Trichinella is an intracellular nematode that infects a wide variety of animals. The complete life cycle of the parasite is completed in a single host via the invasion of intestinal epithelial and skeletal muscle cells. A recent study of proteases throughout the life of Trichinella spiralis found that excretion-secretion (ES) and crude extracts of muscle stage larvae show substantial serine protease activity against structural proteins, whereas newborn larvae and adult worms principally degrade hematic proteins. This stage-specific proteolytic activity contributes to the breakdown of both mechanical and humoral barriers within the host during parasite infection. These serine proteases are targets of the antibody response, which can inhibit the protease activity and possibly contribute to the impairment of the parasite in a sensitized host [5,6].

During the invasion of epithelial cells, the larvae released several glycoproteins that bear the highly antigenic sugar moiety, tyvelose (3, 6-dideoxy arabinohexose). Monoclonal antibodies against tyvelose protect against infection, which implicates that tyvelose-bearing glycoproteins play keys roles in intestinal epithelium invasion and niche establishment. With the aim of investigating these glycoproteins at the molecular level, Romaris et al. [7] first isolated glycoproteins by affinity chromatography technique using monoclonal antibodies (mAbs). De novo peptide sequencing combined with cDNA library screening identified that these glycoproteins are serine proteases (TspSP-1). Western blot analysis and immunohistochemistry indicated that these glycoproteins are muscle larvae (ML) stage specific and are synthesized in α stichocytes. Furthermore, the inhibition of epithelial cell invasion and migration by mAbs against TspSP-1 indicated that TspSP-1 could play an important role in degrading cytoplasmic or intercellular proteins, thereby facilitating the movement of the larvae [7]. Subsequently, Nagano et al. [8] also isolated a serine protease, named Ts23-2, from a cDNA library of T. spiralis muscle larvae. The Ts23-2 gene is only transcribed after the completion of cyst formation. The protease activity of the recombinant catalytic domain was confirmed using synthetic peptide substrates, indicating that it is a plasmin-like protease [8].

Recently, another member of this subfamily, named TspSP-1.2, was characterized. The anti-serum against TspSP-1.2 can partially prevent the larval invasion of intestinal epithelial cells. Furthermore, the recombinant TspSP-1.2 protein induced a partial protective immunity in mice. These results indicated that TspSP-1.2 contributes to the larval invasion of host intestinal epithelial cells and could be a potential vaccine candidate against T. spiralis infection [9]. A similar protein (TppSP-1) from Trichinella pseudospiralis muscle larvae was identified by Cwiklinski et al. [10]. Analysis of the deduced amino acid sequence found that the histidine residue of the catalytic triad in TsSP-1 was replaced with an arginine residue in the TppSP-1. This could lead to the loss of proteolytic activity, and the role in the T. pseudospiralis-host interaction needs further research [10]. Trap et al. [11] identified another putative serine protease by screening a library from T. spiralis adult-newborn larvae mixed stage with a radioisotope-labelled DNA probe. TsSerP contains 2 trypsin-like serine protease domains flanking a hydrophilic domain. Northern blot analysis of the expression profile for TsSerP genes demonstrated that it was expressed in all life cycle stages of the parasite. Western blot analysis using soluble and E-S antigens found that it was not detected in ES products. Immunolocalization showed that TsSerP is expressed on the peripheral regions and the esophagus of T. spiralis muscle larvae and adult worms. Thus, TsSerP may be involved in the parasite’s moulting process and digestive function [11].

Liu et al. [12] identified a newborn larval stage-specific serine protease gene (NBL1) via a subtractive cDNA library of T. spiralis newborn larvae. It includes 2 regions, a catalytic domain and a C-terminal domain. Epitope mapping using truncated variants of rNBL1 indicated that the C-terminal part of NBL1 is the main immunodominant region. NBL1 showed encouraging potential in the early detection of Trichinella infection and protective immunity against T. spiralis infection in pigs [13]. Based on the high immunogenicity of the C-terminal domain, we hypothesized that during the newborn larval invasion of the host, it may divert the immune response away from the functional regions of NBL1 to contribute to host invasion.

The multiple serine proteases identified at different stages of T. spiralis indicated the existence of a superfamily of serine proteases in T. spiralis. Each member of serine protease families may have different functions in parasite infection, which depends on stage-specific expression, location, and the presence of a regulation domain of the serine protease. Further studies are required to fully understand their functions in parasite-host interplay.

