• Echinococcus granulosus

• Homeobox genes

• microRNAs

• Fatty acid binding proteins

Alvite G, Chalar C, Martínez Debat C, Esteves A (2016) Insights into Key Molecules of Echinococcus granulosus. J Vet Med Res 3(5): 1065.

Cystic echinococcosis represents a major public health and economic issue in many countries and is considered a neglected disease by the WHO [1,2]. Five countries in South America, Argentina, Brazil, Chile, Peru and Uruguay, are classified as countries with high infection rates [3,4]. The livestock economy in this region is largely based on cattle and sheep, and dogs are often fed the viscera from home slaughter, which may be infected with larval form, continuing the cycle of the parasite to the adult stage. An FAO report shows that production losses in the Southern Cone of Latin America because of cystic echinococcosis range between 77 and 115 million dollars [5].

Ten different Echinococcus granulosus (Platyhelminth, Cestoda) strains (G1-G10) have been identified that show several differences with respect to intermediate hosts, morphology, maturation time in the definitive host, organ localization and protoscolex production by the metacestode. The most common strain is G1, which infects sheep and has a worldwide distribution [6-10]. It is not possible to be precise about differences in infectivity of the different genotypes of E. granulosus for humans [11].

The developmental process of E. granulosus has several interesting traits: the existence of sexual and asexual reproductive phases, a regeneration-like process in the neck of adult worms that gives rise to the strobilate condition, and a particularly interesting feature: the dual ability of the protoscolex to turn into an adult worm after ingestion by the definitive host or in a new metacestode within the intermediate host (secondary cystic echinococcosis) [12,13]. This fact highlights a remarkable biological plasticity.

In the span of the past twenty years, different expressed molecules from E. granulosus have been characterized [14]. Recently, significant progress has been made with E. granulosus genome data and with transcriptome and proteome analysis [15-17]. The whole of the information currently available should allow for a better understanding of the molecular mechanisms involved in parasite biology. This fact will have significant implications for the search for new ways of disrupting parasites and will contribute to identifications of new drug and vaccine targets. In the present review, we focus on the advances made with molecules significantly involved in the parasite biology and offer insights into new actors.

E. granulosus homeobox-containing genes

The homeobox, a 180 base pairs DNA sequence, encodes for a highly conserved DNA-binding motif known as the homeodomain. Transcription factors with the homeodomain regulate developmental programs by activating or repressing diverse groups of target genes in a temporal, spatial and tissue specific manner. Most homeodomains are 60 amino acids in length, although some are larger because of insertions. The domain is globular and consists of three defined α-helices, a fourth more flexible helix and an N-terminal extension. Helices I and II lie parallel to each other and across from the third helix, which is the recognition helix that confers the homeodomain DNA-binding specificity [18,19]. The amino acid in the position 9 of the third helix (position number 50 of the homeodomain) provides critical high-affinity contacts with bases in the target site and serves to classify the homeodomain [20,21]. Other residues seem to be essentially invariant and constitute the signature of the homeodomain: leucine (Leu16), phenylalanine (Phe20), tryptophan (Trp48), and phenylalanine (Phe49). However, they can be substituted with amino acids of similar properties. The N-terminal arm sequence offers considerable variation, but basic residues are often present and achieve contacts with bases in the minor groove, aiding the binding specificity. Since the consensus homeodomain sequence was originally described [20], many genomes have been sequenced, and more divergent homeodomains have been encountered. However, compared to the previous profile, the overall pattern of amino acid conservation remains essentially the same, although individual positions now show more variability due to more divergent sequences [22].

Homeobox genes have been subdivided in various ways, but the classification schemes generally share the goal of seeking the evolutionary diversification [22,24]. A more recent comprehensive categorization of human homeobox genes provided a framework in which homeobox genes are classified into 11 homeobox gene classes (ANTP, PRD, LIM, POU, HNF, SINE, TALE, CUT, PROS, ZF and CERS), subdivided into gene families[18]; 96 homeobox gene families are thought to have existed at the origin of Bilateria [24]. Gene families are defined to contain all genes that derive from a single gene in the most recent common ancestor of bilaterian animals, and a gene class contains one or more gene families [18]. This classification scheme may be widely applicable to homeobox genes in other animal genomes [22]. It is now known that there are approximately 100 homeobox genes in protostome species. The completion of the genome sequence of the nematode Caenorhabditis elegans has shown 103 homeobox genes, of which 70 are orthologous to human homeobox genes [25] and can be grouped into similar classes. As with the human homeobox genes, the ANTP class is the largest among them and is subdivided into HOXL (Hox-Like) and NKL (NK-Like). The first group encompasses genes most similar to the Hox genes, those genes found in the “Hox cluster”. These genes are expressed in particular subdomains along the anteroposterior axis so that they specify segment identity. The order in which individual Hox genes are expressed along the anteroposterior axis of the embryo reflects the physical order of the Hox genes within the Hox cluster in the genome, a characteristic known as spatial collinearity [26-28]. Collinearity may also be temporal: the genes at the 3’ of the cluster are expressed before those at the 5 ‘end [29]. The Hox cluster can be subdivided into three groups according to the gene position and gene function, both in Drosophila and mammalian species: the anterior, medial and posterior genes. It has been suggested that clustering is an intrinsic property of Hox genes. Nevertheless, cloning of Hox genes from many bilaterians revealed a variety of cluster structures as well as cases with a complete absence of clustering [30]. Furthermore, it has been argued that the integrity of the gene complex is not an absolute requirement for the establishment of Hox expression patterns [28]. The Hox cluster is not the only homeobox gene cluster. There is a smaller cluster with three genes, called the ParaHox cluster, grouped with a few additional Hox-related genes into the HOXL genes. Additionally, there is the NK cluster initially discovered in Drosophila (see below).

