Entamoeba Stage Conversion: Progress and New Insights.

Abstract

Entamoeba histolytica, an anaerobic protozoan, is an important global health problem. This parasite has a biphasic life cycle consisting of a dormant cyst stage which is environmentally resistant and transmits the infection, and the proliferative trophozoite stage which is motile and causes invasive disease. The stage conversion process remains poorly understood despite being central to amoebic biology. In this review, we will highlight recent progress in our understanding of Entamoeba stage conversion including dissecting transcriptome analysis in development, characterization of transcriptional networks, demonstration of epigenetic regulation, and role of small molecules that regulate Entamoeba development.

Introduction

The protozoan parasite Entamoeba histolytica causes an estimated 50 million cases of invasive disease annually and is the second leading parasitic cause of death worldwide [1]. The disease is spread by contaminated food and water and is especially common in developing countries. The infectious cycle of E. histolytica begins with the ingestion of the cyst, a non-dividing, quadri-nucleate form that is able to survive in the environment due to a protective, chitin-containing cell wall [2]. After ingestion, the cyst undergoes excystation in the small intestine to produce the invasive trophozoite form. Disease caused by E. histolytica begins with the adherence of trophozoites to colonic mucins and epithelial cells leading to asymptomatic colonization or invasive disease [3].The most common disease caused by E. histolytica is amebic colitis, but infection may spread to other organs, particularly the liver, lung, and brain [4,5]. Some trophozoites then encyst, allowing cysts to be excreted in the stool and go on to infect new hosts; the factors influencing this transition are unclear. In endemic countries, individuals can get repeated episodes of invasive disease, necessitating repeated treatment [6].

Due to the lack of an established in vitro method for encystation in E. histolytica, the established model for Entamoeba development is in the related reptilian parasite, Entamoeba invadens. In E. invadens, encystation in vitro is triggered using osmotic shock and removal of glucose or other carbon source [7–9]. High levels of cyst formation require the addition of either 5% serum, or the serum glycoprotein mucin, which acts as a ligand for the Entamoeba membrane lectin and stimulates trophozoite aggregation [10]. Regulated excystation is also established and is achieved by exposure to water, bicarbonate and bile [11]. In this review, we will discuss some recent important developments in study of E. invadens stage conversion, including advances in our understanding of transcriptional regulation and the external triggers that initiate encystation.

Transcriptomic analysis of Entamoeba development

Application of modern techniques for studying molecular changes at a large scale have been vital for enhancing our understanding of Entamoeba stage conversion. Two key studies have looked at transcriptomic changes during E. invadens stage conversion: De Cádiz et al. [12] which used DNA microarray technology, and Ehrenkaufer and Weedall et al. [13] which employed SOLiD platform RNA-seq. Both groups looked at mid-encystation (8–24h) and mature (>48h) cyst timepoints. The two groups focused on some different timepoints, with De Cádiz et al. investigating several early encystation timepoints (0.5 and 2h) while Ehrenkaufer and Weedall et al. omitted these early times but analyzed two excystation time points (2 and 8h). As a result of these differing platforms, and differing analysis, the overall percentage of genes determined to be regulated were different between the two studies: ~65% of expressed genes changed significantly in at least one encystation time point using microarray, versus ~29% using RNA-seq.

Despite these differences, clear patterns emerged from examining the developmentally regulated genes in these datasets. In addition to the expected cyst wall related proteins, numerous genes encoding potential signaling molecules such as kinases, small GTPases and phospholipase D increased expression during encystation. Additionally, both studies identified several developmentally regulated transcription factors, including a SHAQKY-Myb domain protein whose E. histolytica ortholog was previously shown to be developmentally regulated [14]. These proteins may be involved in transduction of initial encystation signals or regulation of the terminal differentiation program. Some gene families of interest and their numbers of upand down- regulated genes are shown in Table 1.

Table 1: Examples of gene families regulated during encystation in E. invadens.

