1 Microbiology, Cardiff School of Biosciences, Main Building, Cardiff CF10 3TL, Wales, UK
2 The Institute of Molecular Cardiobiology, Johns Hopkins Medicine, 720 Rutland Ave, 844 Ross Building, Baltimore, MD 21205-2195, USA
3 Cultech Biospeciality Products Ltd, York Chambers, York Street, Swansea SA1 3NJ, Wales, UK
Correspondence
David Lloyd
LloydD{at}cf.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Allyl alcohol (AA), the focus of the present study, is produced from garlic in two ways: firstly by a self-condensation reaction of allicin and secondly by the reaction between alliin, the precursor of allicin, and water (Lawson, 1996). It is released after ingestion of garlic (Egen-Schwind et al., 1992
) and is present in exhaled air after ingestion of all garlic products, the highest concentration being after ingestion of freeze-dried garlic tablets (Laasko et al., 1989
). Mechanisms, first proposed by Block (1985)
, for the formation of AA in aqueous environments such as those found in vivo result from the transformation of the thiosulphinates such as diallyl trisulphide and diallyl disulphide (Block, 1985
; Block et al., 1992
), with up to one-third of the lost allyl groups during the transformation producing AA (Lawson, 1996
). Recent investigations with rats reported that similar degradative metabolites are formed in vivo (Germain et al., 2002
).
Garlic as an antibacterial (Sharma et al., 1977; Skyrme, 1997
) has been the focus of detailed investigations recently on Helicobacter pylori (O'Gara et al., 2000
; Ross et al., 2001
). Differential inhibitory effects against Escherichia coli and Lactobacillus casei, whereby a 10-fold greater sensitivity was evident in the former (Rees et al., 1993
) have been studied in more detail with respect to the different membrane structures of Gram-positive and -negative bacteria (Cottrell, 2003
). Antiprotozoal studies include those on Entamoeba histolytica (Mirelman et al., 1987
; Reuter, 1994
) and Giardia intestinalis (Harris et al., 2000
); the anticandidal effects (Ghannoum, 1988
, 1990
; Lemar et al., 2002
, 2003
) of garlic similarly include a wide range of ultrastructural lesions affecting cytoplasmic membranes, organelles and cytoskeletal organization. The widespread efficacy of this plant extract as an antimicrobial has been linked to the ease by which these molecules pass through cell membranes and react biologically at the low level of thiol bonds in amino acids (Miron et al., 2000
). That no example of acquired microbial resistance to garlic has been reported may also stem from its diverse modes of action and the multiplicity of intracellular targets that each bioactive component inactivates. Work with G. intestinalis (Harris et al., 2000
) and Candida albicans (Lemar et al., 2002
) has suggested that two of the simplest garlic constituents, diallyl disulphide and AA, are amongst the most potent; the former can be isolated by a process of steam distillation (Wertheim, 1844
). More recent investigations, however, show that diallyl trisulphide is still more potent (Davis, 2005
). A well-researched mechanism of AA toxicity is by its inhibition of alcohol dehydrogenase after its conversion to the toxic aldehyde acrolein (Rando, 1974
). Acrolein (crotonaldehyde) and AA were the most toxic of 20 low-boiling-point compounds tested on the yeast Candida utilis (Sestakova et al., 1976
). More recent investigations have indicated that AA itself is not toxic to cells of the methylotrophic yeast Pichia pastoris, but it is toxic when oxidized via an alcohol oxidase pathway to acrolein (Johnson et al., 1999
). In rats, in vivo, AA is metabolized by liver alcohol dehydrogenase to acrolein (Mapoles et al., 1994
). Severe damage to the microtubules of rat hepatocyte mitochondria after exposure to AA (Vengerovskii et al., 1989
) was observed concomitantly with the depletion of glutathione (Nagelkerke et al., 1991
). However, more recent studies revealed that 100 µl AA per kg body weight in rats fed by gastric tube was not toxic to hepatocytes as determined by RNA extraction (Tygstrup et al., 1997
). These studies do not preclude this derivative from possible clinical use at the in vivo concentration required to be an effective antifungal. Effects of AA on Saccharomyces cerevisiae include disruption of cellular kinetics, redox balance and electrophoretic mobility (Wills & Phelps, 1978
). Acrolein is also known to deplete intracellular stores of glutathione and cause peroxidation of cellular lipids. This affects the permeability of the membrane and consequently undermines the viability of the cell (Glascott et al., 1996
).
