Introduction

Trichinella is a genus of nematode that infects a wide variety of vertebrate hosts. Larvae at a muscle stage are an infective form of Trichinella spp. In the host stomach, larvae are released with the aid of host gastric juice and develop into adult worms in the host intestine, and the female begins to produce the second generation of larvae, which migrate through the whole body of the host. Infection causes satellite cell proliferation (Matsuo et al. 2000; Wu et al. 2001) and transformation of muscle cell to the nurse cell in the capsule (known as cyst; Jasmer 1990; Despommier et al. 1990). Two clades in genus Trichinella have been identified: the encapsulated clade and the nonencapsulated clade (Zarlenga et al. 2006). Muscle larvae in encapsulated species develop a thick collagen capsule, and the nonencapsulated species develop only a very thin collagen capsule (Xu et al. 1997).

Muscle cell transformation is likely initiated by excretory–secretory (E–S) products released from the larvae (Ko et al. 1994). Trichinella spp. produces a variety of biologically active proteins, which may or may not be a part of E–S products. Proteins in E–S products likely affect host cells and tissues for respective purposes, and non-E–S proteins likely are engaged in internal reactions within the parasites. Among them, E–S products of Trichinella spp. have received a great deal of attention not only from an immunological point of view but also from the point of host–parasite interaction because of assumption that the intracellular parasite secretes some functional proteins and alters the host cell in such a way that Trichinella spp. can establish parasitism and survive for a longer period of time.

The 43-, 53-, and 45-kDa glycoproteins in larval E–S products contain tyvelose-containing antigen (Wisnewski et al. 1993; Romarís et al. 2002a; Arasu et al. 1994), which is the major antigen recognized by the host during infection (Appleton et al. 1991). Furthermore, these glycoproteins have been characterized at the molecular level, and it has been shown that these proteins are important for muscle cell transformation, capsule formation, and continuation of parasitism due to Trichinella spp. However, it is completely obscure how Trichinella spp. changes terminally differentiated muscle cells into nurse cell, whose architecture has never been designed in the host genome.

Protein engineering is an extremely hot topic of recent biological studies. Functional proteins have been produced for many purposes including therapeutic usage. Parasite proteins are not an exception. Efforts seeking for such an important protein responsible for host cell transformation in E–S products resulted in the discovery of many functional proteins owing to recent progress in molecular technology. In addition to the 43-, 53,- and 45-kDa glycoproteins, the E–S proteins of Trichinella spp. contain some functional proteins such as heat shock proteins, endonucleases, proteinases, protein kinases, proteinase inhibitors, superoxide dismutases, glycosidases, etc. This article will review the characterization of Trichinella proteins with emphasis on E–S products, including analytic information by means of classical wet laboratory techniques as well as molecular techniques.

Proteomic analysis of Trichinella proteins

To better understand the muscle cell transformation leading to capsule formation, it is critical to examine the nature of Trichinella E–S products that affect the infected muscle cells after being released to the cell cytoplasm. In this section, some landmark papers on the characterization of Trichinella proteins will be reviewed in chronological order.

Early investigators used classical laboratory techniques such as electrophoresis or gel filtration to investigate the nature of E–S products. Despommier and Laccetti (1981) characterized antigenic proteins derived from a large-particle fraction of muscle larvae of Trichinella spiralis in relation to their molecular weights, isoelectric points, carbohydrate contents, antigenicity, and their ability to induce protection in mice. Gel filtration yielded five major peaks of material, while sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) revealed a minimum of 28 proteins ranging in molecular weight from 11 to 200 kDa. Analytical isoelectric focusing on SDS-PAGE yielded 37 bands of protein, while the periodic acid-Schiff reaction performed on a similar gel revealed 22 glycoproteins. Most proteins were within a pI range of 4.0–7.0, while all of the glycoproteins had pI ranging from 4.0 to 6.5. Secreted proteins of muscle larvae, adult males, and newborn larvae of T. spiralis were relatively few in number and were different for each stage, whereas somatic proteins demonstrated an unresolved smear in all cases (Parkhouse and Clark 1983).

T. spiralis and Trichinella pseudospiralis are independent species in the genus Trichinella. These two species are similar but different in terms of host responses. By high-power resolution (two-dimensional Western blot analysis), Wu et al. (1999) showed that antigenic peptides of T. spiralis consisted of about 100 peptide spots, and the molecular weight of these peptides ranged from 22 to 80 kDa, and pI ranged from 4 to 7. Antigenic peptides of T. pseudospiralis consisted of about 20 to 30 peptide spots, and the molecular weights ranged from 25 to 80 kDa, and pI ranged from 4 to 7. The E–S products of T. spiralis and T. pseudospiralis were highly cross-reactive with each other.

