New Leading Compounds as Possible Drug Targets in Cysticercosis

#### **Chapter 5**

## *Taenia solium* microRNAs: Potential Biomarkers and Drug Targets in Neurocysticercosis

*Matías Gastón Pérez*

#### **Abstract**

MicroRNAs (miRNAs) found in animals, plants, and some viruses belongs to the heterogeneous class of non-coding RNAs (ncRNAs), which posttranscriptional regulates gene expression. They are linked to various cellular activities such as cell growth, differentiation, development and apoptosis. Also, they have been involved in cancer, metabolic diseases, viral infections and clinical trials targeting miRNAs has shown promising results. This chapter provides an overview on *Taenia solium* and *Taenia crassiceps* miRNAs, their possible biological functions, their role in host– parasite communication and their potential role as biomarkers and drug targets.

**Keywords:** miRNAs, small noncoding RNAs, neurocysticercosis, biomarkers, drug target

#### **1. Introduction**

#### **1.1 Overview of miRNAs: definition, biogenesis, and functions**

MicroRNAs (miRNAs) are a major class of small noncoding RNAs (ncRNAs) found in animals, plants, and some viruses, which negatively regulate gene expression at the messenger RNA (mRNA) level [1]. The first known miRNA (lin-4) was found in the free-living nematode *Caenorhabditis elegans* [2]. Seven years later let-7 was identified, and together with lin-4 were found to regulate developmental timing of *C. elegans* larvae [3]. By definition, miRNAs are small RNA molecules incapable of encoding proteins, but possessing important structural, catalytic, and regulatory functions comprise one of the more abundant classes of gene regulatory molecules in multicellular organisms and likely influence the output of many protein-coding genes. According to the latest miRBase release (v22.1, October 2018, http://www.mirbase.org/), there are miRNAs from 271 species, expressing 38589 mature miRNAs.

It is well established that miRNA biogenesis is a complex process classified into canonical and non-canonical pathways. The canonical biogenesis is the dominant pathway by which miRNAs are processed [4]. This pathway includes three main steps: (i) In the nucleus, miRNA genes are transcript by RNA polymerase II as part of much longer RNAs called pri-miRNAs which contain one or a few stem-loop structures composed of approximately 70 nucleotides each (**Figure 1**). Sometimes miRNAs are transcribed as one long transcript called clusters, which may have similar seed regions, and in which case they are considered a family [5]. About half of all currently identified miRNAs are intragenic and processed

#### **Figure 1.**

*Canonical microRNA biogenesis and mechanism of action. The biogenesis begins with the generation of the pri-miRNA transcript by RNA polymerase II in the nucleus. The microprocessor complex, composed of Drosha and DiGeorge syndrome critical region 8 (DGCR8), cleaves the pri-miRNA to produce the precursor-miRNA (pre-miRNA). After translocation into the cytoplasm by exportin 5, pre-miRNAs are processed by Dicer to form the mature miRNA/miRNA\* duplex. Following processing, miRNAs are assembled into the RISC complex. Only one strand of the duplex is stably associated with the RISC complex. The mature miRNA directs repression of mRNA containing complementary miRNA binding sites within the 3'UTR.*

mostly from introns and relatively few exons of protein coding genes, while the remaining are intergenic, transcribed independently of a host gene and regulated by their own promoters [6] (ii) Then, the microprocessor complex, consisting of an RNA binding protein DiGeorge Syndrome Critical Region 8 (DGCR8) and a ribonuclease III enzyme, Drosha trims the pri-miRNA to liberate a pre-miRNA hairpin which is actively transported to the cytoplasm by 5 (XPO5)/RanGTP complex (**Figure 1**) (iii) Its final maturation is processed in the cytoplasm where Dicer RNase III endonuclease cleaves the pre-miRNA into a single-stranded mature miRNA, removing the terminal loop (**Figure 1**) [4]. The directionality of the miRNA strand determines the name of the mature miRNA form [5]. Subsequently, with assistance from chaperone proteins (HSC70/HSP90) the mature miRNA is loaded into proteins of the Argonaute (Ago) family and assembles the RNA induced silencing complex (RISC) together to exert its further physiological functions (**Figure 1**). We defined the unloaded strand as passenger or star strand. The start strand that contains no mismatches are cleaved by AGO2 and degraded (**Figure 1**). Also, miRNA duplexes with central mismatches or non-AGO2 loaded miRNA are unwound and degraded. After being incorporated into the RISC, the mature miRNA induces posttranscriptional gene silencing by tethering RISC to be partially complementary to the target mRNA predominantly found within the 3′-untranslated regions (UTR). Targeting can also be facilitated by additional sequence elements, such as an unpaired Adenosine in the mRNA target sequence, corresponding to the nucleotide 1 in 5′ end of the mature miRNA. On the other hand, non-canonical miRNA biogenesis pathways are grouped into

#### Taenia solium *microRNAs: Potential Biomarkers and Drug Targets in Neurocysticercosis DOI: http://dx.doi.org/10.5772/intechopen.97305*

Drosha/DGCR8-independent and Dicer-independent pathways. In general, these pathways make use of different combinations of the proteins involved in the canonical pathway, mainly Drosha, Dicer, exportin 5, and AGO2 [4].

It has been estimated that miRNAs regulate the expression of approximately one-third of the protein-coding genes [1]. Each miRNA can have many target mRNAs and a single mRNA can be regulated by multiple miRNAs [7]. Given this vast majority of mRNA targets regulated by miRNAs, aberrant miRNA expression profoundly influences a wide variety of cell regulation pathways important to cell proliferation, apoptosis, and stress responses.

Single miRNA gene can generate multiple miRNA isoforms (isomiRs). For example, the inconsistent choice of the strand loaded into AGO can generate two different functional miRNAs from both strands of the pre-miRNA duplex [8]. Imprecise cleavage of pri-miRNA by Drosha or pre-miRNA by Dicer can generate heterogeneous 5′ or 3′ ends. Another way to generate isomiRs is by RNA editing which may have a functional impact when the seed nucleotide is altered [4]. Exonuclease activity could remove nucleotides from miRNA 3′ end, and terminal nucleotide-transferase could add nucleotides to the miRNA 3′ end generating different isomiRs.

For almost a decade, some of the miRNA genes have been categorized into different groups, named miRNA families, based on the mature miRNA sequence and/or structure of pre-miRNAs [9]. Thus, means that microRNAs are grouped into families based on their targeting properties, which depend primarily on the identity of their extended seed region (miRNA nucleotides 2–8) [10]. Interestingly, it has been observed that miRNA genes in the same miRNA family are non-randomly colocalized and well organized around genes involved in infectious, immune system, sensory system and neurodegenerative diseases, development and cancer [11]. As with paralogous proteins, members of the same seed families often have at least partially redundant functions, with severe loss-of-function phenotypes apparent only after multiple family members are disrupted [12].

Biological functions of individual miRNAs have been extensively explored and have revealed the important role of miRNAs in many biological functions such as developmental timings, cell differentiation, embryogenesis, metabolism, organogenesis, and apoptosis [13]. Thus, miRNAs have been introduced as therapeutics or as targets of therapeutics for the treatment of disease [14]. Also, the existence of extracellular miRNAs has been widely reported these molecules as potential biomarkers for a variety of diseases. At present, miRNAs-mediated therapies for treatment of cancer and chronic hepatitis C virus (HCV) infection have shown promising results in human Phase I clinical trial [15].

#### **2. miRNAs in** *Taenia solium* **and** *Taenia crassiceps*

The presence of homologs to Drosha, Dicer, and Pasha (as identified in the *T. solium* Genome Project) [16] and the differences in miRNAs profiles between the *Taenia solium* cysticerci and adults suggest that the process for the synthesis of miRNAs is similar to that described for mammals. Identification and sequencing of miRNAs have been largely facilitated by the newly available high-throughput tools that have generated a growing set of miRNA sequences from parasites. Our group report for the first time the high confidence miRNA repertoire from *T. crassiceps* and *T. solium* and show that miRNAs account for most small RNA expression in *T. crassiceps* cysticerci by small RNA-seq experiments [17]. Since our miRNA identification strategy required the matching of *T. crassiceps* small RNA sequences to the *T. solium* genome, the identified miRNAs are considered common to both species, as was previously considered for other helminth parasites [18, 19]. The percentage of miRNAs in *T. crassiceps* cysticerci

reaches 83% of the total small RNA expression, suggesting important functions of this type of RNA in the biology of taenias [17]. Also, miRNAs were identified and validated by northern blot experiments [17]. This validation is especially important in the case that genomic data from other species is used. Additionally, we experimentally detected pre-miRNAs for the first time in cestodes adding confidence to the miRNA identification procedure performed [17]. The *T. solium* and *T. crassiceps* miRNA catalog includes 41 conserved miRNAs grouped into 30 families [17]. The number of conserved miRNA families is similar to that of *Echinococcus canadensis* [20] and *Mesocestoides vogae* (syn: *M. corti*) [21] (28 conserved miRNA families), providing further evidence for the loss of conserved miRNA families in cestodes [22]. In *T. solium* genome it was reported two miRNA precursors (pre-mir-new-1a and pre-mir-new-1b) arranged in a cluster [17] that were only reported before for the *Echinococcus granulosus* s. s. G1 genotype [23]. The first of these precursors shows expression from both arms in *T. crassiceps*, unlike the *E. granulosus* s. s. G1 genotype that only expressed the 3p arm [17]. Differences in the genome organization of miRNA precursors among cestodes was reported. For example, the cluster miR-7b and miR-3479a found in *T. solium* are not clustered in *Echinococcus* or *M. vogae* [17]. The cluster miR-71/2c/2b, it was found only once in the genome of *T. solium*, as observed in other cestodes analyzed to date [20, 21, 24]. The presence of only one miR-71/miR-2 cluster seems to be a common feature of cestode genomes. The uneven expression found among miRNAs of this cluster was also observed in *Echinococcus* spp. [20, 24] and *M. vogae* [21]. Cluster miRNAs are evolutionally and functionally related and may co-regulate multiple biological processes. Additionally, cluster miRNAs have shown to evolve more rapidly than individual miR-NAs [25]. Current evidence in humans suggests that, various genetic events (deletion, insertion, duplication and base substitution) within a cluster, followed by adaption and neofunctionalization, is the underlying mechanism responsible for the evolution of miRNA clusters [26]. To date, the importance of these differences in genomic arrangement of cestodes is unknown but could potentially influence the expression of the corresponding mature miRNAs.