For both transcriptome studies discussed (Microarray: De Cadiz et al. [12]; RNA-seq: Ehrenkaufer and Weedall et al. [13]) the total numbers of genes significantly up- or down- regulated in at least one developmental time point and the genes regulated in each category listed are shown.

Gene families	Microarray	RNA-seq
Up in encystation
TOTAL	1528	1329
Cyst wall	14	17
Protein kinases	115	81
Cysteine proteases	10	4
DNA repair	19	9
Transcription factors	26	12
BspA proteins	26	3
GTPase signaling	31	18
Down in encystation
TOTAL	2841	1681
Carbohydrate metabolism	20	27
Lipid metabolism	28	22
DNA metabolism	13	18
Virulence	8	8

Genes encoding proteins involved in carbohydrate and lipid metabolism were heavily downregulated in both datasets. This is in agreement with a published metabolic profile during encystation [15], which showed that glycolysis and most other major metabolic pathways not directly related to cyst wall synthesis were decreased during encystation. Known amebic virulence factors such as the GalNAc lectin and rhomboid protease were also found to be downregulated during encystation in both datasets. Importantly, when excystation was examined, many of these downregulated genes were found to recover expression, indicating a return to a typical trophozoite state [13].

Role of small molecules and stress response in encystation

It is presumed that diverse molecular pathways are involved in the regulation of Entamoeba encystation, where uni-nucleated trophozoites transformed into quadri-nucleated mature cysts. Here, we summarize the factors and biomolecules that influence encystation (Figure 1). Environmental factors including glucose starvation and hypo-osmotic shock are the key initiators of E. invadens encystation [9]. Among other factors, addition of 5% adult bovine serum enhances the encystation efficiency; galactose-terminated glycoproteins, such as asialofetuin or mucin, the major glycoprotein comprising the mucus layer of the colon, can substitute for the 5% serum and trigger encystation [31]. Coppi et al. reported that Entamoeba encystation was enhanced when catecholamines (epinephrine and norepinephrine) were added to the encystation medium in micromolar concentrations [32]. In the classical adrenergic signaling pathway, epinephrine is presumed to increase the production of cyclic adenosine monophosphate (cAMP). Frederick and colleagues demonstrated that dibutyryl cAMP can bypass the normal adrenergic receptor-dependent step and would efficiently substitute for the galactose ligand and stimulate E. invadens encystation, suggesting that this catecholamine ligand-receptor system acts downstream of the galactose lectin and upstream of adenylyl cyclase to increase cAMP production [33]. Other pathways that have been shown to play an important role in Entamoeba growth and encystation include Ca⁺² signaling [34,35], cholesteryl sulfate, a common sulfate metabolite in mammals [36], protein kinase C (PKC) [37], and sphingolipids [38,39]. There are multiple indications that lipid signaling is an important part of Entamoeba encystation. Many genes involved in lipid signaling are differentially expressed, including phospholipase D (PLD), which catalyzes the conversion of phosphatidyl choline to phosphatidic acid and has been linked to many important biological processes including vesicle transport and transduction of signals required for proliferation [12,13]. Ehrenkaufer et al. showed that PLD was upregulated during encystation and that inhibition of PLD inhibits stage conversion in E. invadens [13]. These pathways likely interact as calcium levels have been shown in other systems to influence activity of both PLD and PKC [40].

Figure 1: Schematic of regulators of Entamoeba encystation.

Diverse pathways are implicated in the regulation of Entamoeba encystation, where uninucleate trophozoites transformed into quadri-nucleate mature cysts. The pathway and appearance of key regulators during the different stages of encystation (at 8, 24, 48, and 72h) are depicted. Environmental factors, including glucose starvation and hypo-osmotic shock, are the key initiators of E. invadens encystation [9]. Among the signaling molecules, Gal-terminated ligands [10], catecholamine signaling [32], cyclic AMP (cAMP) [33], cholesteryl sulfate [36], Ca⁺² signaling [34, 35], PLD [13], sphingolipids [38] and NAD⁺ [28] have shown to enhance encystation. On the contrary galactose (gal), N-acetylglucosamine (glcN) [31] and short chain fatty acids [8] inhibit encystation. Among the transcriptional regulators of encystation, the roles of three transcription factors: Myb-dr, ERM-BP, and nuclear factor Y (NF-Y) [14,28,27] and their appearances at different time points of development are illustrated here. Epigenetic control by histone acetylation plays a key role in the regulation of Entamoeba differentiation. Encystation in presence of Trichostatin A blocks the activity of HDACs expressed in cysts and modifies the histone acetylation patterns allowing the expression of trophozoite genes and blocking the encystation pathway [8,30].