Recent investigators adapted new molecular techniques. Robinson et al. (2005) have used matrix-assisted laser desorption–ionization time-of-flight (MALDI-TOF) mass spectrometry for identification of the peptide spots of muscle larvae E–S products. In addition, MALDI-TOF mass spectrometry and laser chromatography–mass spectrometry/mass spectrometry (LC–MS/MS) were used to identify the peptide spots, and these data were analyzed by specific Trichinella expressed sequence tag (EST) databases (Robinson and Connolly 2005). They identified 43 out of 52 protein spots analyzed as the major secreted glycoproteins. The 43 protein spots represented only 13 different proteins indicating that there are multiple proteins isoforms present in the E–S products. Also, two major groups of T. spiralis-specific proteins and several T. pseudospiralis-specific proteins were identified using two-dimensional gel electrophoresis and tandem mass spectrometry (Robinson et al. 2007a). Liu et al. (2007) cloned six newborn larva stage-specific and four adult worm stage-specific genes of T. spiralis using suppression subtractive hybridization technique.

A dataset for expressed Trichinella genes may support proteomic methods to identify parasite proteins involved in specific interactions. In addition, genome information provides comparative analysis among life cycle stages relative to biological similarities and differences, some of which may involve host cell interactions. Comparisons with other sequences can also identify putative proteins that are restricted to nematodes or nematode groups, thus providing insight that may be related to parasitism. Mitreva et al. (2004) analyzed the transcripts of Trichinella spp. using ESTs that are produced from cDNA libraries for immature L1, mature muscle larvae, and adult stages of T. spiralis and showed that 10,130 ESTs were grouped into 3,454 gene clusters.

Major Trichinella E–S proteins with unrevealed function

The 43-, 53-, and 45-kDa glycoproteins, which are three major antigenic proteins, have received more attention than other E–S proteins because these three proteins are candidates of immunodiagnostic antigens for trichinellosis and are present in much greater amounts in the E–S products, but the function of the 43-, 53-, or 45-kDa protein remains unknown.

It should be emphasized that the 43-, 53-, and 45-kDa glycoproteins share a unique so-called TSL-1 antigen (Wisnewski et al. 1993; Romarís et al. 2002a; Arasu et al. 1994). TSL-1 antigen is one of the most intriguing glycoprotein antigens, which induce a powerful antibody response in parasitized animals and can be used for immunodiagnostic purposes (Appleton et al. 1991). The unique and critical sugar of TSL-1 antigen is 3,6-dideoxyarabinohexose (tyvelose; Wisnewski et al.1993).

The 43-kDa glycoprotein

In this section, the 43-kDa glycoprotein will be reviewed in terms of its biochemical properties and possible functions.

The early authors Gold et al. (1990) isolated 43-kDa glycoprotein from E–S products of T. spiralis muscle larvae by biochemical methods and provided convincing characterization with respect to their biochemical and immunological properties. This was followed by molecular characterization by Su et al. (1991) who cloned and expressed the 43-kDa glycoprotein (they called the 49-kDa antigen) in Escherichia coli. The predicted 344 amino acid of the 43-kDa glycoprotein have an N-terminally located signal peptide and a potential helix–loop–helix motif (HLH) that resembles HLH domains critical in the function of muscle differentiation factors such as MyoD and myogenin in the main body of the protein (Vassilatis et al. 1992).

The 43-kDa glycoprotein may be responsible for capsule formation that occurs immediately after the entrance of muscle cells by the newborn larvae because the gene encoding a 43-kDa glycoprotein is expressed by either precapsule or postcapsule muscle larvae but not expressed by adult worms (Wu et al. 2002). And the diverse isoforms of the 43-kDa gene appear to be expressed by immature L1 (Mitreva et al. 2004).