The expression profile of *T. crassiceps* cysticerci showed that miRNA expression is highly biased to a few miRNAs: miR-10, let-7, miR-71, bantam and miR-61 [17]. These five miRNAs account for ~90% of miRNA expression [17]. These miRNAs and miR-4989 were also highly expresses in *Taenia solium* cysticerci [27]. Coincidentally, in other reports of small RNAs from cestodes that used the same methodology for miRNA discovery, miR-10, let-7, miR-71 and bantam were the most highly expressed, suggesting important functions in cestode biology [20, 21, 23, 24]. The repertoire of miRNAs in *T. solium* genome included protostomian miRNAs, such as miR-4989 and bantam that are absent in human host [17]. MiR-4989 is a divergent member of the miR-277 family. This protostomian-specific family is known to be involved in amino acid catabolism in *Drosophila*. Recently, miR-4989 was shown to be involved in development of juvenile worms in *S. mansoni*. In the *T. solium* genome miR-4989 target a Cationic amino acid transporter and a basic leucine zipper bZIP transcription factor without orthologs in any model species. Additionally, in the genome of *T. solium* and expressed in *T. crassiceps* cysticerci we found bilaterian miRNAs that are absent in human host, such as miR-71 or divergent from their host ortholog, such as let-7 [17].

The identification and characterization of miRNA targets is essential for understanding the function of these ncRNAs at molecular level. MiR-10 is the most expressed miRNA in *T. crassiceps* and *T. solium* cysticerci. This miRNA is highly conserved across metazoan organisms and is implicated in Hox gene regulation, embryonic development, and cancer [28–30]. Tow ANTP class homeobox genes were found among predicted miR-10 targets in *T. solium* genome [17] as in many others bilaterians [31]. Let-7, a conserved miRNA across evolution, was shown to regulate the developmental timing in *C. elegans* [32] and was shown to be a central

Taenia solium *microRNAs: Potential Biomarkers and Drug Targets in Neurocysticercosis DOI: http://dx.doi.org/10.5772/intechopen.97305*

regulator of mammalian glucose metabolism by targeting several genes of the insulin-PI3K-mTor pathway, including the insulin receptor [33]. MiR-9 is a deeply conserved miRNA across evolution known to be involved in neural development. One possible target for miR-9 is a Slit 2 protein, the ortholog of *C. elegans* slt-1 that is expressed in muscle cells and neurons and is involved in generation of neurons and axon guidance during embryonic and larval development. Also, a Carbonic anhydrase was reported as a putative target gene for miR-9, which is the ortholog of *C. elegans* cah-1 that is expressed in different neurons and head ganglion and is predicted to have a carbonate dehydratase activity. Other relevant target for miR-9 was Peregrin, the ortholog of *C. elegans* lin-49 that is involved in normal larval development. MiR-71 is bilaterian miRNA absent in vertebrates and involved in the promotion of longevity and neuronal asymmetry in *C. elegans,* is the miRNA with more targets predicted in the genome of *Taenia solium.* Some interesting targets are shown in **Table 1**.

Parasite miRNAs that are absent in the host, such as miR-71 or highly divergent (e.g let-7) from their host orthologs may be considered as selective therapeutic targets for treatment and control of helminth parasite infections. In addition, miR-71 is highly expressed in *T. solium* adults stage suggesting that it could be involved in important biological functions in the life cycle of *Taenia* genus. Nevertheless, the characterization of the physiological effects of the presence or absence for each identified miRNA needs more complex approaches in *T. solium*. In this respect, in vitro and in vivo optimization strategies for efficient and long-lasting loss-offunction, such miR-71 silencing reported in *E. multilocularis* [34] are still required for meaningful silencing studies in other metacestodes.

#### **2.1 miRNAs and immune response**

Helminth's parasites modulate immune responses in their host to prevent their elimination and establish chronic infections. Neurocysticercosis (NCC) implicates chronic parasitic disease with different variety of host and parasite interactions [35]. Clinical manifestations are mainly the result of inflammatory response to degeneration of parenchymal cysticercus [36].

*Taenia solium* cysticerci actively prevents this inflammatory response [37], which prolongs its survival in the host. The intensity of NCC symptoms depends primarily on the inflammatory response, which is associated with the Th1 response with high levels of TNF, IFN-γ, IL-17, and IL-23 whereas the Th2 response (antiinflammatory response) is associated with asymptomatic NCC with high level of IL-10, IL- 4, IL5, and IL-13 [38]. In *T. crassiceps* model, the cysticercus growth is controlled by macrophages and the promoting of Th1 and Th2 responses. The production of inflammatory cytokines by macrophages and dendritic cells are blocked by excreted/secreted antigens (E-S antigens). Also, toll-like receptor (TLR) are blocked facilitating cysticercus growth [38]. The miRNA signature of T regulatory (Treg) cells has been characterized and among the miRNAs expressed are mir-21 and mir-31, which have opposing effects on the Treg TF FOXP3 [38]. Also, it was demonstrated that the E-S antigens of *T. crassiceps* cysticerci can modulate proinflammatory responses in macrophages by inducing regulatory posttranscriptional mechanisms, while E-S antigens reduced the production of inflammatory cytokines (IL-6, IL-12, and TNFα), they increased the release of IL-10 in LPS-induced bone marrow-derived macrophages [39]. microRNAs are a key component of macrophage posttranscriptional regulation [40] and it was shown that E-S antigens of *T. crassiceps* cysticerci induced upregulation of miR-125a-5p, miR-762, and miR-484, which are predicted to target canonical inflammatory molecules and pathways in LPS-induced bone marrow-derived macrophages.


#### **Table 1.**

*Interesting miR-71 targets in* Taenia solium *genome.*

*Taenia solium* E-S antigens have been implicated in immune modulation and it is also known that the intimate association between host and parasite and the immune response is highly controlled at the post transcriptional level [41]. Target prediction of miR-10 and miR-125 found in *T. solium* cysticerci are potentially involved in macrophage IRF/STAT pathways, such as CD69 and TNF. Also, miR-9 was found to be related in the classical activation of macrophages [38]. Mir-10 and miR-125 were also implicated in expression of cytokine receptors, cell activation markers and cell adhesion molecules that activate macrophages to secrete TNF involved in IFNsignaling pathway. Furthermore, potential miR-10 targets such as IL12 and IL23, could interfere with the IL-12 family signaling pathway with a probably Tregs induction. In addition, let-7 showed predicted targets, such as IL10 that encodes cytokines involved in M2 polarization [42]. These suggest an important role in the polarization of macrophages. Macrophages in cysticercosis promote a transient Th1 protective response with classical activated macrophages that is changed by parasite products to a Th2 permissive response with alternatively activated macrophages [43].

Already knowing that the more abundant miRNAs (miR-10-5p, let-7-5p) putatively have target genes of immune response and that macrophages in murine cysticercosis promote Th1 or Th2 responses it was demonstrated that synthetic miR-10-5p and let-7-5p were internalized into the cytoplasm of murine peritoneal macrophages in vitro [27]. Interestingly, the down regulation of the expression of pro-inflammatory cytokines, such as Il6, Il1b, and TNF, IL-12, was reported when activated macrophages were incubated with IFN-γ and miR-10-5p or let-7-5p.

Taenia solium *microRNAs: Potential Biomarkers and Drug Targets in Neurocysticercosis DOI: http://dx.doi.org/10.5772/intechopen.97305*

Moreover, in macrophages activated with IL-4 these miRNAs reduced the expression of cytokines involved in M2/Th2 differentiation. These results were important, because murine resistant to cysticercosis display high levels of TNF, IL-12, IL1-β, and NO during early infection (Th1 response), which is associated with the elimination of larvae [44]. On the other hand, high levels of pro-inflammatory cytokines (IL-6 and TNF) cause damage to the microglia promoting autoimmune and neurodegenerative diseases [45, 46]. This tissue damage is also observed in human NCC at the beginning of larvae degeneration and in pig NCC when they are treated with praziquantel [47]. In contrast, viable larvae are associated with a long initial asymptomatic phase that correlate with undetectable inflammation in the SNC, presumably due to *T. solium* larvae factors prevent inflammation [48].

The striking ability of helminth parasites in conferring protection from diseases of immune dysregulation has increased the attention into the immunomodulatory mechanisms evoked by these parasites. Administration of E-S antigens of *T. crassiceps* in experimental ulcerative colitis, autoimmune encephalomyelitis and type 1 diabetes shown positive results [49, 50]. The ability of *T. crassiceps* to prevent inflammatory responses was demonstrated to be dependent on a population of macrophages that produced markers of alternative activation (M2) [51]. Excreted/secreted *T. crassiceps* products decreased the production of inflammatory cytokines (IL-12, TNFα, and IL-6) in LPS-induced macrophages but has a limited role in inducing directly the production of M1 and/or M2-associated molecules. The immune-modulatory ability of these E-S antigens was further associated with increased levels of specific microRNAs, which are predicted to target numerous inflammatory mRNAs involved in the TNF and NF-κB signaling pathways [39].

#### **3. miRNAs in drug response**

In 2010 Devaney and collaborators [52] speculated that the link between changes in miRNA levels and drug resistance in cancer cells may also be a feature of drug resistance in parasitic nematodes. On the other hand, few data have been published in connection with drug resistance in cestodes [53]. Our group study the miRNA expression profile of *T. cr*assiceps cysticerci incubated for 24 h with sublethal doses of praziquantel (PZQ ), one of the main antiparasitic agents used for cysticercosis and taeniasis [17].

The experiments showed that the overall miRNA profile remained unchanged under PZQ treatment, except for miR-7b that showed a sixfold enhanced expression [17] under PZQ treatment. One of the predicted miR- 7b targets was calponin, a calcium binding protein that inhibits myosin. This may be related to the expected alteration of intracellular calcium concentration produced by PZQ, a drug binding and inhibiting voltage-gated calcium channels, a key molecule for the regulation of calcium level inside the cell. Also, other targets of miR-7b are involved in several pathways such as amino acid and nucleotide metabolism, vesicular transport, signaling pathways, cell adhesion, cell growth, cell death and interaction with neuroactive ligands, suggesting the importance of this miRNA in parasite biology [17]. Calponin is one of miR- 7b predicted targets linked to calcium binding protein that inhibits myosin and may be related to the intracellular calcium concentration produced by PZQ. Other predicted targets of miR-7b are involved in several pathways such vesicular transport, signaling pathways, cell adhesion, cell growth, cell death and interaction with neuroactive ligands, suggesting the importance of this miRNA in parasite biology [17]. In these experiments other miRNAs showed differences in expression levels during treatment with PZQ, such as miR-31. This miRNA showed a decrease in the level of expression in cysticerci treated with PZQ and therefore the


#### **Table 2.**

*Interesting drug response miRNAs and targets in* Taenia solium *genome.*

genes regulated by the miRNA would be overexpressed compared to the cysticerci that did not receive treatment with PZQ. Predicted target genes for this miRNA include: ABC transporters (transporters responsible for expelling different drugs out of the cell), thioredoxins (involved in drug metabolism), and the voltage-gated L-type calcium channel subunit alpha-1D, which is a probable site of action for praziquantel (PZQ ) [54] **Table 2**. Additionally, other *T. solium* miRNAs were found to have targets related to flow, metabolism, and drug action [17].

These results prepare the way for continue with more studies in order to understand the response of miRNAs to drug treatment and the influence that these ncRNAs may have on drug action and/or drug resistance.

