In this study, we performed sequence alignments between the proteins of T. solium cysticercus and adult by comparing them to the T. asiatica database, and these proteins were analyzed by bioinformatics. We found the proteins differed starkly between the two developmental stages, Although the survival of cysticercus and adult depended on the energy metabolism in their hosts. Their main energy source, carbohydrates, might be catabolized by aerobic respiration or by two complementary anaerobic pathways, those of lactate fermentation and malate dismutation . Additionally, both cysticercus and adult relied upon glutathione metabolism to grow and develop in their host. The difference between the two stages was that adult could also survive through pyrimidine metabolism.
Glutathione played a role in antioxidative defense and in maintaining the reducing environment of the cytosol, and many of the known glutathione-dependent processes were directly related to the specific lifestyle of the parasite. For Plasmodium falciparum, proteins involved in GSH-dependent processes were studied not only as factors in the pathophysiology of malaria but also as potential drug targets . According to KEGG, pyrimidine and purine were the raw materials for the synthesis of DNA and RNA. The parasite itself synthesized pyrimidine and formed its own set of survival mechanism. Accordingly, it was plausible then, to generating new leading compounds to treat malaria and schistosomes by targeting the purine and pyrimidine pathways [9, 10]. In our study, glutathione-related enzymes were identified in proteomics of both cysticercus and adult, whereas proteins involved in pyrimidine metabolism were identified only in adult. Hence, the pyrimidine metabolic pathway might be used as a starting basis for the novel treatment of taeniasis, which also showed the differences in growth and development between cysticercus and adult.
Comparative analysis of mitochondrial genomes is often been used in phylogenetic studies. The complete nucleotide sequence of T. solium mitochondrial DNA (mtDNA) has been determined . The sequence is 13,709 base pairs in length and contains 36 genes: 12 for proteins involved in oxidative phosphorylation, namely adenosine triphosphatase subunits 6 and 8 (ATP6 and 8), 2 for ribosomal RNAs, and 22 for transfer RNAs. The mitochondria of most metazoan animals contained their own circular mitochondrial DNA (mtDNA), which usually contains the genes for large- and small-subunit ribosomal RNAs (LrRNA and SrRNA, respectively), 22 transfer RNAs (tRNA), and 11–12 proteins involved in oxidative phosphorylation: adenosine triphosphatase sub units 6 and 8 (ATP6 and 8), cytochrome b, cytochrome c oxidase subunits 1-3(Cox 1-3), and NADH dehydrogenase subunits 1-6 and 4 L (ND 1-6 and 4 L). The arrangement of these genes within the molecule is useful for deducing evolutionary relationships among distantly related taxa. Our obtained results for the proteomic identification of ribosomal proteins and cytochrome proteins were consistent with the above, as well as for the enzymes involved in pyrimidine metabolism in adult,
which indicated that T. solium had a strong ability to synthesize DNA during its growth and development. Yet other researchers, by analyzing the genomes of four species of tapeworms, found many genes lost from the genome in course of adapting to a parasitic life history, albeit some genes that could improve the survival rate of parasites were nonetheless retained and amplified at the same time. For example, the tapeworm genome lacked genes related to the synthesis of fatty acids and cholesterol de novo [12, 13]. Over time, tapeworms have lost their ability to synthesize essential fats and cholesterol essential for larval development, which they instead obtained from their hosts. Nevertheless, there were numerous lipid elongating enzymes and fatty acid transporter genes, and many fatty acid-binding proteins and apolipoprotein B antigens were expressed, which might be related to the parasite consuming a substantial amount of nutrients in the process of its growth and development. These proteins were identified here in the proteomics of both cysticercus and adult. This showed that the tapeworm was constantly evolving to be adapt to its environment, and it was precise because these proteins played a corresponding function that the tapeworm could survive and became a target for disease treatment.
However, the more puzzling thing was that cysticerci reached their final location in the parenchyma of the brain, usually followed by a period of several months or years until the onset of disease symptoms. During this period, affected host individuals possibly remained asymptomatic through active evasion and suppression of host immunity, as the development of symptoms was known to strongly depend on the degree and intensity of the host response , Another main, related point was the oxidative stress response of ROS, and the activity of GST (glutathione S-transferase), a group of enzymes that could protect cells from ROS damage. Tapeworms typically had only one cytochrome P450 gene, suggesting their ability to oxidize many xenobiotics and steroids was substantially lower than that of their hosts. Uniquely, tapeworms and flukes have merged two key enzymatic functions for redox homeostasis in one single enzyme: thioredoxin glutathione reductase (TGR). The gene encoding TGR was essential for parasite survival, and TGR was a validated drug target in flukes.For downstream of TGR’s gene, there were thioredoxins, glutaredoxins, and mu-class glutathione S-transferases (GSTs). Moreover, GSTs had immunogenicity and could be served as a specific target for the treatment of cysticercosis. Functional annotation of differentially expressed proteins between the two stages of T. solium revealed that the number of proteins identified as involved in the immune process was greater in cysticercus than in adult, of which 25 proteins were involved in the former’s elicited immune response. This indicated that the immunosuppression caused by the cysticercus was more severe than that of adult. Accordingly, these findings provided the necessary data for better supporting the experimental verification of the immune-influencing mechanism of cysticercus, and could point to new possible targets for devising vaccines and therapeutants. Therefore, in summary, cysticercus and adult relied on different ways to resist the immune response of the host during their growth and development, and likewise severity of the ensuing disease that manifests also differed between them. Some important proteins in cysticercus and adult were detailed in the next section below, most of which were promising targets for specific antigens and candidate vaccines in the field of parasites. In follow-up work, we would plan to further screen the related proteins that have not been verified by immunity to elucidate and confirm their function.