In this study we investigated the biocidal mechanisms of AA on C. albicans by assessing its effects on cell physiology, and also on morphology using optical and electron imaging.
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METHODS |
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Garlic preparations.
Garlic powder (provided by Cultech Biospeciality Products, Swansea, Wales, UK) was prepared to the required concentration in sterile growth medium. After 30 min the garlic powder extract (GPE) was centrifuged at 3900 g for 10 min and the supernatant passed through a sterile 0·2 µm filter (Millipore). Stock suspensions were prepared on the day of testing in PBS (pH 5·6) or appropriate media as stated. Concentration ranges for biocide assays for AA (010 mM) were prepared in sterile growth medium (AA was purchased from Sigma-Aldrich). MIC values were determined by the microtitre broth dilution method (Cottrell, 2003).
Ultrastructural investigations
Scanning electron microspcopy.
Cells were fixed in 3 % (v/v) glutaraldehyde in PBS (pH 7·4) and dehydrated in increasing concentrations of ethanol (10 %, v/v, increments, to 100 %). A drop was placed onto a round glass coverslip for critical-point drying (CPD 030; Balzers). The sample was mounted onto an aluminium slab (10x10 mm JEOL type; Agar Scientific) using silver paint (Agar Scientific). Sputter coating was performed in a sputter coater (S150B; Edwards) and the image viewed using a scanning electron microscope (5200LV; JEOL).
Transmission electron microscopy.
Cells were fixed in cacodylate buffer, pH 6·9, containing 1 % (v/v) paraformaldehyde and 2 % (v/v) glutaraldehyde, at 4 °C for 1 h. The cells were centrifuged and dehydrated in successive washes of ethanol for 30 min. The pellet of cells was embedded in Spurr resin and ultrathin sections obtained (Ultratome III; LKB). The sections were mounted onto a 0·5 % Pioloform (in chloroform)-coated 3·05 mm copper grid then stained with 2 % aqueous uranyl acetate and 2 % lead citrate before imaging on a transmission electron microscope (1210; JEOL).
Whole-cell respiration.
Organisms in growth medium were harvested in exponential phase. Cells were washed and resuspended in 500 µl PBS pH 6·4. Measurement of glucose-supported O2 consumption was performed at 30 °C in a thermostatically controlled closed electrode system (Rank) using a Teflon membrane electrode. Cell suspensions (40 µl) were injected into 2 ml PBS (pH 5·6) and continually stirred at 200 r.p.m.
Redox investigation: preincubation and conditions for microscopic examination.
Early-stationary-phase cells were harvested and resuspended in PBS (pH 5·6). Samples (0·5 ml) were preloaded with 200 nM tetramethylrhodamine ethyl ester (TMRE) at 37 °C for 30 min before sample removal to the observation chamber and supplemented with 10 mM glucose. The dish containing the yeasts was equilibrated with unrestricted access to atmospheric oxygen on the stage of a Nikon E600FN upright microscope which was maintained at 30 °C. AA or GPE was subsequently added to the incubation mixture.
Fluorescent probes for two-photon laser scanning microscopy.
The cationic potentiometric fluorescent dye TMRE was used to monitor changes in mitochondrial membrane potential, m. The large negative potential gradient across the inner mitochondrial membrane results in the accumulation of TMRE within the matrix compartment according to its Nernst potential (Loew et al., 1993
). Production of reactive oxygen species (ROS) was monitored with the ROS-sensitive fluorescent probe 5-(-6)-chloromethyl-2',7'-dichlorohydrofluorescein diacetate (CM-H2DCFDA, 10 µM). The acetate group of CM-H2DCFDA is hydrolysed by esterases when it enters the cell and the molecule is trapped inside as the non-fluorescent 5-(-6)-chloromethyl-2',7'-dichlorodihydrofluorescein (CM-H2DCFH). CM-H2DCFH was chosen because, unlike underivatized dichlorodihydrofluorescein, it is well retained in cells (Xie et al., 1999
) and, in this case, in the mitochondrial matrix (Aon et al., 2003
). Oxidation of CM-H2DCFH by ROS, particularly by hydrogen peroxide (H2O2) and hydroxyl radical (Vanden Hoek et al., 1997a
, b
), yields the fluorescent product carboxymethyldichlorofluorescein (CM-DCF), and indirectly measures mitochondrially produced
that has dismutated to H2O2 through the action of superoxide dismutase (Chance et al., 1979
; Turrens et al., 1985
).