#### **4. miRNAs as potential biomarkers**

A biomarker is described as a feature that is objectively measured and evaluated as an indicator of many biological processes. Hunting for helminths biomarkers capable of providing diagnostic, prognostic, or therapeutic information has become a necessary but challenging work in cestodes research. MiRNAs were reported in blood – plasma, serum and other fluids like urine and saliva. This attribute has raised the interest of their use as potential biomarkers and diagnostic tools [55]. In the case of cestodes diseases the use of pathogen miRNAs as biomarkers promises the advent of highly specific and non-invasive diagnostic tools, since the miRNA

Taenia solium *microRNAs: Potential Biomarkers and Drug Targets in Neurocysticercosis DOI: http://dx.doi.org/10.5772/intechopen.97305*

repertoire of *T. solium* present a set of unique or divergent miRNAs with respect to the corresponding host homologs.

The small size and the stability of miRNAs are two important features that permit the circulation of these molecules in biological fluids. The formation of protein– miRNA allows circulating miRNAs escape of degradation [5]. Also, the majority of miRNAs detectable in serum and saliva are found inside extracellular vesicles (EV) that could avoid miRNA degradation and serve as transport particles to facilitate miRNA actions in neighboring cells [56]. The term EV groups includes several types of vesicles among which microvesicles and exosomes are the most thoroughly characterized. In helminths parasites EVs are the preferred extracellular compartment under study and miRNAs as the most thoroughly characterized RNA biotype [57]. The identification and sequencing of *T. solium* miRNAs is a must for their use as diagnostic tools. Among these contexts, the potential of miRNAs being involved in cestodes diseases as biomarkers has been investigated. It was demonstrated that the miR-10 and let-7 families are present in the EV from cestodes [57–61]. These two miRNAs are highly conserved throughout evolution and are present in bilaterians where they play fundamental roles in regulating stem-cell division and differentiation and embryonic development. The metacestode larval stage of *E. multilocularis* presents a morphological barrier to the secretion of EV towards the extra-parasite milieu and hence, ex-RNAs secreted in vitro are mostly detected in the EV-depleted fractions [57, 61]. Interestingly, the opposite is observed in the mestacestodes of *T crassiceps* and *M. vagae*, a parasites models of *T. solium*, that do not have such a structure [61] but parasite ex-RNA detection in patient biofluids is still in a very early phase of study.

The extensive use of next generation of technologies such as miRNA microarrays and high-throughput deep sequencing techniques, translating biomarker into practice with increased diagnostic and therapeutic sensitivity and specificity would be less of a problem [62]. With respect to the use of ex-RNAs as biomarkers in NCC, to date, no laboratory assay from plasma, serum or cerebrospinal fluid has been performed. Furthermore, patient samples from different geographic regions together with specificity assessment with samples of patients would also provide a more realistic view of the potential of ex-RNAs as biomarkers of NCC.

#### **5. miRNAs as potential drug target**

The hypothesis that many *T. solium* miRNAs have crucial roles in development, host–parasite interaction and immune response, and also the absent of some miRNAs in the host has led to considerable interest in the therapeutic targeting of miRNAs in NCC. The main approaches commonly taken are: i) miRNA inhibition by antisense oligonucleotides, miRNA sponges or small-molecule inhibitors ii) miRNA upregulation with miRNA mimics [63]. Mirna sponges' strategies rely on the expression of mRNAs containing multiple artificial miRNA-binding sites, which act as decoys. The overexpression of mRNA- sponges selectively sequesters endogenous miRNAs and thus allows expression of the target mRNAs [64]. Approaches that are based on small molecules generally rely on reporter-based assay systems for compound library screening and have identified small molecules that could specifically inhibit miRNA expression, such as azobenzene (which affects human miR-21 expression) and several diverse compounds that inhibit human miR-122 [65]. Considerably more attention has been paid to antimiRs, particularly to those that target miRNAs directly to specifically inhibit miRNA function and upregulate miRNA targets. In practice chemical modification of oligonucleotides is required to increase resistance to serum nucleases, to enhance binding affinity for targeted

miRNAs and to improve the pharmacokinetics and pharmacodynamics profile in vivo. Other limitations are associated with rapid clearance, immunotoxicity an low tissue permeability. The delivery of artificial miRNAs or of blocking counterparts that could interfere with key processes in parasites has been already postulated by several authors, and some potential targets are already characterized. MiRNA manipulation in parasites has been also proposed as a new strategy for control against schistosomiasis and cystic echinococcosis [38, 66].

#### **6. Conclusions**

For the better understand of the pathophysiology of parasitic diseases at the molecular level is crucial identify and characterize parasite-specific miRNAs and their targets in hosts. The significant advance in biomedical research of miRNAs as target drugs and biomarkers is expected to be widely translated in the field of parasitology in the coming years. Why not think about miRNAs as a profitable approach to better diagnose and properly treat NNC? There is an increasing number of studies that are being done in *T. solium* and cestodes miRNAs, however translational research of miRNA still remains a challenge.

Regarding the neglected diseases, researchers have dedicated decades to the development of new drugs and identifying new biomarkers of disease progression but most researches are limited to academia indicating a gap between basic science and clinical application. Also, the use of miRNAs as a biomarker or potential drug target are poorly explored compared with cancer, neurological disorders, metabolic, cardiac and circulatory diseases.

It is expected that in future years the biological knowledge acquired on miRNAs, especially in biomedical research, could be widely translated into NNC since miR-NAs could hold great potential as therapeutic and diagnosis targets for the control of diseases.

#### **Acknowledgments**

I would like to thanks Dr. Mara Rosenzvit and all the members of the BMHid laboratory (https://impam.conicet.gov.ar/bmhid/).

#### **Author details**

Matías Gastón Pérez Institute for Research in Medical Microbiology and Parasitology (IMPaM-UBA-CONICET), Buenos Aires, Argentina

\*Address all correspondence to: mgperez@fvet.uba.ar; matiasperez@conicet.gov.ar

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Taenia solium *microRNAs: Potential Biomarkers and Drug Targets in Neurocysticercosis DOI: http://dx.doi.org/10.5772/intechopen.97305*

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#### **Chapter 6**

## Development of New Drugs to Treat *Taenia solium* Cysticercosis: Targeting 26 kDa Glutathione Transferase

*Rafael A. Zubillaga, Lucía Jiménez, Ponciano García-Gutiérrez and Abraham Landa*

#### **Abstract**

*Taenia solium* causes neurocysticercosis, a parasitic infection of the central nervous system in humans. The costs of management, treatment, and diagnosis of patients with neurocysticercosis are high, and some patients do not respond to the currently available treatments. Helminth cytosolic glutathione transferases (GSTs) are essential enzymes involved in the regulation of immune responses, transport, and detoxification. In *T. solium*, three cytosolic GSTs with molecular masses of 26.5 (Ts26GST), 25.5 (Ts25GST), and 24.3 kDa (TsMσGST), classified as mu-alpha, mu and sigma GST-classes, respectively, constitute the main detoxification system, and they may be immune targets for the development of vaccines and new anthelmintics. We performed a successful virtual screen, and identified I7, a novel selective inhibitor of Ts26GST that showed a non-competitive inhibition mechanism towards substrate glutathione with a Ki of 55.7 mM and mixed inhibition towards the electrophilic substrate 1-chloro-2,4-dinitrobenzene with a Ki of 8.64 mM. Docking simulation studies showed that I7 can bind to a site that is adjacent to the electrophilic site and the furthest from the glutathione site. This new inhibitor of Ts26GST will be used as a lead molecule to develop new effective and safe drugs against diseases caused by *T. solium*.

**Keywords:** Glutathione transferase, Inhibitor, *Taenia solium*, Neurocysticercosis

#### **1. Introduction**

#### **1.1 Neurocysticercosis**

*Taenia solium* is a cestode parasite in humans. Adult parasites cause taeniasis, and the larvae cause cysticercosis. Larvae located in the central nervous system cause neurocysticercosis (NCC), with a wide spectrum of clinical manifestations that depend on factors such as the location, number of larvae, and the intensity of host immune response [1, 2]. The disease may be asymptomatic or present with nonspecific symptoms, such as epilepsy, cognitive impairment, migraine-type headache, intracranial hypertension, and neurological deficits, among other symptoms [3, 4].

#### **1.2 Clinical spectrum**

According to the location of the larvae, NCC is classified into parenchymal NCC and extraparenchymal NCC. In the parenchymal NCC, the most frequent symptoms are seizures, which can occur at any stage of the cysticercus (viable or calcified) [5], and neurological signs, such as sensory deficits, language, and gait disturbances, as well as involuntary movements. Such manifestations have been reported in up to 15% of patients [3]. In the extraparenchymal NCC, cysticerci are usually found in the subarachnoid and ventricular locations. Hydrocephalus is observed in a significant number of cases of subarachnoid NCC, and neurological alterations associated with the obstruction of the cerebrospinal fluid flow have been observed in patients with ventricular NCC; the blockage of the cerebral aqueduct due to the presence of cysticerci in the fourth ventricle may result in the loss of consciousness or even death [3, 6, 7].

#### **1.3 Treatment**

NCC is a disease transmitted by food, which causes many disability-adjusted life years. In Mexico, the cost of management, treatment, and diagnosis of patients with NCC was approximately U.S. \$52 million in 2015 [8]. In addition to these costs, a study in Peru estimated that two-thirds of patients who develop symptoms lose their jobs, and the sequelae make it impossible for 60% of them to return to work [9]. Treatment should be individualized according to the characteristics of the disease and location of cysticerci, but in general, it consists of a mixture of surgical intervention (recommended for cases of intraventricular or spinal NCC), antiparasitic and anti-inflammatory drugs, and drugs for the management of symptoms [10]. The antiparasitic treatment for NCC includes praziquantel or albendazole. Praziquantel is a pyrazino-isoquinoline derivative that affects calcium channels on the parasite's surface and causes muscle contractions, paralysis, and tegument damage [11]. Maximum serum levels of praziquantel are obtained in 1.5–2 h after administration [12]. Praziquantel is metabolized in the liver, and its mild side effects include gastric disturbances, dizziness, drowsiness, fever, headache, increased sweating, and sometimes allergic reactions; however, these reactions disappear when the drug is withdrawn [13]. Albendazole is a benzimidazole compound that leads to the selective degeneration of cytoplasmic microtubules, affecting the formation of ATP, and glucose intake, which depletes parasite of the energy source [14]. Maximum serum levels of albendazole are achieved in 2 to 3 h after ingestion. This drug penetrates the cerebrospinal fluid better than praziquantel [15]. Side effects in humans are mainly related to liver toxicity (increased liver enzymes), hematological effects, hair loss, and general symptoms that dissipate when treatment is withdrawn [14]. The use of antiparasitic drugs can cause adverse effects arising from the inflammatory reaction induced when cysticerci are damaged; therefore, the use of corticosteroids in addition to treatment is recommended. However, prolonged use of corticosteroids increases the risk of opportunistic infections, skin disorders, depression, osteopenia, among others [13]. Several drugs, including benzimidazole, praziquantel and nitazoxanide, have been evaluated for their ability to control swine cysticercosis in animals intended for consumption. Of these, oxfendazole has been shown to have close to 100% efficacy after a single dose in intramuscular cysticercosis, but the efficacy was lower in swine neurocysticercosis [16].

