Cryptic antifungal compounds active by synergism with polyene antibiotics

Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e5, 2015 www.elsevier.com/locate/jbiosc

Cryptic antifungal compounds active by synergism with polyene antibiotics Hiroshi Kinoshita,1 Mariko Yoshioka,1 Fumio Ihara,2 and Takuya Nihira1, 3, * International Center for Biotechnology, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan,1 National Institute of Fruit Tree Science, 2-1 Fujimoto, Tsukuba, Ibaraki 3058605, Japan,2 and MU-OU Collaborative Research Center for Bioscience and Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand3 Received 1 May 2015; accepted 5 August 2015 Available online xxx

The majority of antifungal compounds reported so far target the cell wall or cell membrane of fungi, suggesting that other types of antibiotics cannot exert their activity because they cannot penetrate into the cells. Therefore, if the permeability of the cell membrane could be enhanced, many antibiotics might be found to have antifungal activity. We here used the polyene antibiotic nystatin, which binds to ergosterol and forms pores at the cell membrane, to enhance the cellular permeability. In the presence of nystatin, many culture extracts from entomopathogenic fungi displayed antifungal activity. Among all the active extracts, two active components were purified and identified as helvolic acid and terramide A. Because the minimum inhibitory concentration of either compound was reduced four-fold in the presence of nystatin, it can be concluded that this screening method is useful for detecting novel antifungal activity. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Antifungal compounds; Polyene antibiotics; Helvolic acid; Terramide A; Entomopathogenic fungi]

Polyene antibiotics are circular molecules that exhibit antimicrobial activity and include 3 to 8 conjugated double bonds in their structure; nystatin and amphotericin B are two examples. Most of the polyene antibiotics were discovered from actinomycetes (1) as fungicides against many eukaryotes, and have been used for medical treatment (2). Polyene antibiotics interact with ergosterol in the fungal cell membrane, make pores, and increase the in- and out-flow of many materials, thereby exerting antifungal activity (3e6). However, when prescribed for humans, these agents often carry serious side effects such as renal dysfunction, electrolyte imbalance, and hypotension, which prevent their further use in medical treatment (2,7). New antibiotics which display high antifungal activity but low toxicity are thus in high demand to deal with the increase of systemic fungal infection. Despite exhaustive screening of antifungal compounds, the majority of isolated compounds target the biosynthesis and/or function of the fungal cell wall/membrane (8e10), possibly because antifungal compounds targeting components other than the cell wall/membrane have been overlooked due to their difficulty in penetrating the fungal cell wall/membrane and exhibiting their properties. Based on this assumption, we constructed a bioassay system to discover novel antifungal activities by increasing the cell permeability with polyene antibiotics. We considered that compounds which have been overlooked due to their inability to penetrate the fungal cell/membrane would thus reveal their antifungal activities in the presence of polyene compounds. In this research, we screened antibiotics active against Candida

* Corresponding author at: International Center for Biotechnology, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: þ81 6 6879 7452; fax: þ81 6 6879 7454. E-mail address: [email protected] (T. Nihira).

albicans (the most common fungal pathogen in humans), which causes systemic infections, especially in susceptible immunocompromised patients (10). MATERIALS AND METHODS Strains and media C. albicans OUT6266 was used as the indicator strain for bioassay. C. albicans was cultivated in Sabouraud medium [glucose 40 g, Bacto peptone (BD Biosciences) 10 g, H2O up to 1000 ml] at 120 rpm at 28
C for 18 h, and was collected by centrifugation at 3500 rpm g for 10 min. The cells were washed three times with sterilized H2O in a volume equal to that of the medium. After adjusting the cell concentration to 2.5  107 cell/ml in 20% glycerol solution, the cells were stored at 80
C as the seed culture for bioassay. SMY medium [maltose 4%, Bacto yeast extract (BD Biosciences) 1%, Bacto peptone 1%] and A3M [glucose 0.5%, glycerol 2%, soluble starch 2%, Pharmamedia (Traders Protein) 1.5%, Bacto yeast extract 0.3%, HP-20 (Mitsubishi Chemical Co.) 1.0%] media were used to cultivate entomopathogenic fungi for metabolite analysis. potato dextrose agar [PDA (BD Biosciences) 39 g L1 ] was used for the bioassay. Determination of the polyene concentration used for bioassay Serially diluted solutions of nystatin (Sigma) and amphotericin B (WAKO) were prepared with dimethyl sulfoxide (DMSO). Hygromycin B was dissolved with dH2O and sterilized by filterization. C. albicans (1 ml of the stock culture) was inoculated into 250 ml of PDA and spread onto Petri dishes. A series of polyene solutions (200 ml) with or without hygromycin B solution were added to penicillin cups and set on the plates. The inhibition zone was measured after cultivation at 30
C for 24 h. Preparation of metabolites from entomopathogenic fungi Mycelium stored on a PDA slant at 4
C were inoculated into 5 ml of SMY medium and cultivated at 120 rpm at 28
C for 4e10 days until they grew. The preculture (0.6 ml) was inoculated into 30 ml of SMY or A3M medium in a 100 ml flask and cultivated without shaking at 25
C under light conditions (MLR-352 growth chamber; SANYO). Samples of the culture broth (2 ml) were taken at the 14th day and 21st day, and extracted with 2 ml each of ethyl acetate. The solvent layer was evaporated in vacuo and the residue was used as the extract of the culture supernatant. At the 21st day, the remaining culture broth was extracted with an equal volume of n-butanol, and the solvent layer was evaporated in vacuo for use as the extract of the whole broth. Bioassay in the presence of nystatin An amount of extract equivalent to two ml of culture was dissolved with 390 ml of methanol and used as a metabolite sample. Each sample (190 ml) was added to a paper disc (F8 mm) with or without

