Regulation of gene expression in the protozoan parasite Entamoeba invadens: identification of core promoter elements and promoters with stage-specific expression patterns

Abstract

Developmental switching between life-cycle stages is a common feature among many pathogenic organisms. Entamoeba histolytica is an important human pathogen and is a leading parasitic cause of death globally. During its life cycle, Entamoeba converts between cysts (essential for disease transmission) and trophozoites (responsible for tissue invasion). Despite being central to its biology, the triggers that are involved in the developmental pathways of this parasite are not well understood. In order to define the transcriptional network associated with stage conversion we used Entamoeba invadens which serves as a model system for Entamoeba developmental biology, and performed RNA sequencing at different developmental time points . In this study RNA-Seq data was utilized to define basal transcriptional control elements as well as to identify promoters which regulate stage-specific gene expression patterns. We discovered that the 5’ and 3’ untranslated regions of E. invadens genes are short, a median of 20 nucleotides (nt) and 26 nt respectively. Bioinformatics analysis of DNA sequences proximate to the start and stop codons identified two conserved motifs: (i) E. invadens Core Promoter Motif - GAAC-Like (EiCPM-GL) (GAACTACAAA), and (ii) E. invadens 3’- U-Rich Motif (Ei3’-URM) (TTTGTT) in the 5’ and 3’ flanking regions, respectively. Electrophoretic mobility shift assays demonstrated that both motifs specifically bind nuclear protein(s) from E. invadens trophozoites. Additionally, we identified select genes with stage-specific expression patterns and analyzed the ability of each gene promoter to drive a luciferase reporter gene during the developmental cycle. This approach confirmed three trophozoite-specific, four encystation-specific and two excystation-specific promoters. This work lays the framework for use of stage-specific promoters to express proteins of interest in a particular life-cycle stage, adding to the molecular toolbox for genetic manipulation of E. invadens and allowing further dissection of factors controlling Entamoeba developmental biology.

An NAD+-dependent novel transcription factor controls stage conversion in Entamoeba

“…To find out whether the ERM motif is functional in the context of a full-length promoter, we used a luciferase reporter construct driven by a cyst-specific promoter that contains the WT-ERM. We have previously demonstrated that this construct increases expression during encystation ( Manna et al, 2014 ). A Mut-3-ERM-Luciferase construct was generated by changing CA to TG at fourth and fifth positions.…”

“…All the oligonucleotides used in EMSA are listed in Figure 1—source data 2 . The cyst-specific promoter construct, C4, has a WT-ERM, a luciferase reporter gene and 3’ regulatory region ( Manna et al, 2014 ). ERM was mutated by changing CA to TG at fourth and fifth position by site-directed mutagenesis kit (Agilent-QuikChange Lightning).…”

Ultra-structural analysis and morphological changes during the differentiation of trophozoite to cyst inEntamoeba invadens

“…Our results showed that the increase in HC patches was an early event during encystation. It could be related to up-or down regulation of stage specific genes which takes place in cells committed for encystation [21,22].…”

Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens

“…Therefore, we unfortunately did not find genes that could function as stage-specific triggers. However, we know that trigger silencing is dependent on promoter activity (20), and thus, the recent identification of stage-specific promoters in E. invadens means that we can use this approach to develop stage-specific trigger silencing (57). Expressing the trigger under the stage-regulated promoters will help regulate the exact stage at which transcription occurs and at which small RNAs can be generated.…”