Immunolocalization and Evaluation of Thioredoxin Glutathione Reductase Role in Diagnosis of Human Schistosomiasis

CONTENTES

I. INTRUDUCTION AND AIM OF THE WORK

II. REVIEW OF LETRATURE
1. Schistosomiasis
2. Antioxidant
3. Thioredoxin Glutathione Reductase (TGR)
4. Diagnosis of Schistosomiasis
5. Antibodies
6. Immunolocalization

III. MATERIALS AND METHODS

IV. RESULTS

V. DISCUSSION

VI. SUMMARY

VII. REFERENCES

VIII. ARABIC SUMMARY

INTRUDUCTION AND AIM OF THE WORK

Schistosomiasis is still considered the most important public health problem in tropical and sub-tropical countries; it was considered the second major parasitic disease in the world after malaria. It affects at least 200 million people, while 800 million are being exposed to the risk of infection. It was also estimated that 20 million individuals suffer from severe consequences of this chronic and debilitating disease responsible for at least 500.000 deaths per year (Tan et al., 2007; Simeonov et al., 2008; Angelucci et al., 2009 ; Farias et al., 2010; Collins et al., 2011).

Despite the availability of an effective drug and the implementation of successful control programs, the number of infected cases has not decreased during the last decades (Corstjens‎‎ et al., 2008 ).

The Schistosoma (S.) parasites can survive for up to decades in the human host due in part to a unique set of antioxidant enzymes that continuously degrade the reactive oxygen species produced by the host's innate immune response. Two principal components of this defense system have been recently identified in S. mansoni as thioredoxin/glutathione reductase (TGR) and peroxiredoxin (Prx) and as such these enzymes present attractive new targets for anti-schistosomiasis drug development (Simeonov et al., 2008).

Thioredoxin reductase (TrxR) is an enzyme belonging to the flavoprotein family of pyridine nucleotide-disulfide oxidoreductases (Maggioli et al., 2004). It was recently discovered that in S. mansoni, specialized TrxR and Glutathione reductase (GR) enzymes are absent, and instead are replaced by a unique multifunctional enzyme, TGR (Alger and Williams, 2002; Rai et al., 2009).

The researchers chose this enzyme because adult worms need to make antioxidants (chemicals that prevent oxygen from damaging cells) to protect themselves against the human immune response. Antioxidant production in these worms depends on TGR; in mammalian cells, two specialized enzymes do its job. The researchers reasoned, therefore, that TGR might be an essential parasite protein and a potentially important drug target.

The ribonucleic acid (RNA) silencing experiment shows that TGR is essential for parasite survival, and the biochemical analysis indicate that TGR and its mammalian counterparts have different substrate specificities. Thus, it should be possible to find compounds that inhibit TGR but have much less effect on the mammalian enzymes (Kuntz et al., 2007).

Such improved understanding of the organisms responsible for neglected tropical diseases (NTDs) presents opportunities for new drug development. However, private-sector biopharmaceutical interest in NTDs has traditionally been limited due to high risk and low expected return-on-investment of these projects, though this is beginning to change with the advent of increased philanthropic and public private government partnership funding (Hopkins et al. , 2007). A significant problem that remains, however, is the significant gap in technologies, expertise, and cultures between academic and biopharmaceutical organizations.

The main burden of disease occurs in sub-Saharan Africa, where individuals are continuously exposed to new infections while in contact with cercaria-contaminated fresh water (Corstjens‎‎ et al., 2008; Ward et al., 2011 ).

The need to control schistosomiasis is acute and efforts have been ongoing for years on three main fronts: prevention (via establishment and maintenance of sources of safe potable water), development of a vaccine, and use of drugs to treat the infection (WHO, 2006). This necessitates rapid and accurate diagnosis.

Correct diagnosis is fundamental for controlling schistosomiasis, including case detection and resulting community treatment, assessment of morbidity and the evaluation of control strategies (Peng et al., 2008).

Diagnosis of the infection is classically based on the detection of parasite eggs in urine or in feces. However, this method has several disadvantages. The number of excreted eggs is often low and shows a high day-to-day fluctuation. Therefore, stool or urine examination needs to be repeated several times. Alternatively, detection of antibodies is a highly sensitive and specific method to diagnose schistosomiasis. High antibody responses are generally seen with travelers originating from areas where schistosomiasis is not endemic. However, in immigrant travelers with a life-long history of exposure, antibody responses are mostly moderate to low. Some may even become serologically negative, while still excreting viable eggs. In addition, antibody levels are not associated with the actual worm burden and remain unaffected by treatment of the infection. Consequently, serology mostly gives straightforward answers for patients tested within months after their first exposure, but data are difficult to interpret for those who have a history of previous infection. A sensitive, serum- or urine-based test demonstrating active Schistosoma infection would be valuable in these cases (Corstjens‎‎ et al., 2008 ; Stothard et al., 2011).

Assays for the detection of Schistosoma circulating antigens (adult worm gut-associated antigens (GAA)) seem very promising, as serum levels of circulating anodic antigen (CAA) are related to actual worm burden and rapidly decrease following drug treatment (Van Lieshout et al., 2000). The current monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) determines serum CAA levels for all human Schistosoma species with virtually 100% specificity (Deelder et al., 1989); its value in epidemiological studies dealing with populations with moderate- to high-intensity infections has been demonstrated previously (Polman et al., 2002). However, the CAA-ELISA still lacks sensitivity when testing light infections, e.g., in the group of international travelers (van Lieshout et al., 1997). Moreover, due to the relatively high complexity of the test, it lacks robustness if performed only occasionally for single case identification. This may hamper implementation of the CAA-ELISA within clinical routine diagnostic settings (Corstjens‎‎ et al., 2008 ).

Antibody detection assays though very sensitive particularly in individuals from endemic areas (Van Lieshout et al., 2000), do not, however, differentiate between active and past infection and do not correlate with intensity of infection (Mott and Dixon, 1982).

On the other hand, detection of circulating antigens has been shown to be a sensitive and specific alternative to parasitology and antibody-detection methods in diagnosis of active parasitic infection (De Jong et al., 1988; Fu and Carter, 1990; Van Lieshout et al., 1995). The use of monoclonal antibodies (mAbs) in circulating schistosomal antigen assays has greatly improved their specificity and sensitivity (De Jonge et al., 1988; Van Lieshout et al., 1995). It is well established that the sensitivity and specificity of immunoassays are critically dependent on specificity and purity of antigen (Van Lieshout et al., 1995).