#### **1.4 New drugs**

Patients who do not respond to therapy with the currently available drugs have been reported. Several factors have been proposed that may be involved in this

*Development of New Drugs to Treat* Taenia solium *Cysticercosis: Targeting 26 kDa Glutathione… DOI: http://dx.doi.org/10.5772/intechopen.97342*

lack of sensitivity to the treatment: differential response according to the state of development of cysticerci, low penetration of the drug into the subarachnoid space, variability of albendazole sulfoxide levels in plasma in individual patients, or interference of corticosteroids with the activity of anti-helmintics [17, 18]. This has led to the search for new drugs that could improve the effectiveness of the anti-helminthic therapy. Therefore, cytosolic glutathione transferases (cGSTs) have been selected as targets for the development of vaccines and drugs against this parasite [19–22].

#### **2. Overview of glutathione transferases**

#### **2.1 The catalytic reaction**

GSTs (EC 2.5.1.18) are a multiprotein family highly expressed in all cells [23, 24]. They are part of phase II detoxification process and catalyze the conjugation of glutathione to a variety of endo- and exo-electrophilic substrates [25]. This conjugation produces soluble compounds and substrates for cellular export proteins, such as P-glycoprotein and multidrug resistance-related protein 1 [26]. The general reactions (GSH + RX → GSR + HX) comprise a nucleophilic attack, aromatic substitution, epoxide ring opening, reversible Michael addition, isomerization or peroxidation. Although nucleophilic attack can also be directed to nitrogen atoms in nitrate esters, sulfurs in organic thiocyanates or disulfites, and oxygen in organic hydroperoxides [25, 27–29].

#### **2.2 Cellular distribution and GST classes**

GSTs can be grouped into three subfamilies according to their cellular location: mitochondrial GSTs, microsomal GSTs or MAPEGs (membrane-associated proteins in eicosanoid and GSH metabolism), and cytosolic or canonical GSTs. In humans, genes encoding all expressed GSTs from a given subfamily are clustered on the same chromosome [30]. The mitochondrial GST subfamily includes a unique kappa (K) class. This class has very high peroxidase activity, and its location suggests an important role in β-oxidation of fatty acids and in lipid peroxidation. Moreover, it is also a key regulator of adiponectin biosynthesis and may function as a chaperone [31–34]. Microsomal GSTs are divided into four groups (I–IV). They share less than 20% sequence identity and are involved in eicosanoid metabolism, such as the synthesis of prostaglandins, thromboxanes, leukotrienes (inhibitors of inflammation), glutathione metabolism, and activation of some lipoxygenases [33, 35–37]. In the subfamily of cGSTs, members of the same class have more than 40% amino acid sequence identity, whereas sequence identity between classes is below 25%. cGSTs are divided into: (1) organism-specific GST classes, which include several GSTs, such as lambda (L), phi (F), and tau (U) in plants; delta (D), epsilon (E) in insects; beta (B) in prokaryotes; and 2) ubiquitous classes in any organism, including mu (M), alpha (A), pi (P), theta (T), sigma (S), zeta (Z), and omega (O) classes. Each of them displays distinct catalytic and non-catalytic binding properties, and their functions are very versatile and involve detoxification, signal modulation, catabolism of aromatic amino acids, ion channel modulation, chemotherapy resistance, prostaglandin and steroid hormone synthesis, and transport of molecules such as bilirubin, heme, steroids, hormones, and bile salts [25, 27, 29, 31, 33, 38–43].

#### **2.3 Structural characteristics**

All cGSTs are dimers with 24–27 kDa monomeric subunits containing ~250 amino acid residues on average. They share the same tertiary and quaternary

structures, and each subunit has two distinct functional domains. The first domain is the G site, which is located at the N-terminal region and is responsible for GSH binding. This domain is highly conserved in all classes and has a thioredoxin-like fold constructed by three helices and four sheets (βαβαββα). Activation of GSH occurs at the G-site by different amino acids, depending on the class, and is either a tyrosine (Y) found in M, P, A, and S-classes, a serine (S) found in T, Z, F, U, and D-classes, or a cysteine (C) to O, and B-classes. The activation allows a nucleophilic attack on the electrophilic compounds, allowing conjugation or thiol transfer. The first two amino acids, tyrosine and serine, promote the formation and stabilization of the thiolate anion of GSH, lowering its pKa to 6.2. This is achieved through hydrogen bond donation of the hydroxyl group, which makes GSH ready for conjugation. The C residue is used for thiol transfer, and it forms mixed disulfides with GSH. The N-terminal domain consensus sequence SNAIL/TRAIL is localized in the region between residues 68 and 77, and appears in all mammalian cGSTs [25, 29, 31, 44, 45]. The second domain is the H site, which is localized in the C-terminal region. This domain binds the electrophilic substrate, and it is constituted exclusively by α-helices. The number of helices varies from four to seven, depending on the class. This variation has been used to explain the wide range of electrophilic substrates for detoxification and specificity among classes. For example, the M-class has very efficient catalysis with molecules containing oxiranes and unsaturated carbonyl groups, whereas A-class acts on 4-hydroxyalkenals and peroxides [20, 25, 31, 33, 45]. Although GSTs do not present specificity for their hydrophobic substrates, they seem very specific for the γ-glutamyl portion of GSH, and there is evidence that a peptide portion in the conjugate binds to ATP pumps or the multidrug resistance-associated proteins to be exported [46, 47]. Furthermore, in these domains, there are also conserved motifs that identify GST classes. For example, the primary and secondary structures that form the mu-loop or α9-helix are characteristic of M, and A-GST classes [20, 45, 48].

#### **2.4 Alternative functions of GSTs**

Besides their catalytic role, ligandin activity has been identified in GSTs because they bind toxic non-substrate ligands, such as hemin, bilirubin, bile salts, steroids, thyroid hormones, fatty acids, drugs (albendazole and praziquantel), and members of the MAPK protein kinase family, which are involved in processes such as the production, storage, and rapid transport of prostaglandins out of cells, intrinsic and acquired drug resistance, cell survival and apoptosis, contributing to passive detoxification or intracellular transport in cells. The ligandin site is different from the G and H sites, and the above-mentioned toxic non-substrates are able to inhibit the catalytic activity of GSTs [49–52]. Another striking property of the GST enzyme is its translocation from the outside to the inside of various cells. This internalization occurs through endocytosis mediated by receptors or by the GST-fold structure, and it is independent of GST function as an enzyme [53, 54].

#### **2.5 GSTs in platyhelminthes**

In these parasites, GSTs also act as xenobiotic detoxifying enzymes, catalyzing conjugation of GSH (active detoxification) or, in the case of ligandin, transporting toxic substrates (passive detoxification) and acting as protective antigens to the host [23, 55]. Finally, many reports on vaccination experiments have described reductions in parasite burden, fecal egg counts, tissue egg densities, and female fecundity in experimental cysticercosis, schistosomiasis, and fascioliasis [23, 56–60]. The World Health Organization has recommended the use of *Schistosoma japonicum*

GST (SjGST) as a vaccine antigen in the form of a DNA vaccine (pcDNA/sjGST) in nanoparticles combined with pIL-12 [61, 62].

#### **2.6 GSTs in** *T. solium*

In the cestode *T. solium*, GST activity has been identified in the microsomal fraction, and it was noncompetitively inhibited by triphenyltin chloride and bromosulfophthalein [63]. Moreover, three cGSTs classes have been identified according to the classification of mammalian GSTs [20]: (i) a moderately abundant S-class GST denoted as TsMσGST, (ii) the least abundant M-class GST named Ts25GST (previously referred to as SGSTM1), which has a high capacity to conjugate reactive carbonyls, the secondary products of lipid peroxidation, and (iii) the most abundant M and A-class GST named Ts26GST (previously referred to as SGSTM2). The characteristics and properties of these enzymes are listed in **Table 1**.

The specific antibodies produced against each TsGST (TsMσGST, Ts25GST, and Ts26GST) showed that they are not antigenically related to each other, nor to trematode, cestode, or mammalian GSTs [19, 20, 64]. Interestingly, these specific antibodies recognized the homologous GST class in *T. saginata*, *T. taeniaeformis*, and *T. crassiceps*. On the other hand, immunizations of a murine model of cysticercosis with the SGSTF fraction purified from cysticerci (comprising both Ts25GST and Ts26GST) or with recombinant Ts26GST alone were highly effective in reducing cysticerci load by 90% and 74%, respectively, whereas the use of the native and recombinant Ts25GST as immunogens afforded lower protection rates, 46% and 25%, respectively [19].

The aforementioned result as well as the known lack of catalase and low activities of CYP450 and glutathione peroxidase have led us to postulate that GSTs are the major detoxification system for this parasite. In addition, the properties of cGSTs as immunogens and vaccination candidates make them attractive targets for the development of new drugs against this parasite [19, 20, 22, 64].

Anti-helminthic compounds such as mebendazole and praziquantel inhibited Ts26GST and TsMσGST *in vitro*, but they did not reach plasma concentrations *in vivo* that would allow effective inhibition of enzyme activity [20, 64, 65, 66]. To date, a


#### **Table 1.**

*Cytosolic glutathione transferases from* Taenia solium.

non-toxic inhibitor for GST has not been developed, but ethacrynic acid, haloenol lactone, disulfiram, and curcumin are potent inhibitors of human GST-P1 [67, 68]. A new generation of drugs, such as modified ethacrynic acid, γ-glutamyl-S-(benzyl) cysteinyl-R(−)-phenyl glycine diethyl ester (TER 199), and prodrug (TER 286), provide a starting point for development of novel powerful and specific inhibitors against human GST-P1. However, the clinical side effects have limited their application [24].

### **3. Kinetic and structural properties of Ts26GST**

#### **3.1 Kinetic mechanism of Ts26GST in the CDNB conjugation reaction**

Ts26GST is a bisustrate enzyme that exhibits a higher affinity for glutathione (GSH) than for 1-chloro-2,4-dinitrobenzene (CDNB), unlike other two cGSTs of

**Figure 1.**

*Alignment of the amino acid sequences of Ts26GST with representatives of different human GST classes. The percent identity matrix shows that Ts26GST is most related to human M-class GST (m1).*

*Development of New Drugs to Treat* Taenia solium *Cysticercosis: Targeting 26 kDa Glutathione… DOI: http://dx.doi.org/10.5772/intechopen.97342*

*T. solium* (see **Table 1**). Furthermore, the kinetic curves for both substrates showed positive cooperativity, indicating that the binding of the first substrate stabilizes the right conformation of Ts26GST to bind the second substrate [21]. This positive cooperativity, previously described for the GSTs of *P. falciparum* and in classes P1 and Z1 of mammals, allows the parasite to adapt to changes in the amounts of toxic molecules secreted by the host's immune cells or induced by oral drugs, and to inactivate them through efficient processing of these substrates [69].

