Taenia solium cysticerci's extracellular vesicles Attenuate the AKT/mTORC1 pathway for Alleviating DSS-induced colitis in a murine model.

The excretory-secretory proteome plays a pivotal role in both intercellular communication during disease progression and immune escape mechanisms of various pathogens including cestode parasites like Taenia solium. The cysticerci of T. solium causes infection in the central nervous system known as neurocysticercosis (NCC), which affects a significant population in developing countries. Extracellular vesicles (EVs) are 30-150-nm-sized particles and constitute a significant part of the secretome. However, the role of EV in NCC pathogenesis remains undetermined. Here, for the first time, we report that EV from T. solium larvae is abundant in metabolites that can negatively regulate PI3K/AKT pathway, efficiently internalized by macrophages to induce AKT and mTOR degradation through auto-lysosomal route with a prominent increase in the ubiquitination of both proteins. This results in less ROS production and diminished bacterial killing capability among EV-treated macrophages. Due to this, both macro-autophagy and caspase-linked apoptosis are upregulated, with a reduction of the autophagy substrate sequestome 1. In summary, we report that T. solium EV from viable cysts attenuates the AKT-mTOR pathway thereby promoting apoptosis in macrophages, and this may exert immunosuppression during an early viable stage of the parasite in NCC, which is primarily asymptomatic. Further investigation on EV-mediated immune suppression revealed that the EV can protect the mice from DSS-induced colitis and improve colon architecture. These findings shed light on the previously unknown role of T. solium EV and the therapeutic role of their immune suppression potential.

A novel, sequencing-free strategy for the functional characterization of Taenia solium proteomic fingerprint.

The flatworm Taenia solium causes human and pig cysticercosis. When cysticerci are established in the human central nervous system, they cause neurocysticercosis, a potentially fatal disease. Neurocysticercosis is a persisting public health problem in rural regions of Mexico and other developing countries of Latin America, Asia, and Africa, where the infection is endemic. The great variability observed in the phenotypic and genotypic traits of cysticerci result in a great heterogeneity in the patterns of molecules secreted by them within their host. This work is aimed to identify and characterize cysticercal secretion proteins of T. solium cysticerci obtained from 5 naturally infected pigs from Guerrero, Mexico, using 2D-PAGE proteomic analysis. The isoelectric point (IP) and molecular weight (MW) of the spots were identified using the software ImageMaster 2D Platinum v.7.0. Since most secreted proteins are impossible to identify by mass spectrometry (MS) due to their low concentration in the sample, a novel strategy to predict their sequence was applied. In total, 108 conserved and 186 differential proteins were identified in five cysticercus cultures. Interestingly, we predicted the sequence of 14 proteins that were common in four out of five cysticercus cultures, which could be used to design vaccines or diagnostic methods for neurocysticercosis. A functional characterization of all sequences was performed using the algorithms SecretomeP, SignalP, and BlastKOALA. We found a possible link between signal transduction pathways in parasite cells and human cancer due to deregulation in signal transduction pathways. Bioinformatics analysis also demonstrated that the parasite release proteins by an exosome-like mechanism, which could be of biological interest.

Discovery of a new non-substrate inhibitor of the 26.5 kDa glutathione transferase from Taenia solium by virtual screening.

The inappropriate use of anthelmintics, such as praziquantel and albendazole, has generated resistance and the need to develop new drugs. Glutathione transferases, GSTs, are bisubstrate dimeric enzymes that constitute the main detoxification mechanism against electrophiles, drugs and oxidative damage in Taenia solium. Therefore, GSTs are important targets for the development of new anthelmintics. In this work, we reported a successful virtual screen aimed at the identification of novel inhibitors of a 26.5 kDa GST from T. solium (TsGST26). We found that a compound, i7, able to inhibit selectively TsGST26 concerning human GSTs, showing a non-competitive inhibition mechanism towards substrate glutathione with a Ki (GSH) of 55.7 µM and mixed inhibition towards the electrophilic substrate 1-chloro-2,4-dinitrobenzene with a Ki (CDNB) of 8.64 µM. These results are in agreement with those of docking simulations, which showed i7 binds a site adjacent to the electrophilic site and furthest from the glutathione site.