Aim of the work

This study aims to purify the TGR secreted from the S. mansoni, preparation of anti-TGR polyclonal antibodies, study the localization pattern of TGR in different stages of the parasite, and different organs of S. mansoni infected mice and finally to evaluate the diagnostic potential of purified TGR for the diagnosis of human S. mansoni.

II REVIEW OF LETRATURE

Schistosomiasis is a chronic and debilitating disease that is caused by parasitic trematode worms (schistosomes). It continues to threaten millions of people, particularly rural areas in the developing world (Chitsulo et al ., 2000; Engels et al ., 2002) . Of the estimated 200 million infected people, more than half have symptoms only, while 20 million exhibit severe disease manifestations and 200,000 deaths each year with a further 700 million people at risk of infection (Rutitzky et al., 2005; Lea et al., 2008; Milligan and Jolly, 2011; Wilson et al., 2011 ).

In the 21st century, the control of parasitic infections, which are a major cause of disability, mortality and economic losses in many developing countries, remains as one of the most important challenges for medicine ( WHO, 2006 ). Even though an effective treatment exists, it does not prevent re-infection, and the development of an effective vaccine still remains the most desirable means of control for this disease (Rofatto et al., 2009; Farias et al., 2010). Among the general population in the delta of Egypt, the prevalence of S. mansoni declined from 14.8% in 1993 to 2.7% in 2002 and continued to decline thereafter, reaching 1.5% in 2006. Similarly, the prevalence of S. haematobium declined from 6.6% in 1993 to 1.9% in 2002, then to 1.2% in 2006 (Salem‎‎ et al., 2011).

A large number of schistosomes are known; however, only five appear to be primarily responsible for human infections. These include S. mansoni, S. japonicum, S. intercalatum, S. mekongi and S. haematobium (Ross et al., 2002). Infection with the former 4 species is associated with chronic hepatic and intestinal fibrosis, while infection with S. haematobium can lead to ureteric and bladder fibrosis and calcification of the urinary tract (Utzinger et al., 2001; Ross et al., 2002; Wichmann et al., 2006). S. mansoni is a major causative agent of schistosomiasis, which constitutes a severe health problem in developing countries (Levano et al., 2007).

All these species require specific intermediate host snails to complete their life cycle. The geographical distribution of schistosomiasis is dependent on the distribution of the intermediate snail host. Schistosomiasis in Egypt is either caused by the helminthes S. haematobium or S. mansoni (Talaat et al., 1999; Salem‎‎ et al., 2011). The Biomphalaria species are freshwater snails which have a wide distribution and are significant both medically and economically as intermediate hosts for the Schistosoma parasite (El-Ansary et al., 2006).

Schistosome parasites have a complex lifecycle involving snail intermediate and human definitive hosts. Humans become infected when contacting water containing cercariae released by infected snails. After penetration, cercariae remain in the skin for several days, then enter the general circulation and are carried to the lungs, where they reside for several further days before finally entering the liver. Once in the liver, parasites undergo rapid growth, development and sexual differentiation and locate a mate. After pairing, adult parasites migrate to the mesenteric venules (S. mansoni and S. japonicum) or the venules of the urogenital system (S. haematobium) of their human host where they commence egg production (McManus and Loukas, 2008; Sayed et al., 2008).

Humans are infected when they enter or come in contact with Schistosoma‎ -infected water. Schistosomiasis is primarily a disease due to extreme poverty-people get infected because they do not have access to save water supplies and proper sanitation. The disease is maintained under these conditions because infected people release Schistosoma‎ eggs in their excreta. After reaching water, the eggs hatch into larvae that infect aquatic snails, where they develop further until they release a free-swimming immature infective parasite stage (cercariae) (Bergquist et al., 2005).

Infection is characterized by the presence of adult worms within the vasculature of their hosts, where they can reside for many years. The worms are covered by an unusual dual lipid bilayer through which they import nutrients. How the parasites import other vital molecules, such as water, is not known (Faghiri and Skelly, 2009).

The initiation of infection of the human host by Schistosoma parasites involves penetration of skin by a multicellular larva (0.1 mm) called a cercariae. Cercariae have forked tails that propel them through fresh water. Depending upon their specific vertebrate host, cercariae can respond to a variety of stimuli, including motion, light and shadow, chemical gradients, and heat. Upon contact with human skin, cercariae are stimulated by the lipid on the surface of skin to begin penetration. Initial penetration involves mechanical entry into the superficial, cornified layer of skin, which presents little barrier in the aquatic environment. However, further entry requires degradation of intercellular bridges between epidermal cells, the dermal/epidermal basement membrane, and the extracellular matrix of the dermis. Ultimately, the larvae, which have now shed their tails and are called schistosomula, enter small vessels in the superficial dermis, where they complete their lifecycle ( Knudsen et al., 2005 ).

Proteins secreted by cercariae play key roles in facilitating skin invasion and evading the immune response of the host. Microscopic and biochemical analyses have identified three potential sources of proteins released by cercariae. First, a carbohydrate-rich surface glycocalyx is released upon entry. This glycocalyx has protected the organism from osmotic shock in fresh water, but is a potent activator of complement, and must be jettisoned prior to entry into the bloodstream (Fishelson et al., 1992; Skelly and Shoemaker, 2000). Second, proteolytic secretions are produced by the set of two groups of acetabular “glands” which are in fact clusters of cells with cytoplasmic processes extending into the anterior end of the organism. Acetabular cells release their contents beginning at the earliest stages of skin invasion and, at least in human skin, well into the superficial dermis. The escape glands, a third potential source of secretions, appear to release their contents late in invasion as the organisms enter dermal vessels (Dorsey et al., 2002).

