Variation of the 2D Pattern of Brain Proteins in Mice Infected with Taenia crassiceps ORF Strain.

Some parasites are known to influence brain proteins or induce changes in the functioning of the nervous system. In this study, our objective is to demonstrate how the two-dimensional gel technique is valuable for detecting differences in protein expression and providing detailed information on changes in the brain proteome during a parasitic infection. Subsequently, we seek to understand how the parasitic infection affects the protein composition in the brain and how this may be related to changes in brain function. By analyzing de novo-expressed proteins at 2, 4, and 8 weeks post-infection compared to the brains of the control mice, we observed that proteins expressed at 2 weeks are primarily associated with neuroprotection or the initial response of the mouse brain to the infection. At 8 weeks, parasitic infection can induce oxidative stress in the brain, potentially activating signaling pathways related to the response to cellular damage. Proteins expressed at 8 weeks exhibit a pattern indicating that, as the host fails to balance the Neuro-Immuno-Endocrine network of the organism, the brain begins to undergo an apoptotic process and consequently experiences brain damage.

Analysis of the expression of cytoskeletal proteins of Taenia crassiceps ORF strain cysticerci (Cestoda).

The Taenia crassiceps ORF strain is used to generate a murine model of cysticercosis, which is used for diagnosis, evaluation of drugs, and vaccination. This particular strain only exists as cysticerci, is easily maintained under in vivo and in vitro conditions, and offers an excellent model for studying the cytoskeletons of cestodes. In this study, several experimental approaches were used to determine the tissue expression of its cytoskeletal proteins. The techniques used were microscopy (video, confocal, and transmission electron), one-dimensional (1D) and two-dimensional (2D) electrophoresis, immunochemistry, and mass spectrometry. The tissue expression of actin, tubulin, and paramyosin was assessed using microscopy, and their protein isoforms were determined with 1D and 2D electrophoresis and immunochemistry. Nineteen spots were excised from a proteomic gel and identified by liquid chromatography-tandem mass spectrometry and immunochemistry. The proteins identified were classic cytoskeletal proteins, metabolic enzymes, and proteins with diverse biological functions, but mainly involved in detoxification activities. Research suggests that most noncytoskeletal proteins interact with actin or tubulin, and the results of the present study suggest that the proteins identified may be involved in supporting the dynamics and plasticity of the cytoskeleton of T. crassiceps cysticerci. These results contribute to our knowledge of the cellular biology and physiology of cestodes.

Sigma-Class Glutathione Transferases (GSTσ): A New Target with Potential for Helminth Control.

Glutathione transferases (GSTs EC 2.5.1.18) are critical components of phase II metabolism, instrumental in xenobiotics' metabolism. Their primary function involves conjugating glutathione to both endogenous and exogenous toxic compounds, which increases their solubility and enables their ejection from cells. They also play a role in the transport of non-substrate compounds and immunomodulation, aiding in parasite establishment within its host. The cytosolic GST subfamily is the most abundant and diverse in helminths, and sigma-class GST (GSTσ) belongs to it. This review focuses on three key functions of GSTσ: serving as a detoxifying agent that provides drug resistance, functioning as an immune system modulator through its involvement in prostaglandins synthesis, and acting as a vaccine antigen.

Biochemical characterization and gene structure analysis of the 24-kDa glutathione transferase sigma from Taenia solium.

Taenia solium can cause human taeniasis and/or cysticercosis. The latter can in some instances cause human neurocysticercosis which is considered a priority in disease-control strategies and the prevention of mental health problems. Glutathione transferases are crucial for the establishment and long-term survival of T. solium; therefore, we structurally analyzed the 24-kDa glutathione transferase gene (Ts24gst) of T. solium and biochemically characterized its product. The gene promoter showed potential binding sites for transcription factors and xenobiotic regulatory elements. The gene consists of a transcription start site, four exons split by three introns, and a polyadenylation site. The gene architecture is conserved in cestodes. Recombinant Ts24GST (rTs24GST) was active and dimeric. Anti-rTs24GST serum showed slight cross-reactivity with human sigma-class GST. A 3D model of Ts24GST enabled identification of putative residues involved in interactions of the G-site with GSH and of the H-site with CDNB and prostaglandin D2. Furthermore, rTs24GST showed optimal activity at 45 °C and pH 9, as well as high structural stability in a wide range of temperatures and pHs. These results contribute to the better understanding of this parasite and the efforts directed to fight taeniasis/cysticercosis.

