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 cells; organisms either synthesize PA or acquire them from the environment. How nematodes that parasitize the gut satisfy their PA requirement has not been resolved. The primary regulatory enzyme in PA biosynthesis in most animals is...

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Veröffentlicht in:Experimental parasitology 1997-11, Vol.87 (3), p.171-184
Hauptverfasser: Klein, R.D., Favreau, M.A., Alexander-Bowman, S.J., Nulf, S.C., Vanover, L., Winterrowd, C.A., Yarlett, N., Martinez, M., Keithly, J.S., Zantello, M.R., Thomas, E.M., Geary, T.G.
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container_end_page 184
container_issue 3
container_start_page 171
container_title Experimental parasitology
container_volume 87
creator Klein, R.D.
Favreau, M.A.
Alexander-Bowman, S.J.
Nulf, S.C.
Vanover, L.
Winterrowd, C.A.
Yarlett, N.
Martinez, M.
Keithly, J.S.
Zantello, M.R.
Thomas, E.M.
Geary, T.G.
description Polyamines (PA) are essential for viability and replication of all cells; organisms either synthesize PA or acquire them from the environment. How nematodes that parasitize the gut satisfy their PA requirement has not been resolved. The primary regulatory enzyme in PA biosynthesis in most animals is ornithine decarboxylase (ODC). This enzyme has recently been characterized in free-living nematodes and in the parasitic species,Haemonchus contortus.Nematode and mammalian ODC are reported to differ in subcellular localization, kinetics, and sensitivity to inhibitors. We cloned anH. contortuscDNA that encodes a full-length ODC (sequence data from this article have been deposited with the GenBank Data Library under Accession Nos. AF016538 and AF016891). This cDNA was functionally expressed in strains ofEscherichia coliandSaccharomyces cerevisiaethat lack ODC and are dependent upon exogenous PA for survival. Expression of nematode ODC reversed the PA-dependence phenotype of both microorganisms. The complemented yeast strain was used to develop a nutrient-dependent viability screen for selective inhibitors of nematode ODC. The antiprotozoal drug stilbamidine isethionate was identified as active in this screen, but biochemical characterization revealed that this compound did not inhibit ODC. Instead, like other cationic diamidines, stilbamidine probably inhibits yeastS-adenosylmethionine decarboxylase. Nonetheless, the activity in the screen of the known ODC inhibitor difluoromethylornithine (DFMO) validates the concept that specific recombinant microorganisms can serve as the basis for extremely selective and facile screens.
doi_str_mv 10.1006/expr.1997.4213
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organisms either synthesize PA or acquire them from the environment. How nematodes that parasitize the gut satisfy their PA requirement has not been resolved. The primary regulatory enzyme in PA biosynthesis in most animals is ornithine decarboxylase (ODC). This enzyme has recently been characterized in free-living nematodes and in the parasitic species,Haemonchus contortus.Nematode and mammalian ODC are reported to differ in subcellular localization, kinetics, and sensitivity to inhibitors. We cloned anH. contortuscDNA that encodes a full-length ODC (sequence data from this article have been deposited with the GenBank Data Library under Accession Nos. AF016538 and AF016891). This cDNA was functionally expressed in strains ofEscherichia coliandSaccharomyces cerevisiaethat lack ODC and are dependent upon exogenous PA for survival. Expression of nematode ODC reversed the PA-dependence phenotype of both microorganisms. The complemented yeast strain was used to develop a nutrient-dependent viability screen for selective inhibitors of nematode ODC. The antiprotozoal drug stilbamidine isethionate was identified as active in this screen, but biochemical characterization revealed that this compound did not inhibit ODC. Instead, like other cationic diamidines, stilbamidine probably inhibits yeastS-adenosylmethionine decarboxylase. Nonetheless, the activity in the screen of the known ODC inhibitor difluoromethylornithine (DFMO) validates the concept that specific recombinant microorganisms can serve as the basis for extremely selective and facile screens.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>9371082</pmid><doi>10.1006/expr.1997.4213</doi><tpages>14</tpages></addata></record>
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subjects Amino Acid Sequence
Animals
Base Sequence
cloning
Cloning, Molecular
DNA, Complementary - genetics
Drug Evaluation, Preclinical - methods
Enzyme Inhibitors
Escherichia coli - genetics
expression
Genetic Complementation Test
Haemonchus - enzymology
Haemonchus - genetics
Haemonchus contortus
Helminth Proteins - drug effects
Molecular Sequence Data
nematode
ornithine decarboxylase
Ornithine Decarboxylase - genetics
Ornithine Decarboxylase Inhibitors
Polyamines
Polyamines - metabolism
Recombinant Proteins - biosynthesis
Saccharomyces cerevisiae - genetics
screen
Selection, Genetic
Sequence Homology, Amino Acid
Stilbamidines - pharmacology
yeast
title Haemonchus contortus:Cloning and Functional Expression of a cDNA Encoding Ornithine Decarboxylase and Development of a Screen for Inhibitors
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