T. muris is a parasitic nematode of mice in which an infective larva invades host intestinal mucosa and develops into an adult worm. The anterior portion of an adult worm embeds within a syncytial tunnel derived from host cecal epithelium. There are 2 major serine peptidases with specific activity for collagen-like molecules in the ES antigens of T. muris adult worms. Interestingly, the activity of both serine peptidases was not observed in worm extract, which suggests that the enzymes are present in the adult worm as inactive precursors or inactivated by endogenous peptidase inhibitors. The ability for degradation of the basement membrane by live worms suggested that these peptidases could be involved in the invasive process. These peptidases also could play important roles in the production and subsequent maintenance of the parasites' syncytial habitat. Moreover, authors speculated that they could contribute to the pathology of trichuriasis by disrupting the integrity of epithelial cell membranes [14].

The intestinal mucus barrier plays a significant role in the expulsion of gastrointestinal nematodes. The mucin is pivotal during the formation of the mucus layer. A recent study found that serine proteases secreted by T. muris can degrade the major intestinal mucin Muc2 and depolymerize the mucin network. Thus, it was suggested that serine proteases secreted by T. muris could contribute to the modification of the parasitic niche to prevent clearance from the host or to facilitate efficient mating and egg laying [15]. Given its essential role in the intestinal stage, this serine protease could be an attractive drug target and should be characterized at the molecular level.

Anisakiasis is a gastrointestinal tract disease, which is caused by the consumption of raw or undercooked seafood that contains larvae of the nematode A. simplex. After ingestion, A. simplex larvae penetrate the mucosa, submucosa, and muscularis of the host stomach and intestine and may migrate to the omentum, liver, pancreas, or gall bladder. Sakanari and McKerrow [16] found that the secretion of infective larvae contains trypsin-like serine proteases that degrade connective tissue extracellular matrix (ECM). Judy et al. [17] isolated 4 serine protease genes of A. simplex using degenerate oligonucleotide probes based on the consensus regions of mammalian serine proteases. One of these genes is 67% identical to the rat trypsin II gene. Alignment of these 2 genes revealed that the intron-exon junctions are conserved between nematodes and rats, confirming the structural and functional similarity of the 2 genes. Thus, serine proteases of infective larvae may be involved in the degradation or digestion of host tissue [17].

Meanwhile, Stephen et al. [18] purified 2 serine proteases from the infective larvae of A. simplex using different affinity chromatography approaches. The 26 kDa-protease is similar to the trypsin of the domestic pig, Sus scrofa. The second serine protease was similar to the extracellular serine protease of the pathogenic bacterium Dichelobacter nodosus, which can degrade elastin, keratin, and collagen. The mechanism involved in the tissue destruction caused by the 2 serine proteases is far from being decrypted and needs further research [18].

A. suum, also known as the large roundworm of pigs, is an intestinal nematode. During the process of A. suum sperm activation, sperm differentiates from immature spermatids into mature and motile spermatozoa. Recently, study of sperm activation indicated that serine protease is responsible for A. suum sperm activation. This serine protease was purified and identified by de novo sequencing. The purified protease showed strong activity in sperm activation, and it can be inhibited by the serine protease inhibitor PMSF. Finally, the full length cDNA named As-TRY-5 was cloned by RACE-PCR using the degenerative primers based on the peptide sequence. Sequence comparisons indicated that As-TRY-5 shares a high degree of homology with trypsin-like serine proteases of eukaryotes. A further study using a serine protease inhibitor (As-SRP-1) that was released by the activated sperm indicated that during the spermatogenesis process, the activity of As-TRY-5 was regulated by this serine protease inhibitor. This could be significant during postcopulatory sexual selection [19].

Onchocerca volvulus is an important filarial nematode that causes subcutaneous filariasis of humans and affects the eyes and skin. The infective larvae, male worms, and microfilariae migrate through the host tissue. A proteolytic activity study indicated that there is a 40 kDa neutral elastase in ES products of microfilariae, which can degrade components of the dermal extracellular matrix, collagen type IV, fibronectin, and laminin but cannot degrade intact immunoglobulins. Based on this proteolytic activity, authors suggested that the elastase of microfilariae plays an important role in the degradation of elastic fibres of the host tissue. Moreover, the elastase proteolytic activity is also present in males, but absent in ES products of females. This is correlated with the different behavior of adult worms; the adult males are able to migrate from 1 nodule to another, whereas adult females only reside in nodules [20]. In addition, a stage-specific 43-kDa serine elastase was also found in infective larvae of Onchocerca lienalis. The specific elastase of L3 larvae most likely contributes to L3 migration from the blackfly bite site to different tissues of the body where the adults will develop and form nodules [21]. Blisterase is a subtilisin-like serine protease and plays important roles in nematode biology including the cuticle production and maintenance, neural signalling, and nematode development. Thus, it is a potential drug target for controlling parasite infection. Catherine et al. [22] isolated blisterase from a cDNA library of the infective larvae (L3) of O. volvulus. A fragment of blisterase was cloned and expressed in insect cells with maximal activity at a neutral pH. However, the roles of the blisterase in the O. volvulus-host interaction remain unknown [22].

