Recent advances in Entamoeba biology: RNA interference, drug discovery, and gut microbiome

In recent years, substantial progress has been made in understanding the molecular and cell biology of the human parasite Entamoeba histolytica, an important pathogen with significant global impact. This review outlines some recent advances in the Entamoeba field in the last five years, focusing on areas that have not recently been discussed in detail: (i) molecular mechanisms regulating parasite gene expression, (ii) new efforts at drug discovery using high-throughput drug screens, and (iii) the effect of gut microbiota on amoebiasis.

Entamoeba histolytica is a unicellular protozoan parasite transmitted to humans via contaminated food or water and is the causative agent of amoebiasis 1,2 . The infectious cycle begins with ingestion of the cyst, which is able to survive in the environment outside the human host 3,4 . After ingestion, parasites excyst as trophozoites in the small intestine and eventually reach the colon, where they adhere to colonic mucins and epithelial cells. The most common disease attributed to E. histolytica is amoebic colitis, whose symptoms range from asymptomatic colonization of the colon to severe, bloody diarrhea. In a subset of patients, extra-intestinal disease occurs following dissemination of the parasite to the liver, lung, or brain 5 . Owing to unknown factors, some trophozoites encyst, allowing them to be excreted in the stool and to go on to infect new hosts. Although E. histolytica encysts naturally inside the human host, no culturing method has been able to induce encystation in laboratory conditions. However, Entamoeba invadens, a reptilian amoeba, can successfully be induced to undergo encystation in the laboratory and is the accepted model to study amoebic stage conversion in vitro.
Amoebiasis is widespread among infants in developing countries because of poor hygiene and sanitation, contamination of food and water with feces, and malnourishment 6-8 . For instance, in an urban slum of Dhaka, Bangladesh, one-third of infants are infected with E. histolytica within the first year of life [8][9][10] . Consequently, amoebiasis prevalence is higher in developing countries, such as the Indian subcontinent, tropical and central regions of Africa, and South America 11,12 . However, recent reports also identified amoebic infections in east Asian developed countries and Australia 13-16 . In developed countries, E. histolytica infection is typically seen in new immigrants and travelers returning from regions where amoebiasis is endemic, and in Japan there is a relatively high incidence of disease in homosexual men 13,15-17 . These findings suggest that amoebiasis can also be a re-emerging disease in developed countries.
In this review, we will highlight a few topics that have emerged in the study of Entamoeba in the last five years. A number of excellent reviews have recently been published on the immune response to Entamoeba 18 , immune evasion mechanisms by the parasite 19 , trogocytosis (nibbling of host cells by the amoeba) 20-22 , and virulence determinants 23 . Thus, we will focus on topics for which recent reviews are not available.

Molecular methods regulating parasite gene expression
Elucidating the molecular mechanisms regulating gene expression Regulation of gene expression is a complex process requiring the simultaneous coordination of large-scale cellular processes (for example, DNA replication and chromosomal segregation) as well as more local processes (for example, euchromatin stabilization and RNA polymerase recruitment). The processes governing transcription and gene expression in Entamoeba remain poorly understood. Recent efforts have elucidated mechanisms for stabilizing ribosomal RNA during encystation 24 as well as under stress conditions 25 . Forward-genetic screens have helped determine the target genes regulated by specific signaling transduction pathways 26 . Additionally, transcriptome analyses in Entamoeba have helped to identify cis-elements and trans-acting factors involved in regulating gene expression 27,28 . However, despite ongoing efforts, only a handful of DNA motifs and transcription factors have thus far been characterized 29 .
In a follow-up to their initial report characterizing the transcription factor EhPC4 (E. histolytica positive cofactor 4) and its role in regulating the expression of genes involved in cell migration 30 , Hernández de la Cruz et al. recently identified a new role for EhPC4 in regulating DNA replication and genome stability 31 . E. histolytica trophozoites exist in cultures as polyploid cells (a subpopulation of cells having either a single polyploid nucleus or multiple nuclei), whereas cysts contain four haploid nuclei. In trophozoites, heterogeneous DNA content is due to genome re-duplication and uncoupling of nuclear division and cytokinesis 32,33 . Therefore, the recent data presented by Hernández de la Cruz et al. are important because they are among the first to identify a protein involved in polyploidy and genetic heterogeneity in Entamoeba. The complexity of DNA organization and structure in Entamoeba has posed a challenge toward unraveling aspects of parasite biology that regulate the flow of information, which arguably influences all other aspects of parasite biology (that is, metabolism and development). Importantly, polyploidy has posed some limitations on parasite genetic engineering, and further molecular dissection of this pathway could aid in the development of improved genetic tools, which can be applied to the study of parasite biology.

