Schistosomes feed on host erythrocytes, using proteases to catabolise haemoglobin from ingested red blood cells, into dipeptides or free amino acids. These peptides diffuse into and/or are taken up by gastrodermal cells lining the gut, and become incorporated into schistosome proteins. While the identity and roles of the enzymes involved in this pathway are unclear, the gut-associated schistosome cathepsin D aspartic protease is thought to play an integral role, and thus has been targeted for development of novel anti-schistosomal interventions. The research presented in this thesis specifically aims to characterise developmental, biochemical and immunological attributes of the cathepsin D aspartic protease from Schistosoma japonicum, in order to further elucidate its role in the physiology of the schistosome.
A starting point for the research was the expression and purification of recombinant forms of the protease. An inactive denatured proenzyme form of the protease (designated rSjASPl) was expressed in Escherichia coli cells using a bacterial expression system, and an enzymatically active, glycosylated, mature form of the protease (designated rSjASP2) was expressed in insect cells, using a baculovirus expression system.
Developmental expression of the protease was explored. Transcripts encoding the cathepsin D gene were detected in all developmental stages of the parasite using RTPCR. Enzyme assays using a synthetic fluorogenic peptide and human haemoglobin as substrates, detected the presence of cathepsin D-like aspartic protease activity in extracts of all developmental stages, and this activity was completely inhibited by the addition of pepstatin. In rank order, the levels of activity were as follows: adult females > mixed-sex adults > adult males , eggs, miracidia > cercariae, schistosomula. In addition, western blotting with rabbit rSjASPl antibodies, detected the presence of an immunoreactive band of ~41 kDa in extracts of all the developmental stages.
The substrate specificity of rSjASP2 was determined by examining its ability to cleave human IgG, complement C3 and serum albumin in vitro. rSjASP2 cleaved IgG in a time and dose-dependent manner, with optimal cleavage at pH 3.6 - 4.5. Amino terminal sequencing of the major cleavage fragments of IgG identified an Fab fragments from the VHl domain, and 2 cleavage sites in the CH2 domain below the hinge region. The PI and P r residues at the CH2 cleavage sites were Phe254-Leu255 and Leu325- Thr326, indicating a preference by the schistosome protease for bulky hydrophobic residues flanking the scissile bond. No cleavage of the IgG light chain was detected, however r,SjASP2 cleaved the human serum proteins C3 and albumin into numerous small fragments.
The immunogenicity and vaccine efficacy of schistosome cathepsin D were examined after immunising mice with rSjASPl and rSjASP2. Mean total worm burdens were significantly reduced in vaccinated mice by 21-38%, and significant reductions in female worm burdens were also recorded (22-40%). Vaccination did not reduce fecundity; rather, increased egg output per female worm was recorded in vaccinated animals, suggesting a crowding effect. Vaccinated mice developed high levels of antibodies (predominantly IgGl, IgG2a and IgG2b isotypes), but there was no obvious correlation between antibody levels and protective efficacy. In addition, immune sera from vaccinated mice did not inhibit the in vitro degradation of human haemoglobin by rSjASPl, and passive transfer of immune serum or cathepsin D antibodies before and after parasite challenge, did not significantly reduce worm or egg burdens in recipient animals.
Cellular immune responses were also measured in mice vaccinated with rSjASFl and rSjASP2. Both rSjASPl and rSjASP2 induced significant prohferation of splenocytes from vaccinated mice, and expression of IFN-y, IL-4 and IL-10 mRNA in these cells was detected using RT-PCR. Secretion of IFN-y, IL-4 and IL-10 by splenocytes from vaccinated mice was confirmed and quantified using ELISA. IFN-y was the most abundant cytokine produced, followed by IL-4 and IL-10 in rank order, indicating that vaccination of mice with the schistosome protease induces a mixed Thl/Th2 cytokine response.
Overall, these results suggested the schistosome cathepsin D aspartic protease is a constitutively expressed enzyme, with a broad substrate specificity for host serum proteins ingested with the blood meal. High levels of enzyme expression in adult female worms (known to be more voracious blood feeders than male worms) supports a role for the protease in parasite metabolism, and release of the schistosome protease into the host's circulatory system may stimulate the production of both Thl and Th2 antibody and cytokine isotypes. Vaccination studies with recombinant forms of the protease suggested these humoral and cell-mediated responses may provide partial protection against schistosome infection, however low levels of protection in vaccinated mice, suggested the protease on its own, may not be an effective vaccine for schistosomiasis.