Perspective Chapter: Parasitic Platyhelminthes Nuclear Receptors as Molecular Crossroads

Abstract

Thanks to the increasing availability of the parasitic Platyhelminthes genomes in recent years, several studies have been directed to the identification of the nuclear receptors set expressed by these organisms. Nevertheless, important gaps in our knowledge remain to be addressed, concerning their mechanism of action, ligands, co-regulator proteins, and DNA binding sequences on target genes. The proposed review chapter will be an account of research into the nuclear receptors field of parasitic Platyhelminthes. Several in vitro effects of host steroid hormones on Taenia and Echinococcus species were observed, however, the classical mammalian estrogen, androgen, or progesterone receptors could not be identified in databases. Nonetheless, novel nuclear receptors and related proteins and genes, are being identified and characterized. The elucidation of their target genes as well as ligands in parasitic Platyhelminthes could allow discovery of new and specific pathways differing from those of their hosts. In this sense, these parasitic proteins seem to be good putative targets of new drugs.

1. Introduction

Since the biochemical identification of the first nuclear receptor (NR) more than 60 years ago [1], the study of these proteins has been increasing. In particular, the cloning of the first NR was a milestone [2], ushering in a new chapter in research into the regulation of cell function and metabolism [3]. NRs are transcription factors that modulate numerous physiological processes such as metabolism, development, reproduction, and inflammation [4, 5, 6], through the regulation of target genes transcription by binding to specific DNA response elements [3, 6]. Unlike other transcription factors, the activity of nuclear receptors can be modulated by the binding of specific ligands, these being mainly small lipophilic molecules that easily penetrate biological membranes [7], providing a direct link between the cellular signals and the transcriptional responses of a cell. These lipophilic ligands can be fatty acids, steroids, retinoids, and phospholipids. This protein family also contains “orphan” members for which no ligand has yet been identified [8].

Despite the diversity of functions presented by the different NRs, they share a common modular structure, with various degrees of conservation among their respective domains. A typical nuclear receptor contains an N-terminal domain (NTD or A/B region), a highly conserved DNA binding domain (DBD or C region), a poorly conserved hinge domain (region D), a ligand-binding domain (LBD or E region), and a C-terminal region (F region) [9, 10]. The A/B region is a poorly structured domain that shows a low percentage of conservation at size and sequence level and may not be present in some NRs [6]. This domain is regulated by the interaction with co-regulatory proteins and also contains an autonomous transactivation region 1 (AF-1, Activation Factor 1) independent of ligand binding [11]. The DBD is the most conserved region compared to the other domains [12] and it is responsible for the binding of the NR to specific DNA sequences, named response element (RE) [13]. Structural studies have determined that the DBD has two subdomains that each contain four cysteine residues that coordinate a zinc ion to create the typical DNA-binding zinc finger motif [14, 15, 16]. The hinge domain is the region with the lowest sequence conservation and it constitutes a flexible linker between the DBD and the LBD [10], giving the connecting domains some independent mobility [17]. The LBD regulates the receptor activity through ligand binding and direct interaction with co-regulatory proteins [18, 19]. This region contains functionally related interaction surfaces: a dimerization surface, which mediates interaction with another LBD [20]; a hydrophobic ligand-binding pocket (LBP) that interacts with lipophilic small molecules [21]; and an activation function surface called AF-2 (Activation Function-2), essential for the ligand-dependent transcription activation [22, 23]. Finally, the F domain is a poorly conserved region, and even many members of the family lack this domain. However, when this domain is present, its deletion or mutation alters transactivation, dimerization, and the receptor response after ligand binding [24].

