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.

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Steven H. Isabelle. / "May 1995." / Includes bibliographical references (p. 157-161). / Supported in part by the Department of the Navy, Office of the Chief of Naval Research as part of the Advanced Research Projects Agency's RASSP Program. N00014-93-1-0686 Supported in part by the U.S. Air Force Office of Scientific Research. AFOSR-91-0034-C

The main aims and objectives of this study was to determine if plant essential oils (PEOs) and polyphenol-rich fruit extracts (PRFEs) could reduce the viability of Giardia duodenalis trophozoites, Trypanosoma cruzi epimastigotes and Cryptospordium parvum oocysts in vitro. All PEOs tested reduced epimastigote and trophozoite viability at a concentration of 0.02% v/v, with titrations of the PEOs showing a concentration dependant decrease in viability. The minimum inhibitory concentrations (MICs) of PEOs demonstrated that myrtle and elemi oil were the most active PEOs (trophozoites = 0.005% v/v; epimastigotes = 0.00125% v/v) with the terpenes, α-pinene and limonene, constituents of these oils, being responsible for their action. Incubation of palmarosa oil and its terpene, geraniol, with C. parvum oocysts caused the almost complete excystation of oocysts (in the presence of increased temperature and time), with geranium oil and its terpene, citronellol, being nearly as effective. PRFEs reduce trophozoite viability, with 4 members of the Rosaceae Family causing complete reduction at 167 μg ml-1, possibly through their ellagitannin content. Cloudberry extract was found to have an MIC comparable to the drug metronidazole (67 μg ml-1). The historical use of blueberries for the treatment of diarrhoeal diseases was demonstrated by the ability of blueberry PRFE, pressed juice and drink to kill trophozoites. Protein expression was both inhibited and upregulated in several proteins in whole cell lysates of PEO treated trophozoites, indicating a supplemental intracellular mode of action. Both PEOs and PRFEs cause morphological changes to epimastigotes and trophozoites through flagellar truncation and internalisation, swelling and rounding of the cell body, cytoplasmic condensation and the formation of large membrane protrusions. These indicate an action on the membrane itself with possible changes in osmoregulation. Both PEOs and PRFEs can be considered to be candidates for novel drug discovery for the treatments of cryptosporidiosis, giardiasis and American trypanosomiasis.