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Title: A family of cysteine-rich proteins is involved in the formation of the oocyst wall of Toxoplasma gondii

item POSSENTI, A - Istituto Superiore Di Sanita
item CHERCHI, S - Istituto Superiore Di Sanita
item POZIO, E - Istituto Superiore Di Sanita
item Dubey, Jitender
item SPANO, F - Istituto Superiore Di Sanita

Submitted to: International Journal for Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/20/2010
Publication Date: 11/1/2010
Citation: Possenti, A., Cherchi, S., Pozio, E., Dubey, J.P., Spano, F. 2010. A family of cysteine-rich proteins is involved in the formation of the oocyst wall of Toxoplasma gondii. International Journal for Parasitology. 40:1639-1649.

Interpretive Summary: Toxoplasma gondii is a single-celled parasite of all warm-blooded hosts worldwide. It causes mental retardation and loss of vision in children, and abortion in livestock. Cats are the main reservoir of T. gondii because they are the only hosts that can excrete the resistant stage (oocyst) of the parasite in the feces. Humans become infected by eating undercooked meat from infected animals and food and water contaminated with oocysts. This paper reports molecular characterization of Toxoplasma oocyst. The results will be of interest to biologists, parasitologists, public health workers, and veterinarians.

Technical Abstract: Among apicomplexan parasites, the coccidia and Cryptosporidium spp. are important pathogens of livestock and humans and the environmentally resistant stage (oocyst) is essential for their transmission. Little is known of the chemical and molecular composition of the oocyst wall. Currently, the only parasite molecules shown to be involved in oocyst wall formation are the tyrosine-rich proteins gam56, gam82 and gam230 of Eimeria spp. and the cysteine-rich proteins COWP1 and COWP8 of Cryptosporidium parvum. In the present study, we searched the ToxoDB database for the presence of putative Toxoplasma gondii oocyst wall proteins (OWPs) and identified seven candidates, herein named TgOWP1 through TgOWP7, showing homology to the Cryptosporidium COWPs. We analysed a cDNA library from partially sporulated oocysts of T. gondii and cloned the full-length cDNAs encoding TgOWP1, TgOWP2 and TgOWP3, which consist of 499, 462 and 640 amino acids, respectively. The three proteins share 24% sequence identity with each other and their overall structure is markedly similar, being based on the presence of an N-terminal leader peptide followed by tandem duplications of a 6-cysteine amino acid motif closely related to the Type I repeat of COWPs. Using antisera to recombinant TgOWP1, TgOWP2 and TgOWP3, we showed by Western blot that these molecules are expressed in T. gondii oocysts but are not detectable in tachyzoites. The solubilization of TgOWP1-3 strictly depended on the presence of reducing agents, consistent with a likely involvement of these proteins in multimeric complexes mediated by disulfide bridges. Additionally, TgOWP1, TgOWP2 and TgOWP3 were localized by immunofluorescence to the outer layer of the oocyst wall, thus representing the first validated molecular markers of this structure in T. gondii.