falciparum subtilisin 1 is inhibited in exactly the same fashion. Subtilisins are further implicated in the formation of the oocyst wall of Eimeria through analogy with their known role in the formation of the cuticle of nematodes. Thus, the assembly of collagens to form the cuticle involves a inhibitor order us number of molecular events that strikingly resemble our model of oocyst wall formation pathways, first, collagens are the re sult of degradation of proproteins by a subtilisin like prote ase, and, second, these collagens are subsequently bonded together by di and tri tyrosine crosslinks. A failure in either of these steps, results in a malformed cu ticle and parasite death. Subtilisins are currently being further investigated as potential candidates in the catalytic cleavage of the oocyst wall precursor proteins.

Conclusion Eimeria tenella possesses a large number of genes coding for proteolytic enzymes, which display a remarkable pattern of stage specific expression. As in other apicomplexan para sites such as P. falciparum and T. gondii, expression of many of these genes is upregulated in the asexual, invasive stages, possibly indicating important roles in host cell inva sion, remodelling and egress. However, expression of al most one third of the protease genes identified in the E. tenella genome is upregulated or confined to the sexual gametocyte stage of this parasites lifecycle, some of these appear to be unique to Coccidia and may play key roles in the formation of the resilient oocyst wall, a defining feature of this group of important parasites.

Methods Data base mining Eimeria tenella genome sequences and gene models were downloaded from GeneDB. The genome of E. tenella was produced by the Parasite Genomics Group at the Well come Trust Sanger Institute and has been provided prepublication. The E. tenella genome database was searched for genes predicted to code for proteins with peptidase ac tivity. All auto annotated peptidase genes identified were manually curated by performing BLAST analysis against apicomplexan genome sequence databases and against vari ous protein databases such as the protein data bank, Swiss Prot and non redundant protein se quence databases. In addition, signature protein motifs for the protein sequence of each gene were identified through Pfam, InterproScan and the MER OPS databases.

Further gene sequence manipulations, such as translation into amino acid sequences and ClustalW alignments, were per formed using the DNASTAR Lasergene 9 Core Suite. After the bioinformatic information was collated, genes were assigned a five tiered level of confidence for gene function using an Evidence Rating system giving an overall score of ER1 5, where ER1 indicates extremely reli able experimental data Carfilzomib to support function and ER5 indi cates no evidence for gene function.