Unveiling Taenia solium kinome profile and its potential for new therapeutic targets.

Background: Helminth infections cause widespread morbidity and are a significant global disease burden. One among them is Neurocysticercosis, a central nervous system infection caused by the larvae Taenia solium, leading to epilepsy. Helminths are strong immune modulators and can survive for a long time in adverse host environments. Kinases are molecular switches and are essential to initiate/propagate signaling cascades and are detrimental to the regulation of homeostasis. They have been implicated in the progression of many diseases and are potentially lucrative drug targets.Objective: To identify kinases in T. solium proteome and prioritize them as drug targets.Methodology: A Hidden Markov Model (HMM) was used to curate and classify kinases into families based on sequence homology to model organisms followed by phylogenetic analysis of each family. To predict potential drug targets, kinases were identified based on a homologically lethal relationship to C. elegans but non-lethal to humans. Kinases thus selected were searched for matching ligands in SARFkinase and DrugBank databases.Result and conclusion: T. solium kinases make up 1.8% of its proteome, CMGC is the largest kinase family and RGC is the smallest and catalytically inactive family. We predict 23-potential kinases to be drug targets for T. solium.[Figure: see text].

Cloning, expression, purification, and kinetic characterization of mitochondrial thioredoxin (TsTrx2), cytosolic thioredoxin (TsTrx1), and glutaredoxin (TsGrx1) from Taenia solium.

We report the complete coding sequences of mitochondrial thioredoxin (TsTrx2) and glutaredoxin (TsGrx1) from the cysticerci of T. solium. The full-length DNA of the TsTrx2 gene shows two introns of 88 and 77 bp and three exons. The TsTrx2 gene contains a single ORF of 423 bp, encoding 140 amino acid residues with an estimated molecular weight of 15,560 Da. A conserved C64NPC67 active site and a 30-amino acid extension at its N-terminus were identified. An insulin reduction reaction was used to determine whether it was a functional recombinant protein. The full-length DNA of the TsGrx1 gene shows one intron of 39 bp and a single ORF of 315 bp, encoding 105 amino acid residues with an estimated molecular weight of 12,582 Da. Sequence analysis revealed a conserved dithiol C34PYC37 active site, GSH-binding motifs (CXXC, Lys and Gln/Arg, TVP, and CXD), and a conserved Gly-Gly motif. The r-TsGrx1 kinetic constants for glutathione (GSH) and 2-hydroxyethyl disulfide (HED) were determined. In addition, cytosolic thioredoxin (TsTrx1), as reported by (Jiménez et al., Biomed Res Int 2015:453469, 2015), was cloned and expressed, and its catalytic constants were obtained along with those of the other two reductases. Rabbit-specific antibodies showed immune cross-reactions between TsTrx1 and TsTrx2 but not with TsGrx1. Both TsTGRs as reported by (Plancarte and Nava, Exp Parasitol 149:65-73, 2015) were biochemically purified to obtain and compare the catalytic constants for their natural substrates, r-TsTrx1, and r-TsTrx2, compared to those for Trx-S2E. coli. In addition, we determined the catalytic differences between the glutaredoxin activity of the TsTGRs compared with r-TsGrx1. These data increase the knowledge of the thioredoxin and GSH systems in T. solium, which is relevant for detoxification and immune evasion.

Molecular characterization of the Taenia solium Tso31 antigen and homologous of other Taenia species from Peru.

Several studies have been performed to determine specific antigens for the diagnosis of tapeworms. One of these antigens is Tso31, which is used to differentiate Taenia solium and Taenia saginata in human feces. The aim of the present work was the molecular characterization of this protein in different tapeworm specimens collected in Peru: T. omisa (n = 6), T. hydatigena (n = 7), T. taeniaeformis (n = 4), T. pisiformes (n = 1), T. multiceps (n = 7), and T. solium (n = 10). Total DNA was extracted from each proglottid using a commercial DNA kit for tissue. A nested PCR was used to amplify a fragment of the previously described oncosphere-specific protein Tso31 gene. The nested PCR products were analyzed by 1.5% agarose gel electrophoresis and visualized after ethidium bromide staining. All nested PCR-positive products were sequenced and their sequences were compared. Of all the tapeworms analyzed, only T. solium and T. multiceps amplified the Tso31 gene. All sequences were identical for each species. Our T. solium Tso31 showed 100% similarity when compared with published GenBank sequences. The difference between T. solium and T. multiceps Tso31 samples was 8.1%. In conclusion, our results show that the tsol31 gene is not exclusive to T. solium.