T. spiralis and T. pseudospiralis are similar but different in terms of host response, including morphology of the capsule, immunological responses, and expression of genes and E–S proteins. Infection causes muscle cell degeneration, which is restricted around the worm in the case of T. spiralis infection, but the affected area spreads over the entire length of muscle cell in the case of T. pseudospiralis infection (Matsuo et al. 2000; Wu et al. 2001). These different pathological changes in muscle cell could be attributed to the differences of the E–S products released to the host cells. The homolog of the 43-kDa glycoprotein of T. spiralis exists in the E–S products of T. pseudospiralis (Vassilatis et al. 1996a). Comparison of the amino acids sequence of the T. pseudospiralis glycoprotein with the T. spiralis 43-kDa glycoprotein indicated that the two proteins are very similar (about 84% homology), but the molecular weight of the homologous glycoprotein from T. pseudospiralis was 38 kDa (Nagano et al. 2004).

The 43-kDa glycoprotein of T. spiralis is secreted in the host muscle cell and may influence and alter the host cells that may lead to the nurse cell formation. Therefore, the location of the 43-kDa protein in the host muscle cell is critical. Vassilatis et al. (1992) reported that the antibodies against the 43-kDa recombinant protein strongly reacted with the nurse cell nuclei and the nematode itself. However, Jasmer et al. (1994) reported that antibodies which specifically recognized 43-kDa glycoprotein failed to bind detectably with in situ and isolated host nuclei and nuclear extracts. In addition, immunodominant epitopes of specific 43-kDa glycoprotein of T. spiralis could not be detected in hypertrophic nuclei of injected muscles (Ko et al. 1994). The protein immunologically related to the 43-kDa glycoprotein but not to the 43-kDa glycoprotein itself was detected in the nuclei of mature nurse cells (Vassilatis et al. 1996b).

Recently, it has been reported that the 43-kDa glycoprotein may have deoxyribonuclease IIα (DNase IIα) activity. DNase IIα, which is an acidic endonuclease, was found in lysosomes and nuclei, and it is also secreted. Its Caenorhabditis elegans homolog is required for digesting DNA of apoptotic cell corpses and dietary DNA. The homologs of DNase IIα with differences at the purported active site histidine residue were detected in the 43-kDa glycoprotein of T. spiralis and T. pseudospiralis (MacLea et al. 2003). This study was expanded by Jasmer and Kwak (2006) who investigated the ability of a 43-kDa glycoprotein to interfere with mammalian skeletal muscle gene expression. They constructed a plasmid containing the gene of 43-kDa glycoprotein, and the gene was expressed as a recombinant protein in C2C12 myoblasts. They showed that the effects of transfection of the 43-kDa gene to the cell lines resulted from similarities of the 43-kDa glycoprotein to DNase IIα.

The 53-kDa glycoprotein

In this section, the 53-kDa glycoprotein will be reviewed in terms of its biochemical properties and possible functions.

Zarlenga and Gamble (1990, 1995) first identified genes encoding the 53-kDa glycoprotein of T. spiralis and elucidated the complete sequence and characteristic of the 53-kDa gene. The homolog of the T. spiralis 53-kDa glycoprotein is present in T. pseudospiralis E–S products (Wu et al. 1998), and the complete sequences and characteristics of the genes encoding the 53-kDa glycoprotein of T. pseudospiralis have been determined (Nagano et al. 2004). The amino acid sequence of the T. pseudospiralis 53-kDa glycoprotein shows a low homology (about 68%) to that of the T. spiralis 53-kDa glycoprotein.

The gene encoding the 53-kDa glycoprotein of T. spiralis is expressed by postcapsule larvae and adult worms but not by precapsule larvae and newborn larvae (Wu et al. 2002). And the 53-kDa glycoprotein is present in the β-granules but not in the α-granules (Romarís et al.2002a). Type I stichocyte granules, which are present within adult stichocytes, resemble β-granules of muscle larvae, and the two granules share antigenicity (Takahashi et al. 1992). Therefore, the 53-kDa glycoprotein, which is present within β-granules, could be expressed in adult worms. These data indicate that the 53-kDa glycoprotein is not responsible for muscle cell transformation nor capsule formation, but the protein plays some role in the continuation of parasitism and modulation of host immune response.