A very interesting study of Fishelson et al. (1992) elucidated how the parasitic blood fluke S. mansoni synthesizes, stores, and releases a serine proteinase during differentiation of its invasive larvae. In situ hybridization with a cDNA probe allowed to localize the proteinase mRNA in acetabular cells, the first morphologically distinguishable parasite cells that differentiate from embryonic cell masses present in the intermediate host snail. Antiproteinase antibody binding showed that the proteinase progressively accumulated in these cells and was packaged in vesicles of three morphologic types. Extension of cytoplasmic processes containing proteinase vesicles formed “ducts” which reached the anterior end of fully differentiated larvae. During invasion of human skin, groups of intact vesicles were released through acetabular cytoplasmic processes and ruptured within the host tissue. Ruptured proteinase vesicles were noted adjacent to degraded epidermal cells and dermal-epidermal basement membrane, as well as along the surface of the penetrating larvae themselves. These observations are consistent with the proposed dual role for the enzyme in facilitating invasion of host skin by larvae and helping to release the larval surface glycocalyx during metamorphosis to the next stage of the parasite (Dzik, 2006).

Cercariae invade the mammalian host by penetrating the skin, and they are stimulated to do so by the secretion of certain fatty acids and their derivatives by the mammalian skin (Stirewalt et al., 1983; Milligan and Jolly, 2011 ). During penetration, the cercariae lose its tail and transform into schistosomula. Migration of schistosomula from the skin to the hepatic portal system is entirely intravascular taking between 8 and 20 days to be completed (Miller and Wilson, 1980). Schistosomula of S. mansoni and S. haematobium have similar migratory patterns through human skin (He Y et al., 2002). After entering the vascular system, schistosomula migrates through the right part of the heart to the lungs, where it changes shape into much longer, slender organism during the next 72 hours (hr) (Sturrock, 1993). From the lung, schistosomula are carried to the left part of the heart and the systemic circulation to the splanchnic vasculature of the hepatic portal system, eventually reaching the sinuses of the liver, where worms remain for a period of 3 weeks (wk) of development then transform into adult worms (Combes, 2001; Milligan and Jolly, 2011 ).

The adult worms lodge in blood vessels of the intestinal or urinary system. After mating, female worms produce eggs that are deposited in the liver, bladder or other tissues, depending on the infecting species and released in excreta to complete the cycle (Bergquist et al., 2005). After adult worms reach full fecundity, schistosome eggs can be found in stool around 6 wks after cercarial exposure and it is commonly held that females of S. mansoni produce up to 100–300 eggs per day (Stothard et al., 2011). Not all Schistosoma eggs are excreted from the body, and up to 50% can embolize to other body areas, leading to host immune reactions and granuloma formation, which progresses to irreversible fibrosis and severe portal hypertension (Capron and Dessaint, 1992; Ward et al., 2011).

Parasites are designed by evolution to invade the host and survive in its organism until they are ready to reproduce. Parasites release a variety of molecules that help them to penetrate the defensive barriers and avoid the immune attack of the host. Secretion of antioxidant enzymes is believed to protect the parasite from reactive oxygen species which arise from the infection-stimulated host phagocytes. Aside from superoxide dismutase, catalase (rarely found in helminthes), and glutathione peroxidase (selenium-independent, thus having a poor activity with H2O2), Prx are probably the major H2O2-detoxifying enzymes in helminthes. Secretion of antioxidant enzymes is stage-specific and there are examples of regulation of their expression by the concentration of reactive oxygen species surrounding the parasite. The majority of parasite-secreted molecules are commonly found in free-living organisms, thus parasites have only adapted them to use in their way of life (Dzik, 2006).

Pathogenesis of hepatic schistosomiasis:

The pathology of schistosomiasis consists, essentially, of a series of chronic inflammatory lesions, produced in and around blood vessels by eggs or their products, and sometimes by dead adult worms (Abdel-Hadi and Talaat 2000). If the ova continue to be deposited in sufficient numbers and over several years, they will ultimately lead to a progressive fibrosis of the portal tracts and urinary bladder, in addition to the occurrence of hepatic, intestinal, genitourinary, neural and pulmonary lesions (Olds and Dasarathy, 2000). The periportal fibrosis is the major pathological consequence of S. mansoni infection (Talaat et al., 2007)

Acute schistosomiasis, or Katayama fever, is a serum sickness-like syndrome that occurs 3 to 9 wk after infection. This period coincides with the onset of egg production. Among people living in endemic areas, the acute phase may pass undiagnosed. It has been suggested that in endemic conditions where exposure to infection occurs early in life, symptoms would be inconspicuous or ill-defined, and infection is not suspected (Jordan et al., 1993).

This acute stage also coincides with the migratory stage of the maturing schistosomula in the lungs and liver, maturation of male and female worms, or early oviposition in mesenteric veins (Boros, 1989). Antigens are released in large amounts during the migration, maturation, or death of schistosomula which trigger serum complement, immediate-type and immune complex-mediated hypersensitivity reactions (Boros, 1989). Acute schistosomiasis, or Katayama syndrome, can present as fever, malaise, myalgia, fatigue, non-productive cough, diarrhea (with or without blood), haematuria (S. haematobium), and right upper quadrant pain (Gray et al ., 2011).

Chronic schistosomiasis is the stage of infection usually observed in endemic areas but most individuals are asymptomatic. S. mansoni eggs are present in the stool and the level of egg excretion is relatively stable (Lambertucci et al., 1983). Asymptomatic infection may become symptomatic after recurrent exposure and reinfection or with continuous long term egg deposition without treatment. Records of the life span of S. mansoni in humans range from 3 to 30 years (Arnon, 1990).

Chronic and advanced disease results from the host’s immune response to Schistosoma eggs deposited in tissues and the granulomatous reaction evoked by the antigens they secrete (Gray et al., 2011). Most persons infected with schistosomes do not suffer from severe hepatosplenic disease (caused by S. mansoni and S. japonicum) or bladder calcification and hydronephrosis (caused by S. haematobium), but from less dramatic morbidities such as anemia, fatigue, malnutrition, or impaired cognitive development (Secor, 2005).

The chronic liver is usually little shrunken in size, but it may not be reduced in weight. Adhesions of the liver to the under surface of the diaphragm may be present (Elwi, 1976). The left lobe of the chronic liver is often affected than the right (Prata, 1982).

