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Production of ammonia is difficult to find among the various studies of amino acid metabolism in protozoa. Several studies suggest that catabolism of arginine to ammonium is important for the growth of trichomonads. Trichomonads are amitochondriate zooflagellates that thrive under microaerophilic and anaerobic conditions. The authors were able to detect accumulation of ammonium ions and ammonia in cultures of Tritrichomonas foetus and Trichomonas vaginalis, including those resistant to metronidazole. Ammonium ions and ammonia were detected using the indophenol colorimetric method. Cells incubated overnight under an ambient oxygen gas phase had 0.9 mM soluble ammonium (NH(4)(+) and NH(3)) or a 20 % greater concentration of ammonium relative to sterile growth medium that had been incubated similarly. Production of ammonia itself was confirmed by analysis of a wick that was moistened with sulfuric acid (20 mM) and placed above the liquid in sealed cultures of a strain of Trichomonas vaginalis. The wicks from these cultures captured the equivalent of 0.048 mM volatile ammonia (NH(3)) from the liquid as compared to 0.021 mM volatile ammonia from sterile medium after overnight incubation. Intact trichomonads, 0.7 x 10(6) cells ml(-1) equivalent to 0.7 mg protein ml(-1), incubated in Doran's buffer with or without (1 mM) L-arginine produced significant amounts of soluble ammonium (0.07 mM and 0.04 mM, respectively) during 60 min. The results indicate that ammonium ions and the more irritating ammonia are significant metabolites of trichomonads. In addition, based upon end-product amounts, it appears that the rate of arginine metabolism is of the same order of magnitude as that for carbohydrate metabolism by trichomonads.

Populations of soil amoebas were monitored in two salt marshes in Staten Island, NY for 2 years. One site, Gulfport Reach on the Arthur Kill, has been highly impacted by numerous oil spills. In particular, in 1990 a massive no. 2 fuel oil spill from a ruptured pipe flooded the area; its sediments had total petroleum hydrocarbon (TPH) concentrations in the range 800-46,000 ppm. A reference site 11 km away, on the Atlantic coast, had low TPH levels. Amoeba population densities were in general higher in the impacted sediments. In laboratory microcosm experiments, sediment samples from unimpacted sites were treated with added fresh (unweathered) hydrocarbons (no. 2 fuel oil) and cultured; these also yielded higher amoeba numbers than untreated controls. Four distinct amoeba morphotypes were monitored. Changes in population levels of total amoebas were correlated in the two sites, particularly for morphotype 2 (r = 0.83). The ratios of total amoebas to total bacterial numbers were also correlated (r = 0.85) between the sites. This suggests the amoebas may function as generalists, and that their trophic relation to bacterial prey is not much affected by the presence of petroleum hydrocarbons, but rather may reflect regional parameters such as ambient temperature or other physical factors.

The effects of 5'-deoxy-5'-(hydroxyethylthio)adenosine (HETA), a trypanocidal analog of 5'-deoxy-5'-(methylthio)adenosine (MTA), on polyamine synthesis and S-adenosylmethionine (AdoMet) metabolism were examined in bloodstream forms of Trypanosoma brucei brucei. HETA was cleaved by trypanosome MTA phosphorylase at the same rate as the natural substrate, MTA, in a phosphate-dependent reaction. Fluorine substitution at the 2-position of the purine ring increased activity by approximately 50%, whereas substitution with an amino group reduced activity to about one-third of the control. HETA was accumulated by trypanosomes with internal concentrations of 100-250 microM and >800 microM after a 15-min incubation with 1 and 10 microM, respectively. Trypanosomes preincubated with HETA metabolized it at a rate of 21.9 nmol/hr/mg protein. Preincubation of cells with HETA at 1 or 10 microM inhibited spermidine synthesis from [3H]ornithine by 22-37%, and increased the cytosolic levels of AdoMet by 2- to 5-fold and that of MTA by up to 8-fold. S-Adenosylhomocysteine (AdoHcy) levels also increased 1.5- to 7-fold in treated cells, whereas decarboxylated AdoMet decreased 65%. Preincubation of trypanosomes with HETA for 4 hr also reduced the incorporation of [35S]methionine in trichloroacetic acid-precipitable material by 50-60%, and reduced the methyl group incorporation into protein from [U-14C]methionine by 65-70%. Thus, HETA interferes with a series of biochemical events involving the participation of AdoMet and methionine in polyamine synthesis, protein synthesis, and transmethylation reactions.