Advances in amoebic RNA interference and gene regulation
The RNA interference (RNAi) pathway is an important basic biological process for regulating gene expression and genome stability as well as a robust tool for genetic manipulation 34-36 . Multiple pathways exist for biogenesis and function of small RNAs; however, all mature small RNAs ultimately associate with an Argonaute (Ago) protein to form an RNA-induced silencing complex, which mediates gene silencing 37-39 . Silencing occurs via target RNA cleavage, translational repression, or transcriptional gene silencing (TGS) 40 . In the case of TGS, RNAi components mediate gene silencing by recruiting histone modification enzymes to targeted loci. Post-translational modifications of the amino terminal tails of histones alter the condensation state of chromatin, regulating the accessibility of DNA-binding sites for components of the transcriptional machinery 41 .
Studies in model systems have provided much of what is known about RNAi 42,43 , although data from non-model organisms have uncovered important variations 44-46 . E. histolytica has a robust and non-canonical endogenous RNAi pathway, which regulates gene expression 44,47 . Entamoeba has an abundant population of 27nt small RNAs that have 5′-polyphosphate (polyP) termini, indicating that they are not Dicer products-an observation made only in amoeba, Caenorhabditis elegans, and parasitic nematodes 43,44,48 . The repertoire of non-canonical RNAi proteins was recently expanded with the characterization of EhRNaseIII, a minimal and non-canonical Dicer-like protein in E. histolytica. Having a single RNaseIII domain and devoid of all domains typically associated with Dicer enzymes in other systems, EhRNaseIII is capable of processing double-stranded RNA into smaller RNA fragments that productively contribute to gene silencing 49 .
Investigating the endogenous RNAi pathway in Entamoeba, Morf et al. 50 discovered that a gene to which abundant small RNAs map can "trigger" silencing of other genes fused to it 51 . This has been an important advance for the community, as methods for genetic manipulation in Entamoeba have previously been limited and technically challenging. Recently, the trigger-silencing approach was adapted for use in E. invadens, which should allow this to be a robust system for genetic analyses of developmental pathways and developmental control 52 . More importantly, in E. histolytica, the trigger-silencing approach was used to demonstrate that RNAimediated gene silencing in amoeba involves repressive epigenetic histone modifications 53 . Future efforts to dissect the machinery responsible for the RNAi and trigger-gene silencing will be important as a means to improve and refine the silencing methodology and to identify aspects of the machinery that may be novel to Entamoeba.

