1 Haskins Laboratories, Pace University, New York, NY 10038, USA
2 Department of Chemistry and Physical Sciences, Pace University, New York, NY 10038, USA
3 Department of Biology, Pace University, New York, NY 10038, USA
Correspondence
Thomas E. Gorrell
tgorrell{at}pace.edu
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ABSTRACT |
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INTRODUCTION |
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Studies indicate that trichomonads require arginine for growth (Kidder, 1951). Trichomonads have an arginine dihydrolase pathway that may supplement the cells' ability to form ATP (Linstead & Cranshaw, 1983
; Yarlett et al., 1996a
). Protozoa are regarded, along with bacteria and metazoa, as primarily ammonotelic (Kidder, 1967
; Yoshida & Camargo, 1978
). Maroulis et al. (2003)
have suggested that trichomonads rely upon the transport of inorganic ions (e.g. potassium ions) during hyperosmotic stress to maintain the cell volume; presence of ammonium was not mentioned. Knodler et al. (1994)
did not see changes in the ammonium content of spent medium after growth of Tri. vaginalis. Studies of the metabolism of ammonium and the more-basic and toxic compound ammonia are difficult to find in the literature describing the metabolism of amino acids by protists (Cazzulo, 2003
; Cazzulo et al., 1985
; Gutteridge & Coombs, 1977
; Honigberg, 1967
; Knodler et al., 1994
; Marr, 1979
). In this study, we were able to show the accumulation of significant amounts of soluble ammonium (
) and volatile ammonia (NH3) by cultures of trichomonads after growth in complex media. Production of soluble ammonium from L-arginine indicated that there is a greater metabolic rate through the arginine dihydrolase pathway than suggested previously for Trt. foetus and most likely Tri. vaginalis.
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METHODS |
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Cells of trichomonads and Try. brucei brucei were counted using a Neubauer haemocytometer. Tri. vaginalis grown in this study, from vaginal exudates and from an in vivo mouse (intraperitoneal) model, have an ovoid shape as compared to the amoeboid forms seen during growth on agar plates, attached to the vaginal epithelial mucosa, and in an in vivo mouse subcutaneous model (Nielsen & Nielsen, 1975; Warton & Honigberg, 1979
; Yarlett, 2000
). It is not clear if there are metabolic differences between these forms. Cultures of Trt. foetus KV1-1MR-100 and RU393 were examined microscopically using a 40x objective for microbial contaminants. Heat-fixed slides stained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) had defined spherical nuclear DNA but no extranuclear DNA that would be indicative of contamination with mycoplasma.
Metabolic studies and analytical methods.
For studies of the metabolism of arginine, cells were harvested by centrifugation, washed in Doran's buffered solution (74 mM NaCl, 1·6 mM KCl, 0·6 mM CaCl2, 30 mM NaH2PO4; pH 6·4) and resuspended to 107 cells (equivalent to 1 mg protein) (ml buffer)1 based upon protein determinations for strain C1-NIH. In this buffer, cells remain motile and are metabolically active (Müller & Gorrell, 1983; Yarlett et al., 1996a
). Cells were incubated with or without 1 mM L-arginine, for 60 min at 37 °C. Cells were cooled to 4 °C and removed by centrifugation before analysis of soluble ammonium in the supernatant fluid. Protein content was determined by the Bradford method.
Soluble and volatile ammonium.
The indophenol colorimetric method of Berthelot (1859) cited in Chaney & Marbach (1962)
and modified by Weatherburn (1967)
was used to determine the amounts of ammonium ions (
) and ammonia (NH3). Absorbance was measured using a Beckman DU-640 spectrophotometer [
=0·0124 A625 nmol1 (ml NH4Cl)1]. Reaction mixtures contained 1 ml of phenate reagent consisting of 1·1 % (v/v) phenol, 0·005 % (w/v) sodium nitroprusside and 1 ml alkaline hypochlorite [0·8 % (v/v) sodium hypochlorite (Clorox) and 0·6 % (w/v) sodium hydroxide].
