**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* [53–56], 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 developmentspecific 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 [64–67]. 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 ligandbinding 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

*Perspective Chapter: Parasitic Platyhelminthes Nuclear Receptors as Molecular Crossroads DOI: http://dx.doi.org/10.5772/intechopen.102648*

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.