More than 900 nuclear receptor genes have been identified throughout the animal kingdom [25, 26]. The NRs have a common ancestral origin and a high conservation rate in all animal taxa and therefore are considered strong phylogenetic markers of animal evolution [27]. This protein group shows an interesting complexity probably driven by gene duplication and gene loss [28], for example, 2 members have been identified from sponges, 48 in mammals and up to more than 250 in nematodes [29, 30, 31, 32]. Phylogenetic studies demonstrated that NRs emerged long before the divergence of vertebrates and invertebrates, during the earliest metazoan evolution [33]. The nomenclature currently used to name the NRs is based on phylogenetic relationships, generated from conserved DBD sequence alignment and the construction of phylogenetic trees. This classification, which was approved by the Nuclear Receptor Nomenclature Committee in 1999 [34], subdivides the nuclear receptor family into six subfamilies (NR1-NR6). The subfamily NR0 was added later and includes atypical nuclear receptors that contain only DBD (NR0A, identified in arthropods and nematodes) or only LBD (NR0B, present in some vertebrates) [34]. In the last decades, the existence of a new NR subgroup called 2DBD-NR was evidenced in parasitic Platyhelminthes; whose members present two DBDs and one LBD [35, 36]. This new group has not yet been included in the classification system described by the NR Nomenclature Committee [21, 34]. However, recent publications already classify it as a subfamily NR7 [37]. Furthermore, nuclear receptors can be classified according to their mechanism of action into four types (I-IV). This classification groups the NRs according to the signaling mechanisms, taking into account the subcellular site where NR-ligand binding occurs (cytosol or nucleus) and the mode of DNA binding (homodimer, heterodimer, or monomer) [6]. Briefly, type I NRs reside in the cytosol and upon ligand binding are trafficked into the nucleus where they typically bind to palindromic REs in promoters as a homodimer. Type II NRs are localized in the nucleus and generally form heterodimeric complexes with RXR; in their unliganded state, are inactive and upon ligand binding, they activate by the co-regulators exchange. Type III NRs are similar to Type II, however, these receptors bind to direct repeat REs as homodimers. Type IV NRs have a similar mechanism of action to Type II and III NRs but instead, bind to DNA as a monomer and recognize extended half-sites within RE [6].

Platyhelminthes are a phylum of bilaterian, unsegmented, soft-bodied invertebrates, but also, they are acoelomates and lack specialized circulatory and respiratory organs. These characteristics make these organisms have a flattened shape that allows the exchange of gases and nutrients throughout the body [38]. Platyhelminthes are traditionally divided into four classes: Rhabditophora, Monogenea, Cestoda (tapeworms), and Trematoda (flukes). The class Rhabditophora includes all free-living flatworms, while all members in classes of Monogenea, Trematoda, and Cestoda are parasitic flatworms [39]. The Platyhelminthes or flatworms include more than 20,000 species [40, 41].

Parasitic Platyhelminthes are a large group of parasites that can affect both human and animal health, causing neglected diseases such as Schistosomiasis, Paragonimiasis and Cestodiasis that can be fatal and are difficult to treat. These infections generally lead to pain, physical disabilities, etc., impeding economic development through human disability and billions of dollars of lost production in the livestock industries [42, 43].

In the last decade, the advent of genome projects has allowed the identification of the nuclear receptors expressed in the different parasitic Platyhelminthes [21, 44]. Nevertheless, only a few NRs have been characterized in these organisms and their biological function continues to be unknown. The first parasitic Platyhelminthes NRs were identified in Schistosoma mansoni [45, 46, 47, 48] and after this, NRs were identified in the genomes of 33 Platyhelminthes species [44, 49]. The number of NRs varies from 15 to 61 in Platyhelminthes, 18–23 NRs are present in Monogenea, 15–20 NRs are present in Cestoda, 21–22 NRs are found in Trematoda, and 27–61 members are identified in Rhabditophora [49]. In this chapter, we performed a systematic review of characteristic and underlying mechanisms of parasitic Platyhelminthes nuclear receptors hoping to provide directions and ideas for future research.

2. Parasitic Platyhelminthes nuclear receptors

2.1 Subfamily 1

The most characterized proteins of this group are SmTRα and SmTRβ from S. mansoni. Both proteins share the consensus structure of TR receptors, including a conserved N-terminal signature of TRs in the A/B domain as well as the specific sequence CEGCKGFFRR of the NR1 subfamily. SmTRs can form a heterodimer with RXR (SmRXR1), similarly to vertebrate members of this family [50].