Soluble ammonium ( and NH3) was measured by placing a 5 µl sample of the liquid from overnight cultures in a sealed glass scintillation vial that contained a wick moistened with (20 mM) sulfuric acid to trap ammonium released by the addition of 2·5 mM boric acid pH 9·5 and subsequently incubated overnight at room temperature. The wick (0·5 cmx1 cm) was cut from a piece of Whatman filter paper. The wick was then assayed with the indophenol method for soluble ammonium. Similar results were obtained by directly adding the reagents for the formation of indophenol to the 5 µl sample of culture (Weatherburn, 1967
). Trichomonads for these experiments were grown in 5 ml of TYM medium using screw-capped culture tubes, or in 1 ml of TYM medium using 24-well tissue culture plates. For aerobic incubation, tubes and plates were incubated in an ambient atmosphere. Cysteine in the medium would maintain the cultures in the tubes under lower redox condition than those incubated in the multi-well plates. The direct indophenol method was used in preliminary studies to determine the concentration of urea in the medium by incubating (37 °C, 20 min) the medium (4 µl) in 0·2 ml of 100 mM sodium phosphate, 26 mM EDTA buffer pH 7·1 with or without commercially available urease (0·08 mg; urea amidohydrolase EC 3.5.1.5; Sigma).
Volatile ammonia (NH3) production was confirmed by analysis of a wick that was moistened with sulfuric acid and placed above the liquid during the growth of the cells (5 ml) in sealed glass scintillation vials (20 ml). The wick was then assayed with the indophenol method for volatile ammonia. Try. brucei brucei was grown in 10 ml of medium using 50 ml plastic tissue culture flasks.
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RESULTS AND DISCUSSION |
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The amounts of soluble ammonium detected in trichomonad cultures were greater than expected from Knodler et al. (1994) but comparable to values that can be calculated from studies of amino acid metabolism by trichomonads (Zuo et al., 1995
; Knodler et al., 1994
; Rowe & Lowe, 1986
). Knodler et al. (1994)
detected less soluble ammonium in spent media for Tri. vaginalis compared to fresh media, whereas their studies showed that spent medium from cultures of Crithidia and Giardia, another amitochondriate protozoan, had increased levels of soluble ammonium. Studies show greatest loss of the basic amino acids arginine and lysine (Zuo et al., 1995
; Knodler et al., 1994
) from the medium and the greatest increase for alanine and proline; these changes seem to be dependent on the protozoan strain (Zuo et al., 1995
; Knodler et al., 1994
). Putrescine also accumulates in the medium (Yarlett, 1988
). There may be strain-dependent variations among Tri. vaginalis isolates for the production of amino acids. Strain C1-NIH did not produce alanine (Steinbuchel & Müller, 1986
; ter Kuile, 1996
). Glutamate was removed from the growth medium in the studies of Zuo et al. (1995)
, whereas Knodler et al. (1994)
detected increased amounts of glutamate. Chyle et al. (1971)
detected several isoenzymes for glutamate dehydrogenase. Biochemical characterization (Turner & Lushbaugh, 1988
) of the glutamate dehydrogenase activity indicated the deamination reaction had a pH optimum of 8·0 and a Km for glutamate about equal to that of the intracellular concentration of glutamate. Cells of Tri. vaginalis have a methionine gamma lyase (Coombs & Mottram, 2001
) which may explain the decreased amounts of methionine seen in spent medium (Knodler et al., 1994
). This enzyme produces ammonia and methanethiol and is distinct from other methionine gamma lyases. Marr (1979)
suggested that protozoa such as Leishmania fix ammonia into amino acids for detoxification purposes.
