Entamoeba histolytica: functional characterization of the -234 to -196 bp promoter region of the multidrug resistance EhPgp1 gene.

The multidrug resistance EhPgp1 gene is constitutively expressed in drug resistant trophozoites from Entamoeba histolytica. It has been demonstrated that two CCAAT/enhancer binding sites located in the EhPgp1 gene promoter control its transcriptional activation. However, functional assays of the 5' end of its promoter showed that region from -234 to -196 bp (38 bp) is also important for the EhPgp1 gene transcription. Here, we demonstrated that in the 38 bp region putative cis-activator sequences are located. In silico analysis showed the presence of GATA1, Gal4, Nit-2, and C/EBP consensus sequences. Additionally, we identified three specific DNA-protein complexes, which were competed by a C/EBP, GATA1, and HOX oligonucleotides. Finally, we partially purified three proteins of 64.4, 56.7, and 27.4 kDa. Further investigations are currently in progress to determine the identity of these nuclear factors and how they are interacting with the EhPgp1 gene promoter.

Cyclosporin A inhibits calcineurin (phosphatase 2B) and P-glycoprotein activity in Entamoeba histolytica.

Cyclosporin A (CsA) inhibits the proliferation of several protozoan parasites through blocking the activity of calcineurin (Cn) or P-glycoproteins (Pgp). We report here, that inhibition of the proliferation of Entamoeba histolytica trophozoites, the causal agent of human amebiasis, is due to interference of the phosphatase activity of Cn, in a similar fashion to the effect of this immunosuppressive drug on T lymphocytes. The non-immunosuppressive CsA analog PSC-833, which binds Pgp without interfering the function of Cn, did not inhibit the proliferation of HM1:IMSS trophozoites. Moreover, phosphatase activity of amebic Cn, detected using the phosphopeptide RII, was drastically affected by incubation with CsA, but not with PSC-833. On the other hand, both drugs were also tested on clone C2 trophozoites, which grow in the presence of emetine due to over-expression of Pgp. The effect of CsA was similar to that observed on HM1:IMSS trophozoites, whereas PSC-833 only affected the proliferation and viability of clone C2 when the trophozoites were grown in the presence of 40 microM of emetine, suggesting an interference of the Pgp activity. This suggestion was confirmed by results from experiments of Pgp-dependent effux of rhodamine from pre-loaded trophozoites, in the presence of either of these drugs. Therefore, CsA inhibition of E. histolytica trophozoite proliferation is more likely due to Cn than Pgp activity inhibition.

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/EBPbeta 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/EBPbeta 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.

The Entamoeba histolytica EhPgp5 (MDR-like) protein induces swelling of the trophozoites and alters chloride-dependent currents in Xenopus laevis oocytes.

Entamoeba histolytica, the protozoan responsible for human amoebiasis, presents the multidrug resistant phenotype due to the expression of the E. histolytica P-glycoproteins EhPgpl and EhPgp5. Here, we studied the protein EhPgp5 encoded by the EhPgp5 gene in emetine-sensitive trophozoites transfected with the pEhNEOPgp5 plasmid carrying the EhPgp5 gene. The transfected trophozoites increased their drug resistance slightly, but became bigger and globular. To investigate other EhPgp5 functions further, we microinjected the EhPgp5 mRNA in Xenopus laevis oocytes. Microinjected oocytes expressed EhPgp5 protein in their membranes and exhibited an ion current not present in the control oocytes. The antisense EhPgp5AS transcript, co-injected with the EhPgp5 mRNA, abolished the exogenous current, showing its specificity. Exogenous current was outward during depolarizing pulses. Reduction of the extracellular Cl- concentration displayed a reversible decrease of the current amplitude. Niflumic acid, 4,4-diisothiocyanatostilbene-2, 2'-disulfonic acid, and other Cl- channel blockers abolished the exogenous current, which was poorly modified by verapamil and changes in osmolarity of the medium. Our results suggest that the EhPgp5 protein could function as a Cl- current inductor and as a coadjuvant factor to avoid drug accumulation in the cell.

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.

Physiology and molecular genetics of multidrug resistance in Entamoeba histolytica.

Entamoeba histolytica presents the evolutionarily conserved multidrug-resistance (MDR) phenotype, discovered in mammalian cells. MDR cells overexpress the membrane P-glycoprotein, which excludes unrelated drugs from the cytoplasm. E. histolytica mutants exhibit cross-resistance to unrelated drugs, which are pumped out from the cytoplasm. In drug-resistant trophozoites, the constitutively expressed EhPg1 gene appears to be up-regulated by a C/EBP-like factor and a multiprotein complex that were not found in drug-sensitive trophozoites. The drug-induced EhPgp5 gene, on the other hand, appears to be up-regulated by AP-1 and HOX factors. Here we review the main physiological and molecular facts of the MDR phenotype in E. histolytica. Copyright 1999 Harcourt Publishers Ltd.