Glutathione was monitored intracellularly by production of the fluorescent adduct glutathione-bimane (GSB) (Kosower & Kosower, 1987) as a result of the reaction of the cell permeant monochlorobimane (MCB, 50 µM) with reduced glutathione (GSH) catalysed by glutathione-S-transferase (GST) (Cortassa et al., 2004
). Reduced nicotinamide nucleotides were monitored by their autofluorescence under the imaging conditions optimized previously (Aon et al., 2003
).
Image acquisition and analysis.
Images were recorded using a two-photon laser scanning microscope (Bio-Rad MRC-1024MP) with excitation at 740 nm (Tsunami Ti : Sapphire laser, Spectra-Physics) as described before (Aon et al., 2003). Briefly, owing to the overlap in the cross-sections for two-photon excitation of the three fluorophores of interest (Xu et al., 1996
) (TMRE, CM-DCF, and NADH or GSB) this wavelength permitted recording of
m, ROS production, and NAD(P)H or GSH simultaneously. The red emission of TMRE was collected at 605±25 nm; the green emission of CM-DCF was recorded at 525±25 nm; and the blue emission of GSB detected at 480±20 nm. NADH emission was collected as the total fluorescence <490 nm. At 3·5 s or 30 s intervals as indicated, 512x512 pixel 8-bit greyscale images of the three emission channels were collected simultaneously and stored.
While determining kinetics of GSB production in cells, about 10 images were recorded to get the cellular background of NADH before acute addition of 50 µM MCB. This was necessary because the NAD(P)H and GSB emissions were collected at similar wavelengths, i.e. 480490 nm (Aon et al., 2004; Cortassa et al., 2004
). At steady state, the NAD(P)H fluorescence levels (n=14) represented 31±1·2 % of the maximal GSB fluorescence levels attained (n=14), indicating an approximately threefold increase with respect to the background. Images were analysed offline using ImageJ software (Wayne Rasband, National Institutes of Health; http://rsb.info.nih.gov/ij/).
Fluorometric kinetic studies.
The effect of AA and GPE on purified GST (from rabbit liver, Sigma cat. no. G8261, 6 U ml1 final concentration) as a function of GSH concentration was investigated. Increasing concentrations of GSH (in PBS pH 5·6) in the absence and presence of AA (0·5 or 1·0 mM) and GPE (5 or 10 mg ml1) were added to 3 ml PBS (pH 5·6); a subsequent addition of 50 µl GST was used to commence the reaction. Monochlorobimane (MCB, 2 µM final concentration; max excitation 391 nm;
max emission 491 nm) was used as previously described (Aon et al., 2003
), and fluorescence measured using a Cary Eclipse fluorescence spectrophotometer (Varian).
Analysis of intracellular ROS using flow cytometry.
Fluorescence distributions were obtained from 100 000 events per cell sample through a bandpass filter FL1 using a FACScalibur flow cytometer (BD Biosciences). Forward light scatter and side scatter were measured and used for gating the data collection. The fluorescence of gated cell populations was analysed using validated analysis software, WinMDI 2.8. Intracellular events in a population of C. albicans were analysed using CM-H2DCFH as a marker for ROS as described previously (Aon et al., 2003).
Materials.
TMRE, CM-H2DCFDA and MCB were purchased from Molecular Probes. All other reagents, including enzymes, were from Sigma-Aldrich unless specified.
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RESULTS |
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Flow cytometric analysis
The fluorogenic compound CM-H2DCFH is oxidized by ROS to give a fluorescent product (see Methods). Control suspensions of C. albicans loaded with this fluorogen showed only autofluorescence at 530 nm, whereas cells treated with AA and GPE fluoresced much more brightly. The fluorescence intensities increased as the concentration of either challenge agent was increased (Fig. 7).
