ABSTRACT
Plasmodium falciparum is the causative agent of the deadliest human malaria. New molecules are needed that can specifically bind to erythrocytes that are infected with P. falciparum for diagnostic purposes, to disrupt host-parasite interactions, or to deliver chemotherapeutics. Aptamer technology has the potential to revolutionize biological diagnostics and therapeutics; however, broad adoption is hindered by the high failure rate of the systematic evolution of ligands by exponential enrichment (SELEX). Here we performed parallel SELEX experiments to compare the impact of two different methods for single-strand recovery on the efficiency of aptamer enrichment. Our experimental results and analysis of SELEX publications spanning 13 years implicate the alkaline denaturation step as a significant cause for inefficient aptamer selection. Thus, we applied an exonuclease single-strand recovery step in our SELEX to direct aptamers to the surface of erythrocytes infected with P. falciparum. The selected aptamers bind with high affinity (low nanomolar Kd values) and selectivity to exposed surface proteins of both laboratory parasite strains as well isolates from patients in Asia and Africa with clinical malaria. The results obtained in this study potentially open new approaches to malaria diagnosis and surveillance.
Subject(s)
Erythrocytes/parasitology , Malaria, Falciparum/diagnosis , Plasmodium falciparum/metabolism , SELEX Aptamer Technique/methods , Aptamers, Nucleotide/genetics , Aptamers, Nucleotide/metabolism , DNA, Single-Stranded/metabolism , Exonucleases/metabolism , High-Throughput Nucleotide Sequencing/methods , Humans , Malaria, Falciparum/blood , Malaria, Falciparum/metabolism , Malaria, Falciparum/parasitology , Plasmodium falciparum/geneticsABSTRACT
The altered permeability characteristics of erythrocytes infected with malaria parasites have been a source of interest for over 30 years. Recent electrophysiological studies have provided strong evidence that these changes reflect transmembrane transport through ion channels in the host erythrocyte plasma membrane. However, conflicting results and differing interpretations of the data have led to confusion in this field. In an effort to unravel these issues, the groups involved recently came together for a week of discussion and experimentation. In this article, the various models for altered transport are reviewed, together with the areas of consensus in the field and those that require a better understanding.
Subject(s)
Cell Membrane Permeability/physiology , Erythrocytes/parasitology , Malaria, Falciparum/parasitology , Animals , Anions/metabolism , Cell Membrane Permeability/drug effects , Dantrolene/pharmacology , Erythrocytes/physiology , Furosemide/pharmacology , Humans , Ion Channels/physiopathology , Malaria, Falciparum/physiopathology , Membrane Transport Modulators/pharmacology , Nitrobenzoates/pharmacology , Oxidation-Reduction , Patch-Clamp Techniques , Plasmodium falciparum/physiologyABSTRACT
The histone deacetylase inhibitors (HDA-CIs) butyrate and trichostatin A activate gamma-globin expression via a p38 mitogen-activating protein kinase (MAPK)-dependent mechanism. We hypothesized that down-stream effectors of p38 MAPK, namely activating transcription factor-2 (ATF-2) and cyclic AMP response element (CRE) binding protein (CREB), are intimately involved in fetal hemoglobin induction by these agents. In this study, we observed increased ATF-2 and CREB1 phosphorylation mediated by the HDACIs in K562 cells, in conjunction with histone H4 hyperacetylation. Moreover, enhanced DNA-protein interactions occurred in the CRE in the (G)gamma-globin promoter (G-CRE) in vitro after drug treatments; subsequent chromatin immunoprecipitation assay confirmed ATF-2 and CREB1 binding to the G-CRE in vivo. Enforced expression of ATF-2 and CREB produced (G)gamma-promoter trans-activation which was abolished by a 2-base pair mutation in the putative G-CRE. The data presented herein demonstrate that gamma-gene induction by butyrate and trichostatin A involves ATF-2 and CREB1 activation via p38 MAPK signaling.
