Unité de Recherche en Biologie Parasitaire et Fongique, Laboratoire de Parasitologie et Mycologie Médicales, CHU La Milètrie, 86021 Poitiers Cedex, France
Received 12 November 2001; returned 8 February 2002; revised 12 March 2002; accepted 14 March 2002.
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Abstract |
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Introduction |
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Inhibition of the matrix metalloproteases (MMPs), which also degrade these constituents, has been the subject of intense research in recent years. Some compounds are effective on gelatinases and others on collagenases, but they do not necessarily inhibit only one class of enzyme.3 For example, antibiotics such as tetracycline, gentamicin and cefalothin are known for their ability to inhibit the activity of some collagenases originating from a variety of cellular sources.4,5 Galardin, a synthetic peptidyl hydroxamate inhibitor of MMPs, prevents the degradation of collagen caused by some bacterial agents.6 Some fatty acids, such as elaidic or oleic acids, also have inhibiting effects on MMPs. Oleic acid (cis-9-octadecenoic acid) plays a role in the inhibition of lung metastasis by colon carcinoma in mice.7 It has been proven that oleic acid reduces the activity of gelatinase A (MMP-2), which is first secreted from cells as a zymogen and then activated to degrade ECM components.8 A recent study provides in vitro and ex vivo evidence that fatty acids could contribute to regulate the ECM breakdown by inhibiting gelatinases A and B, involving fibronectin type II repeats.9 By acting as MMP inhibitors, fatty acids such as oleic acid could be considered as a novel class of modulators of metastasis formation.
The aim of the present study was to look for an inhibitory effect of doxycycline, cefalothin, gentamicin, galardin, and elaidic and oleic acids on the 95 kDa metallopeptidase of C. albicans, in relation to the ability of the enzyme to degrade ECM components.
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Materials and methods |
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C. albicans strain 2091, obtained from the Pasteur Institute (Paris, France), was grown for 48 h at 37°C on Sabouraud dextrose agar slants (Sanofi Diagnostics Pasteur, Marnes-La-Coquette, France). The cells were harvested in Tris-buffered saline (TBS; 140 mM NaCl, 10 mM TrisHCl, pH 7.2), washed three times at 4°C by centrifugation and suspended to a final concentration of 109 cells/mL in the same buffer. The cells were disrupted for 10 min in a cell homogenizer MSK (B. Braun, Melsungen, Germany) with glass beads (0.45 0.55 mm) with cooling under CO2.
Disrupted yeasts were centrifuged at 100 000g for 30 min at 4°C. The supernatant fluid represented the cytoplasmic extract and was used for the purification steps, which were performed as previously described using a high-performance liquid chromatography system (Laboratoires Merck Clevenot, Nogent-sur-Marne, France).1 Purification was confirmed by SDSPAGE of the final sample containing the enzyme activity.
Peptidase assays
The enzyme activity was assayed, as described previously, against the fluorogenic substrate L-Arg7-amido-4-methylcoumarin (AMC) (Sigma, St Louis, MO, USA).1 Briefly, 10 µL of purified enzyme were incubated with 20 mM of substrate in 190 µL of a 50 mM TrisHCl buffer, pH 7.4, containing 150 mM NaCl. After a 15 min incubation at 37°C, the fluorescence was measured at 430 nm with excitation at 380 nm.
Inhibition of the purified 95 kDa metallopeptidase by the test agents
Antibiotics were kindly provided by the manufacturers: doxycycline, Elerté (Aubervilliers, France); cefalothin and gentamicin, Panpharma (Fougères, France). Galardin was provided by Calbiochem (San Diego, CA, USA) and elaidic and oleic acids by Sigma.
The inhibitory effect of doxycycline (4.3, 2.15 and 0.43 mM), gentamicin (4.3, 2.15 and 0.43 mM), cefalothin (2.5 and 0.5 mM), galardin (3 nM, and 300, 30 and 3 µM), elaidic acid (7 and 0.7 mM) and oleic acid (20 µM) on the metallopeptidase activity was tested after a 4 h incubation of the enzyme with each compound. Oleic acid was first tested at 20 µM during different times of incubation, then in the concentration range of 020 µM for 4 h at 37°C.