Histologically, the liver has a fibroelastic consistency. The cut surface is reddish or brownish in color. The most striking feature is the thick fibrous scar-like and cord like appearance of the portal tracts which may measure few millimeters to one centimeter or more in thickness. The thickened fibrous portal tracts may have a round, triangular, or ellipsoidal shape depending on the plane of the section. Few short fibrous septa may proceed from the thickened portal tracts into the surrounding parenchymatous liver tissue, but the latter is not divided into nodules surrounded by fibrous tissue as in genuine form of cirrhosis; fibrous septa extend from Glisson’s capsule inward giving the liver typical nodular appearance in this subcapsular area of the liver as those of cirrhosis. The lumens of the portal veins in small and medium sized portal tracts are indistinguishable, but those in the large portal tracts may be dilated. The main trunk of the portal vein may show some fibrous thickening or thrombosis (Prata, 1982; Grimaud and Borojevic, 1986).

Also, the portal tracts in chronic schistosomiasis are of all orders, small, medium, and large are evidenced by fibrous tissue which may contain Schistosoma eggs and granulomas in all stages of development. A variable amount of mononuclear cell infiltration may be seen in the portal tracts and their periportal extensions, sometimes with no relation to Schistosoma eggs (Grimaud and Borojevic, 1977). The liver parenchymal cells are usually not affected and there is no evidence of regeneration, but there may be focal areas of necrosis, probably produced by intrahepatic thrombi or ischaemia after massive gastrointestinal haemorrhage (Prata, 1982).

A high prevalence of hepatomegaly was recorded concurrent with S. haematobium infection (Nafeh et al., 1992). In addition, splenomegaly is common in schistosomiasis due to lymphocytic proliferations and increased portal pressure (Olds and Dasarathy, 2000).

Antioxidants:

1- Role of the antioxidant enzymes in survival of schistosomes:

S. mansoni, a causative agent of schistosomiasis, resides in the hepatic portal circulation of their human host up to 30 years without being eliminated by the host immune attack. Production of an antioxidant "firewall," which would neutralize the oxidative assault generated by host immune defenses, is one proposed survival mechanism of the parasite (Sayed et al., 2006; Rai et al., 2009).

Living in an aerobic environment, worms must have effective mechanisms to maintain cellular redox balance. Additionally, worms must be able to evade reactive oxygen species generated by the host's immune response (Kuntz et al., 2007).

There are two paradigms that exist in Schistosoma‎ immunology. The first is that the schistosomule stages are the most susceptible to immune killing and the second is that the adult stage, through evolution of defense mechanisms, can survive in the hostile host environment. One mechanism that seems to aid the adult worm in evading immune killing is the expression of antioxidant enzymes to neutralize the effects of reactive oxygen and nitrogen species. Investigators have shown the effectiveness of cells that release reactive oxygen species such as monocytes, macrophages, eosinophil’s, and platelets against schistosomula stages of S. mansoni in an antibody-dependent manner (Cook‎‎ et al., 2004).

In vitro assays have shown the effectiveness of antibody-dependent cell cytotoxicity and reactive oxygen and nitrogen species in killing schistosomule stages of the parasite, with a minimal effect on adult worms (LoVerde, 1998).

In vitro cytotoxicity assays as well as passive transfer experiments have demonstrated the importance of these cells in association with immunoglobulin (Ig) E and certain isotypes of IgG in rats, primates, and humans on the larval stages (Capron, 1987; Butterworth et al., 1992). A common defense mechanism against immune attack is the expression of antioxidant enzymes (Mei and LoVerde, 1997; LoVerde, 1998). In general, these enzymes work to protect an organism from oxidative damage caused by the reactive oxygen species and other molecules associated with host toxic responses.

2- Level of antioxidant enzymes and parasite survival:

Effective protection of an invading parasite from host-produced reactive oxygen species (ROS) would depend on levels of scavenger enzymes in the invasive forms of the parasite. Studies on Nippostrongylus brasiliensis infection showed that the upregulation of superoxide dismutase, catalase, and glutathione peroxidase is correlated with persistence in the host. On the other hand, increased ROS production by peritoneal leukocytes has been correlated with the rejection of N. brasiliensis (Smith and Bryant, 1989).

Newly excysted juvenile (NEJ) flukes of Fasciola (F.) hepatica are relatively resistant to killing by free radicals in comparison to schistosomula of S. mansoni. This resistance could, in part, be due to the significant activity of oxidant-scavenging enzymes of NEJ flukes (Piedrafita et al., 2000).

3- Stage-specific expression of antioxidant enzymes and parasite survival:

Oxidant-resistant adult worms and muscle larvae had several times more of glutathione peroxidase (GPX) and about four times more of superoxide dismutase (SOD) than the oxidant-sensitive newborn larvae. Newborn larvae were partially protected against oxidant damage when mixed with adult worms. Similarly, NEJ flukes of F. hepatica expressed 2.5−20 fold lower levels of superoxide dismutase and glutathione S-transferase (GST) activity relative to immature or adult parasites. Incubation of NEJ flukes with inhibitors of peroxidases and inhibitors of glutathione metabolism increased their killing by LPS-stimulated rat phagocytes (Piedrafita et al., 2000). Studies of the developmental regulation of localization of GPX and superoxide dismutase in the trematode S. mansoni (Mei and LoVerde, 1997) showed that these enzymes were found to be associated only with the adult tegument and gut epithelium.

LoVerde (1998) hypothesized that, adult worms protect themselves against oxidant damage by producing antioxidant enzymes. To begin to test this hypothesis, he reasoned that the antioxidant enzymes should be developmentally regulated. He went on to demonstrate that expression of the Schistosoma‎ antioxidant enzymes (Cu-Zn SOD; GPX) is developmentally regulated such that the lowest levels of gene expression (as measured by transcription) and enzyme specific activity were in the larval stages, the most susceptible to immune killing, and highest in adult worms, the least susceptible to immune elimination (Maizels et al., 1993; Mei and LoVerde 1997; LoVerde, 1998).

In vitro studies have demonstrated that schistosomula are sensitive (95% killed) to oxidative killing, whereas adult worms exhibit much greater resistance (2% killed) to oxidative killing (Mkoji et al., 1988a).