Kinetic analyses performed at different concentrations of GSH and CDNB produced intersecting double-reciprocal plots that provided evidence of ternary complex formation during enzymatic conjugation [70]. Furthermore, because the intersection occurred on the abscissa, the mechanism proceeds through the random sequential binding of co-substrates [71].

To determine the GST class to which Ts26GST belongs, various class marker substrates and inhibitors were tested. Ts26GST conjugates the A-class markers cumene hydroperoxide and ethacrynic acid better than the M-class marker 1,2-dichloro-4-nitrobenzene [20]. However, Ts26GST is more sensitive to the M-class inhibitors cibacron blue and triphenyltin chloride than to bromosulfophtalein, an A-class inhibitor. This enzyme is also sensitive to the anthelminthic mebendazole, displaying a non-competitive inhibition pattern, which suggests that at least two molecules bind to Ts26GST [21].

#### **3.2 Structural similarity of Ts26GST to human cGSTs**

Multiple amino acid sequence alignments of Ts26GST with all classes of human cGSTs are shown in **Figure 1**. It can be seen from the percent identity matrix that the primary structure of Ts26GST is closely related to M-class (42% sequence identity) and A-class (27% sequence identity) but is more distant from other human GST classes. The G-site of Ts26GST belongs to class Y, with Y8 being the catalytic residue that activates GSH. This site also has the essential conserved residues for γ-glutamyl binding: P(57), Q(68), and S(69). The last two residues are part of the (Q )SHVIT sequence, which in mammalian GSTs constitutes the consensus motif (Q )SNAIL /(Q )TRAIL. Notably, amino acid variation in this consensus motif is one of the markers for distinguishing between mammalian and

#### **Figure 2.**

*Modeled structure of Ts26GST. (A) The domain with the site where glutathione binds, is highlighted in green, and the domain with the hydrophobic site, to which electrophilic substrates bind, is highlighted in gray. (B) The structure of Ts26GST in white is compared to human A-class GST structure (blue) and M-class structure (brown).*

parasite cGSTs [20]. Ts26GST has ligandin activity and is internalized by macrophages, suggesting an important role in transport and the parasite–host relationship [72, 73].

A homology model for Ts26GST was built from the structure of *Fasciola hepatica* M-class GST (PDB ID 2FHE), whose sequence has 47% identity, with 96% query coverage [70]. The analysis of this model with PROCHECK showed that 91.5% of residues are in favored regions in the Ramachandran plot, with no residues in the disallowed region. In addition, verification with ERRAT yielded an Overall Quality Factor of 93.55 and the Verify3D score was 95.18. A comparison of the Ts26GST model with M and A-class human GST structures is shown in **Figure 2**. It is clear that Ts26GST does not have the classical mammalian mu-loop or the canonical α9-helix observed in A-class GSTs.

### **4. Structure-based discovery of Ts26GST selective inhibitors**

#### **4.1 Search and selection of cavities with non-conserved residues as potential targets**

Knowing the structure of the target whose activity we wish to inhibit is an essential step for the discovery and optimization of specific inhibitors. Furthermore, if the target is a parasitic enzyme, and the host has orthologs, knowing and comparing their structures allows us to take advantage of their differences and design more specific inhibitors [74]. Different strategies have been used to find appropriate inhibitors, and we decided to look for a non-competitive inhibitor that cannot be displaced by excess substrate, i.e., the one that would not bind to either the G-site or the H-site. Thus, we focused our search on the area of the dimer interface, trying to find a site whose occupation would alter the architecture of at least one of the substrate sites and prevent catalysis. Furthermore, the binding of a molecule in this interfacial region could destabilize the quaternary structure of this enzyme, which is only active as a dimer. Likewise, we assumed that the site has a predominantly hydrophobic surface and contains a considerable fraction of non-conserved residues with respect to its human orthologs. Using the MOE's Site Finder tool [75], we found only one site that met all these requirements; its location is shown in **Figure 3**.

#### **Figure 3.**

*Putative binding site for TS26GST inhibitors whose occupancy could produce non-competitive inhibition. Just one subunit is represented with van der Waals surface for clarity. Bound GSH and CDNB molecules are shown in orange and magenta, respectively, whereas the spheres represent the space that the ligand could occupy.*

*Development of New Drugs to Treat* Taenia solium *Cysticercosis: Targeting 26 kDa Glutathione… DOI: http://dx.doi.org/10.5772/intechopen.97342*

#### **4.2 Virtual screening with a commercial diverse library set**

Once a potential inhibitor binding site has been located, we must find molecules that conform to its surface and interact favorably to form stable complexes. To explore how to cover this site in the chemical space, we used Enamine's library of non-redundant organic compounds called the Discovery Diversity Set, which consists of 50,240 drug-like compounds, and performed virtual screening using AutoDock Vina [76]. The scores of the best candidates were verified using MOE's Dock Tool [75].

#### **4.3 Assortment of candidates**

The best putative binders for Ts26GST were selected using the conventional criteria: the highest docking scores, the highest number of hydrogen bonds, and Lipinski's rule of five [77], but in addition, those ligands were prioritized that established the lowest number of contacts with conserved residues in relation to human GSTs. The best 23 candidates are shown in **Figure 4** and their docking scores obtained using AutoDock Vina and MOE are given in **Table 2**.

**Figure 4.** *Best candidate inhibitors found by virtual screening.*


#### **Table 2.**

*Docking scores of the predicted potential inhibitors determined using Vina and MOE\_Dock. The inhibitory capacity of the compounds was determined by measuring the enzymatic activity of T26GST in the presence of each compound at a concentration of 100 μM, with 5.0 mM GSH and 2.5 mM CDNB. The reaction rate was monitored by ultraviolet–visible absorption at 340 nm and compared with that obtained in the absence of the compound (100% activity).*

#### **Figure 5.**

*Relative position of the substrates GSH (orange) and CDNB (magenta), and the inhibitor I7 (red) in the structure of Ts26GST. (A) This figure was obtained by the superposition of the crystallographic structures of the complex M-class GS-DNB-HsGST (PDB ID: 1XWK) with the modeled complex of Ts26GST-I7, hiding the protein chain of the human GST. (B) Percent residual activity of Ts26GST and three human GSTs in the presence of 100 μM I7.*

*Development of New Drugs to Treat* Taenia solium *Cysticercosis: Targeting 26 kDa Glutathione… DOI: http://dx.doi.org/10.5772/intechopen.97342*

#### **4.4** *In vitro* **assay of selected compounds with the best scores**

The twenty-three compounds previously identified as potential ligands of Ts26GST were purchased and tested for their inhibitory activity using *in vitro* enzymatic assays. **Table 2** shows the residual activity obtained with 5.0 μg of recombinant Ts26GST in the presence of each compound at a concentration of 100 μM. I7 was the best Enamine compound that inhibited enzymatic activity of Ts26GST by 70%. **Figure 5A** shows the location of the I7 binding site, as derived from the docking protocol. We also tested the inhibitory effect of I7 on several human GSTs and observed that it had much smaller or no effect (**Figure 5B**).

#### **5. Conclusions**

Human NCC caused by *T. solium* larvae can be asymptomatic, disabling, and sometimes fatal. Currently, its diagnosis and treatment are expensive, and the approved drugs have associated unwanted effects. The search for the essential targets in *T. solium*, such as GST, and the methodology used to obtain the inhibitor I7 and its derivatives, shows that it is possible to develop safe, specific, and effective drugs that will contribute to eradicating this parasite. We are currently working on the crystallization of Ts26GST and site-directed mutagenesis to verify the location of the I7 binding site.

#### **Acknowledgements**

This work was supported by National Council of Science and Technology of Mexico (National Problems CONACYTPN-594) and (Frontier Science 2019 (7397); General Direction of Academic Staff Affairs at the National Autonomous University of Mexico (DGAPA-PAPIIT-IN217419), and the Direction of Computing and Technologies of Information and Communication (Miztli-LANCAD-UNAM-DGTIC-344). The authors thank the facilities provided by the Supercomputing and Parallel Visualization Laboratory at the Metropolitan Autonomous University, Iztapalapa Campus.

#### **Conflict of interest**

The authors declare that they have no known conflict of interest.

#### **Author details**

Rafael A. Zubillaga1 , Lucía Jiménez<sup>2</sup> , Ponciano García-Gutiérrez1 and Abraham Landa2 \*

1 Department of Chemistry, Metropolitan Autonomous University, Mexico City, Mexico

2 Department of Microbiology and Parasitology, Faculty of Medicine, National Autonomous University of Mexico, Mexico City, Mexico

\*Address all correspondence to: landap@unam.mx

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Development of New Drugs to Treat* Taenia solium *Cysticercosis: Targeting 26 kDa Glutathione… DOI: http://dx.doi.org/10.5772/intechopen.97342*

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Section 4

## The Host-Parasite Interaction

#### **Chapter 7**

## To Be or Not to Be a Tapeworm Parasite: That Is the Post-Genomic Question in *Taenia solium* Cysticercosis

*Diana G. Ríos-Valencia, José Navarrete-Perea, Arturo Calderón-Gallegos, Jeannette Flores-Bautista and Juan Pedro Laclette*

#### **Abstract**

Cestode parasites rely on their host to obtain their nutrients. Elucidation of tapeworm genomes has shown a remarkable reduction in the coding of multiple enzymes, particularly those of anabolic pathways. Previous findings showed that 10–13% of the proteins found in the vesicular fluid of *Taenia solium* cysticerci are of host origin. Further proteomic characterization allowed identification of 4,259 different proteins including 891 of host origin in the parasite's protein lysates. One explanation for this high abundance and diversity of host proteins in the parasite lysates is related to the functional exploitation of host proteins by cysticerci. Supporting this concept is the uptake of host haptoglobin and hemoglobin by the parasite, as a way to acquire iron. Surprisingly, internalized host proteins are minimally degraded by the parasite physiological machinery. Additional proteomic analysis demonstrated that these host proteins become part of the organic matrix of calcareous corpuscles; as 60–70% of the protein content are host proteins. In this review, a collection of available genomic and proteomic data for taeniid cestodes is assembled, the subject of the use and processing of host proteins is particularly addressed; a sketchy and unique cell physiological profile starts to emerge for these parasitic organisms.

**Keywords:** *Taenia solium*, Cestoda, Genome, Proteome, Host proteins, Calcareous Corpuscles

#### **1. Introduction**

Tapeworms are invertebrate metazoans producing zoonotic parasite diseases in animals and humans. These parasites have a worldwide distribution, but they especially affect human populations in developing countries and are considered neglected diseases [1]. Their larvae, known as metacestodes (including forms such as cysticercus in *Taenia solium* or hydatid or alveolar cysts in *Echinococcus* sp.) cause the highest morbidities due to tapeworms [2, 3], since they can produce generalized organ failure or seizures and can even result in patient's death [4–7].