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Please cite this article in press as: Kinoshita, H., et al., Cryptic antifungal compounds active by synergism with polyene antibiotics, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.08.003

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KINOSHITA ET AL.

nystatin (final concentration: 2.9 mg/ml). After drying, the paper disc was put on a plate coated with C. albicans and incubated at 30
C for 24 h. Preparation of metabolites from entomopathogenic fungi cultivated in large scale Preculture (3 ml) prepared by the same procedure for small scale cultivation was inoculated into 150 ml of SMY or A3M medium in 500-ml flasks, and cultivated without shaking at 25
C for 14 or 21 days under light conditions (MLR352 growth chamber; Sanyo). A 10 ml sample of culture broth was taken on the 14th day and extracted with an equal volume of ethyl acetate. The organic solvent layer was evaporated in vacuo and the residue was used as an extract of the culture supernatant. At the 24th day of cultivation, 10 ml of culture broth was subjected to ethyl acetate extraction in the same manner. The rest of the culture broth was extracted with an equivalent volume of n-butanol. The organic solvent layer was evaporated in vacuo and the residue was used as an extract of the whole broth. Analysis of active components from entomopathogenic fungi Cordyceps indigotica HF879 and Torrubiella minutissima HF809 were cultivated in A3M medium for 14 days and in SMY medium for 21 days, respectively. After extraction with ethyl acetate and evaporation in vacuo, 975 mg and 240 mg of residues were obtained from 1.5 L culture of C. indigotica and T. minutissima, respectively. Each extract was analyzed by reverse-phase C18 HPLC [Imtakt Cadenza CD-C18 (F4.6  75 mm)] with a linear gradient of CH3CN from 15% to 85% in 0.1% aqueous HCOOH solution (3 min at 15%, 22 min from 15% to 85%, 7 min from 85% to 15%), at a flow rate of 1.2 ml min1, by monitoring with a photo diode array detector. Samples equivalent to 4 ml of culture broth were loaded. The eluates were collected every 4 min into eight fractions and evaporated in vacuo. The residues were assayed for antifungal activity. The active fractions were further fractionated to determine the active peaks by HPLC using the program described above. The antifungal activity of each distinct peak was also analyzed as described earlier. Purification of active components The extract from C. indigotica HF879 was applied on a Sep-Pak C18 (35 cc) cartridge, and purified by stepwise elution with increasing MeOH concentrations (40 ml of 50%, 20 ml each of 55%, 60%, 65%, 70%, 75%, 80%, 85%, and 90%, and 100 ml of 100% MeOH/H2O). After evaporation of the 80e95% fractions, 15 mg of the residue was further purified by preparative reverse-phase C18 HPLC with a Capcell Pak C18 column (Shiseido: F10  250 mm) with isocratic elution using 60% CH3CN at a flow rate of 4 ml min1. The peak i, detected at around 18.9 min by UV (230 nm), was collected and evaporated in vacuo to dryness, yielding 5.22 mg. In a similar manner, the active component from T. minutissima HF809 was purified with a Sep-Pak C18 (35 cc) cartridge by stepwise elution with increasing MeOH concentrations (40 ml of 20%, 20 ml each of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, and 70%, and 100 ml of 100% MeOH/H2O). After evaporation of the 55e70% fractions, 54 mg of the residue was further purified by preparative reverse-phase C18 HPLC with a Xterra column (Waters: F10  150 mm) with isocratic elution using 25% CH3CN at a flow rate of 4 ml min1. The peak 2, detected at around 7.5 min by UV (210 nm), was collected and evaporated in vacuo to dryness, yielding 2.3 mg. Purification of helvolic acid from Metarhizium anisopliae was carried out according to the previous report (11). MS was measured by FAB, EI-MS (JEOL MS-700 spectrometer, JEOL Ltd., Tokyo, Japan) and NMR was measured in CD3OD by a JEOL ECS400 spectrometer (JEOL Ltd.). Minimum inhibitory concentration C. albicans OUT6266, Saccharomyces cerevisiae ATCC9804, Aspergillus niger NBRC6341, and Rhizopus oryzae NBRC31005 were used as the test strains. The minimum inhibitory concentration was determined by micro-dilution assay (12), while S. cerevisiae was cultivated in YPD medium. A synergistic effect was observed by adding target compounds after 1 h of cultivation with nystatin.

