Ornithine decarboxylase gene deletion mutants of Leishmania donovani

A knockout strain of Leishmania donovani lacking both ornithine decarboxyla se (ODC) alleles has been created by targeted gene replacement. Growth of D elta odc cells in polyamine-deficient medium resulted in a rapid and profou nd depletion of cellular putrescine pools, although levels of spermidine we re relatively unaffected. Concentrations of trypanothione, a spermidine con jugate, were also reduced, whereas glutathione concentrations were augmente d. The Delta odc L, donovani exhibited an auxotrophy for polyamines that co uld be circumvented by the addition of the naturally occurring polyamines, putrescine or spermidine, to the culture medium. Whereas putrescine supplem entation restored intracellular pools of both putrescine and spermidine, ex ogenous spermidine was not converted back to putrescine, indicating that sp ermidine alone is sufficient to meet the polyamine requirement, and that L, donovani does not express the enzymatic machinery for polyamine degradatio n. The lack of a polyamine catabolic pathway in intact parasites was confir med radiometrically, In addition, the Delta odc strain could grow in medium supplemented with either 1,3-diaminopropane or 1,5-diaminopentane (cadaver ine), but polyamine auxotrophy could not be overcome by other aliphatic dia mines or spermine. These data establish genetically that ODC is an essentia l gene in L, donovani, define the polyamine requirements of the parasite, a nd reveal the absence of a polyamine-degradative pathway.