Entamoeba Encystation: New Targets to Prevent the Transmission of Amebiasis

Amebiasis is caused by Entamoeba histolytica infection and can produce a broad range of clinical signs, from asymptomatic cases to patients with obvious symptoms. The current epidemiological and clinical statuses of amebiasis make it a serious public health problem worldwide. The Entamoeba life cycle consists of the trophozoite, the causative agent for amebiasis, and the cyst, the form responsible for transmission. These two stages are connected by “encystation” and “excystation.” Hence, developing novel strategies to control encystation and excystation will potentially lead to new measures to block the transmission of amebiasis by interrupting the life cycle of the causative agent. Here, we highlight studies investigating encystation using inhibitory chemicals and categorize them based on the molecules inhibited. We also present a perspective on new strategies to prevent the transmission of amebiasis.

(e.g., temperature, osmotic pressure, pH, and nutrient deprivation), they can pass through the stringent acidic conditions of the stomach and duodenum and reach the small intestine, where they hatch and become proliferative trophozoites. They then passively move to and colonize the large intestine and proliferate there. The proliferating trophozoites sometimes undergo progression to invasive steps that are closely associated with the clinical manifestations and pathogenesis of severe amebiasis [6,7]. Meanwhile, some proliferating trophozoites differentiate into cysts. The newly formed cysts, together with trophozoites, are then excreted during bowel movements. Only the excreted cysts are a source of infection because the trophozoites are labile to environmental assault. Hence, cysts are solely responsible for transmitting amebiasis, and blocking cyst formation halts the spread of this infectious disease to other individuals.
In this review, we focus on the transition step in the differentiation from the proliferative trophozoite into the dormant cyst: "encystation. " The life cycle of E. histolytica is essentially composed of the trophozoite and cyst stages, which are connected by encystation and excystation (see Fig 1). Encystation and excystation are fundamental cell differentiation processes and are, therefore, important from a biological as well as a medical perspective. Understanding the underlying molecular and cellular mechanisms will not only provide new insights into cellular differentiation but will also provide rationales and potential targets for the development of new preventive measures against amebiasis, such as drugs for blocking transmission (see Fig 1) [8,9]. Here, we take a medical perspective and discuss studies of Entamoeba encystation using inhibitory chemicals, and we categorize these chemicals based on the molecules they inhibit. We also present a perspective for developing transmission-blocking and prophylactic strategies against amebiasis. system [9]-when used within an optimal concentration range (20 and 0.1-10 μg/mL, respectively). Their action is abolished by β-galactosidase pretreatment [13].
These findings indicate that Gal-terminated ligands are important for Entamoeba cell aggregation, a prerequisite for encystation [12,15,16]. They also suggest the presence of a cell surface molecule that mediates cell-cell interaction via binding to Gal-terminated ligands. Both the Gal-terminated ligands and their receptors await identification. However, Entamoeba Gal/Gal-NAc lectin is a candidate for the receptor of Gal-terminated ligands because it is among the best-characterized cell surface proteins of Entamoeba [1,17], it plays important roles in various processes of E. histolytica pathogenicity-including adherence, cytolysis, invasion, and resistance to lysis by complement-and it mediates binding to bacteria, red blood cells, epithelial cells, or mucin-coated tissues [16]. Hence, it is plausible that Entamoeba Gal/GalNAc lectin also participates in encystation [16]. Nevertheless, the following important issue needs to be confirmed: how do Entamoeba Gal-terminated ligands and their receptors commit a cell to encystation? (see Fig 2).
The importance of interactions of Gal-terminated ligands and their receptors has also been demonstrated for the formation of the Entamoeba cyst wall structure. Biochemical analyses identified Jacob as the most abundant glycoprotein in the cyst wall. Jacob can bind to the cyst wall component, chitin (see the "Chitin metabolism" section), and also to purified E. histolytica Gal/GalNAc lectin. Moreover, immunoelectron microscopy revealed that Jacob is released from multiple loci to the surface of encysting E. invadens cells, while low levels of Jacob are seen on the surface of "wall-less cysts" [14]. Another biochemical analysis showed that the protein Jessie can bind to chitin via its chitin-binding domain and also self-aggregate via its unique C-terminal domain [18]. Collectively, these data indicate that a cell surface Gal-lectin, Jacob, and Jessie are important protein components in the formation of an intact cyst (see Fig 2) [ 8,9,14,18]. However, several questions still remain to be answered (see Fig 2); for example, do Jacob and its family members indeed have a Gal residue as their terminal sugar? Are there any Gal-terminated ligands other than the Jacob family? Is Entamoeba Gal/GalNAc lectin indeed a receptor for Jacob and/or other putative Gal-terminated ligands that is needed to form the cyst wall structure?
Ultimately, elucidation of the molecular and cellular mechanisms to which Gal-terminated ligands and Gal-lectin are committed will provide potential targets and help develop strategies to control encystation and also inform new mechanistic insight in encystation.