RESULTS AND DISCUSSION Construction of a bioassay system with C. albicans as the indicator strain We used C. albicans as an indicator strain in the present study; this fungus causes an opportunistic infection, candidiasis, in immunosuppressed patients. First, we determined the optimal concentration of the polyene antibiotics (nystatin and amphotericin B) at which no inhibitory activity against C. albicans was detected. Nystatin at a concentration higher than 5.9 mg/ml showed a distinct growth inhibitory area (Table S2). Based on this result, a concentration of 2.9 mg/ml nystatin was chosen for the bioassay. However, the concentration of amphotericin B as the coexisting polyene was not determined because the effective concentration was too low for practical use (<1.5 mg/ml). Subsequently, this assay system was validated using the aminoglycoside antibiotic hygromycin B (HygB), which inhibits protein synthesis inside the cell. Although a very high concentration of HygB (250 mg/ml) was required to inhibit the growth of C. albicans in the absence of

J. BIOSCI. BIOENG., TABLE 1. Minimal inhibitory concentration of hygromycin in the presence of nystatin. Hyg concentration (mg/ml)

Antibiotics

Hyg Hyg with Nys (2.9 mg/ml)

500

250

125

62.5

31.3

15.6

þþ þþ

þ þ

 þ

 þ

 

 

Degree of anti-Candida activity is expressed by the diameter of inhibition zone (d): moderate: 15 >dS10 mm (þþ), weak: 10>dS7 mm (þ), no inhibition ().

nystatin, the effective concentration decreased 4-fold in the presence of nystatin (2.9 mg/ml) (Table 1), suggesting that HygB was easily localized intracellularly in the presence of nystatin due to the enhanced permeability of the cell membrane. Therefore, this assay system will assist in the discovery compounds which show slight or undetectable activity with a conventional assay system. Screening for anti-C. albicans compounds from the metabolites of entomopathogenic fungi We selected metabolites from entomopathogenic fungi as the source of active compounds against C. albicans. Entomopathogenic fungi which infect and proliferate on host insects are thought to produce various bioactive compounds, and indeed, various bioactive compounds have recently been isolated from these fungi (13e18). Thirty-two representative strains of entomopathogenic fungi (Table S1) from our in-house collection were used for screening. Six different cultivation conditions (with variations in the medium, cultivation time, and extraction method) were used to obtain culture extracts (Materials and methods). Only in the presence of nystatin, 119 out of 192 culture extracts displayed anti-Candida activity (Table 2). These results demonstrated that this screening system was useful to detect anti-Candida compounds that have been overlooked by conventional assay systems. Subsequently, we further evaluated the strains which displayed strong and/or reproducible activity, to purify and identify the active components. Ten of the tested strains (Table S3) were chosen and cultivated in large scale to obtain a sufficient amount of active components for structural identification, and it was found that the productivity of active components did not change significantly even in the case of a five-fold scale-up. Among all samples, the active components in the extracts from Cordyceps indigotica HF879 and Torrubiella minutissima HF809 were selected for further investigation, because they showed the largest inhibitory clear zones in the bioassay. Purification and identification of the active component from C. indigotica To identify the active components in the metabolite of C. indigotica among the many peaks detected in HPLC analysis, a bioassay against C. albicans was conducted for each fraction obtained by sequential fractionation, with fractions 3, 4, and 5 exhibiting significant activity (Fig. 1A and B). The UV absorption spectra of relatively large peaks (peaks a to i) in these fractions were compared with those registered in Dictionary of Natural Products on DVD (CRC Press). As a result, the peaks a to h in fractions 3, 4 and 5 were suggested to be the members of the indigotide family that were recently discovered from C. indigotica (19,20) (Fig. S1, Table S4). To avoid rediscovery of compounds which have already been identified in the same species, we stopped further purification, although the indigotide compounds were not previously shown to have anti-Candida activity. As for fraction 5, we further fractionated the contents, isolated each peak, and evaluated the individual biological activities. Peaks i and h were collected and checked for anti-Candida activity (Fig. 1C). Although both peaks possessed anti-Candida activity, peak i was further purified to determine its chemical structure because peak i showed a different UV spectrum from those of the indigotide family (Fig. S1). The extract of C. indigotica was separated with a Sep-Pak C18 cartridge, and the peak i was present in the fractions eluted

