Extensive study of Plasmodium species and T. gondii has Inhibitors,Modulators,Libraries established that proteases help to coordinate and regulate the lifecycles of these parasites, playing key roles in host cell invasion, general catabolism, host cell remodelling and egress from host cells. These processes are all associated with the asexual stages of apicomplexan parasites. By contrast, relatively little is known about what roles proteases may play in the sexual phase of the apicomplexan lifecycle though it is known that a subtilisin 2 is detected specifically in the gametocyte proteome and expression of falcipain 1 is upregulated in gametocytes of P. falciparum. Moreover, it has been demonstrated that the cysteine protease inhibitor, E64d, or the targeted genetic disruption of falcipain 1 can inhibit oocyst production in P.
falciparum. Likewise, the proteosome inhibitors, epoxomicin and thiostrepin, ex hibit Inhibitors,Modulators,Libraries gametocytocidal activity. In comparison to P. falciparum and T. gondii, pro teases from Eimeria GSK-3 species have been studied far less intensively, despite the economic importance of this genus of parasites. Thus, homologs or orthologs of several classes of proteases found Inhibitors,Modulators,Libraries in P. falciparum and or T. gondii have also been identified in Eimeria species including an aspartyl protease, an aminopeptidase, a rhomboid protease, a subtilisin 2 like pro tease, three cathepsin Cs, a cathepsin L and an orthologue of toxopain, a cathepsin B cyst eine protease. As for P. falciparum and T. gondii, these proteases have been found in the asexual stages of Eimeria and are mostly predicted to play roles in host cell invasion, though expression of some of these enzymes is associated with the sporulation of the devel oping oocyst.
However, it is hypothesized that proteolytic processing of two proteins from the wall forming bodies of the macrogametocytes of Eimeria GAM56 and GAM82 is essential for the subsequent incorporation of tyrosine rich peptides into the oocyst wall. In this study, we screened Inhibitors,Modulators,Libraries the E. tenella genomic data base for genes encoding proteases, classified these into clans and families and designed PCR probes for them. Using cDNA produced from E. tenella stage specific mRNA, we carried out semi quantitative PCR to deter mine the stage specificity of expression of the protease genes, especially to identify protease mRNAs that were upregulated in gametocytes. In order to further resolve which of these may be involved in oocyst wall formation, we carried out a processing assay using gametocyte extracts of E. tenella, whereby a variety of specific prote ase inhibitors were tested for their ability to inhibit the processing of GAM56 into smaller, putative oocyst wall proteins. Results Identification of potential protease genes in Eimeria tenella The genome of E.