4- ROS-induced changes in expression of helminth scavenger enzymes:

Zelck and Von Janowsky (2004) had shown that, stage-dependent expression of SOD, GPX, and GST in S. mansoni is regulated at the transcriptional level. Generation of ROS by xanthine/xanthine oxidase resulted in increased transcript levels for all three enzymes. They compared influence of phagocytic cells of snails (Schistosoma‎ intermediate hosts), susceptible and resistant to infection, on the level of the scavenger enzymes. It appeared that hemocytes from susceptible hosts induced higher levels of these enzyme expressions in Schistosoma‎ sporocysts, compared to hemocytes from resistant hosts. These results indicate that phagocytic cells of resistant snails may directly or indirectly downregulate Schistosoma‎ antioxidant enzyme activity, thus facilitating killing of the parasite (Zelck and Von Janowsky, 2004).

The presence of the parasite in the host hepatic mesenteries puts them under oxidative stress from immune-generated radicals as well as those potentially generated in the parasite during respiration and the breakdown and consumption of host hemoglobin with the concurrent release of toxic heme and ferrous ions. The addition of one electron to O2 produces superoxide, which is rapidly reduced to H2O2 by SOD. The H2O2 formed is itself able to diffuse and cause cellular damage and must be neutralized to prevent the formation the more damaging hydroxyl radical. In many organisms, intracellular H2O2 is eliminated by catalase, GPX 2 and/or thioredoxin peroxidases (Prxs) (Sayed et al., 2006).

5- Antioxidant enzymes secreted by S. mansoni:

Several antioxidant enzymes have been identified in S. mansoni, as cytosolic Cu/Zn superoxide dismutase (SmCT-SOD) and a glutathione peroxidase (SmGPX) ( Cook‎‎ et al., 2004 ). The expression and activity of these enzymes increase as the parasite undergoes development from the schistosomulum to the adult worm (Mei and LoVerde, 1997). These data coincide with in vitro studies on antibody-dependent cell cytotoxicity that show the schistosomule stages of the parasite being most susceptible to oxidant damage, while the adult stage is the least susceptible ( LoVerde, 1998; Mkoji et al., 1988a; Nare et al., 1990).

Vermeire and Yoshino (2007), study the ability of the larval forms of S. mansoni to invade and parasitize their molluscan host, Biomphalaria glabrata. They sought to elucidate the possible mechanisms by which the invading larvae are able to counteract the potentially harmful oxidative environment presented by the host upon initial miracidial infection. This was attempted by examining the gene expression profile of parasite antioxidant enzymes of the linked glutathione (GSH) thioredoxin (Trx) redox pathway during early intramolluscan larval development. Three such enzymes, the Prx (Prx1, Prx2, and Prx3) were examined as to their activity and sites of expression within S. mansoni miracidia and in vitro -cultured mother sporocysts. Results of these studies demonstrated that the H2O2-reducing enzymes Prx1 and 2 are upregulated during early mother sporocyst development compared to miracidia. Immunolocalization studies further indicated that Prx1 and Prx2 proteins are expressed within the apical papillae of miracidia and tegumental syncytium of sporocysts, and are released with parasite excretory-secretory proteins (ESP) during in vitro larval transformation. Removal of Prx1 and Prx2 from larval ESP by immunoabsorption significantly reduced the ability of ESP to breakdown exogenous H2O2, thereby directly linking ESP Prx proteins with H2O2-scavenging activity. Moreover, exposure of live sporocysts to exogenous H2O2 stimulated an upregulation of Prx1 and 2 gene expression suggesting the involvement of H2O2-responsive elements in regulating larval Prx gene expression. These data provide evidence that Prx1 and Prx2 may function in the protection of S. mansoni sporocysts during the early stages of infection.

Trx, TrxR and nicotinamide adenine dinucleotide phosphate (NADPH), the Trx system, is ubiquitous from Archea to man. Trx, with a dithiol/disulfide active site (CGPC) are the major cellular protein disulfide reductases; they therefore also serve as electron donors for enzymes such as ribonucleotide reductases, Prxs and methionine sulfoxide reductases. Glutaredoxins (Grx) catalyze glutathione-disulfide oxidoreductions overlapping the functions of Trx and using electrons from NADPH via GR (Arner and Holmgren et al., 2000).

TrxR is an essential enzyme required for the efficient maintenance of the cellular redox homeostasis (Biterova et al., 2005).

The discovery of this enzyme system, Prxs, represents a major advance towards the understanding of how parasitic nematodes deal with both internal and environmental oxidative stress. Prxs exist as homodimers. They share the property of reducing hydrogen peroxide to water and alkyl hydroperoxides to the corresponding alcohols and have been classified into two families: the 1-Cys and 2-Cys Prxs according to the presence of one or two highly conserved cysteine residues (Dzik, 2006).

The Prxs are distinct from other peroxidases in that they have no cofactors, such as metals or prosthetic groups. Reduction of hydroperoxides by 2-Cys enzymes is accompanied by the formation of an intermolecular disulfide bond which is subsequently reduced by electrons donated by Trx. Trx is regenerated by the system of TrxR and NADPH (Chae et al., 1994). S. mansoni, was shown to possess both bacterial-like (resistant to oxidative inactivation, important in regulating cell signaling pathways), and mammalian-like sensitive Prx (Sayed and Williams, 2004). Schistosomes have abundant SOD but completely lack catalase and have relatively low levels of GPx (Mkoji et al., 1988b; Mei and LoVerde, 1997) . Furthermore, the known S. mansoni GPx is in the phospho-lipid hydroperoxide GPx class (GPx4) with poor reactivity toward H2O2 (Williams et al., 1992; Maiorino et al., 1996). The main function of this GPx class may be to protect biomembranes from oxidative damage.

In most eukaryotes there are two major systems to detoxify reactive oxygen species, one based on the tripeptide GSH and the other based on the 12 kDa proteins Trx. In both systems reducing equivalents are provided by NADPH via dedicated oxidoreductase flavoenzymes. GR reduces glutathione disulfide (GSSG) and drives the GSH-dependent systems (Meister and Anderson 1983; Townsend, et al., 2003), whereas TrxR are pivotal in the Trx-dependent system (Gromer et al., 2004).

As the Schistosomes lack catalase, the main H2O2-neutralizing enzyme of many organisms and their glutathione peroxidases are in the phospholipids class with poor reactivity toward H2O2. Evidence implicates Prx as providing the main enzymatic activity to reduce H2O2 in the parasite (Sayed et al., 2006).