Autocrine Catecholamine System
Catecholamine is a vertebrate hormone synthesized from tyrosine and exerts crucial roles via adrenergic receptor (AR)-mediated signal transduction [19,20]. Coppi and colleagues demonstrated several lines of evidence that an autocrine catecholamine system is involved in Entamoeba encystation [21].
Catecholamines can be a substitute for ABS, a critical component in the in vitro encystation system [11], when used within an optimum concentration range; omitting ABS from the standard encystation medium decreased encystation efficiency to~30%, while the addition of catecholamines to serum-free medium dose-dependently restored the efficiency to over 90%, which can be routinely obtained in the in vitro encystation system. A biphasic effect could be observed for epinephrine and norepinephrine; for example, epinephrine showed maximal activity at 1-10 μM and 1-10 mM.
Furthermore, both β-AR and β1-AR agonists could dose-dependently restore encystation efficiency when added to serum-free medium with a maximal level at 0.1 and 1 μM, respectively. In contrast, an α-AR agonist showed no significant effect in a concentration range of 0.01-10 μM. Consistently, only β-AR and β1-AR antagonists, but not α-AR and β2-AR antagonists, showed negative effects on encystation efficiency when added to standard encystation medium or to serum-free medium supplemented with epinephrine, a catecholamine. These results suggest the participation of the β1-AR-mediated catecholamine signaling pathway in encystation.
Results obtained by quantitative and qualitative biochemical analyses also suggest that Entamoeba trophozoite cells express an AR-like molecule on the cell surface and that they can produce catecholamines that are released into the medium during encystation [21]. Together, these results indicate that the Entamoeba autocrine catecholamine system uses β1-type receptors for encystation.
Nevertheless, the intriguing finding that autocrine regulation of catecholamine release via AR-mediated signal transduction plays a role in the cellular differentiation of a unicellular parasitic organism, Entamoeba, was inconsistent in a number of ways with the reported genome sequence (AmoebaDB). No counterparts of vertebrate ARs are encoded in the Entamoeba genome. Neither are any counterparts of vertebrate enzymes involved in the catecholamine synthetic pathway. These inconsistencies raise two critical questions; why do AR ligands, agonists, and antagonists all show significant, consistent (positive or negative) effects on encystation? How does Entamoeba produce catecholamines?

Cholesteryl Sulfate Synthesis in Sulfur Metabolism
Cholesteryl sulfate (CS) is a common sulfate metabolite in mammals. Recently, CS was shown to play an important role in Entamoeba encystation [22].
Sulfur metabolism provides a variety of sulfur-containing biomolecules, such as methionine, cysteine, Fe-S clusters, sulfolipids, and sulfoproteins [23][24][25]. In Entamoeba, six sulfolipids (including CS) are synthesized as the terminal products of this metabolism [22]. Furthermore, CS enhanced encystation efficiency in the in vitro culture system within an optimum concentration range (50-200 μM). Conversely, inhibition of CS synthesis by chlorate-an inhibitor targeting ATP sulfurylase in sulfolipid synthesis-dose-dependently reduced encystation efficiency at >100 mM; this concentration range is sublethal for E. invadens trophozoite growth (IC 50 , 69.3 ± 6.6 mM). These lines of evidence imply that CS participates in the regulation of encystation and that factors involved in CS synthesis are potential targets for developing means to control encystation.
It is worth mentioning that the idea of CS playing an important role in the regulation of encystation provides an answer to the long-standing enigma of the role of Entamoeba mitosomes [22]. Entamoeba mitosomes are derived from canonical mitochondria and have largely lost typical mitochondrial functions during the course of eukaryote evolution; thus, their raison d'être and function have been a long-standing enigma (see Box 2) [22,[26][27][28][29]. Furthermore, this unique mitosome-linked feature contributes to the adaptation of Entamoeba to its parasitic lifestyle (Box 2) [22].
Hence, elucidation of the underlying molecular and cellular mechanisms modulated by CS will provide not only a new mechanistic insight into encystation but also a new paradigm for linking organellology and evolutionary adaptation to parasitism.