The loss of arginine from the medium (Zuo et al., 1995; Knodler et al., 1994
) and increased putrescine present (Yarlett, 1988
) indicate that ammonium was produced through the arginine dihydrolase pathway. Tri. vaginalis lacks arginase, urease (Linstead & Cranshaw, 1983
) and an arginine aminotransferase. Tri. vaginalis does, however, have an ornithine/lysine aminotransferase (among other aminotransferases) (Lowe & Rowe, 1986
). This enzyme activity may explain the production of proline in the growth medium via ornithine provided by the arginine dihydrolase pathway. Proline may also accumulate from metabolism of glutamate. Ornithine from the pathway can be metabolized to putrescine (Yarlett, 2000
) as detected in vitro and indicated from in vivo studies (Chen et al., 1982
). Tri. vaginalis does secrete proteases (Scott et al., 1995
). Their influence on the available amino acids in cultures is not clear. Each of the strains of two genera of trichomonads used in this study showed increased amounts of soluble ammonia, and at least for Tri. vaginalis volatile ammonia. Production of ammonium was further detected by studies of cell suspensions of Tri. vaginalis and Trt. foetus in a buffered salt solution.
Catabolism of arginine was determined by the detection of soluble ammonium in incubations of Trt. foetus and Tri. vaginalis under aerobic conditions in a buffered salt solution. The results are shown in Table 4. The amount of soluble ammonium produced was greater than expected from previous studies that measured CO2 production from [guanidino-14C-]arginine (Linstead & Cranshaw, 1983
) or [U-14C]arginine (Yarlett et al., 1996a
). Tri. vaginalis and Trt. foetus (Linstead & Cranshaw, 1983
; Yarlett et al., 1996b
) have an active arginine dihydrolase pathway (Fig. 1
). Cells of Tri. vaginalis (Knodler et al., 1994
) have a twofold greater concentration of the intermediates (arginine, citrulline and ornithine) than Trt. foetus (Maroulis et al., 2003
). The metabolic rate for soluble ammonium is less than the specific activity of carbamate kinase, which catalyses the release of a second ammonium in cell extracts. Of these enzymes, the carbamate kinase has been characterized at the molecular level (Minotto et al., 2000
). The arginine deiminase (Yarlett et al., 1996b
) is localized in a membrane-bound particle but sedimented at a lower density than hydrogenosomes. The remaining enzyme activities of the arginine dihydrolase pathway including carbamate kinase, which would remove a second nitrogen as ammonium or ammonia, were found in the non-sedimentable fraction of Tri. vaginalis.
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Tri. vaginalis and Trt. foetus have sufficient concentrations of the other amino acids (12 µmol per 108 cells; Knodler et al., 1994; Maroulis et al., 2003
) to explain the accumulation of ammonium when arginine was not added to the cell suspension. Cells retained the ovoid motile forms as seen in culture. In addition, for Trt. foetus no decrease in protein was detected after 60 min (0·114 mg protein ml1) versus zero time (0·09 mg protein ml1) of incubation in the buffer.
The combined results indicate that trichomonads produce ammonium ions and the potentially more irritable ammonia during growth. Previous work provides the biochemical information to support the idea that the measurement of soluble ammonium from arginine by trichomonads provided a method to detect the metabolic rate of the arginine dihydrolase pathway by intact cells. Anaerobic rumen ciliates (Mah & Hungate, 1965) are thought to produce ammonium by the deamination of amino acids (Coleman, 1979
). Chen et al. (1982)
have detected decreased levels of alanine, putrescine, cadaverine and gamma aminobutyric acid in vaginal fluid after treatment of vaginitis patients with metronidazole. Amounts of ammonium in vaginal fluid were not mentioned (Chen et al., 1982
; Petrin et al., 1998
; Pybus & Onderdonk, 1997
), whereas production of ammonia by bacteria growing on epithelial tissues of the human digestive tract has been studied more extensively (Casiano-Colon & Marquis, 1988
; Verdu et al., 1998
). Further studies of ammonia metabolism by protozoa will most likely reveal interesting variations on the theme of nitrogen and energy metabolism, along with understanding endosymbiotic origins of protozoa.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 26 November 2003;
revised 25 February 2004;
accepted 1 March 2004.
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