Tapeworms produce long-term infections, being able to survive within its host for several years [8], maintaining a dynamic and complex host-parasite relationship [9]. Their lifecycles involve two host (intermediate and final) and include several developmental stages: embryo, larvae and adult stage [10] that can lodge in different tissues of their hosts producing diseases with a wide range of clinical presentations [11].

After description of the genomes of four tapeworms in 2013 [12], molecular studies of these organism have entered an integrative era; including approaches involving genomics, transcriptomics and proteomics [13]. These approaches are presented as promising avenues for the discovery of new pathways to improve our understanding of parasite diseases caused by cestodes, in the hope of developing better surveillance, treatment and control guidelines.

This chapter reviews current perspectives in the study of flatworms; special emphasis is placed on the genomics and proteomics of cestodes and taeniid parasites. The conspicuous and abundant presence of host proteins is particularly considered for taeniid larval forms.

#### **2. The Platyhelminth genome**

Access to massive sequencing technologies allowed characterization of entire genomes for some of the most relevant flatworms; being the free living *Schmidtea mediterranea* the first trematode to be reported in 2008 [14]. The timeline of all flatworms genome projects that have been published to date clearly shows the advent of the post-genomic era of flatworms (**Table 1**). A rapid characterization of the genomes of parasites with medical importance, such as *Schistosoma japonicum, S. mansoni, Clonorchis sinensis*, among others, followed by four cestodes: *Hymenolepis microstoma, Echinococcus granulosus, E. multilocularis and Taenia solium* [12]. Subsequently, the International Helminth Genomes Consortium carried out a project with a goal of 50 helminth genomes. These genomes are currently deposited in the WormBase Parasite database, where users can access 197 genomes [15], including 44 Platyhelminthes: 4 free-living flatworms, 20 trematodes, 19 cestodes and 2 monogeneans (**Figure 1**). This platform also allows searching protein domains and Gene Ontology terms,


#### **Table 1.**

*Statistics of completed genome sequencing for several tapeworms.*

*To Be or Not to Be a Tapeworm Parasite: That Is the Post-Genomic Question in* Taenia solium*… DOI: http://dx.doi.org/10.5772/intechopen.97306*

**Figure 1.** *Timeline of flatworms genome characterization [12, 14–39].*

as well as performing comparative analysis of genes and alignments of RNA-Seq data sets, specific to the life stage genomes, among other useful functions [40, 41].

Other taeniid genomes have been reported outside the International Helminth Genomes Consortium during the past five years: *T. asiatica*, *T. saginata* [31] and *T. multiceps* [34], *E. canadensis* [32], *E. oligarthrus* [35], as well as *Hymenolepis diminuta* [36]; circumstances appear prone to greatly improve our understanding of the biology and evolution in those organisms, as well as to solve old unanswered questions on their host-parasite relationships. Availability of this genomic information allows integrative studies on this ancient lineage of organisms. **Table 1** includes the basic statistics of reported assemblies for several tapeworms of medical or veterinary importance, being *E. oligarthrus* the smallest assembly (86 Mb) and *T. multiceps* the largest one (240 Mb). The average GC content of these genomes is 35-43.7%, similar to trematode genomes [23] but different to bacterial genomes whose GC range content is 13.5%-74.9% [42]. As a reference, GC average content of vertebrates is 46% [43]; mice 41.7% [44] whereas human genome is 40.9% [45].

#### **3. Gene gain/reduction along tapeworm evolution**

The genomic data of the first four tapeworm genomes sequenced [12] permitted identification of reduction events for groups of genes such as Wnt, which corroborated some data that suggested the loss of these genes in trematodes [46]. Moreover, other genes as Nek kinases, peroxisomal genes and ParaHox members, as well as neuropeptides and G-protein coupled receptors (GPCRs) [15].

The loss of approximately 10 Hox gene families in tapeworms during their evolutionary pathway apparently affected the morphology of those organisms, i.e., the lack of eye-cups and gut [12]; Hox genes such as pax3/7, gbx, hbn and rax are mainly involved in neuronal development or eye development [47–50], as well as ParaHox genes in the formation of the digestive tract [51]. Another type of proteins absent in cestodes are those related to germ cells such as piwi, tudor and Vasa, although the latter have been found possible orthologues in the PL10 family [12].

Tapeworms have developed a specialized detoxification system that includes a single cytochrome p450 gene [12, 52], as well as a redox homeostatic system based on thioredoxin glutathione reductase and the expansion of glutathione

#### **Figure 2.**

*Gains and losses of genes in taeniids. Phylogenetic study carried out with the genomes of the cestodes allowed finding important aspects about how these organisms acquired or lost some of their genomic traits to adapt to the conditions of their current environments.*

S-transferases [53–55]. In addition, there was an expansion of some very specific protein families such as non-canonical heat shock proteins, with *Echinococcus* and *T. solium* having the highest number of genetic expansions in the cytosolic clade Hsp70 [12] suggesting that tapeworms have different mechanisms from nematodes to overcome stress [16]. In addition, taeniids have an expansion in some families of antigens such as GP50 [12, 15]. These antigens are useful for diagnostics; for example, coenurosis in goats [56] or cysticercosis in pigs [57]. For diagnosis of human cysticercosis, the use of GP50 as a diagnostic target allows a 100% specificity and 90% sensitivity using serum samples of patients [58, 59]. Some of the main gains and losses of genes in taeniids are summarized in **Figure 2**.

Our current knowledge on cestode's and taeniid's genomes is still limited but the speed of genomic data acquisition can advance significantly in this new era. We envisage a better understanding of these host–parasite interactions, at a molecular/ evolutionary level that can help us unravel events that have permitted the adaptations of these platyhelminths to the host environment.

#### **4. Metabolic adaptations of tapeworms**

A great impact of having available complete tapeworm genomes is the characterization of the metabolic pathways in these organisms. Now we know that taeniid tapeworms cannot synthesize fatty acids and cholesterol de novo [25, 60]. For example, KEGG analysis for fatty acid biosynthesis in *T. solium* clearly shows that most of the components of the pathway are absent (**Figure 3**). Therefore, these parasites cannot carry out biosynthesis of fatty acids and are obligated to acquire host fatty acids through specific transporters [63]. Moreover, no genes related to the β-oxidation pathway were found in *Echinococcus* and *Hymenolepis,* although experimental data suggest that other flatworms do carry out this metabolic process [64] for utilization of lipids as a source of energy. It is clear that their major energy source are carbohydrates such as glucose and glycogen. This is supported by the fact that most enzymes participating in carbohydrate catabolism are expressed.

The synthesis of pyrimidines is also absent for taeniids [65], indicating that they acquire pyrimidines from their hosts. The biosynthesis of purines shows a similar landscape [15]. Parasitic flatworms are considered auxotrophic for eight *To Be or Not to Be a Tapeworm Parasite: That Is the Post-Genomic Question in* Taenia solium*… DOI: http://dx.doi.org/10.5772/intechopen.97306*

#### **Figure 3.**

*KEGG analysis of the fatty acid biosynthesis in* T. solium*. Enzymes available for this pathway are acetyl-CoA carboxylase (6.4.1.2), S-malonyltranferase (FabF), 3-Oxoacyl-[acyl-carrier-protein] synthetase II (FabF) and Ketoacyl-acyl carrier protein (FabG) [green squares] [61, 62].*

of the nine amino acids that are essential for humans (Phe, His, Lys, Leu, Met, Thr, Trp, and Val). Cestodes have a limited ability to synthesize amino acids, as an example, serine and proline are absent in *E. multilocularis* [16]; biosynthesis of lysine and the aromatic amino acids (Phe, Trp and Tyr) are also absent in most cestodes (**Figure 4**). Arginine is also an essential amino acid in helminths including flatworms, as they do not have all the necessary enzymes of the urea cycle to process

#### **Figure 4.**

*KEGG analysis of phenylalanine, tyrosine and tryptophan biosynthesis in* T. solium*. The only enzymes that are present in the* T. solium *genome are indicated in green within boxes [61, 62].*

ornithine, which is the precursor of arginine [15]. In summary, these parasitic organisms rely on their host for the acquisition of fatty acids, nucleosides and most amino acids. Metabolically speaking, they show highly simplified genomes.

#### **5. Taeniid larval tissues contain large amounts of host proteins**

The presence of host proteins in the tissues of the cystic larval forms of taeniids has been known for a long time [66–70]. It has been proposed that the mechanism for the uptake of these proteins is fluid pinocytosis in the cysticerci of *T. crassiceps* [69]. Moreover, in addition of entering the host proteins, these parasites can also secrete them [70, 71]. The biological role of those uptaken host proteins remains elusive, however, uptake of host albumin has been proposed to be involved in the maintenance of host-parasite osmotic pressure [68] and uptake of host immunoglobulins has been proposed as a mechanism of immune evasion and even as a source of amino acids [72].

Recent quantitative estimates indicated that host proteins might represent 11–13% of the protein content in the vesicular fluid of *T. solium* cysticerci, with albumin and immunoglobulins being the most abundant proteins. The use of high-throughput proteomics, allowed identifying 891 proteins of host origin from a total of 4,259 in a *T. solium* cysticerci whole protein extract [73]; thus, host proteins might represent up to 19% of the total protein species in the larval tissue lysates. Moreover, a fraction of these uptaken host proteins are intact and perhaps functionally active in the tissues of taeniid larvae [71].

#### **6. Utilization of host proteins by cysticerci; iron chaperons and IgG**

A known trait of parasitism is the use of the host as a provider of resources; sugars, amino acids, nucleosides, vitamins, coenzymes and/or microelements are good examples of resources that a parasite can acquire from its host. However, considering the abundance and diversity of host proteins present in the tissues of taeniid larvae, a pertinent question would be: are these parasites benefited by the accumulation of host proteins, beyond simply serving as a source of amino acids or as osmotic regulators? We have explored a couple of prospects: the use of host iron chaperones for the management of the parasite's iron necessities, as well as the use of host immunoglobulins as a source of amino acids [70, 71, 74].

Iron is an essential element for virtually all living organisms. Pathogens have evolved mechanisms to uptake iron from their hosts. Usually, iron is uptaken from plasma proteins: hemoglobin (heme prosthetic group) or haptoglobin-hemoglobin complexes, hemopexin (heme prosthetic group), transferrin or lactoferrin (iron), ferritin (iron), etc. In fact, the constant battle between host and pathogens for this element is well-studied [75, 76]. Hepcidin, the hormone that control iron levels in mammals, was first discovered as an antimicrobial peptide [76, 77]. In this light, it is expected that cestodes would acquire iron from their host, however, the mechanism remains elusive. Some evidence have suggested that hemoglobin or the haptoglobin-hemoglobin complexes could serve as an iron source for the cysts [78]. To support this notion, we have documented the immunolocalization of haptoglobin, hemoglobin, hepcidin and ferritin in the cyst; immunoblotting using crude larval extracts confirmed the finding [73]. We also showed that haptoglobinhemoglobin complexes were detected in crude larval extracts in their expected molecular weight, indicating that those complexes are only marginally degraded. In fact, free haptoglobin purified from cysts protein lysates has been shown to retain

*To Be or Not to Be a Tapeworm Parasite: That Is the Post-Genomic Question in* Taenia solium*… DOI: http://dx.doi.org/10.5772/intechopen.97306*

their hemoglobin binding activity, suggesting that the cyst are acquiring iron from those sources. However, future studies are needed to understand how the uptake is performed (is there a specific receptor?), how the heme prosthetic group or iron is removed from those complexes? and which parasite proteins are performing those roles.