Complement plays multiple roles in both innate and adaptive immunity, such as mediating the adherence of myeloid cells to the parasite and subsequently killing parasite and directing cellular recruitment. Rees-Roberts et al. [23] reported that secreted products of Brugia malayi microfilariae can cleave the C5a and completely abolish C5a-mediated chemotaxis of human peripheral blood granulocytes. The inhibition was blocked by a serine protease inhibitor, indicating 1 or more types of serine proteases are responsible for the cleavage of C5a. It has been speculated that serine proteases from B. malayi may suppress the immune system and induce immune tolerance, hindering parasite elimination [23].

Hookworm disease results from infection by a hematophagous nematode of the genus Ancylostoma that lives in the small intestine of the host. Now, more than 1 billion people are infected with this parasite worldwide. Hookworms cause anemia by extracting host blood from lacerated capillaries in the mucosa of the small intestine over an extended period of time. Peter et al. [24] found a 36 kDa elastolytic enzyme with anti-clotting properties in ES products of third-stage infective filariform larvae of Ancylostoma caninum. This elastolytic enzyme interferes with fibrin clot formation and promotes fibrin clot dissolution. The protease can degrade fibrinogen into 5 smaller polypeptides with anticoagulant properties. In addition, this protease can convert plasminogen to a mini-plasminogen-like molecule. This molecule is analogous to leukocyte elastase and could be related to the specific antihemostatic mechanism of the hookworm. According to their results, authors hypothesized that the parasite uses this enzyme to feed on the villous capillaries by preventing the blood from clotting. Thus, this protease is a potential target for chemotherapeutic or immunological intervention [24].

S. carpocapsae is a parasitic nematode of insects and is used as a biological control agent to kill several insect pests and vectors. The infective juvenile can enter the host by mouth or anus and invade the hemocoelium. Invasion has been described as a key factor in nematode virulence and is mediated by proteases. Recently, 2 novel serine protease cDNAs (sc-sp-1 and sc-sp-3) from the parasitic stage were isolated by a degenerate RT-PCR based on conserved motifs near the catalytic histidine of serine protease. The sc-sp-1 expression time frame analysis showed that the sc-sp-1 stage-specifically expressed at parasitic stages. It is mainly expressed in the midgut of the insect (L3), where the nematodes will prepare to invade the insect hemocoelium. Further, analysis of the influence of insect tissue on sc-sp-1 expression showed that different tissues of the insect can induce the expression of sc-sp-1 at different times. This could contribute to the parasite’s ability to sense insect tissues at different time points. The peritrophic membrane of the gut wall and the basal lamina are major barriers of host tissue invasion. The study showed that the sc-sp-1 was highly efficient at destroying the peritrophic membranes and caused epithelium cell detachment from the basal lamina. Thus, the function of sc-sp-1 could be the invasion of hemocoelium through the disruption of the midgut barrier [25]. The sc-sp-3 is a multifunctional chymotrypsin-like protease. It not only shares similar biochemical characteristics with sc-sp-1 but also induces caspase-dependent apoptosis in Sf9 insect cells [26]. Recently, a stage-specific elastase-like serine protease gene (Sc-ELA) was isolated by the suppression subtractive hybridization method during the parasitic stage. Sequence comparison and evolutionary marker analysis revealed that Sc-ELA was a member of the elastase serine protease family with potential degradative, developmental, and fibrinolytic activities [27].

In addition to these serine proteases, Balasubranian et al. [28,29] purified 2 insect immune depression-related serine proteases from the ES products of infective-stage S. carpocapsae. Melanotic encapsulation that is formed by the deposition of multiple layers of hemocytes and⁄or melanin is an important insect defence mechanism against parasites. The trypsin-like serine protease and chymotrypsin-like serine protease can prevent melanotic encapsulation by suppressing prophenoloxidase activity or by disrupting the insect hemocyte F-actin cytoskeleton. Although this experimental evidence did not fully elucidate the exact biological roles of the serine proteases during host immune suppression, it contributes to the understanding of the pathogenesis strategy used by S. carpocapsae. Further biochemical and molecular characterization of Sc-Trypsin and Sc-chymotrypsin is required for a complete delineation of their possible functions in helping parasites to infect and survive within the host [28,29].