The search for homeobox genes from tapeworms started many years ago [31,32]. Few studies and sequences concerning homeobox genes in tapeworms were added over the course of many years, but it has been clear since near the beginning that tapeworm have Hox genes and related families, as other members of Lophotrochozoa [33]. However, some genes that were proposed to be present in the ancestral lophotrochozoan were not found in initial tapeworm surveys. Hence, it was suggested that its reduced complement compared to other lophotrochozoans was caused by gene loss [34]. Recently, great efforts have been made to complete the genome of some tapeworms [35-37], and their precise complement of genes is now available. This greatly facilitates the identification and characterization of homeoboxes genes and their genomic arrangement. Genomic surveys have shown a loss of 24 homeobox gene families in parasitic flatworms and a supplementary loss of 10 families in tapeworms compared with the set of 96 homeobox gene families expected at the origin of Bilateria. This genomic character was interpreted as an adaptation to the parasitic life style [36]. Among the 10 specific homeobox genes that were lost by the tapeworms (Gbx, Hox2, Mnx, Bari, Msx, Hbn, Six4/5, Pax3/7, Phox and Rax), most are implicated in nervous system development.

Regarding Hox genes, a genomic study that gave the first genome-wide picture on the Hox gene family in a flatworm (Platyhelmithes) identified nine Hox genes in seven orthologous groups. The sequences found were homologous to the Hox1, Hox2, Hox3, Hox4, Lox5, Lox4 and Post-2 genes [38]. The same study provided evidence that Hox2 and Hox4 orthologs were located close together on the same chromosome. A very new overview described that in Platyhelminthes the Hox gene set is reduced with Hox5, Hox7, Lox2 and Pst1 being absent. The lack of these genes even in free-living flatworms suggests that their absence would not be related with the parasitism [24]. Cestoda adds the lack of Hox2, hence the tapeworms’ Hox gene set is formed by Hox1, Hox3, Hox4, Lox5, Lox4 and Post2 [24,34,39]. Genome studies in parasitic flatworms have shown that the Hox genes cluster is disrupted. It has been proposed that the parasitic lifestyle and/or the presence of transposable elements could explain this trait [24 and references there in]. RNA-seq data show the presence of non-Hox coding regions flanking the Hox genes in the Hymenolepis genome, confirming the lack of the cluster of flatworm Hox genes [40].

The expression of Hox genes in parasitic flatworms is mainly known from quantitative PCR and RNA-seq data and this points to dynamic patterns throughout their complex life cycles [40]. Expression patterns of Hox genes are virtually unknown in tapeworms. The only expression study known so far was performed in Mesocestoides corti larval stage by PCR [34]. Furthermore, neither these genes along nor any homeobox genes are represented in E. granulosus EST databases, which may indicate that they are not expressed at those particular stages or are expressed at low level. To address these issues, it would be necessary to use in situ hybridization tools to study spatiotemporal expression of individual Hox genes. Their characterization would allow understanding of their roles in the E. granulosus life cycle, also giving insights into their eventual participation in bidirectional development and/or strobilation. Likewise, it could be known if collinear expression of Hox genes is maintained in the absence of the cluster, as has been shown in other animals.

In addition, the NK subclass of homeobox genes was originally recognized by Kim and Nirenberg [41] in a Drosophila genomic library with degenerate oligonucleotides derived from the homeodomain sequence. Four new genes were identified as NK1 to NK4 and were initially placed by Bürglin [20] into two groups: NK-1, which contains NK-1, and NK2, which contains NK2 to NK4. The NK2 genes have become known by their mutant phenotypes: ventral system defective (vnd) for NK2, bagpipe (bap) for NK3, and tinman (tin) for NK4/msh-2. The homeodomains of the NK2 proteins are characterized by presenting a tyrosine at position 54 that is found neither in the NK-1 group nor in any other homeodomain. The classification and nomenclature of NK genes have since been modified with genes contributed by different phyla. For this reason, they commonly appear grouped in different ways, and there are even several denominations for a given gene. According to the systematization published by Holland et al. [18],the human NKL subclass comprises numerous families classified based on homeodomain sequence conservation and additional motifs that contribute to DNA binding and interaction with other proteins. Among others, the Nk1, Nk2.1, Nk2.2, Nk3 and Nk4 gene families can be recognized.

