EhPgp1 gene involved in the multidrug resistance (MDR) phenotype of the emetine-resistant mutant clone C2. To begin the fine characterization of the cis -regulatory sequences of the EhPgp1 gene core promoter, we focused our attention on two highly conserved CCAAT/enhancer binding protein (C/EBP) motifs located at 2 54 and 2 196 bp positions at the EhPgp1 gene promoter. According to the results obtained from electrophoretic mobility shift assays (EMSA), these sites seem to be involved in the expression of this gene (1). The C/EBP family members are important in regulating many eukaryotic genes during differentiation processes. These nuclear proteins have between 150 and 360 amino acids. They present a bipartite DNA-binding domain composed of a positively charged region that contacts the DNA, an amphipathic helix at the conserved carboxy-terminal end that mediates dimerization through the formation of a leucine zipper, and a less well-conserved amino-terminal region containing regulatory and transactivation domains. All C/ EBPs share homology in their basic domain and, as a result, recognize the same DNA motif (T T / G TGG T / A T / A A / T ), forming homoor heterodimers with each other (2). In this report, we present the location and alignment of consensus C/EBP sequences in mdr gene promoters of mammals and protozoan parasites. By EMSA, we detected a nuclear factor able to bind the C/EBP sites in E. histolytica and propose it as a possible trans -regulatory element in MDR phenotype regulation, as described in mammals. Materials and Methods

Cis -Elements Upregulate the Activity of the Entamoeba histolytica EhPgp1 Gene Promoter

EhPgp1 , one of the genes responsible for the MDR phenotype in Entamoeba histolytica , is differentially transcribed in trophozoites of the drug-sensitive clone A and in the drug-resistant clone C2. Our previous results suggested that the expression of this gene is mainly regulated at transcriptional level (1). EhPgp gene promoters have transcription factor binding sequences similar to those described in other organisms, mainly in mammals (1,2). In the EhPgp1 promoter, we found C/EBP, HOX, GATA-1, OCT, and Inr DNA binding sites. These sequences seem to be relevant for the expression of the EhPgp1 gene. Gel shift assays showed complexes formation between DNA promoter fragments and nuclear extracts. Competition assays using the consensus sequences for these transcription factors inhibited the formation of the DNA–protein complexes (1). The identification of the transcriptional regulation factors in E. histolytica genes is fundamental to understand the mechanisms controlling switching of parasite genes. Transcriptional selectivity of the eukaryotic genes is mediated by complex control regions composed of different combinations of elements to allow multiple distinct regulatory factors that function coordinately to potentiate RNA synthesis. However, unique promoter specificity and regulation can be conferred by the particular composition and spatial organization of the multiple elements constituting the complete set used by a given gene. The analysis of binding sequences found in the EhPgp1 promoter showed certain differences with the reported consensus sequences in other organisms. Thus, their functional role needs to be proven by transfection assays. Additionally, their precise positioning on the promoter is also important for the formation of stable complexes that permit the expression of this gene. To investigate the mechanisms regulating differential expression of the EhPgp1 gene, we generated several constructs deleting different sequences in this promoter. The DNA fragments were cloned in front of the CAT reporter gene. Then, the constructs were transfected in sensitive and resistant trophozoites and their ability to drive CAT expression was analyzed.