University of Technology, Sydney. Institute for the Biotechnology of Infectious Diseases.Eimeria is a genus of protozoa within the Apicomplexa, a phylum that includes Plasmodium species (which cause malaria) and Toxoplasma gondii (which cause foetal abnormalities and encephalitis), and many other species of parasites. Eimeria belongs to the subclass Coccidia, and a defining characteristic of this group is their transmission from host to host via oocysts that contaminate food and water. The resilient oocyst wall protects the parasites as they are excreted in the host’s faeces and in the outside world, allowing them to survive for several months between hosts. It is formed from the contents of specialised organelles - wall forming bodies - found in the macro gametocyte stage of the parasites.
Two proteins, EmGam56 and EmGam82, from the wall forming bodies of Eimeria maxima have been studied intensively in recent years (see Belli et al, 2006). Both are processed and/or degraded into smaller tyrosine-rich polypeptides (from 8 to 33 kDa) and incorporated into the oocyst wall. The tyrosine richness of these proteins and the presence of dityrosine in the oocyst wall has led to the proposal that dityrosine cross-linking of these proteins forms a matrix that is crucial component for oocyst wall formation (Belli et al., 2006). The aims of this thesis are to:
[1] deduce the biochemical composition of the oocyst walls using gas chromatography (GC) and mass spectrometry (MS);
[2] determine the structural features of EmGam56 using bioinformatics, circular dichroism (CD), and one-dimensional nuclear magnetic resonance (1D-NMR);
[3] demonstrate that peroxidase-catalysed dityrosine crosslinks can be induced to form between truncated forms of EmGam56.
GC and MS revealed that the Eimeria oocyst wall is composed mainly of proteins (>90%) with small amount of lipids (1.4-7.6%) and carbohydrates (0.3-2.0%). There is little difference between the unsporulated and sporulated oocyst walls of E. tenella and E. maxima. Thus, the structure of proteins like EmGam56 is key to understanding how oocyst walls are constructed.
Bioinformatic analyses indicated that EmGam56 is an intrinsically unstructured protein (IUP), dominated by random coils (52-70%), with some a-helices (28-43%) but few P-sheets (1-11%); this was confirmed by CD and 1D-NMR. Furthermore, the structural integrity of the protein under extreme temperatures (boiling for 40 minutes) and pH (pH 1.3-11) indicated its IUP nature. The intrinsic lack of structure in EmGam56 could facilitate its incorporation into the oocyst wall in two ways: first, IUPs are highly susceptible to proteolysis, explaining the several differently-sized oocyst wall proteins derived from EmGam56; and, second, the flexibility of IUPs could facilitate cross-linking between these tyrosine-rich derivatives.
Peroxidases are key to the formation of dityrosine bonds (see Belli et al, 2006 for a review). An in vitro cross-linking assay was developed using a recombinant 42 kDa truncation of EmGam56. The protein was exposed to various peroxidases and peroxides, the formation of polymers was followed by Western blotting, and the formation of dityrosines was determined by HPLC. Peroxidases from plants or fungi, but not mammals, catalysed rapid formation of polymers. No peroxidase has yet been found in the incompletely annotated E. tenella genome database but peroxidase activity has been detected in the wall forming bodies (Belli et al, 2006). Therefore, future searches for Eimeria peroxidases should focus on plant-like homologous.
The results presented in this thesis support the proposal that dityrosine bonding between proteins is an important factor in the formation of the oocyst wall of coccidian parasites and are consistent with the hypothesis that antibodies stimulated by vaccination with EmGam56 and related proteins could prevent formation of oocysts