Hsp90
In the process of encystation, negative as well as positive factors are involved. Heat shock protein 90 (Hsp90) is such a negative regulator of encystation [30]. Hsp90 is a chaperone that is highly conserved from bacteria to mammals and plays crucial roles via interaction with cochaperones [31][32][33][34]. Protozoan parasites are, however, an exception; they maintain the conserved Hsp90 but lack some co-chaperones. Indeed, E. histolytica has a highly conserved Hsp90 but lacks the co-chaperones p23, cyclophilin 40 (Cyp40), cell division cycle 37 (Cdc37), and full-length Activator of the Hsp90 ATPase-1 (Aha1), although it does possess a novel truncated Aha1 [32,35]. Singh et al. demonstrated that the ATPase activity of purified recombinant E. histolytica Hsp90 (EhHsp90) was inhibited by 17-allylamino-17-demethoxygeldanamycin (17-AAG), a well-known inhibitor of Hsp90 with an IC 50 value of 30.9 μM [35,36]. Furthermore, 17-AAG inhibited the growth of E. histolytica trophozoites with an IC 50 value of 546 nM. These results suggest that 17-AAG targets native EhHsp90 in vivo and that EhHsp90 plays an indispensable role in E. histolytica trophozoites [35].

Box 2. Entamoeba Mitosomes Contribute to Parasitic Lifestyle Adaptation
A role of mitosomes: Recently, Entamoeba mitosomes were shown to atypically compartmentalize sulfate activation and to be necessary for CS synthesis [26]. The regulation of encystation via CS synthesis is, therefore, an important role of Entamoeba mitosomes [22].
Furthermore, interestingly, this feature is not conserved in Mastigamoeba, a nonparasitic close relative of Entamoeba; Mastigamoeba likewise compartmentalizes sulfate activation into mitochondrion-related organelles, which have diversified from canonical mitochondria, but cannot synthesize CS because the cholesteryl sulfotransferase gene is absent in the genome [22,65]. These findings suggest that a unique feature of Entamoeba mitosomes contributes to its adaptation to its parasitic lifestyle.
Hsp90 has recently been shown to play a role in encystation [30]. Encystation efficiency was enhanced by almost two-fold by pretreatment of E. invadens with 600 nM 17-AAG for 24 hours prior to inducing encystation; the concentration used is a sublethal dose for E. invadens trophozoite growth (IC 50 , 711 nM). Consistently, mRNA levels of Hsp90 and co-chaperones are dramatically down-regulated during encystation, suggesting that Hsp90 acts as a negative regulator of encystation. Hsp90, therefore, appears to play versatile roles in the parasitic life cycle of Entamoeba, and elucidation of the underlying molecular and cellular mechanisms in which Hsp90 and co-chaperones participate will inform numerous topics on parasitism as well as differentiation.

