Investigation of the Antifungal Activity of Caledonixanthone E and Other Xanthones Against Aspergillus fumigatus GØrald Larcher1, CØcile Morel2, Guy Tronchin1, Anne Landreau2, Denis SØraphin2, Pascal Richomme2, Jean-Philippe Bouchara1
Abstract
Susceptibility testing was performed according to the National Committee for Clinical Laboratory Standards (NCCLS) M-27A method [3]. However, the close dependence of antifungal activities to experimental conditions [4], led us to modify the culture parameters. Antifungal activity was markedly affected by the pH of the culture medium and the inoculum size. For an inoculum size of 103, 104, or 105 conidia per mL, the MIC80 values were 31, 62 to > 250 mg/mL at pH 7, respectively. At pH 5, no activity (> 250 mg/mL) was detected whatever the inoculum size. In contrast, antifungal activity was more pronounced at pH 3 with MIC80 values of 2, 8 and > 250 mg/mL according to the inoculum size. Interestingly, fluconazole, amphotericin B and the
Affiliation: 1 Groupe d'Etude des Interactions Hôte-Parasite, UPRES-EA 3142, Laboratoire de Parasitologie-Mycologie, Centre Hospitalier Universitaire, Angers, France ´ 2 SONAS, UPRES EA 921, UFR des Sciences Pharmaceutiques et d'IngØnierie de la SantØ, Angers, France Correspondence: Dr GØrald Larcher ´ GEIHP ´ UPRES-EA 3142 ´ Laboratoire de Parasitologie-Mycologie ´ Centre Hospitalier Universitaire ´ 4 rue Larrey ´ 49033 Angers ´ France ´ Phone: +33-02-41-35-34-72 ´ Fax: +33-02-41-35-36-16 ´ E-mail:
[email protected] Received: November 27, 2003 ´ Accepted: February 22, 2004 Bibliography: Planta Med 2004; 70: 569±571 ´ Georg Thieme Verlag KG Stuttgart ´ New York ´ DOI 10.1055/s-2004-827161 ´ ISSN 0032-0943
Letter ¼ Planta Med 2004; 70: 569 ± 571
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In previous works, the isolation of 32 xanthones from the stem bark of the tropical tree Calophyllum caledonicum Vieill. (Guttiferae) was described [1], [2]. In addition, in vitro susceptibility testing performed on 20 out of these 32 compounds showed that caledonixanthone E (1) was the most efficient against Aspergillus fumigatus, an important pathogen in immunocompromised patients. In this work, we attempted to define the in vitro conditions and the mechanism of its antifungal activity.
Letter
Among the different xanthones previously isolated from the stem bark of Calophyllum caledonicum, caledonixanthone E presented the strongest activity (MIC80 = 8 mg/mL) in acidic conditions (pH 3) against the human pathogenic fungus Aspergillus fumigatus. Phase-contrast microscopy studies suggested the assembly or synthesis of cell wall components as the target of the drug. Moreover, the use of fluorescent lectins further supported an impact of caledonixanthone E on the synthesis of chitin, the major structural polysaccharide of the fungal wall. These results suggest that caledonixanthone E may be an interesting model for the design of new antifungal drugs.
bis-triazole derivative D0870 were also reported to be less efficient at pH 5 than at pH 7 [5], whereas more acid conditions (pH 3) led to a marked increase in their antifungal activity. Likewise, polygodial, a sesquiterpene dialdehyde from Polygonum hydropiper, showed a higher activity at acid pH compared to neutral conditions [6].
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The antifungal activity of 1 was then investigated at acid conditions against different human pathogenic fungi. In addition, 1 was compared to 8 other xanthones from C. caledonicum which have not been studied as yet (Table 1). Among these compounds, 1 exhibited the highest activity against A. fumigatus. Moreover, 1 and 1,5-dihydroxy-3-methoxyxanthone (7) presented a relative specificity towards A. fumigatus and A. flavus. An additional species, A. niger, was inhibited by 7-hydroxyxanthone (4). All the species tested except A. terreus were affected by 5-hydroxyxanthone (2), 6-hydroxy-7-methoxyxanthone (3), 7-hydroxy-8methoxyxanthone (5) and 1,3,5-trihydroxy-2-methoxyxanthone (9), whereas 1,5-dihydroxyxanthone (6) and 1,6-dihydroxy-7,8dimethoxyxanthone (8) inhibited all fungi. In addition, microscopic examination showed that exposure of A. fumigatus to 1 led to morphological alterations similar to those re-
Table 1
Inhibition of the synthesis of fungal wall components represents an ideal mode of action for antifungal drugs. Therefore, compound 1 may constitute an interesting model for the design of more efficient structural analogues that we are now investigating.
