RESUMO
Eimeria tenella is the causative agent of coccidiosis in poultry. Infection of the chicken intestine begins with ingestion of sporulated oocysts releasing sporocysts, which in turn release invasive sporozoites. The monoclonal antibody E2E5 recognizes wall-forming body type II (WFBII) in gametocytes and the WFBII-derived inner wall of oocysts. Here we describe that this antibody also binds to the stieda body of sporocysts and significantly impairs in vitro excystation of sporozoites. Using affinity chromatography and protein sequence analysis, E2E5 is shown to recognize EtGAM56, the E. tenella ortholog of the Eimeria maxima gametocyte-specific GAM56 protein. In addition, this antibody was used to screen a genomic phage display library presenting E. tenella antigens as fusion proteins with the gene VIII product on the surfaces of phagemid particles and identified the novel 22-kDa histidine- and proline-rich protein EtGAM22. The Etgam22 mRNA is expressed predominantly at the gametocyte stage, as detected by Northern blotting. Southern blot analysis in combination with data from the E. tenella genome project revealed that Etgam22 is an intronless multicopy gene, with approximately 12 to 22 copies in head-to-tail arrangement. Conspicuously, Etgam56 is also intronless and is localized adjacent to another gam56-like gene, Etgam59. Our data suggest that amplification is common for genes encoding oocyst wall proteins.
Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Eimeria tenella/imunologia , Oocistos/imunologia , Proteínas de Protozoários/imunologia , Esporozoítos/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Western Blotting , Galinhas , Cromatografia de Afinidade , Clonagem Molecular , Coccidiose/parasitologia , Eimeria tenella/crescimento & desenvolvimento , Eimeria tenella/metabolismo , Imunofluorescência , Masculino , Dados de Sequência Molecular , Oocistos/crescimento & desenvolvimento , Biblioteca de Peptídeos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
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RESUMO
Though parasite cyclophilins are promising new drug targets, Eimeria tenella cyclophilins have not been characterized yet. Here, we describe an 89kDa cyclophilin, designated EtCYP89. It is expressed throughout the developmental cycle of E. tenella, both in the intracellular stages in chicken and in extracellular sporulated oocysts and sporozoites. The EtCYP89 protein contains two Ser-rich domains in its NH2-terminus separated by a His-rich stretch. WD40 repeats are localized in the central part of the protein followed by a cyclophilin domain at the COOH-terminus. Both protein and genomic organization of EtCyp89 are conserved in comparison with its ortholog TgCyp81.6 in Toxoplasma gondii, except for the absence of a Ser- and His-rich NH2-terminus in TgCYP81.6. In particular, those 13 residues are conserved which are responsible for binding the anti-coccidial drug cyclosporine A.