Chitin Metabolism
Chitin metabolism may provide potential targets for developing strategies to block the transmission of amebiasis because chitin is present in the cyst wall [5,8,9,15,18] and is not a component of mammalian cells [37,38]. Two independent groups published inconsistent results; one study showed that polyoxin D and Nikkomycin (structural analogs of uridine diphosphate-N-acetylglucosamine, a substrate for chitin synthase) inhibited the cyst formation in a dose-dependent manner when added to the in vitro culture (2-500 and 10-50 μg/mL, respectively) [39]. The other study showed that chitin synthase activity in E. invadens cyst lysates was not inhibited by polyoxin D or Nikkomycin even at 100 μg/mL [40]. This inconsistency raises two questions: Is the Entamoeba chitin synthase a target of polyoxin D and Nikkomycin? Is the Entamoeba chitin synthase a redundant enzyme? The Entamoeba chitin synthase may indeed be redundant because two chitin synthases are encoded in the genome (Amoe-baDB); however, the critical question of what is the target of polyoxin D and Nikkomycin remains unanswered.
The importance of the chitin anabolism during encystation is underscored by a study exploiting the Entamoeba database (AmoebaDB) and a gene knockdown approach [41]. All of the transcripts for the putative genes encoding the enzymes in this pathway are up-regulated during encystation. Moreover, the addition of a double-stranded RNA encoding part of an enzyme in this pathway, glucosamine-6-phosphate isomerase, to the in vitro culture reduces its mRNA level, resulting in significant impairment of chitin synthesis. Furthermore, the addition of glycogen phosphorylase inhibitor to the encystation-inducing in vitro culture reduced the chitin level of cysts; glycogen phosphorylase supplies glucose to chitin synthesis by the breakdown of glycogen. Nevertheless, a causal link between the level of chitin synthesis and encystation efficacy is not currently substantiated.
As well as chitin anabolism, the importance of its catabolism has been demonstrated [42]. The addition of allosamidin, an inhibitor of chitinase, to the culture delayed the progression of the early phase of encystation but could not abolish cyst formation in the in vitro culture. In addition, chitinase activity in E. invadens cell extracts prepared from the encystation-inducing in vitro culture was inhibited by allosamidin with a Ki value of 65 nM. The effect on the ability of cells to form cysts was observed within a concentration range of 80-320 μM, more than three orders of magnitude higher than its Ki value on the enzyme. It is, therefore, necessary to determine whether the target of allosamidin is chitinase. Ultimately, unraveling the involvement of chitin metabolism in encystation is needed.

Proteolytic Systems: Lysosomes and Ubiquitin Proteasome System
In eukaryotes, two proteolytic systems-i.e., the lysosome system and the ubiquitin (Ub) proteasome system (UPS)-control intracellular protein levels within optimal ranges [43,44]. In Entamoeba, these two systems play important roles in encystation as well as in proliferation of trophozoites and pathogenicity [1,45].

(1) Cysteine proteases in lysosomes
Involvement of lysosomes in encystation was shown by using specific irreversible cysteine protease (CP) inhibitors, Z-Phe-Arg-CH 2 F (benzyloxycarbonyl phenylalanyl arginyl fluoromethyl ketone) and E64; CP is known as an important virulence factor in Entamoeba [46][47][48][49]. Sharma et al. described the significant ability of Z-Phe-Arg-CH 2 F to reduce cyst formation efficiency in the in vitro encystation system [46]. Gonzalez et al. demonstrated that the addition of 20 μM E64 significantly delayed the progression of the early phase of encystation but could not abolish cyst formation in the in vitro culture [47,50]. This indicates that CPs only partially contribute to encystation. Alternatively, it implies that the E64 inhibitory effect was not fully exerted in the reported experimental conditions; during incubation, inadequately supplemented E64 was titrated by the turnover of E64-bound CPs and/or by the presence of different types of E64-sensitive proteolytic enzymes, resulting in abrogation of the inhibitory effect. In the studies using CP inhibitors, indirect effects on encystation caused by lysosome dysfunction in trophozoites also needs to be considered [51].
Among 64 CP genes encoded in the E. invadens genome (AmoebaDB), 11 CP mRNAs were up-regulated upon induction of encystation in vitro [52]. In the cysts isolated from amebiasis patients, 8 E. histolytica CP mRNAs were also up-regulated, 2 of which were counterparts of the CP genes detected in the above-mentioned 11 E. invadens genes [52,53]. Collectively, these results indicate that CP plays an important role in encystation, although the nature of this involvement requires elucidation.
(2) Ubiquitin proteasome system The UPS, another essential intracellular proteolytic system, is typically composed of sequential chain reactions, comprising Ub activation, conjugation, ligation, and elongation, as well as protein degradation in the proteasome, a protein complex containing various proteases [44,54]. This proteolytic system also has an important influence on encystation.
Eichinger and colleagues substantiated the involvement of the UPS in encystation [47]. Lactacystin, a specific proteasome inhibitor, blocked cyst formation in a dose-dependent manner with an IC 50 value of 1.25-2.5 μM when added in the in vitro culture. Enrichment of the lactacystin-inhibitable, chymotrypsin-like activity was achieved by column chromatography of the lactacystin-treated E. invadens cell lysate. The enriched fractions show trypsin-like and peptidyl-glutamyl peptide-hydrolyzing (caspase-like) activities. All three of these activities were inhibited by lactacystin at 50 μM, which is characteristic of the proteasome. Furthermore, the fractions display typical features of eukaryotic 20S proteasome core subunits-i.e., molecular masses and isoelectric points of major UPS proteins and typical structures as visualized by negatively stained electron microscopy. These studies confirm that, similarly to other organisms, the proteasome in Entamoeba is the target of lactacystin and that the proteasome plays an important role in encystation.
The transcriptional up-regulation of the Ub gene occurred coincidently with other encystation-specific genes (e.g., gene 122 and chitinase 1) 24 hours after the initiation of encystation in vitro [47]. Moreover, β-lactone and MG-132, specific proteasome inhibitors, also dose-dependently inhibited cyst formation in the in vitro culture, with IC 50 values of~1.1 and~60 μM, respectively [45]. These results further indicate that the UPS plays an important role in encystation. Nevertheless, it cannot be ruled out that overall trophozoite health is affected by proteasome dysfunction that indirectly causes decreased efficiency of cyst formation.
A gene previously identified to encode the E. invadens Ub (AF016643) [47] does not exist in AmoebaDB. Furthermore, a gene annotated as the Ub gene in the AmoebaDB (EIN_063840) does not show high amino acid sequence conservation with other Ubs from different organisms. Therefore, identifying bona fide Entamoeba Ub genes is necessary to address the important issue of how the UPS is involved in encystation.

