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

Abstract

b Entamoeba histolytica, a protozoan parasite, is an important human pathogen and a leading parasitic cause of death. The organism has two life cycle stages, trophozoites, which are responsible for tissue invasion, and cysts, which are involved in pathogen transmission. Entamoeba invadens is the model system to study Entamoeba developmental biology, as high-grade regulated encystation and excystation are readily achievable. However, the lack of gene-silencing tools in E. invadens has limited the molecular studies that can be performed. Using the endogenous RNA interference (RNAi) pathway in Entamoeba, we developed an RNAi-based trigger gene-silencing approach in E. invadens. We demonstrate that a gene's coding region that has abundant antisense small RNAs (sRNAs) can trigger silencing of a gene that is fused to it. The trigger fusion leads to the generation of abundant antisense sRNAs that map to the target gene, with silencing occurring independently of trigger location at the 5= or 3= end of a gene. Gene silencing is stably maintained during development, including encystation and excystation. We have used this approach to successfully silence two E. invadens genes: a putative rhomboid protease gene and a SHAQKY family Myb gene. The Myb gene is upregulated during oxidative stress and development, and its downregulation led, as predicted, to decreased viability under oxidative stress and decreased cyst formation. Thus, the RNAi trigger silencing method can be used to successfully investigate the molecular functions of genes in E. invadens. Dissection of the molecular basis of Entamoeba stage conversion is now possible, representing an important technical advance for the system.

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

“…In order to better understand the role of ERM-BP, we used a trigger-mediated RNA-interference gene silencing approach to downregulate ERM-BP ( Suresh et al, 2016 ). Transcript for EIN_083100 was undetectable in silenced ERM-BP cell lines at 24 hr of encystation, indicating successful silencing of ERM-BP ( Figure 4—figure supplement 1 ).…”

Effect ofPeriostinSilencing on the Autophagy of Osteoblasts

“…Compared with gene knockout technology, RNA interference technology is relatively simple to operate, and gene silencing is reliable (Foda and Singh, 2015;Park et al, 2013;Suresh et al, 2016). In this experiment, POSTN silencing was achieved using RNA interference technology by exogenous lentivirus transfection.…”

The dynamics of ultrastructural changes during Entamoeba invadens encystation

“…In vitro cultures of E. invadens (IP-1) were routinely maintained, and cyst formation was induced as described (Mi-Ichi et al, 2018). Briefly, E. invadens trophozoites suspended in encystation medium (6 × 10 5 cells mL −1 ) were seeded in 96-well culture plates (240 μL per well) and sealed as described using PARAFILM® (Bemis Company, Inc., Oshkosh, WI) (Suresh et al, 2016). Then plates were incubated at 26°C for the period indicated.…”