Secret-AAR: a web server to assess the antigenic density of proteins and homology search against bacterial and parasite secretome proteins.

The secretome refers to all the Excreted/Secreted (ES) proteins of a cell, and these are involved in critical biological processes, such as cell-cell communication, and host immune responses. Recently, we introduced the Abundance of Antigenic Aegions (AAR) value to assess the protein antigenic density and to evaluate the antigenic potential of secretomes. Here, to facilitate the AAR calculation, we implemented it as a user-friendly webserver. We extended the webserver capabilities implementing a sequence-based tool for searching homologous proteins across secretomes, including experimental and predicted secretomes of Mycobacterium tuberculosis and Taenia solium. Additionally, twelve secretomes of helminths, five of Mycobacterium and two of Gram-negative bacteria are also available. Our webserver is a useful tool for researchers working on immunoinformatics and reverse vaccinology, aiming at discovering candidate proteins for new vaccines or diagnostic tests, and it can be used to prioritize the experimental analysis of proteins for druggability assays. The Secret-AAR web server is available at http://microbiomics.ibt.unam.mx/tools/aar/.

A gel-free proteomic analysis of Taenia solium and Taenia crassiceps cysticerci vesicular extracts.

The taeniasis/cysticercosis complex is a zoonosis caused by the presence of the parasite Taenia solium in humans. It is considered a neglected disease that causes serious public health and economic problems in developing countries. In humans, the most common locations for the larval form are the skeletal muscles, ocular system, and the central nervous system, which is the most clinically important. Several glycoproteins of T. solium and Taenia crassiceps cysticerci have been characterized and studied for their use in the immunodiagnosis of neurocysticercosis and/or the development of synthetic or recombinant vaccines against cysticercosis. The aim of this study was to perform a gel-free shotgun proteomic analysis to identify saline vesicular extract (SVE) proteins of T. solium and T. crassiceps cysticerci. After solubilization of the SVE with and without surfactant reagent and in-solution digestion, the proteins were analyzed by LC-MS/MS. Use of a surfactant resulted in a significantly higher number of proteins that were able to be identified by LC-MS/MS. Novel proteins were identified in T. solium and T. crassiceps SVE. The qualitative analysis revealed a total of 79 proteins in the Taenia species: 29 in T. solium alone, 11 in T. crassiceps alone, and 39 in both. These results are an important contribution to support future investigations and for establishing a Taenia proteomic profile to study candidate biomarkers involved in the diagnosis or pathogenesis of neurocysticercosis.

"Omic" investigations of protozoa and worms for a deeper understanding of the human gut "parasitome".

The human gut has been continuously exposed to a broad spectrum of intestinal organisms, including viruses, bacteria, fungi, and parasites (protozoa and worms), over millions of years of coevolution, and plays a central role in human health. The modern lifestyles of Western countries, such as the adoption of highly hygienic habits, the extensive use of antimicrobial drugs, and increasing globalisation, have dramatically altered the composition of the gut milieu, especially in terms of its eukaryotic "citizens." In the past few decades, numerous studies have highlighted the composition and role of human intestinal bacteria in physiological and pathological conditions, while few investigations exist on gut parasites and particularly on their coexistence and interaction with the intestinal microbiota. Studies of the gut "parasitome" through "omic" technologies, such as (meta)genomics, transcriptomics, proteomics, and metabolomics, are herein reviewed to better understand their role in the relationships between intestinal parasites, host, and resident prokaryotes, whether pathogens or commensals. Systems biology-based profiles of the gut "parasitome" under physiological and severe disease conditions can indeed contribute to the control of infectious diseases and offer a new perspective of omics-assisted tropical medicine.