Succinate thiokinase displays a diversity of nucleotide specificity and molecular size throughout Nature. Eukaryotes and Gram-positive bacteria possess distinct 'small' (dimeric) thiokinase enzymes which are specific for adenine (ADP) or guanine (GDP) nucleotides, whereas Gram-negative bacteria contain a single 'large' (tetrameric) enzyme which utilizes both nucleotides. Succinate thiokinase activities, both ADP- and GDP-dependent, were shown to be hydrogenosomal in Tritrichomonas foetus and Trichomonas vaginalis. Surprisingly, the 'small' enzyme was found in T. foetus whereas T. vaginalis contained a 'large' enzyme.

We have determined the primary structure of the [2Fe-2S]ferredoxin of the anaerobic protist Trichomonas vaginalis. This protein, situated in the hydrogenosome, is composed of 93 amino acids. A comparison of T. vaginalis ferredoxin with greater than 80 other ferredoxins shows the closest similarity to [2Fe-2S]putidaredoxin of the aerobic bacterium Pseudomonas putida and a lesser one to mitochondrial [2Fe-2S]ferredoxins of vertebrates. This similarity is reflected in the overall primary structure and in the spacing of cysteine residues coordinating the iron-sulfur center. The primary structure, but not the environment of the iron-sulfur center, also shows similarity with [2Fe-2S]ferredoxins of photosynthetic organisms and halobacteria. We have cloned and analyzed the T. vaginalis ferredoxin gene. The gene is present in a single copy and devoid of introns. It gives rise to a transcript with unusually short 5' and 3' untranslated regions of 16 and 18 nucleotides, respectively. DNA sequence analysis of the gene predicts an additional 8 amino acids at the amino terminus which are absent from the purified protein. This amino-terminal region of the protein is characterized by properties typical of mitochondrial presequences.

We have identified Trichomonas vaginalis strains resistant in vitro to metronidazole, especially under aerobic conditions. Since little is known about the relationship of treatment outcome to metronidazole susceptibility of T. vaginalis, we studied the aerobic and anaerobic susceptibility to metronidazole of 310 clinical isolates of T. vaginalis. Of 199 patients with known outcomes after metronidazole treatment for vaginal trichomoniasis, the geometric mean minimal lethal concentration (MLC) under aerobic conditions for trichomonads associated with cases cured by a single 2-g dose was 24.1 micrograms/ml (n = 146), while that of treatment-resistant isolates (n = 53) was 195.5 micrograms/ml. The corresponding mean anaerobic MLC values were 1.6 and 5.05 micrograms/ml, respectively. The average aerobic:anaerobic MLC ratio was about twofold higher for the resistant isolates. Treatment resistance was more frequent at aerobic MLC values of greater than 25 micrograms/ml or anaerobic values of greater than 1.6 micrograms/ml. Although there was overlap of the metronidazole susceptibility distribution of susceptible and resistant isolates, significant resistance to treatment was common when isolates of T. vaginalis had aerobic MLC values of greater than 100 micrograms/ml or anaerobic MLC values of greater than 3.1 micrograms/ml.

There are currently no laboratory or clinical guidelines for the identification and treatment of disease caused by metronidazole-resistant strains of Trichomonas vaginalis. Fifty-three isolates of T. vaginalis from cases of refractory vaginitis in the United States (26 states) and Canada were tested for aerobic and anaerobic metronidazole susceptibility, and after various dosages of metronidazole, the therapeutic outcomes were evaluated for 31 of these cases. The mean aerobic metronidazole susceptibility of these isolates was 195.5 micrograms/ml (range, 12.5-greater than 1,000), which was about eightfold higher than that seen in isolates that were not resistant to metronidazole. The mean anaerobic susceptibility was 5 micrograms/ml (range, 1.6-25), which was about threefold higher than that of isolates from nonresistant strains. The average aerobic-to-anaerobic ratio of metronidazole susceptibility in the highly resistant isolates was more than 3.5-fold greater than that seen in the nonresistant isolates. White women accounted for 88% of the resistant infections. Of 31 cases that were re-treated and monitored, the highest average dose that failed to achieve a cure was 2.1 g of metronidazole/day given over an eight-day period; 27 (87%) of 31 cases were ultimately cured with an average dosage of 2.6 g of metronidazole/day given over a mean period of nine days. Resistance to treatment with metronidazole varied from mild to severe, and the resistance was occasionally more severe than the susceptibility values indicate.

Trichomonas vaginalis grown in iron-enriched medium contained increased concentrations of iron-sulfur proteins, including ferredoxin and pyruvate-ferredoxin oxidoreductase. The increases in hydrogenosomal constituents correlated with increased in vivo hydrogenosomal metabolism.