The immunogenicity of the 53-kDa glycoprotein using monoclonal antibodies (mAbs) was consecutively studied by Romarís and her colleagues. The native 53-kDa glycoprotein in larval E–S products of T. spiralis contains tyvelose-containing antigen and shows a marked heterogeneity in glycosylation (Romarís et al. 2002a). Some of the epitopes recognized by mAbs are differentially expressed in Trichinella spp. The epitope recognized by mAb US5 on the 53-kDa glycoprotein (another O-glycan/peptide epitope) is present only in T. spiralis, whereas those recognized by mAbs US8 and US9 (peptide epitopes) are present in encapsulated Trichinella species (Romarís et al. 2002a). The 53-kDa glycoproteins from the species Trichinella britovi, Trichinella murrelli, and genotype T8 have higher molecular weight (60 kDa) than the 53-kDa glycoprotein from T. spiralis, Trichinella nelsoni, and genotype T6 and from Trichinella nativa (55 kDa; Romarís et al. 2003). The variation in molecular weight of the 53-kDa glycoprotein is due to small differences in amino acid sequence, leading to change in the number of glycosylation sites. The 53-kDa glycoprotein from T. spiralis bears species-specific epitopes that induce antibody formation during infection. The antigenicity of the 53-kDa glycoprotein is mainly due to protein epitopes, and the antibody response against glycan epitopes is less important (Romarís et al. 2003). The epitope recognized by mAb US9 which is present in all encapsulated Trichinella species is a linear peptide of eight residues and located in the amino-terminal region, while the corresponding epitope recognized by mAb US5 which is present only in T. spiralis is a 47-amino-acid sequence containing two alpha-helix regions flanked by random coils (Perteguer et al. 2004).

The 45-kDa glycoprotein

A novel 45-kDa protein as well as the 43-kDa glycoprotein secreted from T. spiralis was biochemically isolated (Gold et al. 1990). Molecular analysis by Arasu et al. (1994) revealed that the secreted 45-kDa protein is a tyvelose-bearing glycoprotein and is present in the β- and γ-stichocytes of the secretory organs of muscle larvae. The gene encoding this protein belongs to a multicopy gene family present on a single DNA and encodes several larval proteins in the 40-50 kDa range.

The homolog of the 45-kDa protein of T. spiralis exists in the E–S products of T. pseudospiralis, and the 45-kDa protein is composed of two distinct subgroups, tyvelosylated and untyvelosylated (Robinson et al. 2007a). Bioinformatics analysis identified that the secreted 45-kDa protein is a family of trypsin-like serine proteases (Robinson et al. 2007a).

Minor Trichinella proteins with unrevealed function

In E–S products or not E–S products, there are many interesting proteins which have been partially characterized. These minor proteins will be briefly reviewed in the following section.

Nuclear antigens

The so-called nuclear antigens are parasite proteins (approximately 71, 79, 86, and 97 kDa) detected in host cell nuclei by means of immunostaining with antibodies against T. spiralis proteins (Jasmer et al. 1994; Yao and Jasmer 1998). These nuclear antigens can be depleted from the host nuclei of muscle cells by mebendazole treatment, which further strengthens the hypothesis that these nuclear antigens are secreted by parasites (Yao et al. 1998). Nuclear antigens may play a role in regulating the infected cell phenotype, but the functions of these nuclear antigens are unresolved.

Myogenic regulatory factor, MyoD-like protein

A gene encoding a T. spiralis helix–loop–helix protein with homology to the MyoD-like gene was cloned by Connolly et al. (1996). This gene is expressed constitutively during the muscle larval and adult stages. A purified recombinant MyoD-like protein binds to a high-affinity mouse MyoD:DNA-binding site in vitro.

TsJ5 protein

The tsJ5 gene was cloned from the muscle larvae of T. spiralis (Lindh et al. 1998). The TsJ5 protein is not a helix–loop–helix protein but represents a novel protein with properties in common with some myogenic repressors. The tsJ5 gene is expressed at higher levels in encapsulating species than in nonencapsulating species, and the tsJ5 recombinant protein affects the formation of MyoD:DNA complexes in vitro (Kuratli et al. 1999). The TsJ5 protein is found in E–S products, on the cuticular surface and in the body wall muscle of T. spiralis and T. pseudospiralis muscle larvae (Kuratli et al. 2001).

T. pseudospiralis-specific 15-kDa protein

Chung and Ko (1999) reported that the protein with a molecular weight of 15 kDa is present in E–S products of T. pseudospiralis. This protein exists only in the stichocytes of T. pseudospiralis and not in T. spiralis. However, the functions of this protein are unknown.

DNA-binding protein

A DNA-binding protein (approximately 30 kDa) is present in the E–S products of the infective-stage larvae of T. pseudospiralis (Mak and Ko 2001).

