Much attention has focussed on the proteases in ES products because of their potential importance in parasite invasion and nutrition. Proteomic analyis by Yatsuda et al. (2003) detected 224 and identified 107 proteins in adult H. contortus ES products. Among those were several proteases, including zinc metallopeptidases, serine proteases and aspartic proteases. Cysteine proteases have also been detected (Oliver et al., 2006; Yatsuda et al., 2006). This group includes cathepsin B-like proteases (CBLs) and asparaginyl proteinase (legumain). A putative cysteine protease was also identified in T. circumcincta L4 ES products
(Craig et al., 2006), as well as cathespsin F and an O. ostertagi homologous astacin-like metalloprotease in post-infective L3 and L4 ES products (Smith et al., 2009).
Yatsuda et al. (2003) identified four different metallopeptidases, including H11/concortin, in H. contortus ES products; these enzymes were also identified from the intestinal brush border of H. contortus (Munn, 1977; Newlands et al., 2006; Geldorf and Knox, 2008). H11 is usually classified as a hidden antigen which is not presented to the host immune system, but was found in all their ES preparations examined (Yatsuda et al., 2003). H11 degraded
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Figure 1.25: Putative proteolytic cascade for hemoglobin degradation by blood-feeding nematodes. Host hemoglobin (Hb) is initially attacked by aspartic proteases, degraded to smaller peptides by cysteine proteases, then by metalloproteases and finally to free amino acids or small peptides by exopeptidases. APR: aspartic protease; PEPI: parasite pepsinogen-like aspartic protease; Ac: A. canium; CP: cysteine protease; MEP metalloprotease. (Williamson et al., 2003)
Chapter 1: Literature Review 33 fibrinogen and was suggested to function as an anti-coagulant during blood feeding (Geldorf and Knox, 2008). Serine proteases were also proposed to be anticoagulants. Furthermore, serine proteases in ES of Schistosoma mansoni degraded IgE (McKerrow et al., 2006). Serine and metalloproteases of Trichostrongylus vitrinus ES products were shown to degrade fibrinogen, plasminogen and fibronectin (Maclennan et al., 1997) and aspartic proteases of shistosomes and H. contortus degraded hemoglobin (Longbottom et al., 1997; Brindley et al., 2001). S. mansoni asparaginyl proteinases activated cathepsin B, which is also believed to be the case for H. contortus asparaginyl proteinase (Sajid et al., 2003;Oliver et al., 2006).
Cysteine proteases, including CBLs, are the most widely reported protease class from parasitic nematodes. CBLs of H. contortus are encoded by 22 genes (Jasmer et al., 2001) and at least seven CBLs have been identified in ES products (Yatsuda et al., 2006). H. contortus
cysteine proteases present in adult ES products, as well as in live L4 and adult parasites,
were able to degrade a model extracellular matrix consisting of glycoproteins, elastin and collagen (Rhoads and Fetterer, 1996). In addition, cysteine proteases in extracts of
H. contortus digested hemoglobin, fibrinogen, collagen and IgG (Williamson et al., 2003). A putative proteolytic cascade for hemoglobin digestion, shown in Figure 1.25, was proposed to include all protease classes (Williamson et al., 2003;2004).
1.3.3.2 Protease Inhibitors
In addition to proteases, different classes of protease inhibitors have also been identified in a variety of parasites, including Schistosoma spp., Ascaris spp., Brugia malayi, Onchocerca volvulus,
Trichuris suis, T. vitrinus, O. ostertagi and H. contortus. Functions assigned to the inhibition range from regulating parasite proteases, protection against degradation of proteins by host proteases, modulation of the host immune response and/or a role during feeding as anticoagulants (McKerrow et al., 2006; Knox, 2007). A serine proteinase inhibitor (serpin) was identified in ES products of L4 and adult T. vitrinus (MacLennan et al., 2005). Serpins
were also detected in H. contortus adult ES products, but were thought to be of host origin (Yatsuda et al., 2003). Homologues of Ancylostoma caninum anticoagulant proteins of the serine protease inhibitor family have also been detected in H. contortus (Knox, 2007).
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1.3.3.3 Antioxidant Enzymes
The antioxidant enzymes superoxide dismutase (SOD) and glutathione S-transferase have been identified in ES products of adult H. contortus (Yatsuda et al., 2003) and thioredoxin peroxidase in ES of T. circumcincta L4 (Craig et al., 2006). These enzymes are used by the
parasites to protect against oxidative stress generated by their own cellular metabolism or by the host, including potentially damaging reactive oxygen and nitrogen intermediates produced by immune cells. SOD and thioredoxin peroxidase were also found in other parasitic nematodes, including O. volvulus and B. malayi (Henkle-Dührsen and Kampkötter, 2001;Hewitson et al., 2008). In O. volvulus, SOD was located in the intestine, where it could minimise oxidative damage after ingestion of host erythrocytes.