Another aspect related to the host's protein uptake by tapeworm's larvae, is the utilization of these proteins as a source of amino acids. Internalization of IgG has been traced using a metabolically labeled (Leu-3H) IgG produced *in vitro* using a mice hybridoma [71]. Through *in vitro* culture of *T. crassiceps* (a closely related species of *T. solium*) cysts in the presence of (Leu-3H) mice IgG, uptake of the immunoglobulin can be monitored. Metabolic labelling also allowed tracking incorporation of Leu-3H into newly synthesized cyst proteins. The biochemical analyses revealed that within the tissue extracts, no other radiolabeled proteins were found. The two bands corresponding to the heavy (50 kDa) and light (25 kDa) chains remained intact after 3 days of culture. This would imply that these proteins are negligible used as a source of amino acids for the biosynthesis of the larvae's own proteins. Furthermore, the integrity and functionality of the Igs was conserved, as shown by SDS-PAGE and western blots marked with the Igs purified from tissue extracts. This finding led the research into a new direction: If immunoglobulins (and perhaps other uptaken host proteins) are only a minor source of amino acids [71], what is the fate of uptaken host proteins?

#### **7. The calcareous corpuscles as a final deposit for host proteins**

The tracking of metabolically radiolabeled IgG demonstrated that cysticerci do not significantly use these proteins as a major source of amino acids [71]. A possibility was that these proteins could end in the calcareous corpuscles (CC), that are known as a waste of toxic metabolites and other materials. These CC are microscopic calcifications occurring in the lumen of protonephridial canals, resulting after accretion of mineral salts (calcium carbonate and calcium phosphate) on an organic matrix composed by polysaccharides and other macromolecules [79, 80]. The CC

#### **Figure 5.**

*Immunological identification of host IgG and albumin recovered from the protein matrix of calcareous corpuscles of* T. solium *cysticerci. Lanes 1 and 2 correspond to Coomassie blue staining of protein extract from CC and silver staining of porcine serum respectively. Lanes 3 and 5 are western blots of protein extracts obtained from CC, lanes 4 and 6 are gels run with porcine serum, these blots were revealed with an* α*-IgG coupled to HRP (3 and 4) or sheep* α*-albumin and then with a rabbit* α*-sheep IgG coupled to HRP (5 and 6). This figure was originally published in [70].*

are involved in the removal of toxic solutes and regulation of mineral trafficking [81]. Formation of CC has been proposed as a mechanism for protecting cysticerci from calcification [79]. The CC represent about 10% of the dry weight of total larval tissue [82]. It has been estimated that in aged *T. solium* cysticerci, calcareous corpuscles can represent up to 41% of the dry weight [81, 82].

Searching for host proteins in the organic matrix of CC from *T. solium* cysticerci, a mass spectrometry analysis was carried out. A total of 636-760 proteins were identified and quantified, from which 412-508 (60-70%) corresponded to host proteins. *T. solium* proteins in the organic matrix of CC were only 224-252 (30-40%). The remarkable finding that the major protein component in the organic matrix are host proteins, suggests that CC act as a final destination for host proteins. We also showed that intact host proteins can be recovered even after dissolution of CC in a weak acid solution (**Figure 5**). Therefore, these proteins are incorporated into the organic matrix without being degraded [71]. If host antibodies are incorporated into the organic matrix of CC in the form of immune complexes, it is conceivable that cysticerci developed this strategy as a way to diminish exposure of relevant parasite antigens, which could result in a sophisticated mechanism to evade the adaptive humoral immunity of the host.

#### **8. Conclusions**


#### **Acknowledgements**

This report was supported in part by grants A1-5-11306 (CONACYT) and [IN 205820] PAPIIT-UNAM.

#### **Conflict of interest**

The authors declare that there are no conflict of interest associated with the manuscript.

*To Be or Not to Be a Tapeworm Parasite: That Is the Post-Genomic Question in* Taenia solium*… DOI: http://dx.doi.org/10.5772/intechopen.97306*

### **Author details**

Diana G. Ríos-Valencia1 , José Navarrete-Perea<sup>2</sup> , Arturo Calderón-Gallegos1 , Jeannette Flores-Bautista1 and Juan Pedro Laclette1 \*

1 Biomedical Research Institute, Universidad Nacional Autónoma de México, Mexico City, Mexico

2 Department of Cell Biology, Harvard Medical School, Boston Massachusetts, United States

\*Address all correspondence to: laclette@iibiomedicas.unam.mx

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 8**

## Hormones and Parasites, Their Role in *Taenia solium* and *Taenia crassiceps* Physiology and Development

*Marta C. Romano, Ricardo A. Valdez, Martin Patricio, Alejandra Aceves-Ramos, Alex I. Sánchez, Arlet Veloz, Pedro Jiménez and Raúl J. Bobes*

#### **Abstract**

The host's hormonal environment determines the susceptibility, the course, and severity of several parasite infections. In most cases the infection disturbs the host environment, and activates immune responses that end up affecting the endocrine system. In the other hand, a number of reports indicate that parasites have reproductive systems, and some others have shown that these organisms synthetize sex steroid hormones. We have shown that cysticerci, the larval stage of *Taenia solium and Taenia crassiceps* ORF and WFU, synthesize steroid hormones. This capacity was modified by drugs that act inhibiting the steroid synthesizing enzymes, or blocking the parasite's hormone receptors. We have also shown that the cysticerci of *T. crassiceps* WFU and *T. solium* have the capacity to synthesize corticosteroids as deoxicorticosterone and corticosterone. We also reviewed the effects of insulin on these parasites, and the receptors found for this hormone. A deep knowledge of the parasite's endocrine properties will contribute to understand their reproduction and the reciprocal interactions with the host. Likewise, may also help designing tools to combat the infection in clinical situations.

**Keywords:** Parasites, *Taenia*, cysticerci, hormones, steroids

#### **1. Introduction**

Corticosteroids and sex steroids are crucial in vertebrate reproduction, metabolism and immune response, but their role in invertebrates had received a reduced attention, similarly happen with the influence of peptides and protein hormones in parasite's development. Therefore, we review here the parasite-endocrine system interplays.

The interaction between parasites and the host defines the intensity of parasite infections. In many cases, the presence of parasites in the host changes its endocrine equilibrium due to the activation of the immune system response, which finally affects the host endocrine system through the influence of cytokines and growth factors released by the immune cells. These changes sometimes control the infection, but in many cases the immune system of the host cannot reject the parasite invasion and thereafter the organisms succeed, and rapidly multiply in the host. A role for 17-beta-estradiol in immunoendocrine regulation of murine cysticercosis by *Taenia crassiceps* was verified [1].

Some parasite infections disrupt the host endocrine system, to this regards we recently reported that the chronic infection of female mice with *T. crassiceps* WFU disrupted the ovarian folliculogenesis, causing a significant increase in follicle atresia, and a reduction in the number of corpora lutea (**Figure 1**) [2]. We also showed in that study that *T. crassiceps* cysticerci infection increased the female mice serum estrogen concentrations, an effect that augmented with the infection time, and that the infection increases the ovarian expression of the steroidogenic enzymes P450 aromatase and P450-Cyp19 [2]. We and collaborators also shown that the nervous system infection with *T. solium* cysticerci (neurocysticercosis) caused endocrine alterations in male and female patients [3], and Sacerdote et al. [4] showed that brain cysticerci images reduced or disappeared after treatment with raloxifene in a patient diagnosed with neurocysticecosis.

In some cases, the parasite's infection affects the host reproductive behavior, for example, changes in reproductive behavior occurred in *Taenia crassiceps* ORF infected male mice [5].

#### **1.1 Sex steroids effects**

Several reports indicate the host hormonal environment determines the susceptibility, the course, and severity of many parasite infections. Supporting this fact, a clear dichotomy in infection susceptibility between males and females had been observed in some parasitic infections. For example, the rich estrogen environment provided by female mice facilitates *T. crassiceps* ORF cysticerci proliferation [6].

In addition, steroids may directly influence the growth and proliferation of parasites. For example, *T. crassiceps* ORF and WFU cysticerci cell proliferation and metabolism evaluated by <sup>3</sup> H-thymidine and MTT incorporation was increased by the addition of physiological concentrations of testosterone, and 17β-estradiol to the culture media [7] and enhance proliferation of *T. crassiceps* ORF cysticerci, a progesterone like receptor was found in these parasites [8].

**Figure 1.**

*The infection with* Taenia crassiceps *WFU decreased the ovarian corpora lutea of female mice. The number of corpora lutea diminished when the infection progresses.*

*Hormones and Parasites, Their Role in* Taenia solium *and* Taenia crassiceps *Physiology... DOI: http://dx.doi.org/10.5772/intechopen.98531*

Particularly estrogens are important for *T. crassiceps* and *T. solium* cysticerci development. Estrogen synthesis is the result of the transformation of androgens to estrogens by the steroidogenic enzyme P450-Aromatase (Arom) that transforms androstendione and testosterone to the estrogens 17β-estradiol and estrone. Interventions that reduced estrogen synthesis, or affected the binding to its receptors affect the cysticerci proliferation. For instance, the administration of fadrozole, a drug that inhibits Arom, to *T. crassiceps* ORF female infected mice reduced the parasite's load [9].

The presence of steroid receptors in parasites have been documented [10], hence the blockage of steroids receptors might mitigate the effect of these hormones. For example, the expression of an estrogen binding protein similar to nuclear estrogen receptor was shown in *T. crassicpes* ORF cysticerci [11].

Interventions on the sex steroid receptors affect the *T. crassiceps* cysticerci parasite charge. That is the case for the administration of tamoxifen, a competitive antagonist of the estrogen receptor alfa that reduced *in vitro* the proliferation and viability of *T. crassiceps* ORF cysticerci [12] and *in vivo* reduced parasite's load. Likewise, we have shown that the administration of flutamide, an androgen receptor competitor, reduced the parasite proliferation [7].

#### **1.2 Corticosteroids are key hormones in the host-parasite interplay**

Corticosteroids are synthesized in the adrenal cortex and are classified as glucocorticoids, mineralocorticoids and adrenal androgens. Cortisol and corticosterone are the main glucocorticoids and are involved in glucose, lipid and protein metabolism. Aldosterone and dexycorticosterone (DOC) are classified as mineralocorticoids because they participate in the hydro-electrolytic balance, whereas adrenal androgens as dehydroepiandrosterone (DHEA) take part in the pubertal process. DHEA is an estrogen precursor that can be transformed to potent androgens in the testis and is an important immune regulator [13].