Mechanistic understanding of transcriptional gene silencing in Entamoeba
It has been demonstrated in E. histolytica that genes targeted by small RNAs are effectively silenced. The ability to silence genes in E. histolytica was accidentally discovered following efforts to overexpress an amoebapore gene (Ehap-a); this strain, in which Ehap-a is permanently silenced, is the G3 clone 54 . However, the mechanism by which TGS is initiated and maintained in Entamoeba remained unclear. Huguenin et al. first proposed a role for chromatin remodeling in the regulation of gene expression in the G3 clone of Entamoeba 55 . They reported a decrease in methylation of lysine 4 of histone 3 (H3K4) and an overall enrichment of H3 near transcriptionally silent loci. Subsequent analyses revealed that the 27nt small RNAs mediated the silencing of the Ehap-a gene observed in the Entamoeba G3 clone 48 . The small RNAs were shown to have nuclear localization, and an enrichment of EhAgo2-2 was found in close contact with silenced gene loci 48 .
Recently, Foda et al. identified the first epigenetic histone modification involved in RNAi-mediated TGS in E. histolytica 53 . Using a trigger expression vector to induce the expression of small RNAs to a transcriptionally active gene, they observed dimethylated H3K27 (H3K27Me2) deposition at both episomal and chromosomal gene copies. Importantly, their model links RNAi gene silencing and repressive histone-mediated TGS in Entamoeba. Additionally, other active and repressive epigenetic modifications for Entamoeba histones have been described. Interestingly, unlike most eukaryotes, both activating and repressive post-transcriptional histone modifications co-localized, suggesting that the nuclear organization of Entamoeba is atypical 56 . This is not surprising and is consistent with observations that Entamoeba's replication cycle and chromatin organization are characteristically unique 57 .
Toward identifying the biological significance of RNA interference in Entamoeba One aspect that continues to elude clarification is the biological impact of the RNAi pathway in Entamoeba. In other systems, RNAi is reported to regulate diverse biological processes 58 . In Entamoeba, the RNAi pathway has been reported to silence genes relevant to virulence and thus contributes to strain-specific virulence profiles 59 . In an attempt to identify the biological conditions under which gene expression is regulated by RNAi, Zhang et al. 60 investigated the possibility that RNAi in Entamoeba is involved in regulating gene expression in response to oxidative or heat stress or in parasite stage conversion.
In their report, Zhang et al. analyzed small RNA profiles from E. histolytica trophozoites subjected to oxidative stress or heat shock. Although robust small RNA populations are present in each condition, the small RNA populations and the genes to which they map did not change abundance or expression under the various stress conditions. To determine whether the small RNAs controlled gene expression relevant to stage conversion, they generated and sequenced 27nt small RNA libraries from encysting and excysting E. invadens parasites. Similar to E. histolytica, genes targeted by small RNAs in E. invadens are silenced. However, somewhat unexpectedly, they demonstrated that the 27nt small RNAs do not appear to regulate genes that change expression during stage conversion 60 . Adding to the genomic complexity already observed in Entamoeba, there are some notable differences in the small RNA profile between E. histolytica and E. invadens. E. invadens had a larger percentage of small RNA reads mapping to intergenic regions, retrotransposons, and repetitive elements while having a smaller percentage of small RNA reads mapping to open reading frame. Zhang et al. speculated that the large percentage of small RNAs that map to retrotransposons and repetitive elements suggests that RNAi in E. invadens may have roles in preserving genome integrity and stability.
Thus, despite extensive efforts, to date no biological condition(s) that are regulated by RNAi in Entamoeba have been identified. However, maintenance of the pathway across multiple Entamoeba species and the conservation of genes silenced by RNAi indicate a strong selection pressure and an important biological role for this phenomenon in amoebae. It is interesting to speculate that, in this unique parasite, RNAi is central to the flow and regulation of information that go beyond those involved in transcriptional silencing. Growing evidence in fungi, plants, and animals suggests that RNAi plays important roles in regulating numerous nuclear processes, including transposon regulation, heterochromatin formation and propagation, and genome stability 61 . Additionally, it is possible that amoebic small RNAs serve to mediate intercellular communication via their transfer in exosomes or extracellular vesicles or both, as noted in the parasitic nematode Heligmosomoides polygyrus 62 . Given that the parasite maintains a complex and robust endogenous RNAi pathway, there is no doubt that much is yet to be uncovered about its impact on parasite biology.
The regulation of biological processes by RNAi does not need to be confined exclusively to small RNAs. Emphasis thus far has been on the 27nt small RNA population in Entamoeba. However, diverse non-coding RNA species have been found to regulate biological processes in other systems, such as the discovery that long non-coding RNAs (lncRNAs) function as scaffolds to regulate the expression of a large subset of genes in mammals 63 and are centrally involved with X chromosome inactivation 64,65 . As such, there exists a need to expand the breadth and scope of investigations for amoebic processes that are potentially regulated by other non-coding RNA species. As an example, cell division in Entamoeba is poorly understood, especially related to our understanding of the mechanism regulating chromatin pairing and segregation during cell division. Entamoeba exist as multinucleated polyploid cells in vitro and in vivo 66 , suggesting that the parasites are able to mitigate issues arising due to gene copy number. Given the atypical cell cycle in Entamoeba 33 , one could be enticed to hypothesize that amoebic RNA is involved in genome stabilization during cellular division. Consistent with this hypothesis, growing evidence suggests that long and small RNAs can serve as an alternative to DNA-binding proteins for epigenetic regulation of gene expression 58 . Notably, the Bhattacharya group discovered the first lncRNA in Entamoeba 67 and described its role in mediating stress responses. Further research on amoebic lncRNA is poised to help address remaining questions in the field.