Besides the osmotic stress which facilitates E. invadens encystation, heat shock has been noted to have some overlap with encystation, as genes involved in cyst wall formation such as chitinase and Jacob are noted to be upregulated in both conditions [41]. Recent studies by Manna et al. showed that levels of NAD⁺ increase in heat shock in both E. invadens and E. histolytica, as well as in E. invadens encystation [28, 42]. This change regulates activity of the encystation specific transcription factor ERM-BP. Moreover, heat stress can induce the production of quadri-nucleated cyst-like structures, and multinucleated giant cells also observed due to heat stress similar to encystation, supporting the concept that heat-stress response and encystation are related [42]. Finally, a number of molecules such as galactose, N-acetylglucosamine and short chain fatty acids, have been shown to inhibit encystation [8,31]. Altogether, we have now better understanding regarding the small molecules which have an important role in Entamoeba encystation and this knowledge could be utilized to develop and in vitro encystation method for E. histolytica.

Transcription factor regulatory control and epigenetic regulation

At the structural level, the core promoters of E. histolytica contain three conserved elements: a non-consensus TATA element (GTATTTAAA), a GAAC element (AATGAACT), and an Initiator (Inr) element (AAAAATTCA) [16–18]. Furthermore, these features are conserved in E. invadens, the GAAC-like core promoter motif (GAACTACAAA) is likely a combination of the previously reported GAAC and Inr regions [19]. Regarding the basal transcription machinery, a TATA box-binding protein (EhTBP) [20] the TBP-related factor 1 (EhTRF1) [21] and the TBP-associated factor 1 (EhTAF1) [22] have been studied.

In Entamoeba, transcription factors regulate gene expression involved in many important aspects of amebic biology, including virulence, oxidative stress response, cell migration, DNA replication, genome stability and stage conversion [14,19,23–27]. So far, at least three transcription factors that control amebic development, including a Myb gene that is developmentally regulated (EhMyb-dr), an NF-Y complex transcription factor, and a novel transcription factor (ERM-BP) have been identified (Figure 1). EhMyb-dr belongs to the SHAQKY family of Myb genes and binds to a CCCCCC motif; overexpression in E. histolytica trophozoites showed a transcriptional profile that overlaps significantly with amebic cysts [14]. Nuclear factor complex (NF-Y) is formed by NF-YA/NF-YB/NF-YC subunits which bind to CCAAT motif and appears at a later time point of encystation (48h). NF-YB and NF-YC subunits were expressed during encystation, and the silencing of the NF-YC subunit resulted in reduced stability of the NF-Y complex and decreased encystation efficiency [27]. A novel transcription factor recently identified in E. invadens, Encystation Regulatory Motif-Binding Protein (ERM-BP) binds the CAACAAA motif in the promoters of cyst-specific genes in early encystation (24h). The function of ERM-BP is regulated by direct binding of the metabolic cofactor NAD⁺; binding to NAD⁺ changes protein conformation and facilitates ERM-BP binding to the DNA motif. Silencing of ERM-BP caused a decrease in the encystation efficiency and formation of abnormal cysts with defective cyst wall [28].

