Roles of Tyrosine-Rich Precursor Glycoproteins and Dityrosine- and 3,4-Dihydroxyphenylalanine-Mediated Protein Cross-Linking in Development of the Oocyst Wall in the Coccidian Parasite Eimeria maxima

Abstract

The oocyst wall of apicomplexan parasites protects them from the harsh external environment, preserving their survival prior to transmission to the next host. If oocyst wall formation could be disrupted, then logically, the cycle of disease transmission could be stopped, and strategies to control infection by several organisms of medical and veterinary importance such as Eimeria, Plasmodium, Toxoplasma, Cyclospora, and Neospora could be developed. Here, we show that two tyrosine-rich precursor glycoproteins, gam56 and gam82, found in specialized organelles (wall-forming bodies) in the sexual stage (macrogamete) of Eimeria maxima are proteolytically processed into smaller glycoproteins, which are then incorporated into the developing oocyst wall. The identification of high concentrations of dityrosine and 3,4-dihydroxyphenylalanine (DOPA) in oocyst extracts by high-pressure liquid chromatography, together with the detection of a UV autofluorescence in intact oocysts, implicates dityrosine-and possibly DOPA-protein cross-links in oocyst wall hardening. In addition, the identification of peroxidase activity in the wall-forming bodies of macrogametes supports the hypothesis that dityrosine-and DOPA-mediated cross-linking might be an enzyme-catalyzed event. As such, the mechanism of oocyst wall formation in Eimeria, is analogous to the underlying mechanisms involved in the stabilization of extracellular matrices in a number of organisms, widely distributed in nature, including insect resilin, nematode cuticles, yeast cell walls, mussel byssal threads, and sea urchin fertilization membranes.Eimeria maxima is an intestinal parasite of chickens and is one of the causative agents of coccidiosis, contributing to costs on the order of billions of dollars per year to the poultry meat industry (34, 37). The life cycle of Eimeria includes asexual and sexual stages of development that lead to the formation of the infective form of the parasite, the oocyst (23). Prior to excretion, the oocyst is encapsulated by a hard barrier, the oocyst wall, which protects the parasite from the harsh external environment. Once excreted from the host, the oocyst develops further (sporulation) and is passed onto the next host via the fecal-oral route.The oocyst wall is a structure that is vital to the parasite and ensures the organism's survival and transmission to the next host. It is bilayered, composed of a 10-nm-thick lipid outer layer and a 90-nm-thick glycoprotein inner layer arising from the contents of specialized organelles called wall-forming bodies, which are found exclusively in the sexual stage (macrogamete or macrogametocyte) of the parasite (13,26,31). Progress towards understanding the mechanism of oocyst wall formation in Eimeria has been difficult due to the lack of a good culture system for studying the sexual stages of Eimeria and problems associated with isolating large quantities of parasites. However, with the advent of new technologies in proteomics and molecular biology, together with improved methods for the increased ...

Reassessment of the Role of Aromatic Amino Acid Hydroxylases and the Effect of Infection by Toxoplasma gondii on Host Dopamine

“…The function of AAH1 might be revealed using recently described strategies for inducible knockdown (39). Possible alternative activities of the hydroxylases include the production of dityrosine, which has previously been described in the oocyst walls of Eimeria (40,41). Microarray data describe the upregulation of AAH genes in the oocyst stages of T. gondii (42), suggesting they participate in this process.…”

Excystation of Eimeria tenella Sporozoites Impaired by Antibody Recognizing Gametocyte/Oocyst Antigens GAM22 and GAM56

“…This process of sclerotization involves the formation of dityrosine bonds as well as the emergence of covalent bonds between proteins by peroxidase-mediated mechanisms involving L-3,4-dihydroxyphenylalanine (DOPA). Incidentally, sclerotization of the oocyst wall appears to be comparable to other hardening processes in extracellular matrices, such as insect and nematode cuticles, yeast cell walls, mussel byssal threads, and sea urchin fertilization membranes (3). In E. tenella, the WFBII of macrogametocytes and the inner oocyst wall can be labeled specifically by the monoclonal antibody E2E5 (26).…”

“…Only a few WFBII-localized glycoproteins have been characterized for E. tenella (10) and for E. maxima. They have been shown to undergo site-specific proteolysis before incorporation into the mature oocyst wall (1,3,4,5). Moreover, there is compelling evidence for the occurrence of cross-linking of these tyrosine-rich proteins.…”

Mechanics of the Toxoplasma gondii oocyst wall

“…How these different proteins are processed and/or packed to form the oocyst wall is still not clear (8). Current models support a strong contribution of protein-tyrosine cross-linking in the formation and hardening of the oocyst wall in Toxoplasma-related coccidia (8,12,26,28) and results in the development of its typical blue autofluorescence (AF) under UV excitation (10,26,28,29) (SI Appendix, Fig. S1B).…”