In addition to providing protection against oxidative damage, the Trx and GSH systems also play important roles in cell proliferation, redox regulation of gene expression, xenobiotic metabolism, and several other metabolic functions (Townsend et al., 2003; Gromer et al., 2004).

Thioredoxin Glutathione Reductase (TGR)

1- TGR enzyme:

Studies of the Schistosoma‎ life cycle have focused on the fact it can survive for decades in the blood stream of the human host without being severely affected by the immune system and the associated assault by various ROS. Since schistosomes do not have catalase to degrade hydrogen peroxide Mkoji et al., 1988 a&b), other mechanisms must exist within the parasite to degrade ROS.

Two principal components of this defense system have been recently identified in S. mansoni as TGR and Prx (Simeonov et al., 2008). Prx are members of a recently identified family of antioxidants involved in the detoxification of hydrogen peroxide and other hydroperoxides (Chae et al., 1994; Rhee et al., 2005).

S. mansoni parasites survive in humans in part because of a set of antioxidant enzymes that continuously degrade reactive oxygen species produced by the host. A principal component of this defense system has been recently identified as TGR, a parasite-specific enzyme that combines the functions of two human counterparts, GR and TrxR, and as such this enzyme presents an attractive new target for anti-schistosomiasis drug development (Lea et al., 2008).

Kuntz et al. (2007) stated that, the adult worms need to make antioxidants (chemicals that prevent oxygen from damaging cells) to protect themselves against the human immune response. Antioxidant production in these worms depends on TGR . In contrast to their mammalian hosts, platyhelminth thiol-disulfide redox homeostasis relies on linked thioredoxin-glutathione systems, which are fully dependent on TGR, a promising drug target (Bonilla et al., 2008).

Alger and Williams (2002) stated that, adult schistosomes, which reside in the hepatic portal system, are exposed to reactive oxygen compounds through respiration and as a result of the host immune response. To minimize oxidative stress schistosomes must possess adequate mechanisms of detoxification. Major detoxification systems rely on reducing equivalents from the disulfide oxidoreductases glutathione and TrxR. Therefore, maintenance of adequate levels of these thiols in a reduced form is critical. S. mansoni possess an unusual thiol redox system centered on TGR. This enzyme represents an unusual fusion of a pyridine nucleotide disulfide oxidoreductase with a redox active Grx extension. Furthermore, Alger and Williams (2002) predicted that, this is a selenocysteine (Sec) protein. Immunoprecipitation, Western blot (WB) and inhibitor studies show that this protein has TrxR, GR, and Grx activities. Most importantly, they show that TGR appears to be the major, if not the sole enzyme for these activities in adult worms, completely replacing TrxR and GR. This is the first example of an organism with a redox system based exclusively on TGR.

TGR is a multifunctional Sec-containing enzyme that catalyzes the interconversion between reduced and oxidized forms of both GSH and Trx, which are major contributors to the maintenance of redox balance in eukaryotes (Alger and Williams, 2002; Rai et al., 2009).

Using RNA interference Kuntz et al. (2007) found that, TGR is essential for parasite survival; after silencing of TGR expression, in vitro parasites died within 4 days.

Kwatia et al. (2000) and Sayed and Williams (2004) suggest that, the Schistosoma‎ redox system is significantly different from that of the host and that Prxs provide significant, perhaps the vast majority of hydrogen peroxide reducing activity in schistosomes.

In most organisms, including the mammalian hosts of platyhelminths, cellular redox homeostasis, antioxidant defenses and supply of reducing equivalents to several targets and essential enzymes rely on two major pathways: the GSH and the Trx systems, which have overlapping and differential targets and functions (Fernandes and Holmgren, 2004 ; Winyard et al., 2005).

Humans possess two distinct enzymes, GR and TrxR, which specifically recognize GSH and Trx as substrates, respectively (Salinas et al., 2004).

In contrast, platyhelminth parasites lack conventional TrxR and GR, and hence conventional Trx and GSH systems ( Salinas et al., 2004; Kuntz et al., 2007; Cioli et al., 2008). Instead, they rely exclusively on linked thioredoxin-glutathione systems, with TGR being the key enzyme that provides reducing equivalents to both pathways. Another feature of the linked systems in platyhelminths is that cytosolic and mitochondrial TGR derive from a single gene and have identical sequence, once the leader peptide of the mitochondrial variant is removed (Agorio et al., 2003; Angelucci et al., 2009).

It was recently discovered that in S. mansoni, specialized TrxR and GR enzymes are absent, and instead replaced by a unique multifunctional enzyme, TGR (Alger and Williams, 2002). In mammalian cells, two specialized enzymes do TGR job. The researchers reasoned, therefore, that TGR might be an essential parasite protein and a potentially important drug target (Kuntz et al., 2007). In TGR, the presence of the Grx domain, in addition to the sequences highly homologous to TrxR 1, appeared to increase a host of activities by adding those with specificity for the GSH system (Sun et al., 2001).

The reliance on a single enzyme for both GSSG and Trx reduction suggests that the parasite's redox systems are subject to a bottleneck dependence on TGR. The amino acid sequence and domain structure of Schistosoma‎ TGR has similarities to mammalian forms of TrxR and GR, with an additional amino-terminal extension of a Grx domain of ~110 amino acids with a typical CPYC active site (Alger and Williams, 2002).

On the other hand, in schistosomes and other helminthes, TGR represents the only enzyme capable of supplying reducing equivalents to both GSH/GSSG and Trx systems, shuttling electrons to downstream proteins ( Agorio et al., 2003; Rendon, 2004).

2- Structure of TGR:

TGR is a homodimeric enzyme and peculiar fusion of a Grx domain and TrxR domains with a C-terminal redox center containing Sec. Sec is located on a flexible C-terminal arm that is usually disordered in the available structure of the protein and is essential for the full catalytic activity of TGR (Sun et al., 1999; 2001; Bonilla et al., 2008; Angelucci et al., 2010).

TrxR is an enzyme belonging to the flavoprotein family of pyridine nucleotide-disulfide oxidoreductases (Maggioli et al., 2004).