The molecular mapping of Drosophila NK genes has allowed for the definition of a new cluster, named 93DE, which has six genes. Like the Hox cluster, it is located on the right arm of the third chromosome. Not all the Drosophila NK genes are contained within the 93DE cluster; two of them are located elsewhere in the genome [42]. In addition to coding for the homeodomain, all members of the 93DE cluster encode for a short sequence motif located at the amino-terminal region. This domain is similar to the repressive domain Eh1 originally found in the family of transcriptional repressors Engrailed (En) that was renamed as the TN domain [43]. Additionally, these proteins display another preserved motif at the carboxyterminal of the homeodomain called specific domain NK2 (NK2-SD) (Harvey 1996). A limited group of NK genes also contains the so-called acid box that encodes for an acidic domain located before the N-terminal homeodomain region [44]. The commonality among 93DE genes is that they participate in mesoderm differentiation programs [45,46]. In this regard, it has been suggested that NK genes could represent an ancient mesodermic clustering whose chromosomal organization was retained due to a common regulation [47]. However, NK homeobox gene clustering is not conserved in other taxa although total dispersion has not taken place because Tlx-Lbx and NK4-NK3 gene pairs remain linked in chordates [48]. NK subclass members play essential roles in tissue differentiation and organogenesis in vertebrates, and they are involved in mesoderm derivatives specification, as well as in Drosophila [49].

In flatworms, the first searches for homeobox containing genes were performed by DNA hybridization and genomic or cDNA PCR amplifications. These approaches revealed the presence of NK subclass homeoboxes in free living as well as parasitic flatworms, most of them in the NK2 family [32,50]. Identification of these genes was supported by the presence of characteristic amino acidic residues as well as intron position and phylogenetic analysis. Interestingly, NK2 platyhelminth proteins lack conserved peptide domains outside the homeodomain. This also happens with C. elegans NK2 proteins; therefore, it was argued that this should be indicative that the ancestral NK gene did not present any extra conserved domain [51].

A phylogenetic study was performed with all platyhelminth NK genes available. Homeodomain sequences were retrieved from GenBank after querying with prototypical Drosophila NK proteins and only complete homeodomains were considered for analysis. Approximately 30 complete sequences were rescued that could be grouped into NK subfamilies and classified as belonging to NK-1 (7 genes), NK2 (4 genes), NK-3 (5 genes), NK-4 (7 genes), and other NK (4 genes) [52].

In the beginning, the search for homeobox sequences in E. granulosus led to the identification of three genes belonging to the subclass NK. These were named eghbx1, eghbx2 and eghbx3 [32,53]. The eghbx1 homeodomain was similar to S59/NK1 from Drosophila; therefore, it is grouped with NK-1. eghbx2 and eghbx3 homeodomains show high homology compared to NK3/ bagpipe and NK4/tinman (both NK2 genes from Drosophila) and include Phe8 and Glu17, as well as the typical Tyr54. It should also be noted that the eghbx2 and eghbx3 homeodomains, like that of planarian Dth-2, are interrupted between positions 44-45 by the presence of an intron. The location of this intron is shared with several other homeobox genes in Drosophila, C. elegans and mouse. Its presence in flatworms would indicate, according to Garcia-Fernandez et al. [50], the common origin of these genes from an ancient branch of homeobox genes.