Screening S. mansoni female worms using the whole-mount in situ hybridization was conducted to the identification of a gene predicted to encode a homolog of the Drosophila melanogaster nuclear hormone receptor Ecdysone-Induced protein 78c [51]. A second putative member of this group of nuclear receptors is the Smp_248100, an uncharacterized protein from S. mansoni [52]. Primary sequence analysis confirmed that Smp_248100 contains a DBD with high amino acid identity to DBDs from other vertebrates and invertebrate NRs, including HRp6 from D. melanogaster and DAF-12 from Caenorhabditis elegans [52].

In 2011, Förster and collaborators characterized for the first time a cestode NR, named EmNHR1. The isolated Echinococcus multilocularis receptor is homologous to NRs of the DAF-12/HR96 group that regulates cholesterol homeostasis and longevity in metazoans. EmNHR1 gene expression was described in all E. multilocularis larval stages that are involved in the infection of the intermediate host. The authors report that EmNHR1 is related with the TGF-beta signaling pathway and that human and bovine host serums contain a ligand that induces homodimerization of EmNHR1 LBD. Since the serum is an important component in all culture media that enables the E. multilocularis development in vitro [53, 54, 55, 56], it was suggested that this NR could play a role in host cross-communication mechanisms during infection [57].

The second NR characterized in cestodes was EgHR3 of E. granulosus. This protein contains the typical structure with a DBD and an LBD. The EgHR3 expression was especially high in the early stage of adult worm development. Immunolocalization revealed that the protein was localized in the parenchyma of protoscoleces and adult worms [58]. The authors suggested that this protein could participate in development-specific responses to ecdysteroid as was described for insects [59]. On the other hand, ecdysteroids and molecules of the ecdysteroid signaling pathway had been identified in protoscoleces of E. granulosus [60]. With this input, a genomic search allowed the identification of two sequences coding to the following nuclear receptors: E78 (GenBank accession: CDS17388.1) and FTZ-F1 (GenBank accession: CDS15732.1) [61, 62].

2.2 Subfamily 2

Several members of subfamily 2 nuclear receptors were isolated and characterized in Platyhelminthes: SmTR2/4, SmRXR1, SmRXR, HNF4, and HR78. SmTR2/4 is a protein of 223 kDa with extremely large A/B and hinge domains. It shares sequence identity with the DBD of other members of this group of NRs ranging from 69 to 88%, while with de LBD shares from 16 to 38% of similarity. The corresponding gene is expressed in all S. mansoni developmental stages. SmTR2/4 might play a role in the regulation of schistosoma female reproductive development [63].

Homologous proteins of vertebrate retinoid-X-receptor (RXR) were identified in S. mansoni being classified as NR2B4-A and NR2B4-B [64, 65, 66, 67]. Vertebrate counterparts can heterodimerizate with thyroid hormone receptor, retinoic acid receptor, or vitamin D receptor. They bind to DR1 to D5 response elements with the consensus sequence Pu GGTCA [68]. These receptors contain the general basic structure of the nuclear receptors. DNA binding domain sequence of both receptors shares high identity with mouse and human RXRα, and Drosophila USP receptor. Low conservation was observed when ligand-binding domain is analyzed. Long A/B, hinge domains, and C terminal tail (F domain) are characteristics of both S. mansoni receptors. Members of this group usually lack the F domain. Sequence differences in ligand-binding signature and AF-2 motif between SmRXR and SmRXR1 suggest that specific cofactors may be necessary for the transactivation activity. A low level of identity was also found comparing the DBD sequences of both receptors, strengthening the idea that these two NRs differ in the recognition of their target genes. In addition, DNA binding properties also differentiate both receptors, while SmRXR1 binds to a DR1 response element, SmRXR fails to bind to direct repeat response elements on its own. SmRXR probably binds to conventional response elements and dimerizes with SmFtz-F1 [69]. A differential regulation expression was observed between both S. mansoni receptors since SmRXR transcript is expressed at all life cycle stages with highest levels in miracidia and cercaria and much lower in female worms, while SmRXR1 seems to be constitutive.