Structural and biochemical studies of a recombinant 25.5 kDa glutathione transferase of Taenia solium metacestode (rTs25GST1-1).

In this work, we studied a recombinant mu-class glutathione transferase of 25.5 kDa from Taenia solium metacestode (rTs25GST1-1) that follows Michaelis­Menten kinetics with 1-chloro-2,4-dinitrobenzene (CDNB). The kinetic parameters obtained for rTs25GST1-1 with CDNB and GSH were V(max) =12.04 µmol/min/mg and K(m)=1.38 mM, and V(max) =10.20 µmol/min/mg and K(m)=0.90, respectively. The optimal activity was found at pH 8 in the 37-40 °C temperature range. Circular dichroism studies for rTs25GST1-1 at different pH showed that it maintains a typical α-helix structure between pH 6.5-7.5, but loses it between pH 8 and 8.5. Thermal CD assays showed rTs25GST1-1 barely changed its secondary structure. Unfolding/refolding assays showed that rTs25GST1-1 retained its structure up to 40 °C without loss of its activity. Additionally, exposure of rTs25GST1-1 to cumene hydroperoxide did not produce significant changes in its structure and only affected 50% of its activity.

Neuroprotection of Thioredoxin1 in the Brain.

Thioredoxin1 (Trx1) is a ubiquitous antioxidant protein that regulates the cell's redox status. Trx1's thiol redox activity protects neurons from various physiological processes that cause neuronal damage and neurodegeneration, including oxidative stress, apoptosis, and inflammation. Several studies have found that direct or indirect Trx1 regulation has neuroprotective effects in the brain, protecting against, preventing, or delaying neurodegenerative processes or brain traumas. This review focuses on the term neuroprotection, Trx1 localization, and expression in the brain, as well as its modulation concerning its neuroprotective effect in both animal and clinical models of ischemia, hypoxia, hemorrhage, traumatic brain injury, epilepsy, Alzheimer's disease, and Parkinson's disease.

Differential Protein Expression of Taenia crassiceps ORF Strain in the Murine Cysticercosis Model Using Resistant (C57BL/6) Mice.

A cysticercosis model of Taenia crassiceps ORF strain in susceptible BALB/c mice revealed a Th2 response after 4 weeks, allowing for the growth of the parasite, whereas resistant C57BL/6 mice developed a sustained Th1 response, limiting parasitic growth. However, little is known about how cysticerci respond to an immunological environment in resistant mice. Here, we show that the Th1 response, during infection in resistant C57BL/6 mice, lasted up to 8 weeks and kept parasitemia low. Proteomics analysis of parasites during this Th1 environment showed an average of 128 expressed proteins; we chose 15 proteins whose differential expression varied between 70 and 100%. A total of 11 proteins were identified that formed a group whose expression increased at 4 weeks and decreased at 8 weeks, and another group with proteins whose expression was high at 2 weeks and decreased at 8 weeks. These identified proteins participate in tissue repair, immunoregulation and parasite establishment. This suggests that T. crassiceps cysticerci in mice resistant under the Th1 environment express proteins that control damage and help to establish a parasite in the host. These proteins could be targets for drugs or vaccine development.

The effect of thioredoxin-1 in a rat model of traumatic brain injury depending on diurnal variation.