1.3.3.4 Other Enzymes
Fetterer and Rhoads (2000) detected acid phosphatase and phosphorylcholine hydrolase in adult H. contortus ES products. Acid phosphatase was suggested to play a role in intestinal digestion, as the highest tissue concentrations were detected in the worm intestine. This is consistent with the suggestion that acid phosphatase secretion by fourth stage larvae coincides with the onset of feeding (Gamble and Mansfield, 1996). The proposed role for the secreted acid phosphatase was to act in combination with cysteine proteases in extracorporeal digestion and tissue penetration (Fetterer and Rhoads, 2000).
Nucleoside diphosphate kinase has been identified in ES products from adult H. contortus
(Yatsuda et al., 2003), adult T. circumcincta (Craig et al., 2006) and Trichinella spiralis (Gounaris
et al., 2001). Proposed functions range from regulation of host cell proliferation and differentiation, ATP-dependent cytotoxity for macrophages and mast cells and prevention of apoptosis (Zaborina et al., 1999; Punj et al., 2000; Gounaris et al., 2001).
Acetylcholinesterase has been detected in a range of nematode ES products, primarily from adult parasites, including Nippostrongylus brasiliensis, T. vitrinus, Nematodirus battus,
Trichostrongylus colubriformis, as well as T. circumcincta and H. contortus (Knox and Jones, 1990; Mallet et al., 1997; Joshi and Singh, 2000). A role for acetylcholinesterase has been
Chapter 1: Literature Review 35 suggested in the prevention of peristalsis and parasite expulsion (Opperman and Chang, 1992), inhibition of mucus secretion (Philipp, 1984) and alteration of the immune response (Rhoads, 1984). In vitro, T. colubriformis acetylcholinesterase stimulated cell growth at low concentrations and had an inhibitory effect at high concentrations, suggesting a role in intestinal crypt cell repair and renewal during parasitism (Huby et al., 1999).
In their adult H. contortus ES products, Yatsuda et al. (2003) detected the cytosolic enzymes glutamate dehydrogenase (GDH) and glycolytic enzymes including enolase, but no other intracellular proteins such as ribosomal or cytoskeletal proteins, which might have indicated some cell damage. However, several metabolic enzymes are secreted into ES or are present on the helminth surface and do not necessarily indicate cell damage. Skuce et al. (1999) demonstrated that GDH is located in the gut of H. contortus and may be shed into ES products. Similarly, a range of homologous metabolic enzymes, including enolase, have been detected in T. circumcinta L4 and adult ES products (Craig et al., 2006). Several
structural proteins including actin, tropomyosin and paramyosin have been detected in ES products of both these stages and may or may not be derived from degrading parasites in culture.
Enolase has been detected in ES products from Fasciola hepatica, Echinostoma caproni, adult
Schistosoma bovis, T. spiralis as well as in most tissues of adult O. volvulus and infective larvae (Jolodar et al., 2003; Bernal et al., 2004; Pérez-Sánchez et al., 2006; Marcilla et al., 2007). Even though enolase lacks a signal peptide for secretion in most cases, a conserved region at the amino-terminus has been suggested as a signal peptide for T. spiralis and E. caproni. Enolase of F. hepatica, E. caproni and O. volvulus bound human plasminogen in vitro. Host activators convert plasminogen to plasmin, which has proteolytic activities. It was suggested that this might have a role in host tissue invasion by promoting attachment to the mucosa and, through plasmin-mediated proteolysis, might cause degradation of the extracellular matrix (Jolodar et al., 2003; Marcilla et al., 2007).
1.3.3.5 Calreticulin
Suchita and Joshi (2005) found that adult H. contortus ES products contain calreticulin, which prolonged the plasma coagulation time, suggesting a function in the prevention of
Chapter 1: Literature Review 36 blood clotting by binding Ca2+, clotting factors and C-reactive protein (CRP), presumably
aiding blood feeding of the parasite. Another proposed function is the modulation of the host immune response by binding to CRP and complement C1q. Binding to secreted calreticulin may limit the concentration of free CRP, blocking its procoagulant function as well as the activation of the classical complement pathway. This pathway can be activated by CRP-C1q interaction and inhibition may take place by calreticulin binding to CRP or directly to C1q (Suchita and Joshi, 2005; Suchita et al., 2008). Secretion of calreticulin and its interaction with C1q was also shown for Necator americanus and Trypanosoma cruzi (Kasper
et al., 2001;Ferreira et al., 2004).
1.3.3.6 Non-Protein Compounds
Generally, studies have investigated the proteins present in ES products, although a few studies have also reported the release of other components by H. contortus and
T. circumcincta. These include the metabolic endproducts propan-1-ol, acetate and propionate, as well as small amounts of ethanol, lactate and succinate (Ward and Huskisson, 1978;Ward et al., 1981).
Hadás et al. (1998) detected the prostaglandins PGA2 and PGB2 in T. colubriformis and H. contortus larval ES products, as well as PGA2,PGB2, PGD2 and PGF2α in extracts from
larvae and adults of both species. The release of prostaglandins was also reported for
Trypanosoma brucei and T.cruzi, Schistosoma spp., N. americanus and O. volvulus (Daugschies and Joachim, 2000; Kubata et al., 2007). It has been suggested that prostaglandins are released by the parasites to modulate host physiology and/or immune reaction in their vicinity, which might include the induction of inflammation, downregulation of cytokines or decreased gastric secretion.
Chapter 1: Literature Review 37