Hnf4 expression was detected by a single-cell sequence approach in S. mansoni stem cells. RNAi experiments indicated that the gene product could be a regulator of intestinal cell proliferation. Further studies indicated that luminal microvilli were altered and the loss of cathepsin proteolytic activity, an enzyme involved in hemoglobin digestion. These results encouraged the authors to initiate in vivo trials, to assess the digestive capability of hnf4 (RNAi) parasites, finding that the treated parasites failed to ingest or digest red blood cells. Finally, mice receiving hnf4 (RNAi) parasites had morphologically normal livers in contrast to controls infected with native parasites. This key regulator of blood-feeding parasites was proposed as a potential therapeutic target to blunt the pathology caused by adult parasites [70]. Since egg deposition depends on blood digestion, hnf4 is at least indirectly required for parasite growth and egg-induced pathology in vivo.

Four more members of this family were also identified in S. mansoni by cDNA cloning of the entire DBD. They are SmTLL, SmPNR, SmDSF, SmCoup-FII [71]. The expression at mRNA level was examined in egg, adult, female, and adult male. Only SmCoup-TFII was expressed in all stages at similar levels; SmTLL expression was high at the egg stage while SmPNR and SmDSF had a very low expression compared with the other receptors [71].

Finally, the orthologues of fax-1 and NHR236 receptors were recently identified in free-living and parasitic flatworms, respectively. It is the first time that an orthologue of NHR236 has been shown to exist in parasitic Platyhelminthes [49].

2.3 Subfamily 3

For a long time, it has not been possible to identify subfamily 3 NRs in Platyhelminthes [21, 72], so this class of proteins seems to have been lost in this phylum. However, recent genome sequence analysis studies identified several ERRs (estrogen-related receptor) belonging to subfamily 3 [37, 49].

Several reports strongly indicate that host steroid sex hormones affect the biology, and in particular reproduction and growth, of parasitic flatworms. However, to date, it has not been possible to identify steroid hormone receptors similar to those of mammalian hosts in the available genomes. It was demonstrated through in vitro assays that sex steroids act directly on Taenia crassiceps (Cestoda) cysticerci proliferation and viability [73]. Host hormones 17-β estradiol (E2) and progesterone (P4) promote parasite reproduction without affecting their viability. On the contrary, testosterone (T4) and dihydrotestosterone (DHT) significantly inhibit parasite proliferation, generating a deleterious effect. When 17-β-estradiol concentrations increased, the number of T. crassiceps cysticercus buds also increased, and an opposite behavior was observed when tamoxifen (human alpha estrogen receptor antagonist) was tested in cysticerci culture [73]. However, the existence of a T. crassiceps ER-like protein (GenBank: AY596184.1) is controversial since a similar protein could not be identified in any of the published genomes of Taenia and Echinococcus species which are available in WormBase Parasite (https://parasite.wormbase.org/index.html). Although this T. crassiceps protein is not the product of contamination by host cells, functional studies are necessary to demonstrate that it is capable of binding estrogens. Undoubtedly, this parasitic flatworm would have to express one or more proteins responsible for the binding of the hormone and triggering of signaling. Finally, in 2014, two papers showed the inhibition of the survival of Echinococcus granulosus protoscoleces and Echinococcus multilocularis metacestode vesicles after an in vitro tamoxifen treatment and a pharmacological screening, respectively [74, 75]. Nevertheless, the parasitic estrogen receptors or other proteins responsible for these effects have not yet been isolated and characterized.

It was in vitro demonstrated that T. solium cysticerci treatment with P4 increases evagination and growth [76]. The P4 direct effect could be mediated by the presence of a putative progesterone-binding protein in the parasite similar to a nuclear classical progesterone receptor (PR) or a membrane receptor. A nuclear classical progesterone receptor could not be identified in Taenia spp. genomes. However, it was reported that T. solium cells expressed a P4-binding like protein exclusively located at the cysticercus subtegumental tissue. This protein named as membrane-associated progesterone receptor component (PGRMC) was identified by 2D-electrophoresis and sequencing [77]. Molecular docking showed that PGRMC is potentially able to bind steroid hormones such as progesterone, estradiol, testosterone, and dihydrotestosterone with different affinities, and the binding domain to steroids was localized in the C-terminal region. Moreover, the T. solium PGRMC is related to a steroid-binding protein of Echinoccocus granulosus (GenBank: CDS20257.1). A putative mechanism was proposed where progesterone is captured from the external environment and exerts its action upon cysticerci differentiation involving a progesterone membrane receptor and a nuclear PR-like protein [77]. It should be mentioned that the latter protein has not yet been identified in the available genomes of other taeniid cestodes.

