Hydrogen peroxide is a product of oxygen consumption by Trichomonas vaginalis
Alan Chapman*, David J Linstead and David Lloyd
School of Biosciences (Microbiology, Main Building),
Cardiff University of Wales, PO Box 915,
Cardiff, CF1 3TL, Wales UK
*ML Laboratories PLC, 60 London Road, St. Albans, AL1 1NG, UK
Corresponding author (Fax, 44 1222 874305; Email, email@example.com).
The amitochondriate sexually-transmitted human parasitic protozoan Trichomonas vaginalis (Bushby strain) grown anaerobically on complex medium containing cysteine and ascorbic acid consumed O2 avidly (6·9 µM
min1 per 106 organisms) with an apparent Km value of 5·1 µM O2 : O2 uptake was inhibited by O2 > 120 µM. Spectrophotometric assays in the presence of microperoxidase (419407 nm) indicated that H2O2 was produced and that inhibition by high O2 concentrations was again evident. Hydrogenosomes oxidizing pyruvate in the presence of ADP and succinate showed similar patterns of O2 consumption, H2O2 production (33·5 pmol min1 per mg protein), and O2 inhibition. Cytosolic NADH oxidase gave no detectable H2O2, whereas the cytosolic NADPH oxidase produced H2O2 at a rate (43 pmol min1 per mg protein) greater than that of hydrogenosomes. These results are discussed in relation to the oxidative stress experienced by the pathogen in its natural habitat.
An understanding of the oxidative metabolism of the amitochondriate flagellated parasitic protozoan, Trichomonas vaginalis, an extremely common cause of human genitourinary infections, is paramount in devising strategies for chemotherapy (Milne et al 1978; Lloyd and Petersen 1985). The redox-active organelles of trichomonads, the hydrogenosomes (Lindmark and Müller 1973) are responsible for the oxidation of pyruvate (Cerkasov et al 1978). Under anaerobic conditions hydrogenase is active and protons serve as the terminal electron acceptor for a chain of flavin and iron sulphur carriers (Lindmark and Müller 1973; Chapman et al 1986). When exposed to low O2 concentrations (e.g., 20 µM O2) the carbon fluxes through the alternative branching pathways from pyruvate are adjusted (Ellis et al 1992), hydrogenase activity becomes inhibited (Lloyd and Kristensen 1985), and O2 serves as terminal electron acceptor (Lloyd et al 1983). NADH and NADPH oxidation also occurs in hydrogenosome-enriched fractions (Lindmark and Müller 1973; Lloyd et al 1983) with a variety of electron acceptors including O2. T. vaginalis also possesses a highly active cytosolic NADH oxidase (Tanabe 1979; Brown et al 1996) and an NADPH: acceptor oxidoreductase (EC 18.104.22.168) which can use O2 as electron acceptor (Linstead 1983; Linstead and Bradley 1987). As the vagina is not a strictly anaerobic habitat, but rather may have regions where O2 can occasionally reach almost 60 µM (Wagner and Levin 1978), it is important to investigate O2 scavenging as a mechanism of protection against O2 toxicity (Ellis et al 1994) in this microaerophilic protozoan (Paget and Lloyd 1990). In this report we have determined the oxygen affinities of the major oxidases in the Bushby strain, and shown that H2O2 is a minor product, both in the hydrogenosomal and cytosolic compartments.
2.1 Growth of the organisms and preparation of subcellular fractions
T. vaginalis (Bushby strain) was grown with a logarithmic phase doubling time of 3·6 h in modified Diamonds medium as described previously (Chapman et al 1986). Disruption was with a motor-driven PotterElvejhem homogenizer with a Teflon pestle rotating at about 800 rev min1 in a buffer containing 1 mM EDTA (Cerkasov et al 1978). The hydrogenosomal-enriched particulate fraction was obtained by centrifugation at 2·500 gav for 15 min. The cytosolic fraction was prepared by centrifuging cell-free extracts at 94,000 gav for 1 h.
2.2 Dependence of O2 uptake on O2 concentration
This was determined in the specially-constructed
open O2 electrode system (Lloyd and Kristensen 1985; Yarlett
et al 1986) with windows for simultaneous spectrophotometric measurements. Argon (< 3 ppm O2) was used to obtain anaerobiosis of the liquid in the vessel. One hundred per cent O2 air and a standard gas mixture of 5% O2 in N2 was also used (Air Products, Cardiff, UK). Various O2 tensions were obtained by the use of a digital gas mixer to provide O2 dilutions in 5% steps. Apparent Km O2 values were obtained at low O2 concentrations
(050 µM). The evolving gas mixture (150 ml/min) was humidified by passage through water-soaked cotton wool before passage over the liquid vortex in the vessel. O2 concentrations were calculated from percentage dilutions of stock gas mixtures.
Calibration for dissolved O2 and
determination of t1/2 (half times for equilibration) were obtained using
standard gas mixtures in the absence of biological material. Values for the O2
concentration in the liquid phase (TL) in the presence of respiring
cells or subcellular fractions at a series of fixed values of O2 concentration
in the gas
phase (TG) then allowed calculation of respiration rates (from Vr = k(TG TL) where Vr = respiration rate and k = loge 2/t1/2).
