Silencing the cleavage factor CFIm25 as a new strategy to control Entamoeba histolytica parasite

Abstract

The 25 kDa subunit of the Clevage Factor Im (CFIm25) is an essential factor for messenger RNA polyadenylation in human cells. Therefore, here we investigated whether the homologous protein of Entamoeba histolytica, the protozoan responsible for human amoebiasis, might be considered as a biochemical target for parasite control. Trophozoites were cultured with bacterial double-stranded RNA molecules targeting the EhCFIm25 gene, and inhibition of mRNA and protein expression was confirmed by RT-PCR and Western blot assays, respectively. EhCFIm25 silencing was associated with a significant acceleration of cell proliferation and cell death. Moreover, trophozoites appeared as larger and multinucleated cells. These morphological changes were accompanied by a reduced mobility, and erythrophagocytosis was significantly diminished. Lastly, the knockdown of EhCFIm25 affected the poly(A) site selection in two reporter genes and revealed that EhCFIm25 stimulates the utilization of downstream poly(A) sites in E. histolytica mRNA. Overall, our data confirm that targeting the polyadenylation process represents an interesting strategy for controlling parasites, including E. histolytica. To our best knowledge, the present study is the first to have revealed the relevance of the cleavage factor CFIm25 as a biochemical target in parasites.

Targeting the polyadenylation factor EhCFIm25 with RNA aptamers controls survival in Entamoeba histolytica

“…Most conventional means of introducing nucleic acids to cells are based on lipofectamine transfection systems. In the present work, we observed that the dsRNA soaking method (originally used to silence gene expression in E. histolytica ) 29 , 45 , 50 represents an efficient, reproducible, fast, easy-to-implement strategy for blocking a nuclear target protein like EhCFIm25. In the literature, aptamers linked to siRNA (aptamer-siRNA chimeras) are used to deliver both molecules to cells 57 .…”

“…To deliver C4 and C5 aptamers into E . histolytica , we took advantage of our previous observation whereby soaking trophozoites in medium with dsRNA produced in bacteria results in uptake 29 , 45 , 50 . We cloned the cDNA sequences corresponding to the C4 or C5 aptamers into a pL4440 plasmid (Fig.…”

“…In E . histolytica , we have observed that EhCFIm25 silencing alters poly(A) site selection and parasite survival 29 . Since EhCFIm25 controls the selection of poly(A) sites, EhCFIm25 sequestration is expected to modify the stability of the mRNAs produced.…”

“…Furthermore, we recently observed that EhCFIm25 silencing induces cell death and decreases virulence capacity, prompting us to hypothesize that EhCFIm25 may be a relevant target for E . histolytica control 29 . To test this hypothesis, we applied two powerful molecular biology approaches: (i) the development of aptamers that bind to EhCFIm25 and (ii) delivery aptamers to trophozoites via soaking.…”

mRNA Polyadenylation Machineries in Intestinal Protozoan Parasites

“…Moreover, we recently demonstrated that the 25 kDa subunit of cleavage factor Im (EhCFIm25) is necessary for trophozoites survival, indicating that polyadenylation factors are valuable targets for parasite control (Ospina‐Villa et al. , ,b) as it has been reported for other protozoan parasites (Hendriks et al. ; Palencia et al.…”