The above-cited scientific papers point to a better understanding of the host–parasite molecular cross-communication, providing new information which could be useful in designing anti-helminthic drugs. The strategy consists in the designing of new drugs specifically directed to inhibit or block key parasite molecules, such as hormone-binding proteins, transduction proteins, transcription factors, or nuclear receptors involved in the parasite establishment, growth, and proliferation in the host. In addition, it is a requirement that the new drug specifically recognize parasite cells with minimal secondary effects to the host, so the search has to be directed toward molecules that are differentially expressed in the parasitic Platyhelminthes.

2.4 Subfamily 4 and 6

NR4A was the only subfamily 4 member identified in parasitic platyhelminths. Phylogenetic analysis suggested that it is orthologue of Drosophila, Mollusca, and human NR4A receptor [49]. The relative mRNA expression level of SmNR4A5 from S. mansoni was similar in egg, adult female, and adult male [71].

Concerning subfamily 6 of NRs, only one member of subfamily 6 (MlNR6) identified belongs to the free-living flatworm Macrostomum lignano [49].

2.5 Subfamily 5

Until now the only two receptors of subfamily 5 characterized in parasitic Platyhelminthes are Ftz-F1 (Fushi Tarazu-factor 1) NRs from S. mansoni, one called SmFtz-F1 belonging to the NR5B1 group, and the other named SmFtz-F1α classified in the NR5A3 group [48, 49, 78, 79]. In addition, the previously mentioned sequence identified in E. granulosus genome Ftz-F1 (GenBank accession number: CDS15732) also belongs to this subfamily [60, 61, 62].

SmFtz-F1 was the first member of this subfamily to be characterized from a lophotrochozoan [48]. Subfamily 5 only contains orphan receptors that bind to their response element as monomers, the most studied members being mammalian SF-1 (steroidogenic factor-1) and LRH-1 (liver receptor homolog-1), both involved in embryonic development. The first member of the subfamily was isolated from D. melanogaster [80, 81]. SmFtz-F1 has a deduced amino acid sequence of 731 residues and an apparent molecular mass of 78 kDa (GenBank accession number AF158103), while SmFtz-F1α contains 1892 residues and an apparent mass of 207,402 kDa (GenBank accession number AY665680). The length of these receptors differs from orthologue members of the family, however, both proteins conserved the general structure of the nuclear receptors [48, 78]. The hinge region of SmFtz-F1α is particularly long (1027 amino acids). The DBDs of the two NRs share an identity of 55 to 75% to other members of the family, while LBDs are less conserved but contain the typical LBD signatures of the family as well as a high identity with the AF-2 sequence [48, 78]. Both receptors exhibit the expected monomeric DNA-binding ability since the DBD recognizes an SF1 response element-like sequence. However, SmFtz-F1 recognized this response element with a different binding affinity than SmFtz-F1α. The transactivation mechanism is also different between both receptors [79]. On the other side, as was previously mentioned, it was demonstrated that SmFtz-F1 dimerizes with SmRXR [69].

Although Ftz-F1 protein and mRNA expression are detected during all life cycles, expression levels differed according to the developmental stage. The higher expression of SmFtz-F1 was observed in the larval stages of miracidia, sporocysts, and cercaria, while the protein highest level was found in cercaria, schistosomula, and male adult work suggesting a role during host invasion and adaptation. The transcription behavior of SmFtz-F1α makes a difference between the two NRs since the higher mRNA level was detected in the schistosoma egg stage. A similar gonad distribution was also observed in several Ftz-F1 homologues [82, 83].