2.3 Production of H2O2 by T. vaginalis cells and subcellular fractions
Microperoxidase (MPII, Sigma) was used (3·0 µg/ml final concn.) for the detection of H2O2. The absorbance maxima of the free enzyme and the peroxide complex are at 407 nm and 419 nm respectively. The rate of H2O2 production was determined by the rate of change of absorption at 419407 nm in the dual wavelength mode (molar absorption coefficient 78000 M1 cm1).
Figure 1a shows a double reciprocal (LineweaverBurk) plot of the glucose-supported dependence of O2 consumption rates on O2 tension by intact T. vaginalis cells. In a series of experiments at low O2 concentrations (050 µM), the apparent Km value was determined to be 5·12 ± 1·35 µM (SD from 3 experiments). The respiration of whole cells was inhibited by high oxygen concentrations (> 120 µM O2). The extrapolated value for Vmax of O2 consumption was found to be 6·9 µM O2/min/106 cells. H2O2 production by intact cells revealed a similar pattern of inhibition by high O2 concentrations in the liquid phase (figure 1b). Thus both O2 consumption rate and rate of H2O2 production increased as the O2 concentration in solution was raised to a value of about 150 µM, above which, the cellular O2 uptake and rate of H2O2 production decreased. The ratio of H2O2 produced to O2 consumed increased as the O2 concentration increased up to 250 µM (figure 1c).
A similar pattern of O2 uptake, H2O2 production, and inhibition was observed for hydrogenosomes oxidizing pyruvate in the presence of ADP and succinate, as for intact cells. Figure 2a shows the LineweaverBurk plot of hydrogenosomal O2 consumption at low O2 concentrations with an apparent Km for O2 of 11·2 µM. A value of 11·0 ± 3·2 µM (SD from 3 experiments) was obtained in a series of experiments. Inhibition of hydrogenosomal O2 consumption occurred at an oxygen concentration of 110 µM, with an extrapolated Vmax of 79·6 µM O2/min/mg protein. The rate of production of H2O2 (figure 2b) increased up to an O2 tension of 30 µM to a rate of 33·5 pmol/min/mg protein, above this O2-concentration, production rapidly fell, to a rate of 11 pmol/min/mg protein. At 30 µM O2, most of the O2 consumed was converted to H2O2, but at high O2 concentrations, the ratio of H2O2 production to O2 consumption progressively decreased. Almost 14·4% of the O2 consumed was converted to H2O2 at an O2 concentration of 150 µM compared with 65% at a O2 concentration of 30 µM (figure 2c).
The double reciprocal plot obtained for O2 consumption rate against O2 concentration by cytosolic NADPH oxidase (NADPH: flavin oxidoreductase) is shown in figure 3a. The apparent Km for O2 was determined after a series of experiments to be 27·2 ± 5·1 µM (SD from 3 experiments). The Vmax was determined to be 33·1 µM O2/min/mg protein. Cytosolic NADPH oxidase was shown to produce H2O2 (figure 3b). The rate of H2O2 produced (43·5 pmol/min/mg protein) was higher for the NADPH oxidase system than the hydrogenosomal O2 uptake system. This production was decreased at an O2 concentration greater than 90 µM: the ratio of H2O2 production to O2 consumption at low O2 concentrations was twice that of whole cells (figure 3c).
No increase in the rate of H2O2 production was observed when superoxide dismutase (SOD) (20 EU/ml) was added to the reaction vessel containing intact cells or subcellular fractions. The addition of catalase (5 EU/ml) prevented any further H2O2 production; i.e., no increase in absorbance of the microperoxidase complex was observed at 419407 nm after its addition.No H2O2 could be detected when the cytosolic fraction was incubated aerobically with NADH.
The microaerophilic parasite, T. vaginalis (Paget and Lloyd 1990) is finely tuned so as to be able to utilize the very low (< 0·25 µM) levels of dissolved O2 that accelerate hydrogenosomal acetate production (Ellis et al 1992) and hence substrate-level phosphorylation.
Presumably low levels of O2 occur in microenvironments at the mucosal surface of the vagina. Increased blood flow can lead to more oxygenated conditions (Wagner and Levin 1978) and there may be other situations where elevated ambient O2 can increase oxidative stress in the protozoon. We have previously shown that inhibition of O2 consumption occurs when a threshold of O2 concentration is exceeded, and that the critical level of dissolved O2 varies from one strain of T. vaginalis to another (Yarlett et al 1986; Ellis et al 1994). One of these studies (Ellis et al 1994) confirmed earlier reports of the presence of superoxide dismutase activity in this (Linstead and Bradley 1988) and other anaerobic flagellates (Lindmark and Müller 1974). Other O2-defense mechanisms include high levels of intracellular thiols especially methanethiol, propanethiol and H2S (Ellis et al 1994). Therefore the sensitivity of T. vaginalis to O2 at above physiological levels comes about from the production of H2O2, with the consequent likelihood of the generation of other reactive oxygen species in an organism where no catalase can be detected (Honigberg 1978).