New drugs against amoebiasis
At present, there are no vaccines available against amoebiasis. Currently, metronidazole is the drug of choice and is used worldwide to treat invasive amoebiasis in both adults and children [68][69][70][71] . In addition, the luminal amoebicide paramomycin is administered to eradicate cysts from the colon 69,70 . Other nitroimidazole derivatives such as tinidazole and ornidazole have improved dosing schedule with a single 2 g dose once daily for three days 69,70 . Marie and Petri highlighted the use of available anti-amoebic drugs by comparing their efficacy and safety 11 . However, metronidazole has significant associated side effects, including nausea, vomiting, and headaches; furthermore, it has been found to be mutagenic in bacteria and carcinogenic in experimental mammalian models at high doses over long periods 72,73 . Thus, there is an important need for the development of specific, novel, and safe drug(s) to treat amoebiasis.
In an important advance in this direction, Debnath et al. developed an automated high-throughput drug screen and discovered that auranofin is potent against E. histolytica trophozoites both in vitro and in vivo and also is effective against E. invadens cysts 74-76 . Auranofin is a US Food and Drug Administrationapproved oral, gold-containing drug that has been in clinical use to treat rheumatoid arthritis for the last 25 years 76 . It is reported to be effective against several other protozoan parasites 77 such as Giardia lamblia 78 , Trypanosoma brucei 79 , and Plasmodium falciparum 80 and kills the promastigote stage of Leishmania infantum 81 in vitro. It also kills other human parasites, such as Schistosoma mansoni, which causes the disease schistosomiasis 77 , and also is effective against larval worms Echinococcus granulosus 82 . Oral administration of auranofin significantly decreased the parasite number in both the amoebic colitis and the liver abscess models, suggesting its great potential as an anti-amoebic drug 74,83 . Transcriptional profiling identified E. histolytica thioredoxin reductase (EhTrxR) protein as a target of auranofin. It is proposed that a monovalent gold atom, Au, is released from auranofin, subsequently inactivating the EhTrxR protein, which interferes with the redox homeostasis in the parasite and subsequently makes the parasites more sensitive to reactive oxygen-mediated cell killing 84 . Jeelani and Nozaki recently highlighted this unique thiol-based redox metabolism system as a drug target against amoebiasis 85 . E. histolytica trophozoites are microaerophilic in nature and prefer the microaerobic environment in the lumen of the large intestine. However, once trophozoites invade the tissue barrier, they are exposed to a high-oxygen environment 86 . Being a microaerophilic organism, E. histolytica does not contain most of the elements required for eukaryotic oxidative stress defense systems, including catalase, peroxidase, glutathione, and glutathione-recycling enzymes 85,87,88 . However, Entamoeba has several other key redox regulators, which are unique to Entamoeba and which show promise as drug targets 85 . In another approach, Boyom et al. repurposed the open access malaria box compounds and identified effective compounds against E. histolytica 89 , although further characterization and validation are needed before they can be identified as valid drug candidates against amoebiasis.
Efforts have been made by several groups to develop anti-amoebic drugs from natural resources, particularly from plants or herbs which were used in traditional remedies against amoebic infection [90][91][92] . Recent screening of a natural products library from fungi and actinomycetes sources against E. histolytica identified several cysteine synthase inhibitors 93 . However, the toxicity of these compounds against human cell lines precluded their use as an alternate to metronidazole.
In another approach, Shahinas et al. used a target-based drug screen for Entamoeba heat shock protein 90 (HSP90) inhibitors and identified five compounds (rifabutin, rutilantin, cetylpyridinium chloride, pararosaniline pamoate, and gentian violet), which inhibited E. histolytica growth in the micromolar range 94 . HSP90 is an essential chaperone-like protein and a good drug target candidate as the N-terminal ATP-binding domain is structurally diverse and presumably specific for individual organisms. Thus, inhibitors that are highly functional against Entamoeba HSP90 could be highly unique and specific.
The recent interest and efforts to identify new compounds against Entamoeba are important advances. Ideally, drugs that target multiple microaerophilic protists could be identified (as with the recent identification of auranofin) 74 , as a one-drug/multiple-bug approach is most likely to be effective, especially in developing countries where disease burden is high and resources are limited.