Expressions of eghbx1, eghbx2 and eghbx3 genes were studied in the metacestode and adult stages using several methodological approaches. eghbx1 expression, as a single transcript, was detected by Northern blotting in protoscoleces; in situ hybridizations showed the presence of eghbx1 mRNA in both protoscoleces and hydatid cyst germinal layer, with a diffuse pattern in both cases [53]. These results suggested that eghbx1 expression may be associated with a cell type (or types) widely distributed in the whole larval stage. When eghbx3 expression was studied in protoscoleces by whole mount in situ hybridization, the signal was clearly detected at the stalk (protoscolex binding region to the hydatid cyst germinal layer). The expression was particularly strong at the end of the stalk near the protoscolex. Thus, the eghbx3 transcript is associated with a specific region and could play a role in determination and / or differentiation of stalk cells [54]. eghbx3 expression was also found in adult worms’ genitalia [52].The eghbx2 expression was analyzed by RT-PCR, in situ hybridization, immunohistochemistry both at the optical and ultrastructural level, and Western blot studies. RT-PCR detected the presence of a single transcript in protoscoleces and adult worms. Western blot assays from these two developmental stages detected a single protein of apparent molecular weight of 46 kDa, present also in the protoscolex nuclear extract. eghbx2 mRNA and the corresponding protein localize in cells associated with calcareous corpuscles. Immunocytochemistry analyses indicated that eghbx2 protein localizes to the nucleus of cells surrounding the calcareous corpuscles. These results allowed postulating eghbx2 as an NK homeobox gene that belonging to the NK2 family, and acting as transcription factor, would have a role in the determination/differentiation of a specific cell type, the cell that forms a calcareous structure, the so-called calcareous corpuscle. eghbx2 could thus be a parasite cell type-specific marker [55]. As previously reported [32], the closest homology of the eghbx2 homeodomain corresponds to the D. melanogaster NK-3 gene, also called bagpipe. The bagpipe gene is expressed in the dorsal and visceral mesoderm during embryogenesis and is under the control of another gene of the NK family (msh2, also called tinman or NK-4) as well as by segmentation genes [45]. In this sense, it would be important to know whether eghbx2 is under the control of E. granulosus tinman homologous (eghbx3). While sequence homologies do not necessarily indicate functional conservation, it is worth noting that the bagpipe orthologues in vertebrates (known as Bapx1) also have specific roles in the development of calcified structures [56-58]. In this sense, it would be very useful to have data on gene expression of those NK2 genes related to Bapx1 that were identified in a calcareous sponge [59]. In a later developmental stage (the adult Taenia worm), eghbx2 protein is present in subtegumental muscles, as evidenced by immunochemical studies [52].

In summary, the expression patterns hitherto known suggest that the E. granulosus NK genes play a role in the determination and /or differentiation of specific cell types. In addition, consistent with the functions assigned to the subclass members, most of these cells are of mesodermal origin. These aspects may be refined with the input of new sequences and the contribution of other expression studies.

E. granulosus miRNAs

Gene expression regulatory RNAs known as microRNAs (miRNAs) are small ~22 nucleotides (nt) non-coding RNAs that play a major role in regulation of diverse cell and tissue processes during plant and animal development [60,61]. They function as post-transcriptional regulators, partially or completely complementary binding to messenger RNA transcripts (mRNAs), usually resulting in direct degradation or translational repression of target genes [62]. To date, almost thirty thousand miRNAs from more than two hundred species have been registered in miRBase [63]. miRNAs are usually highly conserved throughout the animal kingdom [62]. Its increase in gene number, mainly from the emergence of vertebrates, correlates with the increase in morphological complexity, and thus may have significantly contributed to phenotypic evolution in animals [64].

miRNAs genes and clusters have been shown to be powerful markers in phylogenetic studies [65]. Taking advantage of the availability of several complete genomes [36] and miRNA-specific deep sequencing approaches (see below), some miRNA-based studies have demonstrated that miRNA support the phylogenetic af?liation of flatworms to Lophotrochozoan [66]. Additionally, phylogenetic analyses comparing the presence and absence of 153 conserved miRNAs in platyhelminths with 32 other metazoan taxa support the monophyly of Platyhelminthes (Turbellaria + Neodermata [Monogenea {Trematoda + Cestoda}]) [67].

miRNA gains and (most remarkable) losses have been found in flatworms; in addition to being phylogenetically informative, they could be related to their low morphological complexity and high adaptation to parasitism [36,68]. Several Echinococcus miRNAs are absent from vertebrate hosts since they have a protostomian or a lophotrochozoan origin or because they are Echinococcus-specific. Although many conserved Echinococcus miRNAs belong to miRNA families that are present in the subphylum Vertebrata, many of them are only poorly conserved. The divergence of these sequences found at the nucleotide level with respect to those from other organisms, including other platyhelminths, may indicate an accelerated evolution of these miRNAs in the Echinococcus lineage. This could imply specific roles for these miRNAs in development, survival and/ or host-parasite interaction. Additionally, this may reflect the more complex life cycles of parasitic species and their ability to adapt to different environments [68]. Specifically, the losses of

E. granulosus miRNAs may be associated with the loss of ciliated cells, the gut and sensory organs [69]. Additionally, miRNAs do not seem to be randomly distributed in genomes and are typically found in well-characterized loci. Though Echinococcus lacks typical homeobox-containing gene clusters [24] and a high rate of loss of conserved miRNA loci has been found in nematodes and flatworms [69], miR-10, a repressor of Hox genes that are involved in early embryonic development [70], was also found in a similar distribution pattern in three flatworms, including Echinococcus, and neighboring homeobox gene(s) [71]. In Echinococcus species, miR-153 was also found as a neighbor to homeobox genes, which agrees with previous studies showing the coevolution and localization of this miRNA in close proximity to Hox clusters [72]. These results demonstrate the conservation of miRNA positions on chromosomes in Platyhelminthes.

