title: Eimeripain, a cathepsin B-like cysteine protease, expressed throughout sporulation of the apicomplexan parasite Eimeria tenella.
abstract: The invasion and replication of Eimeria tenella in the chicken intestine is responsible for avian coccidiosis, a disease that has major economic impacts on poultry industries worldwide. E. tenella is transmitted to naïve animals via shed unsporulated oocysts that need contact with air and humidity to form the infectious sporulated oocysts, which contain the first invasive form of the parasite, the sporozoite. Cysteine proteases (CPs) are major virulence factors expressed by protozoa. In this study, we show that E. tenella expresses five transcriptionally regulated genes encoding one cathepsin L, one cathepsin B and three cathepsin Cs. Biot-LC-LVG-CHN₂, a cystatin derived probe, tagged eight polypeptides in unsporulated oocysts but only one in sporulated oocysts. CP-dependant activities were found against the fluorescent substrates, Z-FR-AMC and Z-LR-AMC, throughout the sporulation process. These activities corresponded to a cathepsin B-like enzyme since they were inhibited by CA-074, a specific cathepsin B inhibitor. A 3D model of the catalytic domain of the cathepsin B-like protease, based on its sequence homology with human cathepsin B, further confirmed its classification as a papain-like protease with similar characteristics to toxopain-1 from the related apicomplexan parasite, Toxoplasma gondii; we have, therefore, named the E. tenella cathepsin B, eimeripain. Following stable transfection of E. tenella sporozoites with a plasmid allowing the expression of eimeripain fused to the fluorescent protein mCherry, we demonstrated that eimeripain is detected throughout sporulation and has a punctate distribution in the bodies of extra- and intracellular parasites. Furthermore, CA-074 Me, the membrane-permeable derivative of CA-074, impairs invasion of epithelial MDBK cells by E. tenella sporozoites. This study represents the first characterization of CPs expressed by a parasite from the Eimeria genus. Moreover, it emphasizes the role of CPs in transmission and dissemination of exogenous stages of apicomplexan parasites.
authors.affiliation: INRA, UMR1282, Equipe Pathogenèse des Coccidioses, Infectiologie et Santé Publique, Nouzilly, France.
authors.forename: Anaïs
authors.initials: A
authors.lastname: Rieux
authors.forename: Simon
authors.initials: S
authors.lastname: Gras
authors.forename: Fabien
authors.initials: F
authors.lastname: Lecaille
authors.forename: Alisson
authors.initials: A
authors.lastname: Niepceron
authors.forename: Marilyn
authors.initials: M
authors.lastname: Katrib
authors.forename: Nicholas C
authors.initials: NC
authors.lastname: Smith
authors.forename: Gilles
authors.initials: G
authors.lastname: Lalmanach
authors.forename: Fabien
authors.initials: F
authors.lastname: Brossier
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain; D002403:Cathepsins / Q000145:classification / Q000235:genetics / Q000378:metabolism; D057056:Cysteine Proteases / Q000378:metabolism*; D017931:DNA Primers; D004285:Dogs; D016786:Eimeria tenella / Q000201:enzymology* / Q000254:growth & development; D015971:Gene Expression Regulation, Enzymologic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D060888:Real-Time Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D037521:Virulence Factors / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D037521:Virulence Factors; D002403:Cathepsins; D057056:Cysteine Proteases; C574014:eimeripain protein, Eimeria tenella