In addition to regulation by transcription factors, epigenetic mechanisms such as histone post-translational modifications, specifically histone acetylation, may be involved in the regulation of genes related to the encystation. Hyperacetylation induced by Trichostatin A, an HDAC inhibitor, drastically decreased the percentage of encysting cells in E. invadens [29]. It was recently determined that the negative effects of histone acetylation during encystation of E. invadens are due to down-regulation of expression of subset of genes implicated in synthesis of chitin, polyamines, gamma-aminobutyric and cyst wall proteins. Additionally, in silico analysis and experimental assays suggested that an HDAC class I protein may control those genes during encystation [30]. Together, these data indicate that differential histone acetylation patterns at the promoters of trophozoite- and cyst-specific genes is required for stage conversion (Figure 1). These findings represent an important advance to understanding how molecular signals regulate transcriptional regulatory networks that are involved in the amoebic stage conversion.

E. histolytica encystation

During human infection E. histolytica cysts spontaneously form; however, the internal or external signals that induce a trophozoite to begin the process of encystation are at present unknown. Clinical studies have shown significant variability in the numbers of cysts produced by different patients [43], and it is likely that both host and parasite factors may contribute to these differences. To date, most studies of E. histolytica cysts have been performed using either cysts directly from patients or recent clinical isolates. One of the most important of these was a proteome of Entamoeba cysts derived directly from the stools of infected children [44]. The authors identified 195 proteins which were not found in previously published trophozoite proteomic and EST datasets. Cyst-specific proteins included the cyst wall proteins as expected, but also potential signaling molecules such as trans-membrane kinases and Rab family GTPases. Intriguingly, four DNA repair pathway proteins were found, similar to E. invadens transcriptome results and suggestive of a role during nuclear division. Only parasites which have developed a cyst wall were isolated, hence proteins involved in the early steps of encystation were unlikely to be identified. However, this study was an important advance in our knowledge of cyst formation in the human pathogen.

Historically, after parasites were isolated from human stool, they were cultured in complex media which included agar or egg slants and bacteria derived from the intestinal flora. Under these conditions cultures of recent clinical isolates produce cysts, although with variable efficiency [45–48]. Researchers used these cultures to probe conditions that could enhance cyst production [45] and, more recently, to identify a transcriptome associated with cysts and encysting parasites [49]. Novel cyst-specific genes identified include a SHAQKY Myb domain transcription factor and cysteine proteases. Transitioning trophozoites to growth in axenic medium seems to have resulted in loss of this ability to undergo stage conversion [50]. This process of gradual reduction in encystation capacity as culture conditions diverge from the in vivo situation is illustrated in Figure 2. In addition to the effect of the loss of intestinal flora, it is likely that selection of parasites able to grow in axenic conditions is a contributing factor, as the addition of bacteria to axenic cultures does not cause cyst formation [50]. There have been multiple reports through the years [51–53] of induction of cyst-like structures using various divalent cations and other factors including H₂O₂ [53]. These structures are viable and stain with calcofluor, indicating that cyst wall formation has begun, but are not mature, quadri-nucleated cysts. The physiological relevance of these cyst-like structures is not clear.

Figure 2: Influence of the transition from in vivo growth to axenic in vitro conditions on encystation capacity of E. histolytica.

Parasites growing in the human colon are exposed to colonic epithelial cells, a complex bacterial flora, and numerous other factors such as mucin and short chain fatty acids. Additionally, there is phenotypic and potentially genetic diversity between individual parasites, allowing some to initiate stage conversion while others remain as proliferating trophozoites. Recent clinical isolates grown in xenic culture retain a capacity for spontaneous encystation, but this is lost in the process of adaptation to axenic growth. Cyst-like structures can be generated by addition of divalent cations, but these are not mature cysts. Examples of publications studying encystation utilizing each type of culture are listed.

Despite the challenges posed by the lack of in vitro encystation in E. histolytica, progress has been made in relating results from encystation studies in E. invadens to the human parasite. Multiple transcription factors and signaling molecules, including the above mentioned Myb protein, ERM-BP, and phospholipase D, can be shown to change expression or have functional relevance during stage conversion and stress response in both species [13,14,28,49,54]. Future efforts to further link findings in E. invadens to E. histolytica as well as attempts to develop a true model of E. histolytica encystation will be important in the future for our understanding of this vital biological process.