Blood flukes from the genus Schistosoma are the causative agent of a serious human disease known as schistosomiasis. It is estimated that 240 million people are infected and more than 750 million live at risk of infection globally, mostly in sub-Saharan Africa (Colley et al., 2014). Adult worms reside in pairs inside the mesenteric venous plexus, and the females produce approximately 300 fertilized eggs per day (Moore and Sandground, 1956). Immature freshly laid eggs located in the blood vessel are small and contain only a zygote and vitelline cells (Ashton et al., 2001, Jurberg et al., 2009). They appear to be immunologically inert because, in contrast to mature eggs, they do not invoke macrophage recruitment and granuloma formation (Takaki et al., 2021). During maturation, which takes approximately 6 days (Michaels and Prata, 1968), eggs first adhere to endothelial cells (deWalick et al., 2014) and then migrate from blood vessels to the intestinal wall. However, approximately one-third to one-half of the laid Schistosoma mansoni eggs do not pass through the intestinal wall but end up trapped in the liver (estimates vary in published studies) (Moore and Sandground, 1956, Fan and Kang, 2003). Here, the eggs induce inflammation, eventually leading to the clinical manifestations of chronic schistosomiasis mansoni. Either in intestine or liver, the developing eggs absorb nutrients from surrounding tissues, grow substantially and develop a subshell envelope that exhibits high protein synthetic activity (Ashton et al., 2001, Costain et al., 2018). This envelope was proposed as the site of origin of egg secretions, also termed excretion-secretion products (ESPs). ESPs contain a mixture of proteins (Cass et al., 2007, Mathieson and Wilson, 2010, deWalick et al., 2012, Carson et al., 2020), some of which are potent immunomodulators capable of inducing a Th2 immune response and granuloma formation around the egg (Pearce, 2005, Schramm et al., 2006, Everts et al., 2009, Ittiprasert et al., 2019). Granulomas are structured clusters of specific immune cells around the eggs, which most likely aid the successful migration of the egg into the intestinal lumen while protecting the host from excessive organ damage (Hams et al., 2013, Schwartz and Fallon, 2018). If the egg successfully migrates through the intestinal wall, it is then expelled to the external environment where the miracidium hatches.

An important role in the parasite's survival in the host has been attributed to proteases (peptidases, proteolytic enzymes) (McKerrow and Salter, 2002, Yang et al., 2015). Apart from internal physiological functions, these hydrolytic enzymes are essential for the initiation and maintenance of the infection, nutrient acquisition, migration, interaction with the host and modulation of the host immune response (Atkinson et al., 2009, Kasny et al., 2009, Dvorak and Horn, 2018). Therefore, they have been considered candidate targets for drug and/or vaccine development (Abdulla et al., 2007, Tallima et al., 2017). The action of endogenous proteases is controlled by protease inhibitors. However, it has been shown that protease inhibitors can be secreted by the parasite and are able to inhibit host proteases. Inhibitors take part in protection against host digestive proteases and immunity, modulation of the immune response and interference with hemostasis (Knox, 2007, Ranasinghe and McManus, 2017). In a previous study, we showed that ESPs of S. mansoni eggs exhibit proteolytic activities of serine, cysteine, and metalloproteases (Horn et al., 2014, Dvořák et al., 2016). It can be hypothesized that the proteases responsible for this activity may be important for interactions with the host, e.g., they may mediate the migration of eggs through host tissues. However, no specific protease that plays a role in egg-host interactions has yet been identified and studied in detail.

Our understanding of the biology of S. mansoni eggs, the process of their migration and the associated changes in host physiology can be significantly advanced by comparison with a phylogenetically closely related parasite that does not cause similar pathology. Fasciola hepatica, a cosmopolitan trematode that infects a broad range of mammals including humans, appears to be a relevant candidate for such a comparison. Fasciola hepatica adult worms reside in the biliary ducts of the host, feed on blood and lay eggs that exit the biliary ducts through the intestinal lumen to the external environment. The reason for this comparison of the two species is that S. mansoni eggs are in close contact with host tissues and need to modulate the host's physiological mechanisms to escape from the host. In contrast, F. hepatica eggs do not need to cross any endothelial or epithelial barrier to pass into the external environment. Although newly excysted juveniles and adults of F. hepatica can cause extensive bile duct and liver damage during invasion, migration and feeding (Fürst et al., 2012), the eggs have not been implicated in the pathogenesis of the disease. In only a few cases, F. hepatica eggs have been reported to be trapped in tissue, and this was only due to massive infection (Harrington et al., 2017). Eggs of S. mansoni and F. hepatica differ also in aspects of their development and overall biology. While F. hepatica eggs are more resistant in the environment, where they may develop for ∼14 days to become fully mature and then hatch, S. mansoni eggs are already fully mature once they reach the environment, and rapidly hatch in the water under appropriate temperature and light conditions (Harrington et al., 2017). Another difference between the eggs of the two species is that F. hepatica eggs do not need external nutrients to fully mature, while S. mansoni eggs, when cultured in vitro, cannot fully develop without blood serum (Wilson, 1967, Michaels and Prata, 1968).

In this study, we used RNA sequencing and comparative transcriptomic analysis of S. mansoni eggs to characterize their portfolio of proteases and protease inhibitors, and to determine the dynamics of their expression during egg development. In addition, by comparing proteases and inhibitors from S. mansoni eggs, which cause severe pathology in the host, with those from eggs of non-pathogenic F. hepatica, we sought to identify proteins that might be key to the unique interaction of S. mansoni eggs with host tissues. The results of this methodological approach provide new insights into schistosome egg biology and reveal novel egg molecules with potential roles in egg-host interactions.