The transcriptome is used to study the expression and regulation level of functional genes’ mRNA, an approach that is highly sensitive to the detection of differentially expressed genes and can more accurately convey the entire complex process of expression-driven regulation of the transcriptome. Here, through the transcriptome analysis of T. solium cysticercus, we found that paramyosin, a major egg antigen, in addition to cathepsin L-like cysteine proteinase, 70-kDa heat shock protein, and the H17g or TEG-Tsol surface antigen had potential for developing worm antigens for immunodiagnostics or vaccines . Similar to the results of our proteomic analysis, most of the proteins we identified in the parasite were also involved in cell processes, catalytic activity, and protein binding. Proteomics is the identification of the whole protein content present in a given sample, but when combined with a transcriptome-based perspective, it can yield a more comprehensive understanding of the pathogenic mechanism and treatment of Cysticercus cellulosae. However, proteomics can only detect the static proteins in a sample’s preparation, whereas investigating the transcriptome can reveal the expression and regulation level of mRNA at different time points, letting us observe the related pathways more intuitively.
Identified Proteins of Cysticercus (Down-regulated)
Several reports that investigated the proteome of T. solium across its developmental stages have discovered that cytoskeleton, actin, and paramyosin could be used as targets for applied research on cysticercosis vaccines and potential diagnostic antigens (e.g., enolase, calcium binding protein, small molecule shock protein, 14-3-3 protein, tropomyosin α, α-1 tubulin, β tubulin, annexin B1 and cAMP cyclic adenylate protein dependent kinase) [16–19]. Calcium-binding protein was involved in various regulatory functions of host invasion by parasites, these mainly including members of the calmodulin family (CAM), the calcineurin B-like (CBL) family, and the calcium-dependent protein kinase (CDPK) family: all these harbor a highly conserved helix–loop–helix structure, namely the EF-hand domain. For Plasmodium and Toxoplasma, phosphatidylinositol could also be stimulated by ligands binding to surface receptors (such as G-protein binding receptors [GPCR]), to produce calcium signals, thereby stimulating multiple cellular pathways . A cyclic GMP (cGMP)-regulated kinase of Plasmodium, the protein kinase G (PKG) might regulate the Ca2+ signaling pathway with ookinetes’ movement being controlled, as well as the initiation of gametocytes, and a formation of a fissure in the schizont . Fatty acid-binding proteins (FABP) were a family of proteins with isoforms in parenchymal and tegument cells. They were known to be involved in the uptake and transport of cholesterol and long-chain fatty acids, as well as the binding of triclabendazole , anti-oxidant activity, and immunomodulation . Notably, FABP produced a high level of specific antibodies during Schistosoma infection, which confered a strong immune-protective effect , thus increasing the value of FABP in the design of vaccines. Similarly, for Echinococcus granulosus, FABP was under investigation as a potential drug target .
Glycolytic enzymes were usually used for the development of drug and vaccine. Fructose-1-6-bisphosphate aldolase (FBPA) was a crucial glycolytic enzyme and a plasminogen-binding protein, it was be related to certain non-glycolysis functions, such as host cell adhesion, plasminogen-binding, and parasite invasion of the host. Research has demonstrated that FBPA was expressed on the surface of parasites and involved in parasitic motion and invasion by connecting surface adhesion proteins to the actin-myosin of parasites . Specifically, FBPA could bind to the surface of the cell membrane, and then bind to plasminogen, using the activity of hydrolyzing surface-related proteins to help invade host cells. Hence, it was also considered as a potential vaccine candidate or chemotherapy target for Trichinella spiralis and Giardia infection [27–29].
The damage to the host caused by cysticercosis was more serious than that of adult, and mechanism by which the host immune system was evaded or manipulated remained unclear. But through a functional enrichment analysis, we uncovered 25 proteins in cysticercus that were involved in the immune process but only two such proteins in adult. Some of these proteins have been studied for immunity in other parasitic worms. For example, the Trichinella spiralis ES product thioredoxin peroxidase-2 could induce macrophages towards an M2-like phenotype, both in vivo and in vitro, and CD4+ T cells increased in number after immunization of mice with rTsTPX2 and this mediated the expulsion of worm from the host to protect them, thus suggesting TsTPX2 was a potential vaccine candidate against trichinosis . Also, thioredoxin peroxidase was significantly recognized by melioidosis-positive sera in cysticercus stage, for which strong immunogenic properties rendered it an anticipated vaccine target. Pyruvate kinase was a crucial glycolytic enzyme characterized in Clonorchis sinensis that could promote the development of Th1 and inhibition of dendritic cells. Much like FBPA, it was also viewed as a promising target for drug therapy and vaccine development.
