Determining the role of polyamine metabolism in two human pathogenic protozoa : Tichomonas vaginalis and Giardia intestinalis

This work relates the arginine dihydrolase and polyamine pathways to the synthesis of nitric oxide in Trichomonas vaginalis, a microaerophilic eukaryotic protozoan which is the causative agent of the sexually transmitted disease trichomoniasis. When organisms were grown overnight in the presence of 5mM difluoromethylornithine (DFMO), an inhibitor of polyamine synthesis: a combination of plasma and hydrogenosome membrane potential decreased, electron-dense inclusions appeared in greater numbers in hydrogenosomes, and the oxygen consumption rates of hydrogenosomes increased by approximately two-fold. Upon adding exogenous sources of spermine or spermidine, various effects inflicted by the presence of DFMO were virtually reversed. Based on enzyme assays conducted, polyamine depletion by DFMO also caused changes in activities of enzymes in the arginine dihydrolase pathway. An important addition to the polyamine pathway was discovered in the body of this work: the production of nitric oxide by both Giardia intestinalis, an intestinal parasite that causes giardiasis, as well as Trichomonas vaginalis. Fluorimetric detection by confocal laser scanning microscopy and flow cytometry was performed after preincubation with the NO-specific fluorogen 4-amino-5methylamino-2'7'-difluorescein (DAF-FM). Microscopy indicated population heterogeneity with respect to NO production in freshly-harvested organisms and this was confirmed by the broad distribution of fluorescence intensities in the flow cytograms. Specific activities were determined for two nitric oxide synthases from T vaginalis, one located in the cytosol and the other in the hydrogenosome, and one nitric oxide synthase from G. intestinalis, localized to the granular fraction. Bioinformatic searches confirmed the presence of two NOS genes and contain protein motifs typically associated with NOS sequences. The N- terminal domains of both genes lack a NO synthase motif and instead contain motifs normally associated with various iron sulfur proteins (Fe-hydrogenase). Implications of NO production in the evolution, biology and pathogenicity of these important parasites are discussed