Materials and Methods Antifungal susceptibility testing was performed on strains of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus and Candida albicans with xanthones (purity greater than 99 %) from C. caledonicum as already described [1], [2]. The broth microdilution test factors assessed were inoculum size (103, 104 and 105 conidia/mL) and pH of culture medium (pH 3, 5 and 7). Aspergillus fumigatus cultivated or not in the presence of 1 (8 mg/ mL) was examined by phase-contrast microscopy at a 400 magnification or incubated for 30 min at 37 8C in 200 mL of calcofluor white (1 mg/mL) or FITC-conjugated lectins (100 mg/mL) in phosphate buffered saline (PBS) at pH 7.2. After washing, the fungal
Antifungal activity of xanthones from C. caledonicum against Aspergillus spp. and C. albicans
Compounds
Fungal species A. fumigatus CBS 113.26
A. flavus IHEM 37.19
A. niger IHEM 2 951
A. terreus 5 029.2000
C. albicans ATCC 663.90
Positive control Amphotericin B
8
8
16
16
1
1
8
16
> 250
> 250
> 250
2
31
16
62
125
62
3
16
16
31
125
31
4
31
16
62
> 250
250 62
Xanthones
5
31
31
62
250
6
16
16
31
62
62
7
31
31
125
> 250
> 250
8
16
8
31
62
62
9
31
31
62
125
62
NB: Results which correspond to MIC80 (mg/mL), are mean values of triplicate determinations.
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Letter
ported for lipopeptides such as echinocandins [7], suggesting an inhibition of the synthesis of cell wall components (Fig.1A and B). To further investigate the cell wall changes, A. fumigatus, treated or not by 1, was incubated with fluorescein isothiocyanate (FITC)-conjugated lectins or calcofluor white. A bright and uniform labeling of the hyphal wall was observed with calcofluor white which links b-glucans, as with concanavalin A- and peanut agglutinin-FITC which recognize a-D-mannosyl or b-D-galactosyl residues, respectively. Moreover, labeling was not affected by exposure of the fungus to 1. In contrast, incubation with wheat germ agglutinin (WGA)-FITC resulted in an intense fluorescence all along the hyphal wall for the control (Fig. 2B), whereas a heterogeneous labeling was detected when the fungus was grown in the presence of 1 (Fig. 2A). As WGA recognizes chitin, a structural polysaccharide of the fungal wall, our results suggested an important reduction of the chitin content in hyphae after exposure to 1.
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Yamada H, Tsuda T, Watanabe T, Ohashi M, Murakami K, Mochizuki H. In vitro and in vivo antifungal activities of D0870, a new triazole agent. Antimicrob Agents Chemother 1993; 37: 2412 ± 7 6 Lee SH, Lee JR, Lunde CS, Kubo I. In vitro antifungal susceptibilities of Candida albicans and other fungal pathogens to polygodial, a sesquiterpene dialdehyde. Planta Med 1999; 65: 204 ± 8 7 Kurtz MB, Heath IB, Marrinan J, Dreikorn S, Onishi J, Douglas C. Morphological effects of lipopeptides against Aspergillus fumigatus correlate with activities against (1,3)-beta-D-glucan synthase. Antimicrob Agents Chemother 1994; 38: 1480 ± 9
Fig. 2 Labeling of A. fumigatus CBS 113. 26 with FITCconjugated WGA. Cells were grown in RPMI medium containing (A) or not (B) compound 1. Note the heterogeneous labeling after cultivation in the presence of 1, and the more pronounced fluorescence at the apex of hyphae (arrows). Bars correspond to 5 mm.
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elements were examined under a Nikon microscope equipped for epifluorescence.
References 1
Morel C, SØraphin D, Teyrouz A, Larcher G, Bouchara JP, Litaudon M, Richomme P, Bruneton J. New and antifungal xanthones from Calophyllum caledonicum. Planta Med 2002; 68: 41 ± 4 2 Morel C, SØraphin D, Oger JM, Litaudon M, SØvenet T, Richomme P, Bruneton J. New xanthones from Calophyllum caledonicum. J Nat Prod 2000; 63: 1471 ± 4 3 National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved Standard M27-A. National Committee for Clinical Laboratory Standards, Villanova, Pa: 1997 4 Buchta V, Otcenasek M. Factors affecting the results of a broth microdilution antifungal susceptibility testing in vitro. Zbl Bakt 1996; 283: 375 ± 90
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Fig. 1 Phase-contrast micrographs of drug-induced morphological changes in A. fumigatus CBS 113.26. The fungus was grown in RPMI medium with (A and B) or without (C, solvent control) compound 1. In the presence of 1, an abnormal hyphal growth is observed with swollen germ tubes and tortuous hyphae, larger than usual whereas they are thinner and rectilinear in the control. Bars correspond to 5 mm.