Furthermore, antigens and immunomodulatory proteins observed in cysticercus could engage in and foster a special mechanism of adaptation at this developmental stage, one that has evolved to evade the host immune system, which was a prerequisite for establishing a successful infection. In follow-up work, we would screen for some proteins and use them in related immune function experiments to further understand the immune escape mechanism of cysticercus.
Identified Proteins of Adult (Up-regulated)
Lacking a digestive tract, T. solium instead absorbs nutrients from its host across the body wall. The cortex absorbs all kinds of nutrients by means of diffusion and active transport, and also has the function of secreting and resisting the destruction of host digestive juice. Crucially, T. solium obtains its energy mainly via sugar metabolism. Adults rely mostly on glycolysis, but a few can also obtain energy from the tricarboxylic acid cycle and electron transfer system. Here we uncovered adult up-regulated proteins, most of which were related to metabolism, with just 2 proteins related to immunity. Given that adenosine 5'-triphosphate (ATP) was the most direct energy source used by this parasite, the adenylate energy charge (AEC) reflected its energy balance, which played a fundamental role in regulating the metabolism of the body. However, adenosine monophosphate deaminase (AMPD) could indirectly affect AEC. AMPD belonged to the amide hydrolase superfamily. It was not only part of the purine nucleotide cycle but also independently capable of regulating the dynamic equilibrium of the adenylate kinase reaction. In malaria parasites, the existence of AMPD did not affect its growth and development at any parasitic stage. However, when AMPD was overexpressed, it would lead to the death of the malarial parasite . It was known that cytidine 5-triphosphate (CTP) was the basis for the construction of nucleotides and nucleic acids, and protein glycosylation depends on it . Moreover, it worked as a high-energy molecule shown to be responsible for lipid biogenesis [35, 36] and participated in various cellular communication processes. But CTP was not self-synthesized in Toxoplasma spp., and, when it was absent, the growth and development of these parasites was adversely affected, making it a potential target for disease treatment . Cullin protein was the most typical ubiquitin ligase family and a key tumor-associated protein. It could promote the proliferation of tumor cells and be used as a marker and therapeutic target for tumor prognosis. According to the GO analysis, Cullin protein was also involved in cell cycle progression, signal transduction and transcription, intracellular metabolism, and the corresponding response to stimuli. Yet, surprisingly, this protein has not been studied in parasites, we suggested that it could provide clues for the follow-up research [38–40]. Topoisomerase was a critical type of enzyme for overcoming the problem of chromosome topology that arose during DNA replication, transcription, recombination, and mitosis. Thus it was involved in cell growth, tissue development, and cell differentiation [41–43]. There were some enzymes which participated in a variety of DNA metabolic processes. In parasitic infections, the inhibition of topoisomerase II by different ketone benzene and furan derivatives in cells hindered the basic metabolic process of cells and eventually led to apoptosis. In view of the parasite DNA topoisomerase II, these compounds could be used as potential anti-parasite drugs. Schistosome parasite miRNA could mediate the activity of the frizzled protein (frizzled related protein1 [FRZB2]), which increased liver fibrosis. FRZB2 was a secreted frizzled protein-related protein, which could competitively bind to specific frizzled protein receptors to suppress the signal transduction of Wnt, thereby influencing the severity of liver fibrosis. The FRZB2 gene knockdown would affect S. japonicum morphology, development, and survival, as well as its reproductive capacity [44, 45]. Major vault protein was a highly conserved ribonucleoprotein in lower and higher eukaryotes, whose function remained unknown . Some studies did find that Schistosoma mansoni had an adaptation mechanism to parasitize its hosts in the process of infection. Plasmodium glyceraldehyde-3-phosphate dehydrogenase acted as a glycolytic enzyme and also as a host plasminogen-binding protein, thereby interacting with the host’s fibrinolytic system to establish an important mechanism for that parasite invasion, growth, and development. At the same time, it could be used as a potential diagnostic biomarker for a variety of parasites, such as Plasmodium, T. solium, E. granulosus, and those causing filariasis, as well as S. mansoni, and Babesia microti [47–51]. Proliferating cell nuclear antigen (PCNA)was a protein that acted as a processivity factor for DNA polymerase δ in eukaryotic cells. it was originally identified as an antigen expressed in the nuclei of cells during the DNA synthesis phase of the cell cycle[52, 53]. The distinct mechanism of cell cycle arrest was associated with the up-regulation or down-regulation of TbPCNA. Deregulating the intra-parasite levels of TbPCNAwas a potential strategy for therapeutically exploiting this target in the bloodstream form of Trypanosoma brucei, which showed that PCNA was related to the growth and development of the parasite .
T. solium adult was able to reproduce and survive in the host intestines for escaping the immune response without causing excessive damage to the host. In our study, we found that there were few proteins in adult related to immunity, and most of them were related to growth and development, which also reflected the difference in immune response between adult and cysticercus to the host.