HAEMONCHUS-CONTORTUS - CLONING AND FUNCTIONAL EXPRESSION OF A CDNA-ENCODING ORNITHINE DECARBOXYLASE AND DEVELOPMENT OF A SCREEN FOR INHIBITORS

Polyamines (PA) are essential for viability and replication of all cel ls; organisms either synthesize PA or acquire them from the environmen t. How nematodes that parasitize the gut satisfy their PA requirement has not been resolved. The primary regulatory enzyme in PA biosynthesi s in most animals is ornithine decarboxylase (ODC). This enzyme has re cently been characterized in free-living nematodes and in the parasiti c species, Haemonchus contortus. Nematode and mammalian ODC are report ed to differ in subcellular localization, kinetics, and sensitivity to inhibitors. We cloned an H. contortus cDNA that encodes a full-length ODC (sequence data from this article have been deposited with the Gen Bank Data Library under Accession Nos. AF016538 and AF016891). This cD NA was functionally expressed in strains of Escherichia coli and Sacch aromyces cerevisiae that lack ODC and are dependent upon exogenous PA for survival. Expression of nematode ODC reversed the PA-dependence ph enotype of both microorganisms. The complemented yeast strain was used to develop a nutrient-dependent viability screen for selective inhibi tors of nematode ODC. The antiprotozoal drug stilbamidine isethionate was identified as active in this screen, but biochemical characterizat ion revealed that this compound did not inhibit ODC. Instead, like oth er cationic diamidines, stilbamidine probably inhibits yeast S-adenosy lmethionine decarboxylase. Nonetheless, the activity in the screen of the known ODC inhibitor difluoromethylornithine (DFMO) validates the c oncept that specific recombinant microorganisms can serve as the basis for extremely selective and facile screens. (C) 1997 Academic Press.