The 21- and 28-kDa proteins of T. pseudospiralis and T. spiralis

The proteins with a molecular weight of 21 or 28 kDa are present in E–S products of T. pseudospiralis and T. spiralis. Any related proteins with a significant homology have not been identified by database searches. The expression of these genes is restricted largely to the 30 days postinfection muscle larvae (Nagano et al. 2001a, 2002).

Newborn-larvae-specific protein

The glutamic acid-rich protein is transcribed specifically in the newborn larvae stage of T. spiralis and is present in the periphery of the developing stichocytes within newborn larvae (Zarlenga et al. 2002).

Nematode-specific cysteine–glycine domain proteins

Nematode-specific proteins of T. spiralis have a similarity to a predicted secreted or extracellular C. elegans protein. The region of similarity includes a conserved cysteine–glycine (CCG) domain, which is nematode specific. Two cysteine–glycine domains (Ts-CCG-1 and Ts-CCG-2) are present in the predicted T. spiralis protein, and particularly Ts-CCG-2 protein is present in E–S products (Gare et al. 2004). The Ts-CCG-1 gene is constitutively expressed, and Ts-CCG-2 gene expression is restricted to the muscle larvae.

The proteins with FYVE zinc-finger domain

The FYVE finger domain is a cysteine-rich zinc-finger-like motif that coordinates two zinc atoms. The function of this domain is to target signal-transducing proteins to cell membranes through binding to the membrane lipid phosphatidylinositol-3-phosphate with high specificity. A protein with zinc-finger domain in T. spiralis (approximately 55 kDa) is present in the crude extracts of muscle larvae, adult worms, and newborn larvae (Fu et al. 2005). Another protein with zinc-finger motifs is present only in the adult worms of T. spiralis, and this protein contains putative DNA-binding motifs (Hu et al. 2005).

Rcd1-like protein

Required cell differentiation 1 (Rcd1) was initially identified as a factor essential for the commitment to nitrogen starvation-invoked differentiation in fission yeast. The murine Rcd1 protein was identified as a cofactor of the c-myb proto-oncogene product, and the c-Myb and Rcd1 proteins physically interact with each other and that the c-myb-specific mim-1 promoter is downregulated by Rcd1 (Haas et al. 2004). The Rcd1-like gene was cloned from muscle larvae of T. pseudospiralis (Nagano et al. 2006). The Rcd1-like protein is mainly synthesized in the stichocytes, secreted into the host cell.

Trichinella proteins with defined function

The functions of some proteins of Trichinella spp. have been revealed. Such information is not only useful for cell biological understanding of host–parasite relationships but it also raises the possibility of application to therapeutic treatment. Defining protein function used to be difficult, but recent molecular techniques allow easier identification by homology searching between specimen genes and DNA sequences posted to the GeneBank database.

Proteinases

Among functional proteins, proteinase is one of most well investigated. Proteinases secreted by parasitic organisms may be involved in a wide variety of adaptive functions such as tissue penetration, larval migration, immunoevasion, retardation of blood coagulation, digestion, molting, and degradation of cellular matrix. These proteinases can also serve as immunodominant antigens, stimulating a protective immune response, or as potential targets for chemotherapy (Todorova and Stoyanov 2000).

Several authors have reported proteinases in the E–S products of the infective-stage larvae or adult worms of Trichinella spp. Serine proteinases and metalloproteinases in whole crude extracts and E–S products from T. spiralis muscle larvae were identified by Criado-Fornelio et al. (1992). The proteinases in crude extracts had molecular weights of 48, 54, and 62 kDa, and the proteinases in E–S products had molecular weights of 33, 62, and 230 kDa. de Armas-Serra et al. (1995) reported a class-undetermined proteinase from E–S products of T. spiralis muscle larvae, which was a single polypeptide with an estimated molecular weight of 35 kDa and an isoelectric point of 6.2.

Todorova et al. (1995) showed that the proteinases secreted from adult worms of T. spiralis degraded fibrinogen and plasminogen, and degradation was susceptible to the action of serine, cysteine, and aspartyl proteinase inhibitors. Serine proteinases in adult worms were present in E–S products, and the purified enzymes (approximately 18, 40, and 50 kDa) displayed enzymatic activity (Todorova and Stoyanov 2000).