Enolase: A Potential Target for a Monoclonal Antibody (B4F2)
Segovia-Gamboa et al. reported the isolation of a monoclonal antibody, B4F2, by screening monoclonal antibodies raised against E. invadens intact cysts that could inhibit encystation in the in vitro system [55]. The movement of B4F2's target molecule during encystation was also shown. Moreover, enolase was demonstrated to be a potential target for B4F2. These findings indicate a role of enolase, a glycolytic enzyme, in encystation; however, further study is needed to confirm whether the B4F2 antigen is indeed enolase.

Conclusions and Future Perspectives
Amebiasis is a serious public health problem; therefore, the development of novel strategies to manipulate E. histolytica encystation is important for realizing new preventive measures. Impairing the ability to form cysts can block the spread of the disease because dormancy of the E. histolytica cyst is crucial for disease transmission. This type of approach cannot directly eliminate the causative agent, the trophozoite, from infected patients, so it would not cure amebiasis. However, it could be an effective approach to mitigate the disease because high numbers of asymptomatic patients who do not require clinical treatment are unconsciously spreading the disease (see Fig 1). A combination approach of eliminating the causative agent itself and interrupting its life cycle would be effective against infectious diseases such as amebiasis and would accelerate a reduction in endemicity.
Most of the molecules described and discussed in this review are potential targets for the development of transmission-blocking strategies. Ideal targets are molecules that exist exclusively in the parasite and not in the host. However, enzymes that have human and E. histolytica counterparts can also be considered as targets if they have distinct features. These features include differences of primary structure, which can be useful for candidate selection (Table 1), and enzyme characteristics, such as substrate specificity, catalytic mechanism, and threedimensional structures, which are relevant to drug design. Another important factor is the organs and tissues in which the human enzymes are expressed because this has a large bearing on the most efficient drug delivery system. Thus, it will be necessary to differentiate various characteristics of E. histolytica enzymes from their human counterparts. The studies reviewed also describe the important molecular processes involved in encystation; however, the underlying mechanisms are not completely understood. The processes described appear to function separately, and it is necessary to determine causal connections to produce an orchestrated network for the regulation of cyst formation (Fig 2). As described in this review, chemicals that can target specific molecules participating in cyst formation can be effective; nevertheless, a major concern is whether their effects result directly from inhibition of target molecules that specifically function in encystation or indirectly because the target molecule also plays important roles in trophozoite proliferation. For example, some target molecules may be involved in fundamental processes of cell maintenance throughout the Entamoeba life cycle (see the "Proteolytic systems" section; [56][57][58]). This concern can be addressed using a combination approach of gene knockdown (gene knockout has not been achieved in Entamoeba) and supplementation with the metabolite produced by a target enzyme.
Further molecular characterization and elucidation of the underlying mechanism of encystation, both in vitro and in vivo, are essential to provide a solid scientific basis to enable translation of research knowledge into clinically useful materials (see Fig 2). As the ultimate goal, prophylactic drugs as well as transmission-blocking drugs and vaccines against amebiasis will hopefully be developed in the future.