Effect of Transforming Growth Factor-ß upon Taenia solium and Taenia crassiceps Cysticerci.

Taeniids exhibit a great adaptive plasticity, which facilitates their establishment, growth, and reproduction in a hostile inflammatory microenvironment. Transforming Growth Factor-ß (TGFß), a highly pleiotropic cytokine, plays a critical role in vertebrate morphogenesis, cell differentiation, reproduction, and immune suppression. TGFß is secreted by host cells in sites lodging parasites. The role of TGFß in the outcome of T. solium and T. crassiceps cysticercosis is herein explored. Homologues of the TGFß family receptors (TsRI and TsRII) and several members of the TGFß downstream signal transduction pathway were found in T. solium genome, and the expression of Type-I and -II TGFß receptors was confirmed by RT-PCR. Antibodies against TGFß family receptors recognized cysticercal proteins of the expected molecular weight as determined by Western blot, and different structures in the parasite external tegument. In vitro, TGFß promoted the growth and reproduction of T. crassiceps cysticerci and the survival of T. solium cysticerci. High TGFß levels were found in cerebrospinal fluid from untreated neurocysticercotic patients who eventually failed to respond to the treatment (P = 0.03) pointing to the involvement of TGFß in parasite survival. These results indicate the relevance of TGFß in the infection outcome by promoting cysticercus growth and treatment resistance.

Fasciclin-calcareous corpuscle binary complex mediated protein-protein interactions in Taenia solium metacestode.

BACKGROUND: Neurocysticercosis (NC) caused by Taenia solium metacestode (TsM) is a serious neurological disease of global concern. Diverse bioactive molecules involved in the long-term survival of TsM might contribute to disease progression. Fasciclin (Fas) is an extracellular protein that mediates adhesion, migration and differentiation of cells by interacting with other molecules. We hypothesized that TsMFas might bind to calcareous corpuscle (CC) through its adhesive property and participate in crucial protein-protein interactions, thus contributing to the creation of a symbiotic interactome network. METHODS: Two paralogous TsMFas (TsMFas1 and TsMFas2) were isolated, and their molecular properties were characterized. The co-localization pattern of TsMFas1 and TsMFas2 with CC was determined. CC-TsMFas binary complex was generated by incubating CC with recombinant proteins (rTsMFas1 and 2). In vitro binding assay of CC-rTsMFas1 or CC-rTsMFas2 binary complex with TsM cellular proteins extracted from scolex and neck was conducted. Their binding partners were identified through proteomic analysis. Integrated protein-protein interaction networks were established. RESULTS: TsMFas1 (6072 bp long) was composed of 15 exons (841 amino acid polypeptide) interrupted by 14 introns. TsMFas2 (5201 bp long) comprised of 11 exons (597 amino acids) and 10 intervening introns. These proteins displayed 22% amino acid sequence identity to each other, but tightly conserved Fas-related domains. Several isoforms of Fas1 and Fas2 proteins might have been expressed through post-translational modifications. They showed adhesion activity with other cells. TsMFas proteins were largely distributed in parenchymal regions of the scolex and bladder wall. These molecules were co-localized with CC, a unique organelle found in platyhelminths. Subsequent proteome analysis of CC-Fas binary complex mediated protein-protein interactions revealed seven protein ligands in the TsM cellular proteins. Their functions were mainly segregated into carbohydrate metabolism (enolase, phosphoenolpyruvate carboxykinase, phosphoglycerate kinase and glyceraldehyde 3-phosphate dehydrogenase) and cytoskeleton/cellular motility (actin, paramyosin and innexin nuc-9). Those proteins had direct (physical) and/or indirect (functional) relationships along with their biochemical properties and biological roles. CONCLUSION: Protein repertoires strongly suggest that TsMFas and CC may symbiotically mediate protein-protein interactions during biological processes to maintain efficacious homeostatic functions and ensure the prolonged survival of TsM in the host.

Molecular Cloning of a cDNA Encoding for Taenia solium TATA-Box Binding Protein 1 (TsTBP1) and Study of Its Interactions with the TATA-Box of Actin 5 and Typical 2-Cys Peroxiredoxin Genes.

