Protein Phosphatase PP2C Identification in Entamoeba spp

Entamoeba histolytica is the causative agent of amoebiasis, and Entamoeba dispar is its noninvasive morphological twin. Entamoeba invadens is a reptilian parasite. In the present study, Western blot, phosphatase activity, immunofluorescence, and bioinformatic analyses were used to identify PP2C phosphatases of E. histolytica, E. dispar, and E. invadens. PP2C was identified in trophozoites of all Entamoeba species and cysts of E. invadens. Immunoblotting using a Leishmania mexicana anti-PP2C antibody recognized a 45.2 kDa PP2C in all species. In E. histolytica and E. invadens, a high molecular weight element PP2C at 75 kDa was recognized, mainly in cysts of E. invadens. Immunofluorescence demonstrated the presence of PP2C in membrane and vesicular structures in the cytosol of all species analyzed. The ~75 kDa PP2C of Entamoeba spp. shows the conserved domain characteristic of phosphatase enzymes (according to in silico analysis). Possible PP2C participation in the encystation process was discussed.

High-throughput phenotypic screen identifies a new family of potent anti-amoebic compounds

Entamoeba histolytica is a disease-causing parasitic amoeba which affects an estimated 50 million people worldwide, particularly in socioeconomically vulnerable populations experiencing water sanitation issues. Infection with E. histolytica is referred to as amoebiasis, and can cause symptoms such as colitis, dysentery, and even death in extreme cases. Drugs exist that are capable of killing this parasite, but they are hampered by downsides such as significant adverse effects at therapeutic concentrations, issues with patient compliance, the need for additional drugs to kill the transmissible cyst stage, and potential development of resistance. Past screens of small and medium sized chemical libraries have yielded anti-amoebic candidates, thus rendering high-throughput screening a promising direction for new drug discovery in this area. In this study, we screened a curated 80,000-compound library from Janssen pharmaceuticals against E. histolytica trophozoites in vitro, and from it identified a highly potent new inhibitor compound. Further experimentation confirmed the activity of this compound, as well as that of several structurally related compounds, originating from both the Janssen Jump-stARter library, and from chemical vendors, thus highlighting a new structure-activity relationship (SAR). In addition, we confirmed that the compound inhibited E. histolytica survival as rapidly as the current standard of care and inhibited transmissible cysts of the related model organism Entamoeba invadens. Together these results constitute the discovery of a novel class of chemicals with favorable in vitro pharmacological properties which may lead to an improved therapy against this parasite and in all of its life stages.Author summaryThe parasite Entamoeba histolytica represents a significant challenge in the field of global health. It currently infects and causes disease among millions of people worldwide, particularly those lacking access to clean water. Drugs exist to treat this disease, but nevertheless it persists as a problem, likely at least partly due to problems and downsides inherent to these drugs. Hence the search for new and better ones is needed. We report here our contribution to this search, consisting of testing a large, carefully-curated collection of tens of thousands of chemicals for their ability to kill E. histolytica. This large-scale test resulted in the identification of one of the compounds as potently anti-amoebic, capable of killing the parasite cells at extremely low concentrations. Further experimentation found several chemically-related compounds to also possess this property, and additionally found the first compound capable of killing the infective life stage of another Entamoeba parasite. These results have revealed an entire new family of chemicals with good potential for development as better drugs against this disease.

Encystation of Entamoeba histolytica in Axenic Culture

Entamoeba histolytica is a parasitic protozoan that causes amoebic dysentery, which affects approximately 90 million people each year worldwide. E. histolytica is transmitted through ingestion of food and water contaminated with the cyst form, which undergoes excystation in the small intestine to the trophozoite form that colonizes the large intestine. The reptile pathogen Entamoeba invadens has served as a model for studying stage conversion between the trophozoite and cyst form due to lack of reproducible encystation of E. histolytica in the laboratory. Although much has been learned about encystation and excystation using E. invadens, the findings do not fully translate to E. histolytica due to the extensive genetic and host differences between these species. Here, we present the first reproducible encystation of E. histolytica in vitro. The cysts produced were viable and displayed the four characteristic hallmarks: round shape, chitinous cell wall, tetranucleation, and detergent resistance. Using flow cytometry analysis, glucose limitation and high cell density were key for encystation, as for E. invadens. Entry into encystation was enhanced by the short-chain fatty acids acetate and propionate, unlike for E. invadens. This new model will now allow the further study of E. histolytica stage conversion, transmission, and treatment.

