Hypothetical proteins play a role in stage conversion, virulence, and the stress response in the Entamoeba species.

Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and amoebic liver abscess in humans, affecting millions of people worldwide. This pathogen possesses a two-stage life cycle consisting of an environmentally stable cyst and a pathogenic amoeboid trophozoite. As cysts can be ingested from contaminated food and water, this parasite is prevalent in underdeveloped countries and poses a significant health burden. Until recently there was no reliable method for inducing stage conversion in E. histolytica in vitro. As such, the reptilian pathogen, Entamoeba invadens, has long-served as a surrogate. Much remains unclear about stage conversion in these parasites and current treatments for amoebiasis are lacking, as they cause severe side effects. Therefore, new therapeutic strategies are needed. The genomes of these parasites remain enigmatic as approximately 54% of E. histolytica genes and 66% of E. invadens genes are annotated as hypothetical proteins. In this study, we characterized two hypothetical proteins in the Entamoeba species, EIN_059080, in E. invadens, and its homolog, EHI_056700, in the human pathogen, E. histolytica. EHI_056700 has no homolog in the human host. We used an RNAi-based silencing system to reduce expression of these genes in E. invadens and E. histolytica trophozoites. Loss of EIN_059080 resulted in a decreased rate of encystation and an increased rate of erythrophagocytosis, an important virulence function. Additionally, mutant parasites were more susceptible to oxidative stress. Similarly, loss of EHI_056700 in E. histolytica trophozoites resulted in increased susceptibility to oxidative stress and glucose deprivation, but not to nitrosative stress. Unlike the E. invadens mutants, E. histolytica parasites with decreased reduced expression of EHI_056700 exhibited a decreased rate of erythrophagocytosis of and adhesion to host cells. Taken together, these data suggest that these hypothetical proteins play a role in stage conversion, virulence, and the response to stress in the Entamoebae. Since parasites with reduced expression of EHI_056700 show decreased virulence functions and increased susceptibility to physiologically relevant stressors, EHI_056700 may represent a possible therapeutic target for the treatment of amoebiasis.

Eukaryotic Initiation Factor 2α Kinases Regulate Virulence Functions, Stage Conversion, and the Stress Response in Entamoeba invadens.

Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. This pathogen possesses a two-stage life cycle consisting of an environmentally stable cyst and a pathogenic amoeboid trophozoite. Since infection is acquired by ingestion of cysts from contaminated food and water, this parasite is prevalent in underdeveloped countries. A reptilian pathogen, Entamoeba invadens, which can encyst in culture, has long served as a surrogate to study stage conversion. In the host, Entamoeba species must manage stress, including nutrient deprivation and host immune pressure. In many systems, the stress response is characterized by downregulation of translation, which is initiated by the phosphorylation of eukaryotic initiation factor-2 alpha (eIF2α). In mammalian cells, this phosphorylation is carried out by a family of eIF2α kinases. A canonical eIF2α translational control system exists in Entamoeba species; however, no eIF2α kinases have been characterized. In this study, we identified two eIF2α kinases in E. invadens, EiIF2K-A and EiIF2K-B. Their identity as eIF2α kinases was validated using a heterologous yeast system. We used an RNA interference (RNAi) trigger-mediated silencing system to reduce expression of EiIF2K-A, which also reduced expression of EiIF2K-B. Parasites with decreased kinase expression exhibited decreased phosphorylation of eIF2α and increased sensitivity to oxidative stress. Diminished kinase expression also correlated with an increased rate of encystation, a decreased rate of excystation, and an increase in several virulence functions, erythrophagocytosis and adhesion to host cells. Taken together, these data suggest that EiIF2K-A and EiIF2K-B are authentic eIF2α kinases that may regulate the Entamoeba stress response. IMPORTANCE Entamoeba histolytica is a human pathogen that causes dysentery and affects millions of people worldwide. This parasite possesses a two-stage life cycle: an environmentally stable cyst and the pathogenic trophozoite. Cysts are ingested from contaminated food and water; thus, this parasite in prevalent in underdeveloped countries. Current therapies commonly cause adverse side effects; therefore, new treatments are needed. In the host, Entamoeba experiences stress brought on, in part, by the host immune system. Understanding stage conversion and the stress response of this pathogen may lead to new drug therapies. Using the model organism E. invadens, we identified two kinases similar to those involved in stress and stage conversion in other systems. We determined that these kinases may regulate the oxidative stress response, stage conversion, and virulence. This work is significant, as it will inform future studies on the life cycle and pathogenicity of Entamoeba species.