The present work shows correlation of O2 inhibition of O2 uptake with H2O2 production both at the whole cell level as well as in cytosolic and hydrogenosomal-enriched fractions. Similar dependencies of both O2 consumption and H2O2 generation rates suggests that this product of univalent reduction of the terminal electron acceptor arises at the cytosolic NADPH oxidase as well as at hydrogenosomal oxidases responsible for accepting electrons from pyruvate and NADPH.
The apparent Km for O2 of an organism is an indication of its ability to scavenge oxygen from its surroundings both for useful and protective functions. Its measured (apparent) value in intact cells depends upon (i) the nature of the terminal oxidase(s) (especially its affinity for O2), and (ii) in large cells, the existence of O2 gradients between the plasma membrane and sites of O2 reduction. The ability of intact cells of T. vaginalis to remove O2 from their surroundings is similar to that determined for the cattle parasite Tritrichomonas foetus KV1. Similar apparent Km values for O2 have been obtained for the hydrogenosome-containing anaerobic rumen holotrich Dasytricha ruminantium, and also for the free-living mitochondriate protozoan, Tetrahymena pyriformis (Lloyd et al 1982).
The apparent Km value for O2
obtained for the isolated hydrogenosomal or cytosolic fractions indicate that these
subcellular preparations do not have such high affinities for O2 as those
exhibited by whole cells. The higher values of Km for O2 of
both fractions may reflect either (i) the impairment of the activities of the fractions
during processing and prolonged exposure to O2 during the experiments, or (ii) loss of normal interaction between the hydrogenosome and the cytoplasm. The Vmax values for O2 consumption by hydrogenosomal or cytoplasmic NADPH oxidase obtained in the open system do not agree exactly with the values obtained from the rates of O2 uptake recorded immediately after subcellular fractionation using the closed oxygen electrode. This is possible due to the extended times (about 2 h) in which the subcellular fractions are subjected to varying O2 tensions in the open system; this exposure to O2 may partially inactivate the enzyme system(s) acting as terminal oxidases.
From the results presented here, it is not possible
to distinguish between reduced flavin (Massey et al 1971), an ironsulphur
centre (Misra and Fridovitch 1971), or superoxide as the source of hydrogenosomal or
cytoplasmic H2O2 production. The decrease in the rate of H2O2 production observed at high O2 concentrations can have several alternative explanations: (i) at high O2 concentrations, the terminal oxidase is inhibited; this results in decreased H2O2 production; (ii) H2O2 itself becomes inhibitory to the terminal oxidase at high concentrations, i.e., product inhibition of the oxygen-utilizing systems of either the oxidase itself or an intermediate redox carrier by H2O2 may occur; (iii) oxidative damage to the electron transport systems occurs as a result of H2O2 production. The primary reactant responsible for damage to the terminal oxidase systems may thus be oxygen itself, a product of its partial reduction such as H2O2, or a free radical such as superoxide or hydroxyl species. Because T. vaginalis contains no catalase, partial reduction of oxygen must result in a build-up of H2O2 capable of leading to irrepairable damage to sensitive cellular components including redox components of the electron transport chain.
Inhibition of H2O2 production by intact cells, hydrogenosomes and NADPH oxidase occurred at different oxygen concentrations. H2O2 production in hydrogenosomes was inhibited at a lower oxygen concentration than the overall oxygen consumption; this could indicate that H2O2 is not only produced by the terminal oxidase but also by another electron transfer component in the hydrogenosome. If superoxide radicals are produced by reduction of oxygen either at the terminal oxidase or elsewhere in the respiratory chain, then exogenous SOD added to the respiring system might not produce any effect if endogenous SOD activities are high. Indeed, addition of SOD to the reaction vessel did not increase the rate of change of absorbance when measuring H2O2 production by formation of the microperoxidase-H2O2 complex using intact cells or with either of the subcellular O2 consuming systems.
Production of H2O2 by rat liver or pigeon heart mitochondria accounts for 0·32% of oxygen uptake under energetically-coupled or uncoupled physio-logical conditions respectively (Chance et al 1979). In mitochondria from a nematode worm energiza-tion also decreases H2O2 production (Paget et al 1987). Neither of the two O2 consuming systems of T. vaginalis studied here produce H2O2 as the sole produce of O2 reduction; the NADH oxidase in this organism produces only H2O (Tanabe 1979; Brown et al 1996). At elevated oxygen tensions, the likelihood of H2O2 production increases. The production of H2O2 by T. vaginalis at low O2 (< 20 µM) is probably of little significance in terms of toxicity, and presents no major problem to the organism. However, higher O2 concentrations may evoke a potentially lethal situation as the organism becomes unable to excrete the H2O2 into its surroundings faster than it is produced. The resulting build-up of intracellular H2O2 produces irreversible cellular damage.
AC held a postgraduate studentship provided by the Science and Engineering Research Council.
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MS received 8 October 1998; accepted 20 July 1999
Corresponding editor: A Cornish-Bowden
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