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

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 encystation²⁴ as well as under stress conditions²⁵. Forward-genetic screens have helped determine the target genes regulated by specific signaling transduction pathways²⁶. 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 characterized²⁹.

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 migration³⁰, Hernández de la Cruz et al. recently identified a new role for EhPC4 in regulating DNA replication and genome stability³¹. 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 machinery⁴¹.

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 silencing⁴⁹.

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 it⁵¹. 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 control⁵². More importantly, in E. histolytica, the trigger-silencing approach was used to demonstrate that RNAi-mediated gene silencing in amoeba involves repressive epigenetic histone modifications⁵³. 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 clone⁵⁴. 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 Entamoeba⁵⁵. 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 clone⁴⁸. The small RNAs were shown to have nuclear localization, and an enrichment of EhAgo2-2 was found in close contact with silenced gene loci⁴⁸.

Recently, Foda et al. identified the first epigenetic histone modification involved in RNAi-mediated TGS in E. histolytica⁵³. 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 atypical⁵⁶. This is not surprising and is consistent with observations that Entamoeba’s replication cycle and chromatin organization are characteristically unique⁵⁷.

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 processes⁵⁸. In Entamoeba, the RNAi pathway has been reported to silence genes relevant to virulence and thus contributes to strain-specific virulence profiles⁵⁹. 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 conversion⁶⁰. 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 stability⁶¹. 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 polygyrus⁶². 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 mammals⁶³ 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 vivo⁶⁶, suggesting that the parasites are able to mitigate issues arising due to gene copy number. Given the atypical cell cycle in Entamoeba³³, 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 expression⁵⁸. Notably, the Bhattacharya group discovered the first lncRNA in Entamoeba⁶⁷ and described its role in mediating stress responses. Further research on amoebic lncRNA is poised to help address remaining questions in the field.