Jayne E. Ellis

Antioxidant defences in the microaerophilic protozoan Trichomonas vaginalis : comparison of metronidazole-resistant and sensitive strains

The sensitivity of the microaerophilic protozoan Trichomonas vaginalis to oxygen and products of its reduction, and the antioxidant defences employed by this organism, were investigated. Studies revealed that this amitochondrial flagellate is sensitive to oxygen tensions above those experienced in situ in the vagina (i.e. > 60 microM) and that metronidazole-resistant strains (CDC 85 and IR78) were more sensitive to elevated oxygen levels than a metronidazole-sensitive isolate (1910). In the presence of radical scavengers, inactivation of organisms at 60 microM oxygen was significantly lessened. Investigation of the antioxidant enzymes present in this organism revealed that activities of peroxide-reducing enzymes (e.g. catalase and general peroxidase) were not detectable, but that a cyanide-insensitive, azide-sensitive superoxide dismutase was present in cell extracts. Measurement of thiol-cycling enzymes indicated that NADPH could drive the reduction of oxidized glutathione (thiol reductase); however, the corresponding peroxidase activity was not detected. Analysis of thiols in whole cells of T. vaginalis indicated that glutathione was absent, but high levels of other thiols, propanethiol, methanethiol and H2S, were present. No significant differences were detected in thiol levels or antioxidant enzyme activities on comparison of metronidazole-sensitive and resistant strains. These results indicate that the sensitivity of T. vaginalis to oxygen above physiological levels is due to the lack of adequate peroxide-reducing enzymes and radical-scavenging mechanisms.

Electron transport components of the parasitic protozoon Giardia lamblia

The energy metabolism of the intestinal parasite, Giardia lamblia, involves the iron‐sulphur protein, pyruvate:ferredoxin oxidoreductase. Cell fractionation studies showed that this enzyme is associated with the membranes. NADH and NADPH dehydrogenases were found in both the membrane and cytosolic fractions. EPR spectroscopic studies showed the presence of iron‐sulphur clusters in the membrane fraction and in the cytosolic fraction, non‐sedimentable at 6 × 106 g · min. An acidic, soluble protein fraction was separated from the cytosol. It had an EPR spectrum in the reduced state, characteristic of the 2[4Fe‐4S] type of ferredoxin, with g‐factors at 2.04, 1.93 and l .89. and the midpoint redox potential was estimated to be −360 mV. This species is probably a ferredoxin, like those of anaerobic bacteria such as Closlridium and Desiilfovihrio spp. and also that of Entamoeba histolytica. The protein was readily and irreversibly oxidized to give [3Fe 4S] clusters.

Influence of oxygen on the fermentative metabolism of metronidazole-sensitive and resistant strains of Trichomonas vaginalis

The microaerophilic protozoon Trichomonas vaginals responds to extracellular changes in oxygen concentration: acetate, lactate, ethanol, H2 and CO2 formation, as well as glucose-depletion rates, are affected. All these variables except ethanol production rates, also differed between clinically metronidazole-sensitive (1910) and resistant (IR78 and CDC85) strains. Most interesting were the greatly increased glucose-scavenging rates of resistant isolates and their low specific activities of hydrogenase and H2 formation rates by comparison with the metronidazole-sensitive strain. Results suggest that all three strains of this parasite are well adapted to the O2 levels prevailing in situ (13-56 microM). Thus, vaginal oxygen tensions have more pronounced effects on the balances of fermentation products in the resistant strains, and results indicate that these strains may then use hydrogenosomal pathways to their advantage.

Oxygen affinities of metronidazole-resistant and -sensitive stocks of Giardia intestinalis

The common protozoon, Giardia intestinalis, parasitizes the upper small intestine of man, and is often refractory to treatment by metronidazole. Defective oxygen-scavenging mechanisms have been implicated as a cause of metronidazole resistance of another flagellate Trichomonas vaginalis, where metronidazole is also the most common drug treatment. Oxygen consumption of six clinical isolates of G. intestinalis and one line selected for resistance to metronidazole was measured over 0-50 microM-O2 using an oxygen electrode open for gas exchange. At > 30 microM-O2, inhibition of respiration was demonstrated in all seven stocks. Apparent oxygen affinities (KmO2) were found to range from 0.5 to 5.2 microM-O2; however, isolates from patients who failed to respond to treatment with metronidazole did not have measurably defective O2-scavenging capabilities compared with metronidazole-sensitive isolates. These strains did, however, show elevated NADPH-oxidase activities compared with metronidazole-sensitive strains. Results indicate that biochemical mechanisms of drug resistance in G. intestinalis may be quite different from those operating in T. vaginalis.