Using the analytical approaches to recover and study miRNAs (small RNA libraries construction and sequencing, and genome bioinformatics) a few long non-coding RNAs (lncRNAs) were retrieved. It is worth mentioning that no piRNA (piwi associated RNA) was found expressed either in the metacestode or the protoscolex stages. This observation is in agreement with the absence of a canonical PIWI protein in Echinococcus genomes [73] and with the fact that piRNAs have not been identified in any other platyhelminth parasite so far [74].

Remarkably, 5’ half-tRNAs from host origin have found in the parasite larval stage interfacing with the intermediate host, i.e., cyst walls. Since small RNA fragments derived from tRNAs have emerged as a novel type of regulatory RNAs able to inhibit translation in response to stress, including pathogen-induced stress, it will be very important to analyze the role of these tRNA fragments in the host response to infection [68]. To date, few studies have undertaken the task of analyzing Echinococcus miRNAs. Several bioinformatic and sequencing approaches have been successfully used to retrieve, sequence, identify and determinate the genomic location of many different miRNAs and their putative targets. These findings have been validated in some cases by performing poly-A or stem-loop RT-qPCR [68,71,75]. Gene Ontology (GO) and Kegg Encyclopedia of Genes and Genomes (KEGG) pathway analysis have also given some clues about miRNAs putative functions, which merits further research, specifically about roles in bi-directional development, nutrient metabolism and nervous system development in E. granulosus [68,69].

In conclusion, it has been found that miRNAs are the preponderant small RNA silencing molecules in E. granulosus, suggesting that these small RNAs could be an essential mechanism of gene regulation in this parasite. Differential expression analysis has shown highly regulated miRNAs between life cycle stages of Echinococcus. Some miRNAs are abundantly expressed in larval stage/species analyzed so far, suggesting that they could be essential in Echinococcus larval stages for survival in the intermediate host. Both parasite-specific and divergent miRNAs have also been found, which could constitute useful biomarkers of infection. Although the functions of miRNAs in Echinococcus are unknown, the results obtained so far suggest that miRNAs could have stage-specific functional roles and/or regulate developmental timing [68]. In this sense, several E. granulosus miRNA targets mapping to signaling pathways involved in development such as Wnt, Notch, Hedgehog, Hippo, MAPK and Ras, were found. Additionally, transcription factors with known roles in developmental processes such as nuclear hormone receptors, HMG transcription factors and TALE homeodomain containing protein were also predicted to be targeted by miRNAs. In addition, proteins related to stem cells, such as Nanos, were found to be targeted by miRNAs (Rosenzvit M & Maccharoli N, pers. comm.)

E. granulosus fatty acid binding proteins

Lipids are heterogeneous molecules with essential functions in cell structure and metabolism as well as intracellular and extracellular signaling. They are major components of cell membranes and important fuel sources. Lipids are also involved in enzyme regulation, cell surface recognition, cell interaction, surface antigenic determinants expression and gene expression regulation.

Despite the importance of these metabolites, lipid metabolism has been little studied in parasitic Platyhelminthes. During the 1960s to the 1980s, most effort has been done to determine lipid composition and lipid involvement in energy metabolism in these organisms [76-81]. From these studies, two paradigms emerged that have ruled lipid metabolic studies for years: the lack of fatty acid oxidation [82,83] and the inability of de novo synthesis of fatty acids and sterols [84,85]. The paradigm related to the absence of fatty acid oxidation is being questioned (see below). These essential compounds must be taken up from the host and, consequently, factors known to be involved in the uptake and transport of lipids should be highly expressed, becoming key molecules for survival [86].

In this sense, two groups of lipid binding proteins have been studied: fatty acid binding proteins (FABPs) and hydrophobic ligand binding proteins (HLBPs). Members of both groups of proteins could be involved in the recruitment, transportation and/ or storage of lipids. HLBPs constitute a family of cestode-specific lipoproteins characterized as highly abundant, immunogenic and high molecular mass oligomers [87,88]. However, in this instance, we will focus on fatty acid binding proteins, a multigenic family of small proteins (14-15 kDa) largely distributed along the zoological scale that bind hydrophobic ligands, mainly fatty acids (FA) [89,90].

The first FABP was described in 1972 as a protein from rat jejunum with the ability to bind long chain fatty acids [91]. Since then, 10 types of FABPs have been characterized in mammals [92-94]. Although their general roles have been established, specific functions of most members of this family remain to be elucidated. Each FABP should have structural features enabling them to specifically collect and deposit their ligands, interacting directly with cellular components. In particular, some members would be involved in modulation of cell growth and proliferation and in gene expression regulation [95-99]. Recent studies with FABP1 and FABP4 demonstrated that these proteins could be versatile endocrine hormones and potent integrators of tissue status [100,101].