INTRODUCTION: Traumatic brain injury (TBI) is a public health concern with limited treatment options because it causes a cascade of side effects that are the leading cause of hospital death. Thioredoxin is an enzyme with neuroprotective properties such as antioxidant, antiapoptotic, immune response modulator, and neurogenic, among others; it has been considered a therapeutic target for treating many disorders. METHODS: The controlled cortical impact (CCI) model was used to assess the effect of recombinant human thioredoxin 1 (rhTrx1) (1 µg/2 µL, intracortical) on rats subjected to TBI at two different times of the light-dark cycle (01:00 and 13:00 h). We analyzed the food intake, body weight loss, motor coordination, pain perception, and histology in specific hippocampus (CA1, CA2, CA3, and Dental Gyrus) and striatum (caudate-putamen) areas. RESULTS: Body weight loss, reduced food intake, spontaneous pain, motor impairment, and neuronal damage in specific hippocampus and striatum regions are more evident in rats subjected to TBI in the light phase than in the dark phase of the cycle and in groups that did not receive rhTrx1 or minocycline (as positive control). Three days after TBI, there is a recovery in body weight, food intake, motor impairment, and pain, which is more pronounced in the rats subjected to TBI at the dark phase of the cycle and those that received rhTrx1 or minocycline. CONCLUSIONS: Knowing the time of day a TBI occurs in connection to the neuroprotective mechanisms of the immune response in diurnal variation and the usage of the Trx1 protein might have a beneficial therapeutic impact in promoting quick recovery after a TBI.

Cu,Zn superoxide dismutase: cloning and analysis of the Taenia solium gene and Taenia crassiceps cDNA.

Cytosolic Cu,Zn superoxide dismutase (Cu,Zn-SOD) catalyzes the dismutation of superoxide (O(2)(-)) to oxygen and hydrogen peroxide (H(2)O(2)) and plays an important role in the establishment and survival of helminthes in their hosts. In this work, we describe the Taenia solium Cu,Zn-SOD gene (TsCu,Zn-SOD) and a Taenia crassiceps (TcCu,Zn-SOD) cDNA. TsCu,Zn-SOD gene that spans 2.841 kb, and has three exons and two introns; the splicing junctions follow the GT-AG rule. Analysis in silico of the gene revealed that the 5'-flanking region has three putative TATA and CCAAT boxes, and transcription factor binding sites for NF1 and AP1. The transcription start site was a C, located at 22 nucleotides upstream of the translation start codon (ATG). Southern blot analysis showed that TcCu,Zn-SOD and TsCu,Zn-SOD genes are encoded by a single copy. The deduced amino acid sequences of TsCu,Zn-SOD gene and TcCu,Zn-SOD cDNA reveal 98.47% of identity, and the characteristic motives, including the catalytic site and ß-barrel structure of the Cu,Zn-SOD. Proteomic and immunohistochemical analysis indicated that Cu,Zn-SOD does not have isoforms, is distributed throughout the bladder wall and is concentrated in the tegument of T. solium and T. crassiceps cysticerci. Expression analysis revealed that TcCu,Zn-SOD mRNA and protein expression levels do not change in cysticerci, even upon exposure to O(2)(-) (0-3.8 nmol/min) and H(2)O(2) (0-2mM), suggesting that this gene is constitutively expressed in these parasites.

Physiological and modulatory role of thioredoxins in the cellular function.

Thioredoxins (TRXs) are a class of ubiquitous and multifunctional protein. Mammal cells present three isoforms: a cytosolic and extracellular called thioredoxin 1 (TRX1), a mitochondrial (TRX2), and one specific in spermatozoids (TRX3). Besides, a truncated form called TRX80 exists, which results from the post-translational cleavage performed on TRX1. TRXs' main function is to maintain the reduction-oxidation homeostasis of the cell, reducing the proteins through a thiol-disulfide exchange that depends on two cysteines located in the active site of the protein (Cys32-X-X-Cys35 in humans). In addition, TRX1 performs S-nitrosylation, a post-translational modification of proteins that depends on cysteines of its C-terminal region (Cys62, Cys69, and Cys73 in human TRX1). These modifications allow the TRXs to modulate the protein function and participate in regulating diverse cellular processes, such as oxidative stress, transcription, signaling cascades, apoptosis, inflammation, and immunologic response. This points out the crucial relevance of TRXs for cell function, signaling it as a strategic target for the treatment of many diseases and its possible use as a therapeutic factor.

Evaluation of recombinant glutathione transferase 26 kDa, thioredoxin-1, and endophilin B1 of Taenia solium in the diagnosis of human neurocysticercosis.