TATA-box binding protein (TBP) is an essential regulatory transcription factor for the TATA-box and TATA-box-less gene promoters. We report the cloning and characterization of a full-length cDNA that encodes a Taenia solium TATA-box binding protein 1 (TsTBP1). Deduced amino acid composition from its nucleotide sequence revealed that encodes a protein of 238 residues with a predicted molecular weight of 26.7 kDa, and a theoretical pI of 10.6. The NH2-terminal domain shows no conservation when compared with to pig and human TBP1s. However, it shows high conservation in size and amino acid identity with taeniids TBP1s. In contrast, the TsTBP1 COOH-terminal domain is highly conserved among organisms, and contains the amino acids involved in interactions with the TATA-box, as well as with TFIIA and TFIIB. In silico TsTBP1 modeling reveals that the COOH-terminal domain forms the classical saddle structure of the TBP family, with one α-helix at the end, not present in pig and human. Native TsTBP1 was detected in T. solium cysticerci´s nuclear extract by western blot using rabbit antibodies generated against two synthetic peptides located in the NH2 and COOH-terminal domains of TsTBP1. These antibodies, through immunofluorescence technique, identified the TBP1 in the nucleus of cells that form the bladder wall of cysticerci of Taenia crassiceps, an organism close related to T. solium. Electrophoretic mobility shift assays using nuclear extracts from T. solium cysticerci and antibodies against the NH2-terminal domain of TsTBP1 showed the interaction of native TsTBP1 with the TATA-box present in T. solium actin 5 (pAT5) and 2-Cys peroxiredoxin (Ts2-CysPrx) gene promoters; in contrast, when antibodies against the anti-COOH-terminal domain of TsTBP1 were used, they inhibited the binding of TsTBP1 to the TATA-box of the pAT5 promoter gene.

Genome analysis of Excretory/Secretory proteins in Taenia solium reveals their Abundance of Antigenic Regions (AAR).

Excretory/Secretory (ES) proteins play an important role in the host-parasite interactions. Experimental identification of ES proteins is time-consuming and expensive. Alternative bioinformatics approaches are cost-effective and can be used to prioritize the experimental analysis of therapeutic targets for parasitic diseases. Here we predicted and functionally annotated the ES proteins in T. solium genome using an integration of bioinformatics tools. Additionally, we developed a novel measurement to evaluate the potential antigenicity of T. solium secretome using sequence length and number of antigenic regions of ES proteins. This measurement was formalized as the Abundance of Antigenic Regions (AAR) value. AAR value for secretome showed a similar value to that obtained for a set of experimentally determined antigenic proteins and was different to the calculated value for the non-ES proteins of T. solium genome. Furthermore, we calculated the AAR values for known helminth secretomes and they were similar to that obtained for T. solium. The results reveal the utility of AAR value as a novel genomic measurement to evaluate the potential antigenicity of secretomes. This comprehensive analysis of T. solium secretome provides functional information for future experimental studies, including the identification of novel ES proteins of therapeutic, diagnosis and immunological interest.

Cytokine, antibody and proliferative cellular responses elicited by Taenia solium calreticulin upon experimental infection in hamsters.

Taenia solium causes two diseases in humans, cysticercosis and taeniosis. Tapeworm carriers are the main risk factor for neurocysticercosis. Limited information is available about the immune response elicited by the adult parasite, particularly the induction of Th2 responses, frequently associated to helminth infections. Calreticulin is a ubiquitous, multifunctional protein involved in cellular calcium homeostasis, which has been suggested to play a role in the regulation of immune responses. In this work, we assessed the effect of recombinant T. solium calreticulin (rTsCRT) on the cytokine, humoral and cellular responses upon experimental infection in Syrian Golden hamsters (Mesocricetus auratus). Animals were infected with T. solium cysticerci and euthanized at different times after infection. Specific serum antibodies, proliferative responses in mesenteric lymph nodes and spleen cells, as well as cytokines messenger RNA (mRNA) were analyzed. The results showed that one third of the infected animals elicited anti-rTsCRT IgG antibodies. Interestingly, mesenteric lymph node (MLN) cells from either infected or non-infected animals did not proliferate upon in vitro stimulation with rTsCRT. Additionally, stimulation with a tapeworm crude extract resulted in increased expression of IL-4 and IL-5 mRNA. Upon stimulation, rTsCRT increased the expression levels of IL-10 in spleen and MLN cells from uninfected and infected hamsters. The results showed that rTsCRT favors a Th2-biased immune response characterized by the induction of IL-10 in mucosal and systemic lymphoid organs. Here we provide the first data on the cytokine, antibody and cellular responses to rTsCRT upon in vitro stimulation during taeniasis.