TrxR is a dimeric enzyme with a redox-active disulfide and an FAD in each monomer, and it is a member of a larger family of pyridine nucleotide-disulfide oxidoreductases, which includes the closely related enzymes lipoamide dehydrogenase, GR, trypanothione reductase, and mercuric ion reductase. TrxR1 catalyzes the NADPH-dependent reduction of the active site disulfide in oxidized thioredoxin (Trx-S2) to give a dithiol in reduced thioredoxin (Trx-(SH)2) ( Zhong‎ et al; 1998).

There are two known TrxR types that evolved by convergent evolution: a homodimer of 35 kDa subunits present in prokaryotes, yeast, and plants and a homodimer of 55-65 kDa subunits that occurs in animals and some lower eukaryotes ( Su and Gladyshev, 2004).

The calculated molecular mass of the native TGR was ≈130 kDa. Thus, TGR was not a component of a large molecular weight complex, and its experimental mass was consistent with homodimer composition of the enzyme, which is typical of pyridine nucleotide disulfide oxidoreductases (Sun et al., 2001).

Like all mammalian TrxR isoforms, S. mansoni TGR (SmTGR) is a selenoprotein with a carboxyl-terminal GCUG active site motif, where “U” is Sec. Sec is a highly reactive amino acid that gives unique properties to selenoproteins (Sharma et al., 2009). It is encoded by a dedicated UGA codon in the selenoprotein mRNA and is recoded from translational termination to Sec insertion by a translation machinery utilizing a specialized structural element in the 3′-untranslated region, the Sec is an element, which is also found in the mRNA of SmTGR (Alger and Williams, 2002).

TGR, like GR and TrxR, is a homodimer, with monomers oriented in a head-to-tail manner. Based on biochemical data, the current model of the mechanism of reaction for TGR proposes that electrons flow from NADPH to FAD, to the C156xxxxC redox center (numeration according to E. granulosus TGR), to the C-terminal GC595UG (U is Sec) redox center of the second subunit, and finally to the C31xxC redox center of the Grx domain of the first subunit. The fully reduced enzyme can reduce either oxidized Trx using the C-terminal active site GCUG, or GSSG through the CxxC redox center of the Grx domain (Sun et al., 2001; 2005). Recently, a crystallographic structure of an S. mansoni C-terminally truncated TGR (GCstop) has been solved. Based on the residual GR activity of the mutant, the authors proposed an alternative view in which GSSG could be reduced directly by the CxxxxC redox center of TrxR domains (Angelucci et al., 2008).

Gladyshev et al. (1999) stated that, mammalian TrxR are members of the Type I pyridine nucleotide–disulfide oxidoreductase enzyme family, which also includes several other enzymes such as GR and lipoamide dehydrogenase. The members of this family are homodimers of 50-65 kDa subunits and each subunit contains a flavin adenine dinucleotide cofactor. These enzymes utilize NADPH to reduce natural substrates and the reduction takes place at the disulfide active center located in the N-terminal portion of each enzyme.

Angelucci and others (2008) studied the structural characterization of SmTGR by crystallography, to deepen their understanding of the enzyme’s specificity for a possible rational drug design. In their article they report the first crystal structure at 2.2 A ˚ resolution of a truncated form of SmTGR, lacking the last two amino acids (Sec597- Gly598). The structure reveals the unusual architecture of this recently discovered enzyme and together with functional data update the knowledge of the electron pathways between its domains .

3- Mammalian thioredoxin:

Animal TrxR are NADPH-dependent, FAD-containing proteins that belong to a pyridine nucleotide disulfide oxidoreductase family. To date, three TrxR have been identified in mammals, including TR1 (the cytosolic TR, also called TrxR1, TxnRd1, or TrxRR), TR3 (mitochondrial TR, also called TrxR2, TxnRd2, or TrxRâ), and TGR (Trx and GR, also called TR2). TR1 and TR3 are the major TrxR in the cytosol and mitochondria, respectively, and are ubiquitously expressed in various tissues and cell types. The catalytic mechanism of TR1 has been well-characterized, but there is little information about the reactions catalyzed by TR3 and TGR. Recently, TR1 and TR3 were shown to be essential for mouse embryogenesis ( Sun et al., 2005), although through different mechanisms. TR1 was found to be critical for cell growth (Jakupoglu et al., 2005), whereas TR3 was shown to be essential for heart development (Conrad et al., 2004).

Mammalian cytosolic and mitochondrial TrxR are essential Sec-containing enzymes that control Trx functions. TGR is a third member of this enzyme family. It has an additional Grx domain and shows highest expression in testes (Tansatit et al., 2010).

Three mammalian TrxR genes were previously identified that encode TrxR1 (cytosolic enzyme), TGR (also called TrxR3), and TrxR2 (a mitochondrial TrxR) ( Su and Gladyshev, 2004).

All mammalian TrxR isozymes are homologous to GR and contain a conserved C-terminal elongation with a cysteine-Sec sequence forming a redox-active selenenylsulfide/ selenolthiol active site (Arner and Holmgren , 2000).

Six human TrxR1 isoforms were identified that were derived from a large number of transcripts and differed in their N-terminal sequences. One isoform resulted from exons located 30-70 kb upstream of the previously identified core TrxR1 promoter and was composed of a basic TrxR1 module fused to a Grx domain that contained an unusual active site CTRC sequence. This TR1 form occurred in humans, dogs, and chimpanzees but was inactivated in mice and rats (Su and Gladyshev, 2004).

4- Inhibition of TGR as drug target:

Because of the unusual organization of the Schistosoma‎ enzymatic defense against oxygen radicals, Sayed and coworkers (2008) hypothesized that, the parasite redox pathway would be an effective target for the development of new antischistosomal chemotherapies.

The apparent replacement of two human enzymes by one dual-specificity worm enzyme has created a metabolic and regulatory bottleneck in which the inactivation of a single target, TGR, might have an enhanced deleterious effect on both the maintenance of parasite's redox balance and on its “antioxidant firewall”. Indeed, recent small molecule inhibition and RNA interference experiments have shown that inactivation of TGR has profound effects on S. mansoni survival rates both in culture and in infected mice (Simeonov et al., 2008).