Effect of gut microbiota in amoebiasis
With the recent explosion of data on the human microbiome, it makes sense to dissect the interplay between Entamoeba, a gut resident, with the human colonic microbiome. Epidemiological data suggest that over 500 million people are infected by E. histolytica worldwide 95 . Interestingly, not all individuals are equally susceptible to Entamoeba infection; 90% of the infected individuals are asymptomatic carriers, whereas the remaining 10% show serious intestinal and extra-intestinal diseases such as colitis, dysentery, and amoebic liver abscesses 1 . One possible explanation for this observation is the difference in immunity and gut microbiota between individuals. Recently, Burgess and Petri extensively reviewed the role of microbiota in E. histolytica infection 96  More recently, Burgess et al. demonstrated that introduction of commensal bacteria alters the mucosal immune system and reduces the susceptibility of mice to amoebic infection 102 . Thus, alteration of the gut microbiota provides protection against infection by E. histolytica 102 . In a separate report, an association of anaerobic Gram-negative bacteria, Prevotella copri, and Entamoeba was observed in infected children 97 . P. copri is associated with gut inflammation and the generation of excessive immunity in patients and in animal models, which may facilitate the ability of Entamoeba to establish a productive infection and influence the progression to invasive disease 97,103 . These data suggest that E. histolytica infection is influenced by the inflammatory state of the gut, which is potentially associated with changes in the gut microbiome. These studies may provide insights into why some patients get invasive disease, the factors that contribute to parasite-associated malnutrition and growth inhibition, and how intestinal parasites may change gut flora and impact the systemic immune response.

Summary
In this review, we have highlighted recent advances that have contributed to improving our understanding of biological processes central to the human pathogen E. histolytica. A better understanding of the molecular mechanisms regulating gene expression have been achieved, aided in part by the development of the RNAi trigger-silencing approach. The emerging role of gut microbiota in amoebiasis has only begun to be addressed, leaving many tantalizing questions to be answered. In addition, the repurposing of compounds to identify new drugs against E. histolytica shows great potential to improve the treatment of amoebiasis, a neglected tropical disease. Given the many unique aspects of this parasite's biology and its ongoing impact on human health, much remains to be resolved.

Competing interests
The authors declare that they have no competing interests.

Grant information
The author(s) declared that no grants were involved in supporting this work.