Date Published: August 27, 2008
Publisher: Public Library of Science
Author(s): Carol A. Gilchrist, Duza J. Baba, Yan Zhang, Oswald Crasta, Clive Evans, Elisabet Caler, Bruno W. S. Sobral, Christina B. Bousquet, Megan Leo, Ameilia Hochreiter, Sarah K. Connell, Barbara J. Mann, William A. Petri, John Samuelson
Abstract: The Entamoeba histolytica transcription factor Upstream Regulatory Element 3-Binding Protein (URE3-BP) is a calcium-responsive regulator of two E. histolytica virulence genes, hgl5 and fdx1. URE3-BP was previously identified by a yeast one-hybrid screen of E. histolytica proteins capable of binding to the sequence TATTCTATT (Upstream Regulatory Element 3 (URE3)) in the promoter regions of hgl5 and fdx1. In this work, precise definition of the consensus URE3 element was performed by electrophoretic mobility shift assays (EMSA) using base-substituted oligonucleotides, and the consensus motif validated using episomal reporter constructs. Transcriptome profiling of a strain induced to produce a dominant-positive URE3-BP was then used to identify additional genes regulated by URE3-BP. Fifty modulated transcripts were identified, and of these the EMSA defined motif T[atg]T[tc][cg]T[at][tgc][tg] was found in over half of the promoters (54% p<0.0001). Fifteen of the URE3-BP regulated genes were potential membrane proteins, suggesting that one function of URE3-BP is to remodel the surface of E. histolytica in response to a calcium signal. Induction of URE3-BP leads to an increase in tranwell migration, suggesting a possible role in the regulation of cellular motility.
Partial Text: The early branching eukaryote Entamoeba histolytica is a human parasite that is the etiologic agent of amebic dysentery and liver abscess. Only one of every five infections leads to disease , and the parasite and host factors that control the outcome of infection are not well understood. Alteration in transcription of certain crucial genes may contribute to the expression of a virulence phenotype. Distinct gene expression profiles which may be associated with pathogenicity have been identified by comparing the transcriptome of laboratory-cultured HM-1:IMSS E. histolytica to trophozoites growing in vivo, as well as to that of less virulent strains and recent clinical isolates ,,,,.
In this work the DNA consensus motif recognized by the URE3-BP transcription factor was experimentally defined, and then used to identify a subset of E. histolytica transcripts modulated by inducible expression of URE3-BP. URE3-BP had previously been shown to regulate the expression of two virulence factors in the parasite. The current studies provide a more global picture of its role in control of gene expression. The key experimental approach was the inducible expression of a dominant positive URE3-BP mutant and the subsequent identification of uniquely altered transcripts. The majority (42/50) of transcripts were repressed. Over half (54%) of the modulated genes had a URE3 matrix in the promoter region while the other half was comprised of genes presumably downstream of control by URE3.