With technological advancements, mass spectrometry-based proteomic approaches have rapidly developed. This technology provides a high-throughput method for protein identification and quantification compared to two-dimensional electrophoresis and difference gel electrophoresis. Using two-dimensional electrophoresis, Kim et al. [20] identified eight specific and five upregulated proteins in 8-day-old juveniles compared with spargana and adults. However, we identified 1730 DEPs in the S vs. the E groups and 1308 DEPs in the A vs. the S groups by TMT analysis. These results suggest the global reprogramming of cell metabolism across the three developmental stages, as evidenced by the differential expression of glycolytic, translational, carbohydrate metabolic, enzyme inhibitor, and structural proteins.
Under nutrient-rich conditions with ample ATP, the parasite employs a strategy of converting glutamine into GABA (gamma-aminobutyric acid) as a means to avert the overproduction of NAD(P)H and the leakage of reactive oxygen species from the mitochondrial respiratory chain [21, 22]. In situations where exogenous carbon sources are limited, the accumulated GABA serves as a short-term energy reserve, entering the tricarboxylic acid (TCA) cycle to maintain a robust energy flux [23]. Consequently, the GABA shunt emerges as a pivotal regulator of carbon metabolism, playing a significant role in both nutrient-rich and nutrient-depleted conditions. Notably, our study reveals an overexpression of GABA shunt-related enzymes in adult worms compared to spargana, including succinate-semialdehyde dehydrogenase, dihydropyrimidine dehydrogenase, delta-1-pyrroline-5-carboxylate dehydrogenase, and glutamate dehydrogenase. This finding underscores the importance of the GABA shunt in the adult stage, elucidating its role in central carbon metabolism for adult Spirometra tapeworms.
Calcium ions (Ca2+) play a crucial roles as regulators of the parasite's movement and invasion, whether it resides within or outside host cells [24]. However, the role of Ca2+ ions is facilitated by their interaction with downstream proteins [25, 26]. Among these proteins, annexin, calreticulin (CRT), and calmodulin (CaM) take center stage. Annexins, as calcium-dependent phospholipid-binding proteins, engage in various biological processes linked to biofilm development via their binding to membrane phospholipids [27, 28]. Several studies have highlighted the crucial role of annexins in parasite growth, development, maintenance of cellular structural integrity, and the host's immune response against parasites [29, 30, 31, 32]. For instance, Song et al. [33] emphasized the potential significance of annexin B3 and annexin B38 in the invasion and development of Echinococcus granulosus protoscoleces. In our investigation, we observed the upregulation of annexin A1, annexin A7, annexin A5, and annexin A4 in the sparganum stages, while other annexins (annexin A3, annexin A2, annexin A13, and annexin A11) exhibited upregulation in the egg stage. CRT, a protein known for binding and inactivating pattern-recognition receptors like C1, mannose-binding lectin, and ficolins, plays a critical role in inhibiting the classical and lectin pathways of complement [34, 35]. For instance, Trichinella spiralis-CRT directly binds to host complement C1q, which limits C1q-mediated activation of the classical complement pathway and C1q-induced non-complement activation of macrophages, thereby facilitating immune evasion and survival within the host [36]. In our research, CRT exhibited upregulation in the sparganum stage, indicating that its heightened expression might function to suppress the host's immune response, safeguarding the sparganum. CaM, a protein well-known for its ability to regulate a multitude of physiological processes, including muscle contraction, metabolism, and cell motility through the binding of Ca2 + and the downstream modulation of Ca2 + signals, took precedence in the sparganum group [37, 38, 39]. Here, the expression level of CaM was significantly upregulated compared to the adult and egg groups, suggesting a pronounced role in the sparganum stage. Given its structure and function, the elevated expression of CaM may augment the infiltration capacity and motility of spargana [40], enabling them to migrate swiftly through host tissues and organs. In contrast, the downregulation of calmodulin expression in adults, mainly dwelling in the host gut, can be rationalized by their reduced need to traverse host tissues and organs, similar to findings in studies on CaM in Clonorchis sinensis and Echinococcus granulosus [41]. Despite these insights, the biological functions of CaM in tapeworms remain relatively uncharted, warranting further investigation to grasp the multidimensional roles of CaM comprehensively.
Cathepsin, a prominent cysteine protease, holds paramount importance for the parasite's survival and virulence [42, 43]. Its multifaceted roles encompass aiding parasite migration within the host by cleaving host proteins, facilitating nutrient acquisition by breaking down host proteins into absorbable peptides, and subverting host immune defenses through the cleavage of immunoglobulins [44, 45, 46]. Our study reveals an upregulation of three cathepsin L proteins in the sparganum group in contrast to the adult and egg groups. It's worth noting that prior research has also established that spargana secrete a considerable amount of cysteine proteases [47, 48]. Moreover, cathepsins within the sparganum may be closely tied to the process of host tissue digestion during the development of the disease, given their in vitro hydrolyzing activity on collagen, hemoglobin, and immunoglobulins [49, 50]. Collectively, these findings emphasize the pivotal role of cathepsin L in enabling tissue penetration, optimizing nutrient uptake, and orchestrating immune evasion by spargana. Interestingly, previous studies have reported cathepsin expression exclusively in the sparganum stage within various hosts, including frogs, mice, and snakes, while not in the adult or egg stages [51, 52]. These observations deviate from our study, which identified various cathepsins across the three different developmental stages. This discrepancy may be attributed to the vast array of cathepsin types and their varied roles, highlighting the complexity and versatility of this protease family.
Serine protease inhibitors (serpin) can inhibit the activities of host serine and cysteine proteases, thus enhancing the ability of worms to escape the host immune response [53]. Serpin has been isolated from several parasitic helminths, such as Schistosoma japonicum, Taenia solium, Trichinella spiralis, and Echinococcus multilocularis, evidencing its immunomodulatory role in the parasite-host interaction [54, 55, 56, 57]. Here, we found that the expression level of serpin B8 in sparganum stage samples was significantly higher than in the adult and egg samples. This result suggests that this protein might aid in protecting sparganum from the attack of host enzymes so that it can survive in the host with normal immune function for a long time. Notably, serpin B6 and serpin B10 were upregulated in eggs compared with the adult and sparganum stages. Thus, these proteins may be involved in maintaining the physiology of eggs by regulating endogenous serine proteases.
Parasitic worms lack the capacity for de novo synthesis of fatty acids and thus rely on efficient binding systems to absorb and transport essential fatty acids from their host to fulfill a variety of biological needs [58, 59]. Among these systems, fatty acid-binding proteins (FABPs) appear as crucial mediators, facilitating the binding and transport of fatty acids and other hydrophobic ligands through reversible interactions. These proteins employ great significance in supporting the growth, development, and reproduction of parasites within their hosts [60, 61, 62]. For instance, the SeFABP gene is transcribed across various life stages of S. erinaceieuropaei, with a notable presence in the eggshell, sparganum cortex, and parenchyma, as well as the immature, mature, and gravid proglottids of the adult stage [63]. In our study, the FABPs exhibited an upregulation in the egg and adult stages when compared to the sparganum stage. This observation can likely be attributed to the relatively stable environment characteristic of the sparganum stage, where there is no imperative need for progression to the next life stage. On the other hand, the adult stage is marked by rapid growth, development, and the formation of eggs, demanding a substantial surge in energy requirements. The upregulation of FABPs in the egg stage may be associated with the heightened demand for substantial quantities of fatty acids to support egg development and overall survival. These findings show the pivotal role of FABPs in orchestrating the parasite's metabolic strategies to cope with the varying demands across different developmental stages.