A T. spiralis muscle larvae stage-specific antigenic serine proteinase was cloned and characterized as a secretory tyvelose-bearing glycoprotein using molecular techniques (Romarís et al. 2002b). Nagano et al. (2003) also identified the serine proteinase from T. spiralis muscle larvae and confirmed the proteinase activity of the recombinant protein. They showed that the serine proteinase gene is mainly expressed in muscle larvae, but the proteinase is not present in E–S products. Whereas, Robinson et al. (2005) showed by proteomic analysis that this serine proteinase is present in the E–S products. Recently, a putative serine proteinase composed of two proteinase domains was identified (Trap et al. 2006). This proteinase is expressed at various developmental stages of T. spiralis but not found in E–S products.

These works mentioned above mainly dealt with serine proteinase. A cysteine proteinase secreted from the muscle larvae of T. spiralis was reported by Moczon and Wranicz (1999), and a metalloproteinase in E–S product of T. spiralis was reported by Lun et al. (2003).

Proteinase inhibitors

Serine proteinase inhibitor (serpin) inactivates proteinases by forming complexes with serine proteinase. Possible functions of serpin have been postulated in a number of other systems including modulation and inhibition of host immune responses (Macen et al. 1993). A serpin gene of T. spiralis muscle larvae was cloned by Nagano et al. (2001b). The recombinant protein of the serpin inhibited 82% of the activity of the serine proteinase. The serpin gene was restricted largely to the newborn larvae and muscle larvae, and serpin is found within the stichocytes of muscle larvae in the early stage of infection.

Cystatins comprise a diverse group of cysteine protease inhibitors and important immunomodulatory factors when secreted by parasitic nematodes. A novel cystatin-like protein (46 kDa) secreted from T. spiralis is identified as a new member of the noninhibitory cystatin-related proteins by Robinson et al. (2007b). Cystatin-like protein gene expression is largely restricted to intracellular stages, predominantly in the adult worms. Interestingly, this protein is not present in the E–S products of T. pseudospiralis. This may explain why inflammatory response in T. pseudospiralis infections is less than in T. spiralis infection because the cystatin-like protein of T. spiralis acts as a proinflammatory factor. Sugane and Matsuura (1990) reported DNA sequences of a 46-kDa protein from muscle larvae of T. spiralis without referring its function. But this 46-kDa protein seems to be the cystatin-like protein because the sequences of this protein are completely identical to that of the cystatin-like protein published by Robinson et al. (2007b).

Heat shock proteins

Heat shock proteins (Hsps) may protect parasites against stress or injury and may play an important role in tissue invasion and intracellular survival. They have been documented in a large variety of species.

The Hsps in Trichinella spp. were documented for the first time in both crude extracts and E–S products of the heat-shocked infective-stage larvae using SDS-PAGE (Ko and Fan 1996). The major Hsps in crude extracts of T. spiralis are 20, 47, 50, 70, 80, and 86 kDa, and the major Hsps in the E–S products are 11, 45, 53, and 64 kDa. In T. pseudospiralis, the major Hsps in the crude extracts are 20, 26, 31, 50, 53, 70, 80, and 86 kDa, and in the E–S products 11, 35, 37, 41, and 64 kDa. The 70-kDa Hsp of T. spiralis was detected by Western blotting using mAbs (Martinez et al. 2000). The 70-kDa Hsp of T. britovi was detected in the nuclei of the muscle larvae but not in adult worm nuclei (Vayssier et al. 1999).

Martinez et al. (1999) compared three stimuli, elevated temperature, hydrogen peroxide, and mebendazole for their ability to induce heat shock responses in T. spiralis muscle larvae and observed that the exposure to hydrogen peroxide resulted in the induction of constitutive and higher Hsps. The expression levels of the 50-kDa Hsp from infective-stage larvae of T. spiralis immediately and persistently increased after oxidative and cold shock (Martinez et al. 2002). They investigated the relationship between infectivity and the expression levels of Hsp 70 and Hsp 60. Oxidative stress caused a significant increase in Hsp levels and total loss of infectivity, but cold oxidative stress caused no alterations in either Hsp levels or infectivity (Martinez and Rodriguez-Caabeiro 2005).

Three heat-induced genes in T. spiralis were identified using the suppression subtractive hybridization technique (Mak et al. 2001). These genes are homologous to histone H3 gene, histone H2B gene, and translationally controlled tumor protein (TCTP) gene. The TCTP is a stress- and growth-related protein with antiapoptotic and immunomodulatory activities. After heat shock treatment, the expression levels of histone H3, histone H2B, and TCTP increased, but the RNA level of TCTP did not (Mak et al. 2007). The expression of TCTP may be upregulated at the translational level rather than at transcriptional level during early stage of stress adaptation.