Neurocysticercosis caused by Taenia solium larvae is a neglected disease that persists in several countries, including Mexico, and causes a high disability-adjusted life year burden. Neuroimaging tools such as computed tomography and magnetic resonance imaging are the most efficient for its detection, but low availability and high costs in most endemic regions limit their use. Serological methods such as lentil lectin-purified glycoprotein enzyme-linked immunoelectrotransfer blot antibody detection and monoclonal antibody-based enzyme-linked immunosorbent assays for HP10 antigen detection have been useful in supporting the diagnosis of this disease. We evaluated three T. solium recombinant antigens: glutathione transferase of 26 kDa (Ts26GST); thioredoxin-1 (TsTrx-1), and endophilin B1 (TsMEndoB1) by EITB. These are antigenic proteins antigenic, abundant in excretion/secretion products of the parasite, and do not cross-react with homologous host proteins. Ts26GST and TsTrx-1 showed sensitivity of 79 and 88%, specificity of 86 and 97%, PPV of 83 and 97% and NPV of 82 and 91%, respectively, for neurocysticercosis diagnosis. The recombinant antigens allowed the diagnosis of 70% (Ts26GST) and 80% (TsTrx-1) of patients having only one cysticercus. Further studies on specific regions of these proteins could improve T. solium diagnostics.

The hamster model for identification of specific antigens of Taenia solium tapeworms.

Humans acquire taeniasis by ingesting pork meat infected with Taenia solium cysticerci, which are the only definitive hosts of the adult stage (tapeworm) and responsible for transmitting the human and porcine cysticercosis. Hence, detection of human tapeworm carriers is a key element in the development of viable strategies to control the disease. This paper presents the identification of specific antigens using sera from hamsters infected with T. solium tapeworms analyzed by western blot assay with crude extracts (CEs) and excretion-secretion antigens (E/S Ag) obtained from T. solium cysticerci and tapeworms and extracts from other helminthes as controls. The hamster sera infected with T. solium tapeworms recognized specific bands of 72, 48, 36, and 24 kDa, in percentages of 81, 81, 90, and 88%, respectively, using the T. solium tapeworms E/S Ag. The antigens recognized by these hamster sera could be candidates to improve diagnosis of human T. solium taeniasis.

Release of glycoprotein (GP1) from the tegumental surface of Taenia solium by phospholipase C from Clostridium perfringens suggests a novel protein-anchor to membranes.

In order to explore how molecules are linked to the membrane surface in larval Taenia solium, whole cysticerci were incubated in the presence of phospholipase C from Clostridium perfringens (PLC). Released material was collected and analyzed in polyacrylamide gels with sodium dodecyl sulfate. Two major bands with apparent molecular weights of 180 and 43 kDa were observed. Western blot of released material and localization assays in cysticerci tissue sections using antibodies against five known surface glycoproteins of T. solium cysticerci indicated that only one, previously called GP1, was released. Similar localization studies using the lectins wheat-germ-agglutinin and Concanavalin A showed that N-acetyl-D-glucosamine, N-acetylneuraminic, sialic acid, alphamethyl-D-mannoside, D-manose/glucose, and N-acetyl-D-glucosamine residues are abundantly present on the surface. On the other hand, we find that treatment with PLC releases molecules from the surface; they do not reveal Cross Reacting Determinant (CRD), suggesting a novel anchor to the membrane for the glycoprotein GP1.

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.

Protein expression profile of Taenia crassiceps cysticerci related to Th1- and Th2-type responses in the mouse cysticercosis model.