Taken together these events, it was hypothesized that target genes of both receptors exert different roles during the parasite development and these two receptors also have different ligands or co-activators. Co-activators characterization could start to decipher the transcriptional regulation complex formed by each nuclear receptor. In this sense, the search of transcription regulators of SmFtz-F1 was performed. The transcription co-activator CREB-binding protein (CBP) homologs from S. mansoni, named SmCBP1 and SmCBP2, were characterized. SmCBP1 can interact with SmFtz-F1 and activate the transcription of a reporter gene [84]. On the other side, a specific transcriptional co-repressor protein named SmFIP-1, which interacts with the AF2-AD motif of SmFtz-F1, was identified [85].

Finally, an interesting finding was the identification of the first target gene of SmFtz-F1, the micro-exon gene meg-8.3 [86]. meg-8.3 is expressed exclusively in the worm’s esophageal gland, an enigmatic tissue that has recently been shown to play a critical role in defending the worm from host attack [87].

2.6 New subfamily 7

A very interesting finding for the biology of parasitic Platyhelminthes was the identification in S. mansoni of a new group of NRs that has two tandem DNA-binding domains and one LBD, named 2DBD, lacking in vertebrates [71]. Subsequently, members of this 2DBD subfamily have been identified in some mollusks, in Echinococcus granulosus, and other Platyhelminthes [21, 35, 36, 49]. S. mansoni expresses three 2DBD (Sm2DBDα, Sm2DBDβ, Sm2DBDγ) and homologous sequences were found in other parasitic Platyhelminthes including Monogenea, Cestoda, and Trematoda [21, 49]. 2DBD-NRs have the same P-box sequence (CEACKK) in the first DBD that is not present in another known NR [35, 71]. This characteristic P-box could determine a new target DNA binding specificity [88]. In vitro and in vivo studies show that Sm2DBDα could interact as a homodimer, not interacting with SmRXR or SmRXR1. Homodimer formation implies that four P-boxes may be involved in DNA binding. In addition, Wu and collaborators reported that the three Sm2DBDs are regulated during development and may have a differential role in the different stages [35]. Although the databases of E. granulosus (WormBase Parasite) report three Eg2DBD, our research group has cloned from protoscoleces of E. g. sensu lato, a coding sequence for an Eg2DBDα isoform (GenBank MH092994.2) not reported in existing databases. This transcript was probably originated through mRNA alternative splicing and was named Eg2DBDα.1 [36]. A bioinformatic description of this isoform, including domains structure, putative NLS signals, post-translational modifications, and a 3D model of the two DNA-binding domains, was performed [36].

Recently, molecular docking studies showed that unsaturated long-chain fatty acids, in particular oleic, linoleic, and arachidonic acids, are the Eg2DBDα.1 preferred ligands [89]. It is worth mentioning that this ligand’s preference is similar to that of the EgFABP1 protein, previously characterized and studied by our research group [90, 91]. EgFABP1 is a fatty acid-binding protein which was localized in the nuclei of E. granulosus protoscoleces cells and other subcellular compartments [92]. Parasitic Platyhelminthes FABPs are considered essential proteins for these organisms since they are not able to synthesize fatty acids de novo, so these molecules could participate in host fatty acids uptake and distribution [93]. The interaction between vertebrate FABPs and PPAR nuclear receptors was demonstrated by several reports [94, 95, 96, 97]. Taking into account the aforementioned, a model is proposed in Figure 1, where EgFABP1 could transport host unsaturated long-chain fatty acids (FA) to the nucleus and transfer its ligand to Eg2DBDα.1. In this way, Eg2DBDα.1 could homodimerize or heterodimerize with other NR and bind to specific DNA response elements to regulate the gene expression of its target genes. Since, these fatty acids are probably acquired from the parasite–host, the signaling mechanism proposed involves a possible host–parasite communication mediated by Eg2DBDα.1 and EgFABP1. In addition, it is possible that co-activator and/or repressor proteins participate as part of the transcriptional regulatory complex.

Figure 1.

Schematic model of the putative Eg2DBDα.1 mechanism of action.

Acknowledgments

This work was financially supported by the Sectorial Commission for Scientific Research (CSIC, Grants N° C206-348, and N° C112-347) (UdelaR) and the National Agency for Research and Innovation (Grant N° ANII-FCE 2017_1_136527).

Conflict of interest

The authors declare no conflict of interest.