Many features make parasitic platyhelminth FABPs interesting molecules for study. As mentioned above, these molecules are predicted to play an important role in facilitating the incorporation and intracellular transport of host fatty acids. In addition, their highly immunogenic character confers significant levels of protection against challenge infections, establishing FABPs as vaccine candidates [102,103]. The first isolated platyhelminth FABP was Sm14 from Schistosoma mansoni [104]. Since that time, many other FABPs have been described in this phylum [105], E. granulosus the cestode Echinococcus granulosus [106-107].

EgFABP1 was isolated from a differential screening of protoscoleces cDNA library [106]. The open reading frame analysis showed identities between 20 and 41% with other members of the family, and the phylogenetic studies allowed to be included in the subfamily IV [90,108,109]. A second E. granulosus FABP (EgFABP2) was isolated from a genomic library screened with EgFABP1 cDNA as a probe [107]. EgFABP2 present the highest sequence identity (76%) and similarity (96%) scores with EgFABP1. Three additional sequences (EgrG_000417200; EgrG_000550000, and EgrG_000551000) were deduced from the Sanger Institute GeneDB project [35]. These sequences show subtle differences with the already described members of the family that could be attributed to annotation errors or be variants from typical FABPs. We refer to them as EgFABP1.1, EgFABP3 and EgRBP, respectively. EgFABP1.1 is highly similar to EgFABP1 but several conserved amino acids among Echinococcus FABPs are absent. EgFABP3 has more than the typical number of amino acids and an intron longer than other FABPs, in a not fully consensus position. Finally, EgRBP has ambiguous characteristics: it is annotated as fatty acid and retinol binding protein but it has the conserved ligand binding site of subfamily IV FABPs, not that of retinol; it shows a higher sequence identity with FABPs than retinol binding proteins (RBPs); and it has a molecular mass higher than other members of the family and a long intron (Figures 1,2).

Figure 1: Clustal omega alignment of EgFABPs sequences. The two different levels of shading show residues 80% conserved (dark gray black) and similar ones (light gray). Numbers indicate sequence length.

Figure 2: Intron position comparison of EgFABPs genes. Horizontal lanes represent translated genes. indicates the intron position; numbers below : codon position; numbers above : intron length.

Members of the FABPs family have also been reported in other cestodes: Echinococcus multilocularis, Taenia multiceps, Taenia solium, Taenia saginata, Hymenolepis microstoma and Mesocestoides vogae [35,110,111]. Phylogenetic relationships among cestode FABPs are depicted in Figure 3.

Figure 3: Phylogenetic relationships of cestodes FABPs. Tree derived from Maximun Likelihood analysis of cestodes FABPs sequences from GenDB database: EgFABP1:EgrG_000549850.1; EgFABP2:EgrG_000549800.1; EgFABP1.1: EgrG_000550000.1; EgFABP3: EgrG_000417200; EgRBP: EgrG_000551000; EmuJ_000550000: EmFABP1; EmFABP2:EmuJ_000549800; EmFABP3: EmuJ_000417200; EmRBP: EmuJ_000551000; TsFABP1:TsM_000425500; TsMFABP2: TsM_000802800; TsMFABP2.1: TsM_001185100; TsRBP: TsM_000544100; HmNFABPa: HmN_000764100; HmNFABPb: HmN_000764100; HmNFABPc:HmN_000764800; HmNRBP:HmN_000764600. Sequences from GenBank: MvFABPa ABO93626.3; MvFABPb: ABO93625.3; TmcFABP: ADQ55926.1, TsFABP: OCK35733.1;TsFABP2: OCK39642.1; TpFABP: ADQ55925.1 Each FABP name has the indication of the species: Eg: Echinococcus granulosus, Em: Echinococcus multilocularis, Ts: Taenia saginata, TsM: Taenia solium, Tmc: Taenia multiceps, Tp: Taenia pisiformis; Mv: Mesocestoides vogae, HmN: Hymenolepis microstoma Boostrap values are indicated. Numbers of internal branches are boostrap probabilities (% of same node orientation, 1000 iterations).

Despite low support for some branches, there are well resolved clades. One of these clades includes FABPs type 1 and type 2 from Echinococcus and Taenia species. The types EgFABP1.1 and TsMFABP2.1 could be pseudogenes. A second well-solved clade includes the retinol binding type of proteins, and FABP3. These clusterings could be indicative of functional specialization.

