Characterization of Serine Proteases in Developmental Stages of Eimeria tenella
R.H. Fetterer, K.B. Miska, P.C. Allen, H. Lillehoj and R. C. Barfield.
Animal Parasitic Disease Laboratory,
Animal and Natural Resources Institute,
USDA, ARS, Beltsville, Maryland. USA.
rfettere@anri.barc.usda.gov
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 Eimeria 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. Inhibition of the activity by serine protease inhibitors suggests
that the observed proteolysis was the result of a serine protease. Purification
of the proteolytic activity from unsporulated (0 hr) oocysts by affinity and
anion exchange chromatography yielded a protein with an Mr of 24 kDa. Assessment
of proteolytic activity from in vivo derived merozoites (MZs) demonstrated a
similar pattern of proteolytic activity as seen for oocysts. Excretory/secretory
(ES) products obtained from MZ after 3hr in culture at 41 C were enriched 10
fold in serine protease activity compared to MZ extract. In contrast,
proteolytic activity from soluble extracts of sporozoites (SZs) contained only a
single band of serine proteases activity (24 kDa), but ES had little protease
activity. The invasion of E. tenella SZs 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 AEBSF 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 protease 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 expressed by
E. tenlla oocysts using substractive hybridization methods indicate the presence
of two serine protease, a subtilisin and a rhomboid protease. These findings
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.
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