Somatostatin negatively regulates parasite burden and granulomatous responses in cysticercosis.

Cysticercosis is an infection of tissues with the larval cysts of the cestode, Taenia solium. While live parasites elicit little or no inflammation, dying parasites initiate a granulomatous reaction presenting as painful muscle nodules or seizures when cysts are located in the brain. We previously showed in the T. crassiceps murine model of cysticercosis that substance P (SP), a neuropeptide, was detected in early granulomas and was responsible for promoting granuloma formation, while somatostatin (SOM), another neuropeptide and immunomodulatory hormone, was detected in late granulomas; SOM's contribution to granuloma formation was not examined. In the current studies, we used somatostatin knockout (SOM(-/-)) mice to examine the hypothesis that SOM downmodulates granulomatous inflammation in cysticercosis, thereby promoting parasite growth. Our results demonstrated that parasite burden was reduced 5.9-fold in SOM(-/-) mice compared to WT mice (P < 0.05). This reduction in parasite burden in SOM(-/-) mice was accompanied by a 95% increase in size of their granulomas (P < 0.05), which contained a 1.5-fold increase in levels of IFN-γ and a 26-fold decrease in levels of IL-1ß (P < 0.05 for both) compared to granulomas from WT mice. Thus, SOM regulates both parasite burden and granulomatous inflammation perhaps through modulating granuloma production of IFN-γ and IL-1ß.

[Construction and identification of the Bifidobacterium expression system pGEX-TSOL18/B. longum of Taenia solium].

The TSOL18 gene of Taenia solium was synthesized and cloned into Escherichia coli-Bifidobacteria shuttle vector pGEX-1lambdaT. The recombinant plasmid pGEX-TSOL18 was transformed into Bifidobacterium longum with electroporation. The recombinant plasmid containing TSOL18 gene was identified by restriction endonuclease analysis, PCR and DNA sequencing. The length of synthesized TSOL18 gene was 393 bp. The results indicated that the Bifidobacteria expression system pGEX-TSOL18/B. longum was successfully constructed.

Taenia solium tapeworms synthesize corticosteroids and sex steroids in vitro.

Cysticercosis is a disease caused by the larval stage of Taenia solium cestodes that belongs to the family Taeniidae that affects a number of hosts including humans. Taeniids tapeworms are hermaphroditic organisms that have reproductive units called proglottids that gradually mature to develop testis and ovaries. Cysticerci, the larval stage of these parasites synthesize steroids. To our knowledge there is no information about the capacity of T. solium tapeworms to metabolize progesterone or other precursors to steroid hormones. Therefore, the aim of this paper was to investigate if T. solium tapeworms were able to transform steroid precursors to corticosteroids and sex steroids. T. solium tapeworms were recovered from the intestine of golden hamsters that had been orally infected with cysticerci. The worms were cultured in the presence of tritiated progesterone or androstenedione. At the end of the experiments the culture media were analyzed by thin layer chromatography. The experiments described here showed that small amounts of testosterone were synthesized from (3)H-progesterone by complete or segmented tapeworms whereas the incubation of segmented tapeworms with (3)H-androstenedione, instead of (3)H-progesterone, improved their capacity to synthesize testosterone. In addition, the incubation of the parasites with (3)H-progesterone yielded corticosteroids, mainly deoxicorticosterone (DOC) and 11-deoxicortisol. In summary, the results described here, demonstrate that T. solium tapeworms synthesize corticosteroid and sex steroid like metabolites. The capacity of T. solium tapeworms to synthesize steroid hormones may contribute to the physiological functions of the parasite and also to their interaction with the host.