Intron-exon organization is completely conserved among vertebrate FABP genes. Vertebrate FABP genes are organized in four exons and three introns while Egfabp1 and Egfabp2 E. granulosus genes have two exons and one intron at the same position of the third position described for vertebrate FABP genes [90,112]. The promoter region of Egfabp2 was obtained from a λEMBL3 library. The transcription start site of Egfabp2 is located 13 bp upstream of the translation initiation codon and a canonical TATA box is found 44 bp upstream of the transcription start site. Signal Scan analysis indicates several consensus response elements. It is worth mentioning the presence of an imperfect consensus peroxisome proliferator element (PPRE) and a putative glucocorticoid half-site (GRE) [107].

Echinococcus FABPs share variable amino acid sequence identity (16-45%) with vertebrate FABPs, and they do not contain extensive common protein sequence motifs [90,113]. Despite the sequence variability, crystallographic structure of EgFABP1 is similar to the typical β-barrel structure of vertebrate FABPs [114,115]. Interestingly, all EgFABPs have the highest homology with FABP7 and FABP8, both members of the subfamily IV. This reduced diversity suggests a large repertoire of interactions in the cell which leads us to consider that the diversification of this family is the result of the functional specialization of higher organisms.

The recombinant EgFABP1 has high affinity for unsaturated long-chain fatty acids such as arachidonic acid and oleic acid [116]. The crystallographic structure revealed the presence of a palmitate in a U-shaped conformation, similar to that observed for H-FABP [114]. Moreover, the vertebrate H-FABP residues involved in ligand binding are conserved in EgFABP1, EgFABP2, and EgRBP (Arg107, Arg127, Tyr129). EgFABP1.1 replaces Arg/Leu and Tyr/Phe at positions 127 and 129, respectively, suggesting different binding properties. Docking studies of EgFABP1 (1o8v.pdb) and EgFABP2 (modelled) showed that linoleate, arachidonate and palmitate had higher interaction energies when bound to these templates [117]. Arachidonic acid was also reported as a high affinity ligand for EgFABP1 [116]. It is worth mentioning that arachidonic acid and linoleic acid are both involved in signaling pathways and it was demonstrated that Taenia taeniaeformis, Spirometra erinacei, S. mansoni and Trichobilharzia ocellata can capture and metabolize these metabolites [118-123]. The analysis of internal binding surfaces showed that EgFABP1 and EgFABP2 could have ligand preferences [117]. Subtle conformation differences between apo and holo forms were also observed, suggesting that the ligand could trigger surface signals. Potential energy of binding interaction and the conformation of the ligand pocket also differ upon ligand binding. The authors also observed that the flexibility of the ligand modifies the interaction energy. Taking together these data, it was suggested that the ligand could impose particular functions to the proteins through the exposition of specific surface signals that could lead these proteins to different subcellular compartments [117]. It was reported that certain FABP vertebrate members can expose nuclear localization signals [124,125].

The expression of EgFABP1 is localized at the tegument of the protoscolex, being absent in the germinal layer [106]. At the subcellular level, the protein has a ubiquitous expression since it was identified in cytosolic, nuclear, mitochondrial and microsomal enriched protoscoleces fractions [126]. The cytosolic localization was expected but the mitochondrial localization is unclear, since the fatty acid oxidation pathway is considered inactive in platyhelminths. However, the fatty acid oxidation pathway could occur as an alternative energetic source under glucose deprivation. In addition, β-oxidation enzyme expression was demonstrated in many platyhelminths [82,83,127,128]. Considering these results, we suggest that EgFABP1 could transport their ligands to this compartment and could even be related with fatty acids oxidation in certain circumstances.

Three EgFABP1 isoforms were found in the subcellular fractions studied, with the most abundant being a protein of isoelectric point of 7.7 [126]. Postraductional changes could explain the existence of isoforms. A consensus phosphorylation motif was detected in EgFABP1 [129] and this kind of modification was described for rat H-FABP [130]. None of the other FABPs were identified in these extracts, suggesting that they have a low expression in the larval stage or that their expression is induced under particular conditions.

Similar subcellular distribution was attributed to members of this family of proteins from the cestode Mesocestoides vogae [111]. In this opportunity, the authors demonstrated the in vivo uptake of a fluorescent fatty acid (BODIPY FL C16) as well as a higher uptake activity at the apical region of the larvae. In addition, cytoplasmic and nuclear MvFABPs-BODIPY FL C16 co- localizations were shown, indicating that the fatty acid analogue is targeted to the nuclei. It has been proven that several vertebrate FABPs directly interacted with proxisome proliferator activated receptors (PPARs) at a nuclear level [99,131-133]. Nuclear receptors were identified in Schistosoma mansoni [35,134] and at least fifteen sequences with high scores (269.x 1.2e-26 - 139 x 8.7e-10) were identified through blast-search of the E. granulosus genome using the consensus DNA binding domain of mammalian nuclear receptors. It is noteworthy that phospholipid biosynthetic routes had been described in the rat hepatocytes nucleus [135- 138], and recently, nuclear lipid droplets had been identified as a dynamic neutral lipid store [139]. In summary, the role of EgFABP1 in the nucleus could be related to gene expression regulation, nuclear lipid biosynthesis or lipid droplet formation.

