1 Microbiology (BIOSI 1) Main Building, Cardiff University, PO Box 915, Cardiff CF10 3TL, UK
2 School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, 2052, Australia
3 Department of Chemistry, King's College University of London, Strand, London WC2R 2LS, UK
4 School of Science, Parramatta Campus, University of Western Sydney, Australia
Correspondence
D. Lloyd
Lloydd{at}cf.ac.uk
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ABSTRACT |
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Present address: Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L35 QA, UK.
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INTRODUCTION |
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As the environmental interface for chemiosmotically driven processes, the plasma membrane senses and indicates physiochemical changes in the liquid phase of its immediate surroundings. Thus, perturbation of the plasma-membrane potential provides a sensitive and rapid indication of those stimuli likely to lead to functionally important physiological changes (Humphreys et al., 1994; Lloyd & Hayes, 1995
; Lloyd et al., 2000
). Where these effects are reversible, modification of membrane potential will be observable as a transient (often oscillatory) response. Irreversible effects elicit more profound disturbance (Scott & Rabito, 1988
), often leading to cytotoxic cascades and even cell death by necrotic or apoptotic pathways (Ryu & Lloyd, 1995
; Fernandes & Assreuy, 1997
; Lloyd et al., 2003b
).
In this study, we focus on the effects of reactive oxygen species (Lloyd et al., 2000), as well as of nitrosative stress (Lloyd et al., 2003a
), as two of the most important sources of metabolic perturbation and cellular damage. These agents are implicated in significant early changes in the plasma membrane (Scott & Rabito, 1988
), and thereby the onset of cellular ageing, senescence and cell death. In microaerophilic organisms, it would be expected that these sources of cellular stress might play major roles in cytotoxicity and that targets on the cytoplasmic membrane would be the primary sites of free-radical-mediated effects (Kayahara et al., 1998
). Here, we demonstrate that fluorochrome-based measurements of plasma-membrane potential provide a sensitive and useful approach to the monitoring of cellular stress in these organisms as has previously been shown for a yeast (Dinsdale et al., 1995
) and for bacteria (Mason et al., 1994
; Lloyd et al., 2001
).
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METHODS |
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Organisms were grown to late-exponential phase before being harvested by centrifugation for 2 min at 650 gav in an MSE bench centrifuge. Resuspension was in 150 mM NaCl, 5 mM K2HPO4 and 1·8 mM KH2PO4 PBS (pH 7·2), unless otherwise stated (e.g. where effects of K+ were studied, the buffer used was 140 mM N-methyl-D-glucamine chloride, 1 mM CaCl2, 10 mM HEPES and 11 mM glucose).
Fluorescence microscopy.
Cells were examined using an Olympus BH2 triocular fluorescence microscope. Images were acquired on Fuji ISO400 (daylight) 38 mm film.
Confocal laser-scanning microscopy.
A Bio-Rad MRC confocal system attached to a research microscope (1024-Leica DMRB) was used with an argon/krypton air-cooled laser (emission at all lines, i.e. UV, 488 and 514 nm). Images were obtained with a x63 oil-immersion objective (Numerical Aperture 1·38). Section thickness was 5·5 µm. The 0·3 W laser was used at 10 % power to minimize photobleaching. Unless otherwise stated, organisms were washed and resuspended in 0·31 M mannitol before observation using FITC filters. Images were acquired on a Zip disk and printed using an Epson 750 colour printer.
Flow cytometry.
Cellular fluorescence (green emission, 530540 nm) was monitored by flow cytometry using a Mo-Flo cytometer (Cytomation PTY) with excitation at 488 nm from a water-cooled 200 mW argon-ion laser. In addition, forward light scatter and right-angle side scatter were measured and used for gating data collection. Typically, signals from 50 000 cells were acquired and analysed using CYCLOPS software (Cytomation PTY) for each sample. The flow cytometric histograms shown are representative of at least three independent experiments. For non-axenic cultures of M. punctachora, a selected population was analysed to exclude the contribution of signals from bacteria; cell sorting was used to validate the purity of the cohort selected.
Materials.
DiBAC4(3) was from Molecular Probes (catalogue no. B-436); it was stored at 18 °C in the dark as a 100 µM ethanolic solution. Roussin's black salt, NH4[Fe4S3(NO)7], was synthesized in house. Gramicidin, NaNO2, sodium nitroprusside, Na2Fe(CN5)NO and all other chemicals were from Sigma.
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RESULTS |
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DISCUSSION |
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In this study, we have shown that the plasma-membrane potential provides a sensitive and convenient indicator of the vitality of several microaerophilic protist populations (Lloyd, 1993). Moreover, the use of flow cytometry enables resolution of heterogeneous populations, where damage affects only some of the organisms. It also enables experiments on non-axenic cultures as, for example, with M. punctachora as shown here. This is important to distinguish (a) damaged from intact organisms, and (b) the signals emitted from protists in non-axenic cultures. Assessment using fluorometric measurement of DiBAC4(3) emission intensity has been used to determine injury by oxidative or nitrosative stress. Table 1
indicates their
scavenging abilities as measured by the Km O2 values for O2 consumption and the threshold values, above which O2 becomes inhibitory to these oxygen-uptake systems. Previous work has shown that the plasma-membrane potential of G. intestinalis is maintained at 134±3 mV by a K+ diffusion pathway and an electrogenic H+ pump (Biagini et al., 2000
); in this organism, sensitivity to reactive oxygen species (Lloyd et al., 2000
, 2002b
) and to nitrosative stress (Ryu & Lloyd, 1995
; Lloyd et al., 2002a
, 2003a
) has been demonstrated. In M. punctochora, the mechanisms whereby the plasma-membrane potential is generated appear to be similar to those seen in G. intestinalis (G. Biagini & C. Bernard, unpublished data). Table 1
also compares the physiological characteristics of the microaerophilic species studied with respect to oxidative and nitrosative stress.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 16 October 2003;
revised 23 December 2003;
accepted 21 January 2004.