The intraperitoneal cysticercosis model with the Taenia crassiceps ORF strain in female BALB/cAnN mice has been widely used to study the immune response in cysticercosis. During early infection (2 weeks), the host develops a non-permissive Th1 response, whereas during late infection (8 weeks), molecules from the cysticerci induce a Th2 response that is permissive to parasite growth. The modulation of the Th2 response is induced by molecules excreted/secreted by the larval stage of the parasite. However, there is limited information regarding the response of cysticerci to the mouse immunological environment during infection. The proteomic profiles in T. crassiceps ORF cysticerci when faced with the mouse Th1 and Th2 responses were analyzed through two-dimensional gel electrophoresis (2DE), and the differential expression of proteins was evaluated. Thirteen proteins, whose differential expression varied between 70% and 100%, were selected randomly. Protein identification by MALDI-TOF MS and BLAST showed that the proteins were related to folding, signaling, enzymatic activities, cell-movement regulation, cell-cell interactions, motility, carbohydrate metabolism, detoxification, and redox regulation processes. Notably, some of the proteins can act as antigenic-protective molecules and elicit a weak Th1 response; however, most are involved in the avoidance of the immune system, which leads to a Th2 response, or apoptosis. The findings indicate the process by which T. crassiceps cysticerci responds based on the host environment and provides novel insights into the mechanism by which this facilitates its establishment and persistence in the mouse. Furthermore, these proteins could be used as targets for drug and vaccine development.

Antimicrobial peptides (Temporin A and Iseganan IB-367): effect on the cysticerci of Taenia crassiceps.

Taenia solium infections continue being a health problem in undeveloped countries, and few effective control measures against this parasite are being applied. Antimicrobial peptides (AMPs) belong to the innate immune response and capable of destroying pathogens. We tested the ability of two AMPs, Temporin A (TA) and Iseganan IB-367 (IB-367) to damage T. crassiceps cysticerci in vitro. Doses of 200 and 400 microg/ml of TA and IB-367 caused cysticerci to shrink, lose motility, the formation of macrovesicles in the tegument, as well as decreased evagination properties. These changes were observed as early as 3-6h and became more pronounced over 24h, when the morphological changes of the bladders became evident by both light and electron microscopy. Electron micrographs of cysticerci exposed to peptides showed initial changes as collapsed microvesicles in the tegument, with formation of large vesicles and extrusion of tegumentary tissues into the surrounding media, which led to complete loss of the tegument as well as shrinkage and complete loss of structure of parenchymal tissue after 24h. Peptides administered to cysticercotic mice one month post-infection in a single intraperitoneal dose of 200 or 400 microg, reduced the parasite load by 25% for IB-367, and 50% for TA. The humoral response of infected mice does not appear capable of killing surviving cysticerci. Our studies show that in vitro, AMPs severely damage the tegument and the scolex, and open a new pathway for biological drug design or the development of transgenic animals that over express these peptides capable of killing the cysticerci in vivo.

Characterization of one typical 2-Cys peroxiredoxin gene of Taenia solium and Taenia crassiceps.

The Taenia genus is capable of living for long periods within its hosts. Reports have shown that this successful establishment is related to its efficient defense mechanisms against host immune response and its high tolerance to oxidative stress. In this work, we describe the genomic sequences of one Taenia solium and Taenia crassiceps typical 2-Cys peroxiredoxins (Ts2-CysPrx, Tc2-CysPrx) genes, which are 94% identical in primary sequence with the typical 2-Cys Prxs catalytic motifs. Both genes have the same genomic architecture, showing a TATA box and Initiator (Inr) sequence in their proximal promoter, two exons split by a 67-bp type III intron and one unique transcription start site located inside the Inr. We show that T. crassiceps cysticerci are highly tolerant to H(2)O(2) presenting a lethal concentration 50 of 3.0 mM and demonstrate that the typical Tc2-CysPrx gene is not induced by H(2)O(2), showing a behavior of an antioxidant housekeeping gene. This study describes for first time the gene structure of a typical 2-Cys Prx in the Taenia genus.

Taenia solium and Taenia crassiceps: miRNomes of the larvae and effects of miR-10-5p and let-7-5p on murine peritoneal macrophages.

