Submitted to: Coccidiosis International Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 8/8/2005
Publication Date: 9/15/2005
Citation: Fetterer, R.H., Miska, K.B., Allen, P.C., Lillehoj, H.S., Barfield, R.C. 2005. Characterization of serine proteases in developmental stages of eimeria tenella. Coccidiosis International Conference Proceedings. p. 165.
Technical Abstract: Because of the importance of enzymes of the serine class as targets for novel controls of human diseases, the current study investigates the occurrence and function of serine proteases in Emeria tenella developmental stages. Using gel electrophoresis with casein imbedded gels (zymograms), bands of proteases activity with relative molecular weights (Mr) of 15, 24, 40 and 90 kDa were observed in soluble extracts of oocysts following 0, 12, 24, 48 and 72 hr of sporulation. The proteolytic activity observed both on zymograms and by hydrolysis of FITC-casein in solution was greatly reduced by serine proteases inhibitors and by acidic buffer suggesting that the observed proteolysis was the result of serine proteases. Purification of the proteolytic activity from unsporulated (0 hr) oocysts by affinity and anion exchange chromatography yielded a protein with a Mr of 24 kDa. Assessment of proteolytic activity from in vivo derived merozoites (MZ) demonstrated a similar pattern of proteolytic activity as seen for oocysts. Excretory/secretory products obtained from MZ after 3hr in culture at 41 C were enriched 10 fold in serine proteases activity compared to MZ extract. In contrast, proteolytic activity from soluble extracts of sporzoites (SZ) contained only a single band of serine proteases activity (24 kDa) but ES had little proteases activity. The invasion of E. tenella SZ into cultured cells was inhibited by the irreversible serine protease inhibitor, AEBSF, in a dose dependent manner. AEBSF (1mM) also inhibited the release of the microneme protein MIC2 by SZs into the cell culture media suggesting that AEBS prevents cell invasion by inhibition of a serine protease involved in the release or processing of micronemes. Searches of the E. tenella genome for enzymes homologous to the serine proteases class indicated that trypsin-like enzymes are absent but enzymes related to bacterial subtilisins and a rhomboid protease are present in the genome. Consistent with this finding, analysis of transcripts express by E. tenlla oocysts using substractive hybridization methods indicate the presence of two serine protease, a subtilisin and a rhomboid protease. These finding together suggest that serine proteases are widely distributed in E. tenella developmental stages. The enzymes function during sporulation is unknown but a serine protease may be important for sporozoite cell invasion.