Please cite this article in press as: Kinoshita, H., et al., Cryptic antifungal compounds active by synergism with polyene antibiotics, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.08.003

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TABLE 2. Anti-Candida activity of the extracts from cultures of entomopathogenic fungi. Strain

Species 14-days cultivation

21-days cultivation Extraction solvent

EtOAc a

HF768 HF490 HF849 HF763 HF711 HF489 HF273 HF460 HF603 HF879 HF374-1 HF287 HF875 HF397 HF866 HF420-2 HF870 HF829 HF887 HF616 HF698 HF587 HF281 HF656 HF838 HF654 HF633 HF476 HF809 HF871 HF319

Akanthomyces arachnophilius Akanthomyces cinereus Akanthomyces gracilis Akanthomyces novoguineensis Akanthomyces pistillariaeformis Akanthomyces sp. Aschersonia aleyrodis Beauveria bassiana Cordyceps crinalis Cordyceps indigotica Cordyceps militaris Cordyceps paradoxa Cordyceps prolifica Cordyceps ramosopulvinata Cordyceps sp. Cordyceps takaomontana Cordyceps tubeculata Hirsutella neovolkiana Hirsutella nutans Metarhizium anisopliae Metarhizium flavoridie Nomuraera rileyi Paecilomyces cateniannulatus Paecilomyces farinosus Paecilomyces fumosoroseus Paecilomyces sp. Paecilomyces tenuipes Polycephalomyces sp. Torrubiella minutissima Torrubiella superficialis Lecanicillium lecanii

EtOAc

Medium-1

Medium-2

þ þ þc þ þ þc þ  þc þ þ þ þ þ þ þ þ  þ þ þc þ þ þ þ þ þ þ þþ þ þþþc

 þ  þ þ þ  þ  þ þ  þ þ     þ þc þ      þ þ þþc þ þc

b

n-BuOH

Medium-1

Medium-2

Medium-1

Medium-2

 þþc þc   þc  þ þ þþc þ  þc þ þ þ    þ       þ  þþ þ þþc

 þ þ   þc    þ þ þ þ       þ         þþþc  þ

þ þþc  þ þ  þ þ þ  þc þ þ þ  þ þ þ þ þþc  þ        þþc þþþc

þ þ þc þ þ þ þ þ þ þc þþc þ þ þ þc  þ þ þ þþc  þ þ  þþ þ  þc þþþc þ 

Degree of anti-Candida activity is expressed by the diameter of inhibition zone (d): strong: dS12 mm (þþþ), moderate: 12 >dS9 mm (þþ), weak: 9>dS7 mm (þ), no inhibition (). a Medium-1 is A3M. b Medium-2 is SMY. c C. albicans did not grow inside the inhibition zone.

FIG. 1. Metabolite analysis of C. indigotica. (A) Profile of metabolites extracted from the culture on the 14th day by reverse-phase HPLC. The UV spectra of peaks a to i are shown in Fig. S1. (B) Bioassay against C. albicans. Residues equivalent to 2 ml of crude extracts were applied for the bioassay. (C) Bioassay against C. albicans. Residues equivalent to 1 ml of crude extract were applied for the bioassay. Control, only methanol; , without nystatin; þ, with nystatin (Nys, 0.58 mg).