Neurocysticercosis (NCC), a major cause of neurological morbidity worldwide, is caused by the larvae of Taenia solium. Cestodes secrete molecules that block the Th1 response of their hosts and induce a Th2 response permissive to their establishment. Mature microRNAs (miRs) are small noncoding RNAs that regulate gene expression and participate in immunological processes. To determine the participation of Taenia miRs in the immune response against cysticercosis, we constructed small RNA (sRNA) libraries from larvae of Taenia solium and Taenia crassiceps. A total of 12074504 and 11779456 sequencing reads for T. solium and T. crassiceps, respectively, were mapped to the genomes of T. solium and other helminths. Both larvae shared similar miRNome, and miR-10-5p was the most abundant in both species, followed by let-7-5p in T. solium and miR-4989-3p in T. crassiceps, whereas among the genus-specific miRs, miR-001-3p was the most abundant in both, followed by miR-002-3p in T. solium and miR-003a-3p in T. crassiceps. The sequences of these miRs were identical in both. Structure and target prediction analyses revealed that these pre-miRs formed a hairpin and had more than one target involved in immunoregulation. Culture of macrophages, RT-PCR and ELISA assays showed that cells internalized miR-10-5p and let-7-5p into the cytoplasm and the miRs strongly decreased interleukin 16 (Il6) expression, tumor necrosis factor (TNF) and IL-12 secretion, and moderately decreased nitric oxide synthase inducible (Nos2) and Il1b expression (pro-inflammatory cytokines) in M(IFN-γ) macrophages and expression of Tgf1b, and the secretion of IL-10 (anti-inflammatory cytokines) in M(IL-4) macrophages. These findings could help us understand the role of miRs in the host-Taenia relationship.

Taenia solium glutathione transferase fraction activates macrophages and favors the development of Th1-type response.

Glutathione (GSH) transferase (GST) is an essential enzyme in cestodes for the detoxification of xenobiotics. In Taenia solium, two GSTs (Ts25GST and Ts26GST kDa) were isolated as a fraction (SGSTF) by GSH-Sepharose-4B. Both are located on the tegument. Immunization assays with SGSTF reduced up to 90% of the parasitic load in a murine model of cysticercosis. It prompted us to investigate how SGSTF induces this protective immune response. To test it, we exposed peritoneal macrophages to SGSTF for 24 h; such exposure favored the production of IL-12, TNF, and IL-10 as well as the expression of nitric oxide synthase 2 inducible (Nos2) and CD86, but did not induce the expression of chitinase-like 3 (Chil3). Confocal microscopy showed that the macrophages internalize the SGSTF which co-localized after 1 h with MHC-II in their plasma membranes. Macrophages exposed to SGSTF and co-cultured with anti-CD3 pre-activated T CD4+ cells, enhanced the proliferation of CD4+ cells, induced high interferon-γ (IFN-γ) secretion, and elevated the expression of CD25 and CD69, molecules associated with cell activation. Similar assay using T CD4+ cells from DO11.10 mice and ovalbumin (OVA) peptide+SGSTF as stimuli, showed enhanced cell proliferation and OVA-specific IFN-γ secretion. These data are in-line with those indicating that the P1, P5, and P6 peptides of Schistosoma japonicum 28GST highly promote T-cell proliferation and Th1 response in vitro We found that such peptides are also present on Ts25GST and Ts26GST. It suggests that SGSTF activates peritoneal macrophages to a classically activated-like phenotype, and that these macrophages induce the differentiation of T CD4+ cells toward a Th1-type response.

Cooperative kinetics of the recombinant glutathione transferase of Taenia solium and characterization of the enzyme.

Glutathione transferases (GSTs) are essential enzymes in many organisms due their diverse functions and, in helminths they are the main detoxification system. For Taenia solium, two cytosolic GSTs with molecular masses of 25.5 and 26.5 kDa (Ts26GST) have been found. Ts26GST was cloned to be studied in its recombinant form (recTs26GST). Although the primary structure is related to the mu class, the kinetic parameters for CDNB (V(max)=51.5 micromol min(-1)mg(-1); K(m)=1.06 mM; k(cat)= 22.2s(-1)) are related with some alpha GSTs. The substrate and inhibitor class markers reaffirmed these bimodal characteristics. Inhibition studies with anthelminthics indicate that recTs26GST is sensitive to mebendazole, displaying a non competitive inhibition pattern suggesting that at least two molecules are binding to recTs26GST. On the other hand, the kinetic curves for CDNB and GSH showed a positive cooperativity that was corroborated using fluorometric assays. Those assays indicate that CDNB binding is highly influenced by GSH, probably by modulation of the Ts26GST conformational ensamble.