Fasciola hepatica and F. gigantica are the parasites that cause liver fluke disease (fascioliasis). It is not only an important human disease but it also affects cattle and sheep. Infection causes worldwide economic losses of approximately 2 billion dollars per year. Carmona et al. [35] purified a secreted dipeptidylpeptidase (DPP) from F. hepatica by gel-filtration and ion-exchange chromatography. It was found to be a serine protease that is expressed in newly excysted juvenile, immature, and mature flukes. Authors suggested that the parasite DPP may function in the digestion of host macromolecules into peptides. These peptides could be absorbed as nutrients by the parasite’s intestine, which could profit the parasite [35]. In a recent study, a 60-kDa neutral serine protease designated as serine PIc was separated from F. gigantica. A study of biological characteristics found that the activity and stability of the serine protease depended on divalent cations and temperature. Enzyme activity assays indicated that proteolytic activity increased followed by the development of F. gigantica, which suggests that it has a very important physiological role, but the precise function remains unknown [36].

Schistosomiasis is a serious human disease in the tropics, which affects millions of people. Infection of humans by Schistosoma mansoni begins following the invasion of intact skin by the cercariae. The penetration of the skin is facilitated by secretions from the acetabular and head glands. Disruption of these potential mechanisms by specific drugs/vaccines may provide therapeutic benefits. Thus far, a number of studies have confirmed that cercarial elastase (SmCE), which has a chymotrypsin-like activity, is a major histolytic protease involved in skin invasion. Northern blot analysis indicated that it is stage-specific and is only expressed in cercariae. Further anatomical location of SmCE mRNA in tissue sections of developing larvae showed that it is only synthesized in acetabular gland cells of developing cercariae. This further indicated that SmCE is regulated within a limited developmental frame in a specialized cell [37]. A subsequence protease activities assay indicated that a serine protease with “trypsin-like” activities from secretions of cercariae could also be involved in host invasion [38]. To evaluate the relative roles of these 2 serine proteases in larvae invasion, both of 2 serine proteases were analyzed by southern blot, genomic PCR, and immunolocalization. These results demonstrated that only single SmCE with activities against macromolecular substrates is responsible for human skin invasion, and serine protease with trypsin-like activities is a contaminant from the intermediate host snail [39].

To date, 8 isoforms of S. mansoni stage-specific elastases have been identified based on amino acid and promoter sequence homology. In addition, investigation of SmCE ortholog genes in the related species Schistosoma haematobium and S. douthitti found that multiple CE isoforms exist in both species [40]. By contrast, in another schistosome species, Schistosoma japonicum, no SmCE ortholog was identified. This result indicates that the expansion of the cercarial elastase genes is limited to the human-specific schistosomes [41,42]. To further explore the roles of SmCE gene expansion in S. mansoni, James et al. [43] investigated the profile of transcript and protein expression patterns and substrate preferences of the expanded SmCE gene family. The results revealed that these SmcE isoforms are similarly expressed throughout the S. mansoni life cycle. They have largely similar substrate specificities. According to these results, authors suggested that the majority of protease isoforms share a conserved function for a common pool of substrates [43]. Thus, the expansion of the SmCE gene family is functionally redundant and is a direct increase in the amount of protease. In addition, activity-based profiling showed that the activity of SmCE also presented in 6-week daughter sporocysts, suggesting that 1 or more of the SmCE isoforms could have a novel role in its intermediate host [43].

Proteases have long been hypothesized as aiding parasites in evading the immune response of the host by degrading immune effectors or modulating the cellular immune response. Studies have shown a direct correlation between levels of specific IgE and protective immunity against schistosomes in humans [44,45]. Analysis of the immune evasion ability of parasites showed that extracts from cercarial and schistosomular stages of S. mansoni can cleave human, mouse, and rat IgE, but not human IgA1, IgA2, or IgG1. This cleavage can be inhibited by serine protease inhibitors. This indicates that during the establishment of mature infections, an elastase-like serine protease helps the parasite to evade IgE-mediated protective reactions [46]. A recent study using a highly purified SmCE found that these SmCE cleave IgE at solvent-exposed interdomain regions of the IgE-Fc. This sequence of cleavage is also present in numerous key molecules involved in regulating immunity, including FcγRI, IL-2, IL-10R, IL-12R, and TLR3. Thus, additional studies are required to determine more genuine substrates for SmCE, which will help us to completely understand the roles of SmCE in immune evasion [47].

To confirm such a role in vivo would require analysis of the immune clearance of parasites in a living host following chemical or genetic knockout of the protease. Because the degradation of immune effectors is vital for parasite immune evasion, the inhibition of this degradation pathway offers a valid approach for developing novel chemotherapeutic agents. Thus, further investigation to determine serine protease biological function and the evaluation of its potential as a drug target are needed.