Reduced expression of a rhomboid protease, EhROM1, correlates with changes in the submembrane distribution and size of the Gal/GalNAc lectin subunits in the human protozoan parasite, Entamoeba histolytica.

Entamoeba histolytica is a food- and waterborne parasite that causes amebic dysentery and amoebic liver abscesses. Adhesion is one of the most important virulence functions as it facilitates motility, colonization of host, destruction of host tissue, and uptake of nutrients by the parasite. The parasite cell surface adhesin, the Gal/GalNAc lectin, facilitates parasite-host interaction by binding to galactose or N-acetylgalactosamine residues on host components. It is composed of heavy (Hgl), intermediate (Igl), and light (Lgl) subunits. Igl is constitutively localized to lipid rafts (cholesterol-rich membrane domains), whereas Hgl and Lgl transiently associate with rafts. When all three subunits are localized to rafts, galactose-sensitive adhesion is enhanced. Thus, submembrane location may regulate the function of this adhesion. Rhomboid proteases are a conserved family of intramembrane proteases that also participate in the regulation of parasite-host interactions. In E. histolytica, one rhomboid protease, EhROM1, cleaves Hgl as a substrate, and knockdown of its expression inhibits parasite-host interactions. Since rhomboid proteases are found within membranes, it is not surprising that lipid composition regulates their activity and enzyme-substrate binding. Given the importance of the lipid environment for both rhomboid proteases and the Gal/GalNAc lectin, we sought to gain insight into the relationship between rhomboid proteases and submembrane location of the lectin in E. histolytica. We demonstrated that EhROM1, itself, is enriched in highly buoyant triton-insoluble membranes reminiscent of rafts. Reducing rhomboid protease activity, either pharmacologically or genetically, correlated with an enrichment of Hgl and Lgl in rafts. In a mutant cell line with reduced EhROM1 expression, there was also a significant augmentation of the level of all three Gal/GalNAc subunits on the cell surface and an increase in the molecular weight of Hgl and Lgl. Overall, the study provides insight into the molecular mechanisms governing parasite-host adhesion for this pathogen.

Phosphorylation of eukaryotic initiation factor-2α in response to endoplasmic reticulum and nitrosative stress in the human protozoan parasite, Entamoeba histolytica.

Entamoeba histolytica is an intestinal parasite infecting over 50 million people worldwide and is the causative agent of amebic dysentery and amoebic liver abscess. In the human host, E. histolytica experiences stress brought on by nutrient deprivation and the host immune response. To be a successful parasite, E. histolytica must counter the stress; therefore, understanding the stress response may uncover new drug targets. In many systems, the stress response includes down-regulation of protein translation, which is regulated by phosphorylation of eukaryotic initiation factor (eIF-2α). Previous work has demonstrated that phosphorylation of the E. histolytica eIF-2α (EheIF-2α) increases significantly when exposed to long-term serum starvation, oxidative stress, and long-term heat shock. However, the effects of reagents that are known to induce nitrosative or endoplasmic reticulum (ER) stresses, on EheIF-2α have yet to be evaluated. Nitrosative stress is part of the host's immune response and ER stress can be caused by several physiological or pathological factors. We treated E. histolytica cells with various reagents known to induce nitrosative stress (DPTA-NONOate and SNP) or ER stress (BFA and DTT). We examined the morphology of the ER, tracked phosphorylation of EheIF-2α, and assessed protein translation in control and stressed cells. While all four stress-inducing reagents caused a global reduction in protein translation, only DTT was capable of also inducing changes in the morphology of the ER (consistent with ER stress) and phosphorylation of EheIF-2α. This suggests that DTT authentically induces ER stress in E. histolytica and that this stress is managed by the eIF-2α-based system. This was supported by the observation that cells expressing a non-phosphorylatable version of eIF-2α were also highly sensitive to DTT-stress. Since protein translation decreased in the absence of phosphorylation of eIF-2α (after treatment with DPTA-NONOate, SNP or BFA), the data also indicate that there are alternative protein-translational control pathways in E. histolytica. Overall, our study further illuminates the stress response to nitrosative stress and ER stress in E. histolytica.