Please cite this article in press as: Kinoshita, H., et al., Cryptic antifungal compounds active by synergism with polyene antibiotics, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.08.003

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J. BIOSCI. BIOENG.,

FIG. 2. Metabolite analysis of T. minutissima. (A) Profile of the metabolites extracted from the culture on the 21th day by reverse-phase HPLC. (B) Bioassay against C. albicans. Residues equivalent to 2 ml of crude extracts were applied for the bioassay. , without nystatin; þ, with nystatin (Nys, 0.58 mg).

by 80e95% of MeOH. Fifteen mg of the partially purified compound was obtained from 100 mg of crude extracts, and was further purified by reverse-phase HPLC. Based on the molecular mass of peak i (calcd m/z 567.2981 [MH]) by HRFAB-MS (Fig. S2) as well as the UV spectrum, peak i was assumed to be helvolic acid, which has also been discovered in an entomopathogenic fungus, Metarhizium anisopliae (11). Judging from the fragmentation patterns in EI-MS (Fig. S3), the peak i was identified as helvolic acid (Fig. 3). Purification and identification of the active component from T. minutissima The extract of T. minutissima was analyzed by HPLC-DAD and fractionated in a manner similar to that for C. indigotica. Based on the result that fractions 2 and 3 showed strong activity (data not shown), the contents in fractions 2 and 3 were further fractionated, and two distinct peaks were separated and purified (peaks 1 and 2, Fig. 2A). The results of the bioassay clarified that peak 2 was the major active component in the extract of T. minutissima, because peak 2 showed strong activity only in the presence of nystatin, like the whole extract (Fig. 2B). By separation with a Sep-Pak C18 cartridge, peak 2 was eluted in fractions containing 55%e70% of MeOH, yielding 54 mg of the

partially purified compound, which was further purified by reverse-phase HPLC. The molecular mass of peak 2 was determined to be m/z 258.2 [M] by HREI-MS (Fig. S4), suggesting that the composition was C12H22N2O4. Together with the 1H NMR spectra (Fig. S5), this indicated that the peak 2 was terramide A (Fig. 3) from Aspergillus terreus (21), although no information of its activity has previously been reported. Minimum inhibitory concentration of identified compounds A micro-dilution assay was conducted to determine the minimum inhibitory concentration (MIC) values of the antibiotics isolated from entomopathogenic fungi against several yeasts (Table 3). MIC of nystatin was also measured in liquid culture by micro-dilution to determine the suitable concentration of nystatin to measure the synergistic effect with helvolic acid or terramide A. One fourth of the MIC against each test strain was applied for the synergistic activity assay. Although the MIC of helvolic acid against C. albicans was 64 mg/ml, comparable to the value reported (31.5 mg/ml) in our previous study (22), the concentration decreased by 75% in the presence of nystatin (Table 3). Similarly, the MIC of terramide A against

FIG. 3. Structure of the compounds which showed activity against C. albicans in the presence of nystatin. (A) Helvolic acid from C. indigotica, and (B) terramide A from T. minutissima.

Please cite this article in press as: Kinoshita, H., et al., Cryptic antifungal compounds active by synergism with polyene antibiotics, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.08.003

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TABLE 3. MIC of helvolic acid and terramide A against fungi. MIC (mg/ml)

Strain

Candida albicans Saccharomyces cerevisiae Aspergillus niger Rhizopus oryzae

Nys

Hel

Nys/Hel

TeA

Nys/TeA

2 8 32 16

64 >64 >64 >64

0.5/16 2/64 8/>64 4/>64

32 >64 >64 32

0.5/8 2/64 8/>64 4/8

Nys, nystatin; Hel, helvolic acid; TeA, terramide A.

C. albicans and R. oryzae decreased from 32 mg/ml to 8 mg/ml in the presence of nystatin. Although synergistic effect of nystatin with other antibiotics were evaluated in the previous reports (23,24), only compounds which had been known as antifungal compounds were tested as the counterpart. In this study, our newly constructed assay system detected anti-Candida activity in 58% of the extracts from entomopathogenic fungi. Our subsequent assays revealed the antiCandida activity of terramide A, which has not previously been reported to have anti-Candida activity. We then showed that plausible indigotide-family compounds also showed anti-Candida activity. Finally, these results demonstrated that our method is useful for discovering novel compounds or compounds whose activity has been overlooked, and that the prescribed amount of polyene compounds can be lowered to avoid side effects. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2015.08.003.

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Please cite this article in press as: Kinoshita, H., et al., Cryptic antifungal compounds active by synergism with polyene antibiotics, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.08.003