Cortisol and corticosterone are key hormones in the physiological stress response (in example exercise), and in non-physiological stress situations, such as social isolation, persecution, infections, etc., all circumstances that increase serum corticosteroids levels. It is now generally accepted that prolonged stress conducts to impairment of the immune response.

#### *1.2.1 Corticosteroid use in neurocysticercosis*

Corticosteroids are employed to prevent or modulate the brain inflammation that follows anthelmintic treatment of parasitic cysts with cysticidal drugs as albendazole or praziquantel [14–16]. The absence of corticosteroids administration in the cysticidal treatment initiates an acute immune response to the parasite that conducts to serious clinical symptoms as seizures, brain edema, and death. These side effects are caused by neuroinflammation and are effectively managed with corticosteroids. On the other side, the administration of dexamethasone plus albendazol to Balb/c mice reduced the cysticidal effect of albendazole [17].

#### *1.2.2* In vitro *effects of glucocorticoids on parasite growth and viability*

It had been shown that corticosteroids may directly influence parasite's proliferation and metabolism. For instance, we had shown that corticosterone and dexamethasone increase the capacity of *T. crassiceps* WFU cysticerci to synthesize androgens and estrogens, hormones that favor the parasite reproduction [18].

#### **1.3** *Taenia solium* **and** *crassicpes* **synthesize steroid hormones**

#### *1.3.1 Sex steroids and corticosteroids*

The adult worm of *T. solium* and *T*. *crassiceps* WFU remain attached to the host gut with hooks placed in their head, and develop reproductive units called proglottids, where testis and ovaries gradually differentiate, and finally contain spermatocytes and infective eggs [19]. As stated elsewhere *T. solium* cysticerci is the larval stage of the parasite and is found in the brain or muscle of humans and pigs, whereas *T. crassiceps* WFU cysticerci constitute a useful laboratory model due to their reproduction by budding in the peritoneal cavity of mice. In the last years we have been investigating if *T. solium* and *T. crassiceps* ORF and WFU cysticerci and tapeworms synthesize sex steroids *in vitro.* We found that *T. solium* and *T. crassiceps* ORF cysticerci transform steroid precursors such as progesterone, DHEA, and androstenedione to androgens and estrogens, the capacity to transform precursors to testosterone was related to the developmental stage of the larvae (**Table 1**) [20–22]. These findings demonstrated that *Taenids* are steroidogenic organisms.

Our group have also examined the capacity of *T. solium* and *T. crassiceps* WFU to synthesize corticosteroids. Thereafter, we had incubated *T. crassiceps* cysticerci in the presence of 3 H-progesterone and found an important transformation into DOC, a steroid that has mineralocorticoid functions in vertebrates [23, 24]. The addition to the culture medium of metyrapone, a drug used for the medical control of hypercortisolism in Cushing's syndrome, reduced the cysticerci corticosteroid synthesis [23]. In addition, the parasites synthesized corticosterone, which was measured by radioimmunoassay in the culture media. More recently, we found corticosteroid-like synthesis in *T. solium* and *T. crassiceps* tapeworms [24, 25]. To note, the steroidogenic capacity of *T. crassiceps* is related to the development of the parasite [24]. Besides their effects on the own parasite development and differentiation, the cysticerci and tapeworm's steroidogenic capacity might play a role in the permanence of the parasites in the host tissues and organs, by disturbing the host immune cell response.

#### *1.3.2* Taenia solium *and* Taenia crassiceps *and steroidogenic enzymes. Repurposed drugs affect the capacity of parasites to synthesize hormones*

Tritiated androstenediol and testosterone were recovered from the culture media of *T. crassiceps* incubated with 3 H-DHEA indicating the presence and activity of enzymes from the Δ5 steroid pathway in these tapeworms [26].

The effect of enzyme inhibitors on the steroid synthesis by *T. crassiceps* WFU cysticerci was investigated by [27]. This study demonstrated that fadrozole, a drug that inhibits P450-aromatase, reduced the transformation of 3H-androstenedione to 17β-estradiol (**Figure 2**), while danazol that inhibits 3β-hydroxysteroid dehydrogenase and 17β-hydroxisteroid dehydrogenase, reduced the transformation of 3H-DHEA to androstendiol, testosterone and 17βestradiol. The incubation of cysticerci with tritiated progesterone as a precursor and different concentrations of ketoconazole that inhibits 11β-hydroxilase, 17α-hydroxilase and 17-20 lyase, resulted in the reduction of the synthesis of tritiated 3H-DOC [27].

We have recently shown that *Taenia solium* cysticerci express the enzyme 17β-HSD that belongs to the short chain dehydrogenases/reductase family [28]. Transient transfection of HEK293T cells with Tsol17β-HSD-pcDNA3.1 (+) induced expression of Tsol17β-HSD that transformed 3 H-androstenedione into testosterone (**Figure 3**). In contrast, 3 H-estrone was not significantly transformed into estradiol. Therefore, *T. solium* cysticerci express a 17β-HSD that catalyzes the androgen reduction and belongs to the short chain dehydrogenases/reductase (SDR) protein

*Hormones and Parasites, Their Role in* Taenia solium *and* Taenia crassiceps *Physiology... DOI: http://dx.doi.org/10.5772/intechopen.98531*


#### **Table 1.**

*Synthesis of sex steroids by* Taenia crassiceps *WFU cysticerci. The parasites were incubated by different periods in the presence of 3 H-androstenedione, the culture media was analyzed by TLC. The synthesized steroids are express as percent transformation of the tritiated precursor.*

**Figure 2.**

*Effect of formestane, an inhibitor of P450-aromatase, on the synthesis of 17*β*-estradiol by* T. Crassiceps *WFU cysticerci. The parasites were incubated with 3 H-androstenedione for 24 h. The percent of tritiated 17*β*-estradiol synthesized was determined by TLC.*

#### **Figure 3.**

*Testosterone production by HEK293T cells transfected with Tsol-17*β*HSD-pcDNA3.1(+). After 24 h of transfection with Tsol-17*β*HSD-pcDNA3.1(+) (white bars) or with pcDNA3.1(+) (black bars), cells were incubated with 3 H-androstenedioned for 24 or 48 h. The percent of tritiated testosterone was determined by TLC.*

superfamily [28]. A sequence with an identity of 84% with Tsol-17βHSD and a total coverage has been described for *E. multilocularis*, suggesting the presence of 17β-HSD enzymes in these parasites [29]. However, the expression level and enzyme activity of this species has not been yet investigated.

#### **1.4 Additional hormones studied in** *T. solium* **and** *Taenia crassiceps*

Insulin is a potent metabolic hormone that exerts a wide variety of effects. The main metabolic effect of insulin is to stimulate glucose uptake and utilization in muscle and fat tissue, but this hormone also increases lipogenesis, and even acts on protein synthesis. Insulin signaling through insulin receptors (IR) is an ancient and well conserved pathway in metazoan cells organized as transmembrane proteins with tyrosine kinase activity. To note, the uptake and metabolism of glucose is crucial for *T. solium* and *crassiceps* survival.

We have shown that incubation of *T. crassiceps* cysticerci with insulin increased the reproduction of the parasites and also found that female mice exposed to insulin had larger parasite loads than control mice inoculated with vehicle [30]. In the same study an insulin-like receptor present in *T. solium* and *T. crassiceps* was amplified by reverse transcriptase-polymerase chain reaction.

Using genome-wide screening Wang et al. [31] identified putative insulin-like peptides in several parasitic platyhelmths as *T. solium*. Furthermore, two insulin receptor genes were identified and characterized in *T. solium*. The receptors were found in diverse zones of the parasite and are involved in the uptake of glucose, that is crucial for these parasites [32].

The effect of human chorionic gonadotropin (hCG) on the growth and proliferation of larval stages of *T. crassiceps* (WFU strain) and *T. solium,* and the presence of receptors for this hormone in different developmental phases of both cultured parasites was reported [33, 34].

#### **Figure 4.**

*A. Synthesis of steroids by cysticerci. The larval stage of* Taenia solium *and* Taenia crassiceps *synthesize sex steroids and corticosteroids from tritiated precursors. Sex steroids influence the cysticerci development. The addition of steroidogenic enzymes or receptor blockers to the culture media reduced the steroid synthesis by the parasites. B. The host-parasite interplay, and the immune-endrocrine interactions influence the course of the parasite infections.*

*Hormones and Parasites, Their Role in* Taenia solium *and* Taenia crassiceps *Physiology... DOI: http://dx.doi.org/10.5772/intechopen.98531*

#### **2. Conclusions**

The interaction between parasites and the host defines the intensity of parasite infections. Steroid hormones play an important role in this interplay. Sex steroids and corticosteroids modify *in vitro* the proliferation of *T. solium* and *T*. *crassiceps* ORF and WFU cysticerci. Cysticerci and worms have the capacity to synthesize corticosteroids and sex steroids from tritiated precursors, a fact that suggested they have several active steroidogenic enzymes. One of these enzymes, 17β-hydroxysteroid dehydrogenase like was characterized and cloned in *T. solium* cysticerci. The steroidogenic capacity of these parasites was modified with repurposed drugs that affects steroidogenic enzymes as formestane that acts on P450-aromatase, danazol and ketoconazol (**Figure 4**). Insulin modifies the proliferation of cysticerci, and receptors for insulin had been found in parasites. Steroidogenic enzymes inhibitors, and receptors blockers might be used as therapeutic tools for the control of parasitic infections

#### **Acknowledgements**

Thanks to Rangel-Rivera Omar for his technical contribution with Figures and Table.

#### **Conflict of interest**

The authors declare that they have no known conflict of interests or personal relationships that could have appeared to influence the work reported in this chapter.

#### **Author details**

Marta C. Romano1 \*, Ricardo A. Valdez1 , Martin Patricio1 , Alejandra Aceves-Ramos1 , Alex I. Sánchez1 , Arlet Veloz1 , Pedro Jiménez1 and Raúl J. Bobes2 \*

1 Department of Biophysical Physiology and Neurosciences, CINVESTAV del I.P.N, Ciudad de México, Mexico

2 Immunology Department, Biomedical Research Institute, Universidad Nacional Autónoma de México, Ciudad de México, Mexico

\*Address all correspondence to: mromano@fisio.cinvestav.mx and rbobes@iibiomedicas.unam.mx

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### *Edited by Jorge Morales-Montor, Abraham Landa and Luis Ignacio Terrazas*

This book provides updated information to scientists and clinicians on taeniosis/ cysticercosis, a parasitic infection caused by eating undercooked beef or pork that is a serious health and veterinary problem in many developing countries. It discusses incidence, risk factors, diagnosis, immunology, symptoms, rare manifestations, and advances in treatment including vaccination and novel drug therapies.

Published in London, UK © 2021 IntechOpen © Mailson Pignata / iStock

Current State of the Art in Cysticercosis and Neurocysticercosis

Current State of the Art

in Cysticercosis and

Neurocysticercosis

*Edited by Jorge Morales-Montor,* 

*Abraham Landa and Luis Ignacio Terrazas*