Effect of oleic acid on yeast viability
A C. albicans inoculum of 105 cells/mL was grown in 1 mL Yeast Nitrogen Base (YNB) medium (Difco, Detroit, MI, USA) supplemented with 2% (w/v) glucose. Oleic acid was added to the medium to final concentrations of 20, 10, 5, 2.5, 1.25, 0.6 and 0.3 µM. After 48 h at 37°C, 20 µL of each culture were plated on to Sabouraud agar slants for viable count estimations to determine the fungicidal effects of the agent.
Action of oleic acid on secretion of the metallopeptidase
Yeasts grown in 1 mL culture medium (YNBglucose with oleic acid) for 48 h at 37°C were pelleted by centrifugation (2000g for 10 min). The enzyme activity was measured in 20 µL of the supernatant by L-ArgAMC after incubation for 45 min as described previously. A longer incubation time with the fluorogenic substrate was necessary because of the low quantity of the enzyme in the culture medium.
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Results |
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Discussion |
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Metalloproteases produced by pathogenic microorganisms show a wide variety of pathogenic actions. For example, Pseudomonas aeruginosa produces two metallopeptidases that can digest structural host components and accelerate bacterial invasion.12 The 95 kDa metallopeptidase of C. albicans is able to degrade in vitro some constituents of the ECM. This enzyme, localized in the fungus cell wall and active at pH 7, could facilitate in vivo the metastatic access of the yeast to deep organs after crossing the endothelial barrier.1
Inhibitors of one class of MMPs can also be inhibitors of other such enzymes.3 Although these enzymes differ in substrate specificity, their active sites could be similar in structure.3 Inhibition of MMPs may have therapeutic implications for controlling pathological collagen breakdown and represents a promising approach to the treatment of a variety of inflammatory and malignant disorders.10,11
Some antibiotics have been recognized for their MMP inhibitory capacity. Tetracyclines, such as doxycycline, can non-competitively inhibit MMPs via Ca2+ and Zn2+ chelation, and are able to inhibit the activity of collagenases from a variety of cellular sources.10 MMP activity can also be significantly inhibited by cefalothin in a dose-dependent manner, but not by gentamicin.5 The present study demonstrates that none of the tested antibiotics can inhibit the metallopeptidase of C. albicans at therapeutically effective concentrations.
The effect of galardin has been tested in acute and chronic wounds, and also on collagen degradation by P. aeruginosa.6,10 This compound usually acts on MMPs at concentrations as low as 0.5 nM. In our study, 85% of the activity is inhibited, but using a high concentration of 3 µM. Like the tested antibiotics, galardin is not able to inhibit the C. albicans enzyme at usable concentrations.
Metastatic capacity of tumour cells has been related to the increased expression and activation of gelatinase A (MMP-2).7 Fatty acids such as oleic acid have been proven to have an important inhibitory effect on gelatinase A. This fatty acid, by acting as a MMP inhibitor, could be considered as a novel modulator of metastasis formation. Its precise mechanism of action has not been yet elucidated.8
We report here the inhibition of the C. albicans metallopeptidase by oleic acid at low concentrations. This inhibition reached its maximum after 4 h of contact. Oleic acid acts not only directly on the enzyme, but also on its secretion in the culture medium in a dose-dependent manner, without modifying the viability of the yeast. If we consider that the C. albicans metallopeptidase could play a role in fungal dissemination, its inhibition by oleic acid could be of interest in vivo. Further studies are in progress to investigate the effect of oleic acid in a systemic candidosis model.
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Acknowledgements |
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Footnotes |
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References |
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2 . El Moudni, B., Rodier, M. H., Barrault, C., Ghazali, M. & Jacquemin, J. L. (1995). Purification and characterization of a metallopeptidase of Candida albicans. Journal of Medical Microbiology 43, 2828.[Abstract]
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