The significance of SmTGR as a putative drug target was first demonstrated using an RNA interference approach, which killed 90% of treated parasites in vitro. Moreover, it was demonstrated that SmTGR activity is inhibited by two schistosomicidal drugs used in the past to fight the infection, antimonyl potassium tartrate, and oltipraz, suggesting that the enzyme is the main target of these compounds (Kuntz et al., 2007).

TGR have been recently identified and validated as targets for anti-schistosomiasis drug development. In search of inhibitors of this critical redox cascade, Simeonov et al. (2008) optimized and performed a highly miniaturized automated screen of 71,028 compounds arrayed as 7- to 15-point dilution sets. They identified novel structural series of TGR inhibitors, several of which are highly potent and should serve both as mechanistic tools for probing redox pathways in S. mansoni and as starting points for developing much-needed new treatments for schistosomiasis.

A potentially interesting inhibitor of TGR is an auranofin (AF). In vitro, 10 μM AF causes unpairing of male and female worms after 1h and results in 100% mortality after 9 h of exposure (Kuntz et al., 2007). In the same study it was found that, TrxR and GR activities of TGR in worm homogenates were nearly 100% inhibited after 1 hr and that the GSH:GSSG ratio decreased from 18:1 in control worms to 2.6:1 (85% decrease) after 6 hr treatment. Larval, juvenile and adult stages of the parasite are killed by 5 μM AF in 24 hrs, which acquires significance given that this concentration of the drug is well tolerated by mammalian cells. Indeed, in preliminary experiments, AF administered to infected mice at dosages well tolerated by the host killed 60% of adult schistosomes. This reduction of worm burden would lead to a significant decrease in the pathology and morbidity associated with schistosomiasis (Kuntz et al., 2007).

AF has an important advantage as a schistosomicidal drug, since it has been in clinical use to cure rheumatoid arthritis for 25 years and thus presents a well-known and quite safe toxicity profile (Shaw, 1999; Becker et al., 2000). AF has been demonstrated to be a nanomolar inhibitor of Sec-containing enzymes such as TrxR and TGR (Kuntz et al., 2007), whereas 1000-fold higher concentrations appear to be required to inhibit GR, which lacks the C-terminal Sec (Gromer et al., 1998). These observations suggest that Sec is either the binding site of AF or is essential in displacing the gold from its ligands given that its nucleophilic power is greater than sulfur ( Urig et al., 2006 ).

Phosphinic amides and oxadiazole 2-oxides, identified from a quantitative high-throughput screen, were shown to inhibit a parasite enzyme TGR, with activities in the low micromolar to low nanomolar range. Incubation of parasites with these compounds led to rapid inhibition of TGR activity and parasite death. The activity of the oxadiazole 2-oxides was associated with a donation of nitric oxide. Treatment of Schistosoma‎ -infected mice with 4-phenyl-1, 2, 5-oxadiazole-3-carbonitrile-2-oxide led to marked reductions in worm burdens from treatments against multiple parasite stages and egg-associated pathologies. The compound was active against the three major Schistosoma‎ species infecting humans. These protective effects exceed benchmark activity criteria set by the World Health Organization for lead compound development for schistosomiasis (Sayed et al; 2008).

Rai et al. (2009) stated that, the worm death will not occur until the GSH/GSSG ratio reaches a critical point and the overall worm redox balance is unrecoverable, making it difficult to derive correlations between worm killing and TGR inhibition below a certain level.

5- Redox Pathways in Mammals and S. mansoni:

In mammals figure (I), electrons from NADPH are transferred to an oxidoreductase flavoenzyme, either TrxR or GR. Electrons are then transferred from the oxidoreductase flavoenzyme to the appropriate electron carrier, either Trx-S2 or GSSG converting them to Trx-(SH)2 or GSH, respectively. Trx-(SH)2 and GSH then supply reducing equivalents for a number of different reactions, including those that are Grx-dependent.

In S. mansoni Figure (II), TrxR and GR are replaced with a unique oxidoreductase flavoenzyme, TGR, which provides reducing equivalents for Trx-, GSH- and Grx-dependent reactions (Gromer et al., 2004).

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Figure I: Redox Pathways in Mammals (Kuntz et al., 2007)

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Figure II: Redox Pathways in S. mansoni (Kubtz et al., 2007)

6- Reaction Mechanism of TGR:

The redox activity of the enzyme relies on at least 3 redox sites communicating with one another: (i) The FAD site, composed by the isoalloxazine ring of the flavin and the Cys154-Cys159 couple (characteristic of all the enzymes of the TrxR/GR family); (ii) the C-terminus, constituted by the Gly-Cys-Sec-Gly sequence shared with the majority of TrxR but not with GRs; (iii) the Grx redox site represented by Cys28- Cys31 at the N-terminal portion of the protein (Angelucci et al., 2009).

The organization of the TGR homodimer model (Fig. III) was such that the Cys-Sec motif of one subunit could transfer electrons from the thiolydisulfide center to the Grx domain of the second subunit and vice versa. The conserved C-terminal Gly-Cys-Sec-Gly motif may be considered as an analog of small disulfide substrates (e.g., GSSG for GR or lipoate for LDH), which is linked to a pyridine nucleotide disulfide oxidoreductase portion of the protein. In other words, TGR may be viewed as a fusion of three components, which correspond to Grx, GR, and GSSG in the GSH system (Fig. III). These observations suggested a mechanism whereby electrons were transferred from NADPH to downstream electron donors through several redox centers within TGR, NADPH to FAD to thiolydisulfide center to the C-terminal GSSG-like Seccontaining center to the Cys residue within the Grx domain the to downstream substrate (Fig. III).

The key component in this remarkable electron flow was the Gly-Cys-Sec-Gly tetrapeptide that transfers electrons between redox centers through reversible oxidoreduction coupled with conformational changes. The proposed mechanism is consistent with previous models, which proposed, on the basis of kinetics, site-specific mutations and protein microchemistry studies, that the C-terminal redox motif in mammalian TrxR1 and TrxR from Plasmodium falciparum can be directly reduced by the N terminal thiolydisulfide center. Similarity is also evident to LDH, whose substrate, lipoic acid, contains an internal disulfide that transfers electrons and substrate molecules among three different active centers located within three polypeptides. Lipoic acid is fused to a protein component of the pyruvate dehydrogenase complex via a long hydrophobic arm that delivers this compound to various reaction centers (Sun et al., 2001).

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