Influence of CO2 and low concentrations of O2 on fermentative metabolism of the rumen ciliate Dasytricha ruminantium

The effects of ruminal concentrations of CO2 and O2 on glucose-stimulated and endogenous fermentation of the rumen isotrichid ciliate Dasytricha ruminantium were investigated. Principal metabolic products were lactic, butyric and acetic acids, H2 and CO2. Traces of propionic acid were also detected; formic acid present in the incubation supernatants was found to be a fermentation product of the bacteria closely associated with this rumen ciliate. 13C NMR spectroscopy revealed alanine as a minor product of glucose fermentation by D. ruminantium. Glucose uptake and metabolite formation rates were influenced by the headspace gas composition during the protozoal incubations. The uptake of exogenously supplied D-glucose was most rapid in the presence of O2 concentrations typical of those detected in situ (i.e. 1-3 microM). A typical ruminal gas composition (high CO2, low O2) led to increased butyrate and acetate formation compared to results obtained using O2-free N2. At a partial pressure of 66 kPa CO2 in N2, increased cytosolic flux to butyrate was observed. At low O2 concentrations (1-3 microM dissolved in the protozoal suspension) in the absence of CO2, increased acetate and CO2 formation were observed and D. ruminantium utilized lactate in the absence of extracellular glucose. The presence of both O2 and CO2 in the incubation headspaces resulted in partial inhibition of H2 production by D. ruminantium. Results suggest that at the O2 and CO2 concentrations that prevail in situ, the contribution made by D. ruminantium to the formation of ruminal volatile fatty acids is greater than previously reported, as earlier measurements were made under anaerobic conditions.

Formate and glucose stimulation of methane and hydrogen production in rumen liquor.

Membrane-inlet mass spectrometry was used to investigate the effects of increasing the concentration of the rumen metabolites, formate and glucose, upon CH4 and H2 production during fermentation by unfractionated rumen liquor. Additions of formate up to 3.6 mM stimulated CH4 and then excess H2 production. Each addition caused a large accumulation of H2 (>40 µM), which returned to in situ concentrations after periods of more than 1 h. Glucose additions up to 2.0 mM gave linear increases in CH4 and H2 production. The conversion of substrate carbon into CH4 was found to decrease from 34% to 9% for formate, as concentrations were increased (1.6–3.6 mM); approximately 13.5% of the glucose carbon was converted to CH4.

The influence of ruminal concentrations of O2 and CO2 on fermentative metabolism of the rumen entodiniomorphid ciliateEudiplodinium maggii

The effects of ruminal concentrations of CO2 and O2 on glucose-stimulated and endogenous fermentation of the rumen ciliateEudiplodinium maggii were investigated. The principal metabolic products were butyrate, acetate, lactate, propionate, H2, and CO2.13C NMR spectroscopy revealed glycerol to be an important, but previously unidentified, fermentation product of this organism. Glucose uptake and metabolite formation rates were influenced by the headspace composition during protozoal incubations. Glucose uptake was most rapid in the presence of low O2 in N2 (1–3 µM O2 dissolved in the protozoal suspension). Pathways located in the hydrogenosomes were O2 sensitive, and low O2 concentrations resulted in lowered acetate, H2, and CO2 formation. The presence of high CO2 (65% gaseous headspace by volume) resulted in elevated acetate and butyrate formation; fumarate and propionate were similarly found to accumulate at higher concentration than previously detected in the supernatants. Results suggest that under conditions similar to those prevailing in the rumen (i.e., high CO2),Eu. maggii produces higher levels of important ruminal volatile fatty acids, and thus its relative contribution to rumen metabolism may have been underestimated.

Hydrogen Production by Rumen Ciliate Protozoa

The exact role of rumen protozoa in ruminant nutrition is not well defined, but it is implicit that a group of organisms which is present in all wild and domesticated ruminants and contributes as much as half the biomass of the microbial population must make a significant contribution to the economy of the system. Rumen ciliates are classified into holotrichs, which ferment a wide range of soluble carbohydrates1 and entodiniomorphs, which are principally particle feeders (i.e. cellulolytic and amylolytic).2 Some species of the latter have a limited ability to utilize soluble carbohydrates. The production of hydrogen by rumen ciliates3,4 occurs in a specialized organelle, the hydrogenosome.5,6 At some times oxygen is present in the rumen at low concentrations7 and rumen ciliates show high affinity oxygen consumption. Here we show that four different species of ciliates have oxygen-sensitive hydrogenases, so that the availability of hydrogen for interspecies hydrogen transfer will fluctuate depending on ambient oxgyen concentrations.

Hydrogen and Methanogenesis in Rumen Liquor and in Rumen Ciliate/Methanogen Cocultures

Interspecific hydrogen transfer is necessary for methanogenesis by rumen microorganisms, and as the production of methane represents a loss of carbon from the host ruminant, is a process of great economic significance.1 Ruminal hydrogenogens include bacteria,2 chitridomycete fungi3 and holotrich ciliate protozoa.4 Close physical associations between rumen ciliates and methanogenic bacteria have been demonstrated5 as well as metabolic interactions.6 In this report we show how direct measurements of dissolved hydrogen and methane can provide information on the kinetics and stoichiometries of species interactions both in crude rumen liquor and in a defined methanogenic coculture.