FABPs were originally described as intracellular proteins. Some FABPs were found outside the cells, and growing evidence points to a regulated secretion of them, suggesting relevant physiological activities [140]. EgFABP1 seems not to be the exception since it has been identified in the hydatid fluid as well as an excretory/secretory product using in vivo and in vitro approaches [141,142]. This fact opens a new window that can relate FABP functions to the host. EgFABP1 could interact with host cells close to the cyst wall to uptake fatty acid or to deliver the ligands to host tissue, modulating host immune response. The protein could also be acting in combination with extracellular lipid carriers as HLBPs. These proteins are secreted into host tissue and could be sequestering host FAs.

Important progress has been made to understand the molecular mechanism by which long-chain fatty acids (LCFAs) are taken up by cells and distributed among intracellular compartments. FABPs are candidates to participate in LCFA uptake from membranes at the cytoplasmic face. FA transfer from FABP to artificial membrane and vice versa can occur by two mechanisms: ligand diffusion and subsequent association with the membrane (e.g., L-FABP) or via collisional interaction FABP- membrane with FA transfer occurring during the collision (e.g., H-FABP) [143,144]. EgFABP1 interacts directly with artificial membranes to transfer the ligand via a collisional mechanism [145]. The interaction is mediated by ionic and hydrophobic factors, and it is likely that the initial interactions between the protein and the charged groups of the phospholipids are ionic followed by direct and transient hydrophobic interactions with the apolar region of the membrane.

The collisional ligand transfer mechanism has also been demonstrated for a S. japonicum FABP and several mammalian FABPs [146-148]. The helicoidal region near the ligand portal is involved in this direct interaction [149]. Lys22 of the H-FABP helix I was identified as one residue responsible for this interaction [150]. Instead of Lys, EgFABP1 contains an Arg in the same position, which could establish electrostatic interactions with membrane phospholipids. In addition, EgFABP1 also contains a Lys31 located in the helix II that may be involved in the establishment of electrostatic interactions. Furthermore, EgFABP1 presents a pair of amino acids with bulky hydrophobic side chains exposed to the solvent, Phe27 and Val28 in helix II, which are found in other collisional FABPs and could attract and orient ligands for entry into the protein or be involved in protein interactions with membranes or other proteins [145,151].

Members of parasitic platyhelminth FABPs, Sm14 from Schistosoma mansoni and Fh15 from Fasciola hepatica have been proposed as vaccine candidates [153-156]. Successful preclinical studies along with a good immune response in human assays demonstrate the efficacy of Sm14 as a vaccine, now in phase 2 [157]. EgFABP1 immunogenic properties were also studied [158,159]. Immunization assays in dogs with recombinant Salmonella expressing EgFABP1 demonstrated a good humoral but a poor cellular immune response against the antigen [159].

In this review, we focus on progress made in the knowledge and  understanding  of  the  roles  of  important  molecules involved in the development and regulation of gene expression (homeobox genes and miRNAs) and fatty acid binding proteins from Echinococcus granulosus. Concerning development and gene expression, two aspects deserve special attention: first, the expression patterns of homeobox containing genes have been known to suggest that E. granulosus NK genes play a key role in the determination and/or differentiation of specific cell types, and second, miRNAs are the preponderant small RNA silencing molecules in E. granulosus, suggesting that these small RNAs could be an essential mechanism of gene regulation in this parasite.

The identification of several FABPs in the E. granulosus genome makes us consider that each protein should have specific roles. It is likely that during parasite development, the availability of ligands is not the same or there are different metabolic needs. Each protein could have different signals that could be triggered by the bound ligand. In this sense, specific subcellular localization and/or protein-protein interactions could be established. Future work should be directed toward the generation of tools that allow us to discriminate between each of them, allowing the determination of specific functions.

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While still being considered a neglected disease by the WHO, cystic echinococcosis represents a major public health and economic issue in many countries. Echinococcus granulosus, its causative agent, presents several interesting traits, and its developmental process shows a remarkable biological plasticity. In the past twenty years, several molecules from E. granulosus have been characterized; more recently, significant progress has been made with genomic, transcriptomic and proteomic analysis. In this review, we focus on progress made in the knowledge and understanding of the role played by important molecules involved in development, regulation of gene expression (homeobox genes and miRNAs), and fatty acid binding proteins. These proteins, given the restricted lipid metabolism of this parasite, could be key molecules in controlling this disease.