Hsp 60 is a family of ubiquitous molecular chaperonins that regulate posttranslational folding, assembly, and the targeting of proteins. Some chaperonins are known to be expressed as a response to stress whereas others are constitutively expressed. However, the thermal, cold, acidic, and oxidative treatment did not elicit significant changes in the expression of mitochondrial Hsp 60 of the T. spiralis muscle larvae (Wong et al. 2002). A small heat shock gene of T. spiralis, which had a high sequence identity in alpha crystallin domain, was cloned by Wu et al. (2007b). This small heat shock protein is expressed at various developmental stages of T. spiralis and possesses chaperone activity to suppress the thermally induced aggregation of citrate synthase.

Glycosidases

In extracts of T. spiralis adult worms and muscle larvae, the exoglycosidases, β-N-acetyl-d-glucosaminidase, β-N-acetyl-d-galactosaminidase, alpha-1-fucosidase, alpha-d-glucosidase, and alpha-d-mannosidase, are present at high levels (Rhoads 1985). These glycosidases are detected also in culture fluids of both muscle larvae and adult worms.

The β-N-acetyl-d-hexosaminidase has been detected in the E–S products of various parasitic organisms. Its functional roles have been postulated to be linked to various events including host cell invasion, modification and/or remodeling of cell surfaces, or carbohydrate breakdown for nutritional purposes. The β-N-acetyl-d-hexosaminidase is present in the muscle larvae of T. spiralis, and the enzyme is a glycoprotein with an estimated molecular weight of 100 kDa and is localized on cell membranes and the epicuticle (Rhoads 1988). The secreted glycosidase from T. spiralis with significant activity is only an exo-β-hexosaminidase (Bruce and Gounaris 2006). This enzyme is glycosylated with an apparent molecular weight of 50 kDa, and the glycan is decorated with tyvelose.

Protein kinases

Arden et al. (1997) showed that serine–threonine protein kinases activity is present in E–S products of T. spiralis infective-stage larvae and identified two serine–threonine protein kinases of 70 and 135 kDa in E–S products using autophosphorylation. The major phosphorylated proteins (50- and 55-kDa proteins) contain mainly phosphoserine and appear to represent differentially glycosylated variants of a 35-kDa polypeptide.

Endonucleases

Double-stranded endonuclease activity is present in the E–S products of the infective-stage larvae of T. spiralis and is present in much smaller amounts in the E–S products of T. pseudospiralis. The endonuclease activity is associated with at least three molecular forms, designated approximately as 25, 30, and 58 kDa, respectively (Mak and Ko 1999). Unlike the double-stranded endonuclease, the single-stranded molecule is divalent cation independent and is present in both T. spiralis and T. pseudospiralis E–S products (Mak et al. 2000). The single- and double-stranded endonucleases are likely to be encoded by different proteins and may have different functions.

Thymidylate synthase

Thymidylate-synthase-specific activity is present at a high and constant level in crude extracts from muscle larvae and adult worms of T. spiralis and T. pseudospiralis (Rode et al. 2000). Dabrowska et al. (2004) cloned thymidylate synthase gene from the cDNA of T. spiralis and confirmed enzymatic activity of recombinant thymidylate synthase. The expression level of thymidylate synthase was similar in muscle larvae, adult worms, and newborn larvae.

Macrophage migration inhibitory factor

Migration inhibitory factor (MIF) was first identified as a soluble protein secreted by sensitized lymphocytes, which inhibited the migration of macrophages. Recently, it has been discovered that MIF not only plays a critical role in inflammation but also has endocrine and enzymatic function. Parasites usually possess the ability to escape from host immune attack. Some parasites secrete a homolog of host MIF that has the capability of modifying the activity of human monocytes–macrophages.

The MIF of T. spiralis was detected in E–S products of muscle larvae (Tan et al. 2001). The MIF recombinant protein inhibited migration of human peripheral blood mononuclear cells but had no effect on anti-CD3-stimulated murine T cell proliferation. The homolog of MIF from T. spiralis is present in T. pseudospiralis E–S products (Wu et al. 2003). The MIF gene is expressed in various developmental stages, including in adult worms, newborn larvae, and muscle larvae. The MIF exists in the muscle cells of the body wall and some stichocytes of larvae. The MIF recombinant protein profoundly inhibited the macrophage accumulation around the Sephadex beads transplanted in mice.