Identification of oligo-adenylated small RNAs in the parasite Entamoeba and a potential role for small RNA control

Background The RNA interference (RNAi) pathway is a gene regulation mechanism that utilizes small RNA (sRNA) and Argonaute (Ago) proteins to silence target genes. Our previous work identified a functional RNAi pathway in the protozoan parasite Entamoeba histolytica, including abundant 27 nt antisense sRNA populations which associate with EhAgo2–2 protein. However, there is lack of understanding about the sRNAs that are bound to two other EhAgos (EhAgo2–1 and 2–3), and the mechanism of sRNA regulation itself is unclear in this parasite. Therefore, identification of the entire pool of sRNA species and their sub-populations that associate with each individual EhAgo protein would be a major step forward. Results In the present study, we sequenced sRNA libraries from both total RNAs and EhAgo bound RNAs. We identified a new population of 31 nt sRNAs that results from the addition of a non-templated 3–4 adenosine nucleotides at the 3′-end of the 27 nt sRNAs, indicating a non-templated RNA-tailing event in the parasite. The relative abundance of these two sRNA populations is linked to the efficacy of gene silencing for the target gene when parasites are transfected with an RNAi-trigger construct, indicating that non-templated sRNA-tailing likely play a role in sRNA regulation in this parasite. We found that both sRNA populations (27 nt and 31 nt) are present in the related parasite Entamoeba invadens, and are unchanged during the development. In sequencing the sRNAs associating with the three EhAgo proteins, we observed that despite distinct cellular localization, all three EhAgo sRNA libraries contain 27 nt sRNAs with 5′-polyphosphate (5′-polyP) structure and share a largely overlapping sRNA repertoire. In addition, our data showed that a fraction of 31 nt sRNAs associate with EhAgo2–2 but not with its mutant protein (C-terminal deletion), nor other two EhAgos, indicating a specific EhAgo site may be required for sRNA modification process in the parasite. Conclusion We identified a new population of sRNA with non-templated oligo-adenylation modification, which is the first such observation amongst single celled protozoan parasites. Our sRNA sequencing libraries provide the first comprehensive sRNA dataset for all three Entamoeba Ago proteins, which can serve as a useful database for the amoeba community.

The dynamics of ultrastructural changes during Entamoeba invadens encystation

Entamoeba histolytica infection causes amoebiasis, which is a global public health problem. The major route of infection is oral ingestion of E. histolytica cysts, cysts being the sole form responsible for host-to-host transmission. Cysts are produced by cell differentiation from proliferative trophozoites in a process termed ‘encystation’. Therefore, encystation is an important process from a medical as well as a biological perspective. Previous electron microscopy studies have shown the ultrastructure of precysts and mature cysts; however, the dynamics of ultrastructural changes during encystation were ambiguous. Here, we analysed a series of Entamoeba invadens encysting cells by transmission electron microscopy. Entamoeba invadens is a model for encystation and the cells were prepared by short interval time course sampling from in vitro encystation-inducing cultures. We related sampled cells to stage conversion, which was monitored in the overall population by flow cytometry. The present approach revealed the dynamics of ultrastructure changes during E. invadens encystation. Importantly, the results indicate a functional linkage of processes that are crucial in encystation, such as glycogen accumulation and cyst wall formation. Hence, this study provides a reference for studying sequential molecular events during Entamoeba encystation.

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

Entamoeba Histolytica, a pathogenic parasite, is the causative organism of amoebiasis and uses human colon to complete its life cycle. It destroys intestinal tissue leading to invasive disease. Since it does not form cyst in culture medium, a reptilian parasite Entamoeba invadens serves as the model system to study encystation. Detailed investigation on the mechanism of cyst formation, information on ultra-structural changes and cyst wall formation during encystation are still lacking in E. invadens. Here, we used electron microscopy to study the ultrastructural changes during cyst formation and showed that the increase in heterochromatin patches and deformation of nuclear shape were early events in encystation. These changes peaked at ~20h post induction, and normal nuclear morphology was restored by 72h. Two types of cellular structures were visible by 16h. One was densely stained and consisted of the cytoplasmic mass with clearly visible nucleus. The other consisted of membranous shells with large vacuoles and scant cytoplasm. The former structure developed into the mature cyst while the latter structure was lost after 20h, This study of ultra-structural changes during encystation in E. invadens opens up the possibilities for further investigation into the mechanisms involved in this novel process.

Morphological and Motility Features of the Stable Bleb-Driven Monopodial Form of Entamoeba and Its Importance in Encystation

ABSTRACT Entamoeba histolytica and its reptilian counterpart and encystation model Entamoeba invadens formed a polarized monopodial morphology when treated with pentoxifylline. This morphology was propelled by retrograde flow of the cell surface resulting from a cyclic sol-gel conversion of cytoplasm and a stable bleb at the leading edge. Pentoxifylline treatment switched the unpolarized, adherent trophozoites to the nonadherent, stable bleb-driven form and altered the motility pattern from slow and random to fast, directionally persistent, and highly chemotactic. Interestingly, exogenously added adenosine produced multiple protrusions and random motility, an opposite phenotype to that of pentoxifylline. Thus, pentoxifylline, an adenosine antagonist, may be inducing the monopodial morphology by preventing lateral protrusions and restricting the leading edge to one site. The polarized form of E. invadens was aggregation competent, and time-lapse microscopy of encystation revealed its appearance during early hours, mediating the cell aggregation by directional cell migration. The addition of purine nucleotides to in vitro encystation culture prevented the formation of polarized morphology and inhibited the cell aggregation and, thus, the encystation, which further showed the importance of the polarized form in the Entamoeba life cycle. Cell polarity and motility are essential in the pathogenesis of Entamoeba parasites, and the stable bleb-driven polarized morphology of Entamoeba may also be important in invasive amoebiasis.