Despite considerable recent successes, the control of schistosomiasis remains a major public health consideration in many nations of the developing world. Complicating control efforts are high re-infection rates and the potential development of drug resistance, and these features necessitate the development and application of an effective anti-schistosome vaccine as an adjunct to integrated control. In order to achieve this goal, novel proteins with important roles in the intra-mammalian lifeycle stages of the schistosome parasite must be identified and their biological functions confirmed. Serine protease inhibitors (serpins) are a superfamily of proteins involved in many important biological processes, such as blood coagulation, fibrinolysis, complement activation and inflammation. Serpins are known to play central roles in host immune modulation and/or evasion by pathogens. The goals of this thesis were: 1) To identify, clone and sequence full-length S. japonicum serpin genes and determine their gene expression profiles across key parasite life cycle stages; 2) To express and purify recombinant S. japonicum serpins for biochemical characterisation in order to determine their biological functions; and 3) To investigate the immunogenicity of S. japonicum serpins by determining the anti-serpin antibody profiles in the sera of rodents experimentally infected with S. japonicum.
Data mining of published microarray data and cross referencing the S. japonicum draft genome identified two full-length coding sequences of S. japonicum serpins termed SjB6 (GenBank Accession number AAW26816.1) and SjB10 (GenBank Accession number AAK57435.1). Bioinformatics approaches demonstrated that SjB6 contained an open reading frame of 1038 bp that encoded 387 amino acid residues with a predicted molecular weight of 43.7 kDa, whereas SjB10 contained an open reading frame of 1212 bp, encoding 403 amino acid residues with a predicted molecular weight of 45.7 kDa. Sequence analyses of the primary and secondary structures of both proteins revealed that both SjB6 and SjB10 contained essential structural motifs characteristic of the serpin superfamily. SjB6 and SjB10 were classified as secretory and intracellular serpins respectively, based on the presence of a putative N-terminal signal sequence in the former which was absent in the latter. Sequence annotation of the amino acid residues of the hinge region also indicated that both proteins shared the consensus pattern characteristic of inhibitory serpins.
Gene expression profiles, determined by real-time PCR, showed that SjB6 was expressed highly in the adult worms, schistosomula, miracidia and eggs. Expression was significantly higher in the eggs than the other life cycle stages indicating its stage specificity and a possible role in disease pathology. SjB10 was expressed in all life cycle stages except in miracidia, with the highest level of expression evident in cercariae. The molecular weights of native SjB6 and SjB10 determined by immunoblot assays using soluble worm antigen preparations of S. japonicum were approximately 60 kDa and 64 kDa respectively, sizes significantly larger than those predicted from the amino acid sequences. This may indicate that the SjB6 and SjB10 native proteins undergo significant post-translational modifications.
SjB6 and SjB10 were produced recombinantly using the baculovirus expression system and purified by affinity chromatography and size-exclusion chromatography. In order to gain insight on the biological functions of SjB6 and SjB10 in S. japonicum, the protease inhibition activities of the recombinant proteins (rSjB6 and rSjB10) were determined. The results showed that whereas rSjB6 did not inhibit any of the proteases tested, rSjB10 inhibited pancreatic elastase in a concentration-dependent manner. The data from the gene expression analysis and elastase inhibition assays suggest that SjB10 may play an important role in cercarial penetration of the host skin, potentially in regulating endogenous and host derived serine proteases. Immunolocalisation analysis of SjB10 showed it was mainly localised just below the eggshell of eggs and to the foregut of the adult worms. On the other hand, localising native SjB6 using conventional immunolocalisation studies with parasite tissue sections was unsuccessful. A possible explanation is that SjB6 may be secreted in S. japonicum egg-derived exosomes as many other secreted serpins have been identified in exosomes derived from various cell types. This hypothesis was strengthened by preliminary findings from the proteomic analysis of S. japonicum egg-derived exosomes in which a serpin was identified.
Total IgG antibody levels in sera from the non-permissive rat and susceptible mouse animal models of schistosomiasis were collected in order to evaluate the immunogenicity of the SjB6 and SjB10 serpins by ELISA and western blotting and to determine the host antibody profile against the recombinant proteins. The rat sera showed high reactivity against both proteins in direct contrast to the mouse sera in which no reactivity was observed. These results suggest that the induction of a specific immune response against these proteins might, in part, be responsible for the resistance observed in rats. In summary, the findings presented in this thesis provide new insights on the key biological functions of two schistosome serpins, particularly their roles in host-parasite interaction and their possible involvement in host immune modulation and/or evasion.