Nucleotide-metabolizing enzymes

Tissue damage results in a variety of molecular signals that activate elements of the immune system. Recently, it has been shown that the key regulators of these events are extracellular nucleotides, which signal through purinergic receptors (Gounaris and Selkirk 2005).

Nucleoside diphosphate kinases (NDPKs) play a key role in the maintenance of intracellular pools of deoxynucleoside triphosphates and nucleoside triphosphates (NTPs) via the transfer of phosphate from an NTP donor to an NDP acceptor. T. spiralis secretes an NDPK with molecular weight of 17 kDa, and a possible function of NDPK might lie in the regulation of host cell proliferation and differentiation (Gounaris et al. 2001).

Exoenzymes: apyrase, 5'-nucleotidase, and adenosine deaminase are secreted by T. spiralis (Gounaris 2002). These proteins constitute an enzymatic cascade which catalyzes the degradation of extracellular nucleotides, with a potential physiological role in the regulation of purinergic signaling.

A nucleotidase secreted from T. spiralis catalyzes the hydrolysis of nucleoside 5'-diphosphates and 5'-monophosphates but not 5'-triphosphates (Gounaris et al. 2004). The sequence of the nucleotidase is homologous to 5'-nucleotidases from a wide variety of organisms but contains no sequences specifically conserved in apyrases, suggesting that it is a representative of a new class of secreted nucleotidase.

Prosaposin and GM2 activator protein

The lysosomal degradation of glycosphingolipids with small carbohydrate chains requires the presence of nonenzymatic cofactor like prosaposin and ganglioside GM2 activator protein (GM2AP).

Infective-stage larvae and adult worms of T. spiralis secrete a protein homologous to prosaposin (Selkirk et al. 2004). The protein is secreted in an unprocessed form with an estimated molecular weight of 56 kDa and contains the TSL-1 antigens. The protein is localized to membrane-bound vesicles and more complex multilamellar organelles in diverse tissues including the hypodermis, intestine, and stichocytes. The prosaposin facilitates invasion of intestinal epithelial cells and subsequent migration through this cell layer. GM2AP secreted from T. spiralis was reported by Bruce et al. (2006). This protein does not facilitate degradation of GM2 ganglioside by N-acetyl-β-hexosaminidase A because of the absence of a domain implicated in binding to hexosaminidase.

Enolase

Enolase is an enzyme which catalyzes 2-phospho-d-glyceric acid (2PGA) to phosphoenolpyruvate (PEP) in the glycolytic pathway where ATP is synthesized in the process of metabolizing glucose to pyruvic acid. Enolase of parasites enhances the activation of plasminogen, and plasminogen mediated by enolase contributes to tissue migration of the parasites (Bernal et al. 2004).

The enolase from T. spiralis muscle larvae was identified by Nakada et al. (2005). The recombinant full-length enolase of T. spiralis had no activity in the conversion of 2PGA to PEP but gained enolase activity after cutting off the signal peptide from the full-length protein. The enolase is in crude extracts of muscle larvae but is not present in the E–S products.

Superoxide dismutases

Superoxide dismutase (SOD) is an important antioxidant and catalyzes the conversion of superoxide anion into hydrogen peroxide and molecular oxygen. It exists commonly in most organisms to protect against endogenous oxidative stress.

Copper–zinc SOD activity is present in both crude extracts and E–S products of T. pseudospiralis, and the expression of cytosolic SOD is substantially higher in infective-stage larvae than in adult worms (Wu et al. 2006). The manganese SOD of T. pseudospiralis was reported by Wu et al. (2007a). This manganese SOD is a mitochondrial enzyme because of the presence of mitochondrial transit peptides and maturation cleavage site in this protein. The expression level of manganese SOD is lower than that of copper–zinc SOD in infective-stage larvae. The manganese SOD is highly expressed in the infective-stage larvae but not in adult worms.

Caveolin

Caveolins are integral membrane proteins which play a role in cholesterol homeostasis and transport, endocytosis mechanisms, and regulation of signal transduction in differentiated cells. The caveolin of T. spiralis is an adult stage-specific protein and gradually accumulates on the surface of oocytes and embryos, suggesting that the caveolin of T. spiralis plays some role in oocyte maturation and embryogenesis during development (Hernandez-Bello et al. 2007).

Prolactin

The prolactin-like hormone (23 kDa) of T. spiralis was identified in E–S products and the stichocytes within the muscle larvae (Quintanar et al. 2007).