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DISCUSSION |
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Allicin is considered one of the principal antimicrobial constituents of triturated garlic cloves (Cavallito et al., 1944), and although it has microbicidal activity in vitro (Ankri & Mirelman, 1999
), its half-life in tissues is too short to ensure appreciable pathogen damage (Koch, 1996
). Systemic levels of the metabolic products of allicin are reported to be relatively high (Egen-Schwind et al., 1992
), and researchers have now turned their interest to these constituents as possessing considerable antimicrobial properties. AA is found in exhaled air after ingestion of freeze-dried garlic tablets (Laasko et al., 1989
). As it is a known hepato-toxicant, its use in pure form would have to be administered carefully; however, dosage up to 100 µl AA per kg body weight in rats after administration by gastric tube presented no toxicity to hepatocytes (Tygstrup et al., 1997
). These studies do not preclude the use of this constituent for clinical administration at the in vivo concentration required to produce an antifungal effect. Conversion of AA to acrolein can lead to subsequent inhibition of alcohol dehydrogenases (Rando, 1974
). S. cerevisiae has 20 alcohol dehydrogenases (Kruckerberg & Dickinson, 2004
), but the best understood and possibly the most important enzyme targets involved in AA toxicity are the cytosolically located Adh1 and 2, involved in ethanol formation and growth on ethanol respectively, and Adh3, which is mitochondrial. The functions of Adh3 are not entirely understood, but it has been postulated to be involved in a redox shuttle, transferring mitochondrial NADH to the cytosol (Bakker et al., 2000
). Improvements in methods for measuring mitochondrial redox state (see Chance, 2004
, for a review) have allowed us to detect a decrease in both cytosolic and mitochondrial NADH. Results suggest that although it is possible that all three of these enzymes are targeted by AA, another site of action is involved to produce this effect (Fig. 5ciii
). Depletion of NAD(P)H, the co-substrate for glutathione reductase, would diminish the recovery of the GSH pools in both compartments. As AA depletes glutathione (Glascott et al., 1996
), one might predict an increase in ROS. Confocal imaging and quantitative analysis does indeed indicate the depletion of glutathione and increase in ROS when cells are treated with AA (Figs 5, 6 and 7
). Electron microscopy of cells treated with AA also indicates that mitochondria may be a target for AA (Fig. 3
). However, decreased respiration, as observed with GPE and other garlic constituents (K. M. Lemar and others, unpublished), was not observed for AA. This may be explained by the fact that the alcohol dehydrogenase function is not implicated in glucose respiration, as when organisms are grown in the presence of excess glucose, alcohol dehydrogenase is inactivated (Gonzalez et al., 2000
). The conversion of AA to the toxic aldehyde acrolein would therefore not occur. Exposure to AA may thus impair C. albicans viability through mechanisms not directly associated with mitochondrial function.
The enzyme GST, associated with maintaining redox balance, is a target of a different garlic constituent, diallyl disulphide (DADS) (K. M. Lemar and others, unpublished). Our experiments demonstrated that it is not a target for AA. This is perhaps unsurprising, as the two compounds have quite different structures. However, this observation assists in our understanding of why GPE has such a broad spectrum of activity. The multiple metabolic constituents produced by the plant (many of which remain to be explored), may have many different targets, and hence produce diverse effects on cell processes. Its extensive repertoire of inhibitory constituents makes garlic an interesting potential alternative to single-site-specific antibiotics or synthetic organic compounds for combating C. albicans infections. However, the presence of toxic metabolites, such as AA itself, may limit the dose that can be administered. Previous investigations concerning the antifungal properties of whole garlic extracts compared to some of its breakdown products have shown that they have similar MICs, with similar mechanisms of action, such as glutathione depletion and reaction with thiol groups (Davis et al., 2003). AA may well be an exception due to the activity of the acrolein/alcohol dehydrogenase pathway activity; however its antifungal efficacy in vitro would have to be determined by MIC studies.
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Received 6 April 2005;
revised 27 June 2005;
accepted 30 June 2005.
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