Antifungal Long-Chain Alkenyl Sulphates Isolated from Culture Broths of the Fungus Chaetopsina sp.

• Abstract
• Introduction
• Results and Discussion
• Materials and Methods
• General experimental procedures
• Strain and fermentation
• Extraction and isolation
• Biological activity
• References

Abstract

During a high-throughput screening program focused on the discovery and characterization of new antifungal compounds, a total of 8320 extracts from Fundacion MEDINAʼs collection were screened against a panel of 6 fungal parasitic strains, namely Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis, Candida albicans, and Aspergillus fumigatus. A total of 127 extracts displayed antifungal properties and, after LC/MS dereplication, 10 were selected for further fractionation. Bioassay-guided fractionation from a 1-L fermentation of one of these extracts, belonging to the fungus Chaetopsina sp., led to the isolation of linoleyl sulphate (1), linolenyl sulphate (2), and oleyl sulphate (3) as the compounds responsible for the antifungal activity. These molecules were previously described as synthetic products with the ability to produce the allosteric inhibition of soybean lipoxygenase and human lipoxygenase.

Introduction

Invasive infections caused by fungi have severely increased in recent years, mainly due to the rising number of immunocompromised patients [1]. The therapeutic arsenal of antifungal drugs currently in use is rather limited and the evolving threat of resistant emerging pathogens has turned the development of new antifungal agents, preferably naturally occurring compounds with novel mechanisms of action, low resistance rates, and fewer side effects, into an urgent medical need [2].

During an HTS campaign aimed at discovering new antifungal natural products, a subset consisting of 8320 extracts from MEDINAʼs Natural Extracts Collection were tested against a panel of 6 fungal parasitic strains, namely Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis, Candida albicans and Aspergillus fumigatus. A fungal extract of the strain Chaetopsina sp. (CF-255912) grown in STP medium was identified as bioactive against this panel. This fungus was isolated from leaf litter of the endemic plant Beilschmiedia tawa (A.Cunn.) Kirk (Lauraceae). Samuels included Chaetopsina as anamorphs of species of the Nectria group [3] but, according to more recent studies, Luo and Zhuang determined that Chaetopsina species are anamorphs of either the Cosmospora or the newly described Chaetopsinectria genus [4]. Although no molecules have been so far isolated from Chaetopsina sp., examples of several antifungal agents isolated from Cosmospora sp. or others anamorphs of this genus belonging to Fusarium species exist, such as the cosmosporasides [5] or the parnafungins [6]. In this report, we describe the fermentation, isolation, purification, and structural elucidation of three long-chain alkenyl sulphates ([Fig. 1]) as the compounds responsible for the antifungal activity observed in our Chaetopsina extracts.

Results and Discussion

The producer strain was isolated from leaf litter of the plant B. tawa and identified as Chaetopsina sp. (Cosmospora anamorph) based on a megablast search of NCBIs GenBank nucleotide database. The closest hits using the initial 574 bp of LSU sequence were Chaetopsina fulva [GenBank GU075867; Identities = 569/574 (99 %), no gaps], Chaetopsina pinicola [GeneBank KF777201; Identities = 561/574 (98 %), no gaps] and Chaetopsinectria chaetopsinae penicillatae CBS60892 [GeneBanK GU075865; Identities = 551/574 (95 %), no gaps]. The phylogenetic analysis, according to Luo and Zhuang [4], supported this taxonomic identification (Fig. 1 S, Supporting Information).

Bioassay-guided fractionation from a 1-L fermentation in STP medium of the fungal strain using a combination of low-resolution chromatography and HPLC led to the isolation of three long-chain alkenyl sulphates as the compounds responsible for the observed antifungal activity. Molecular formulae of the three compounds in the series were determined by HRESIMS as C₁₈H₃₄O₄S, C₁₈H₃₂O₄S, and C₁₈H₃₆O₄S, suggesting different degrees of unsaturation within the same structural framework. These molecular formulae were not found in The Dictionary of Natural Products database [7], indicating the potential novelty of these compounds as natural products.

Compound 1 was isolated as a white amorphous solid. A molecular formula of C₁₈H₃₄O₄S was determined from its HRESIMS (m/z 347.2240, [M + H]⁺, calcd. for C₁₈H₃₅O₄S⁺, 347.2251) and ¹³C NMR data. Signals observed in its ¹H and ¹³C NMR spectra ([Table 1]) accounted for the presence in the molecule of two double bonds, one oxygenated methylene, one doubly allylic methylene, two allylic methylenes, one aliphatic triplet methyl group, and nine aliphatic methylenes. The four oxygens and one sulphur atom present in the molecule, as deduced from its molecular formula, suggested the presence of a sulphate group that must be attached to the oxygenated carbon C-1 (δ _C 69.1 ppm). Taking into account this consideration and the number of double bond equivalents suggested by the molecular formula, compound 1 must be a linear molecule having two double bonds included in the carbon chain. COSY correlations observed for the methyl group, the olefinic protons, and the oxygenated methylene ([Fig. 2]) were in agreement with this proposal. The remaining task to complete the full structure of the molecule was therefore the location of the two double bonds in the carbon chain. The proton chemical shift of methylene H-11 (2.78 ppm) was indicative of a doubly allylic position of this group and, indeed, COSY correlations to the protons H-10 and H-12, and HMBC correlations to carbons C-9, C-10, C-12, and C-13 corroborated this proposal and identified fragment A ([Fig. 2]) in the structure of compound 1. Key correlations observed in the HMBC spectrum ([Fig. 2]) definitely established the position of this fragment in the aliphatic chain. Thus, a 3-bond correlation was observed between the methyl H-18 protons and carbon C-16. A second correlation between the allylic methylene H-14 was also observed to C-16, and this correlation was considered to also be a 3-bond correlation due to the absence of HMBC correlations between the olefinic proton H-13 and C-16. Finally, the configuration proposed for the two double bonds was based on the high-field NMR chemical shift of the allylic carbons C-8, C-11, and C-14 (28.2, 26.5, and 28.2, respectively), all below 30 ppm, indicative of a Z configuration in this kind of olefinic system [8]. This linear nonconjugated polyunsaturated chain is the same as that displayed by linoleic acid. Compound 1 is thus linoleyl sulphate, previously described as an allosteric inhibitor of SLO and 15-HLO [9], [10].

Compound 2 was isolated as a white amorphous solid. Its molecular formula was determined as C₁₈H₃₂O₄S from HRESIMS data (m/z 345.2091, [M + H]⁺, calcd. for C₁₈H₃₃O₄S⁺, 345.2094), containing an additional unsaturation with respect to compound 1. The ¹H NMR shifts ([Table 1]) showed, in this case, the presence of six olefinic protons, one oxygenated methylene, two doubly allylic methylenes, two allylic methylenes, one aliphatic triplet methyl group, and six aliphatic methylenes. This compound was degraded when spectra were recorded in methanol, so an additional set of NMR experiments was recorded in DMSO-d ₆. The HMBC correlation observed between the methyl H-18 protons and the olefinic carbon C-16, and COSY correlations of the protons H-17 to H-16 and H-18 established the position of the three double bond system in the aliphatic chain ([Fig. 3]). Additionally, the proton chemical shift of the terminal methyl group H-18 (0.97 ppm), slightly deshielded in comparison with the corresponding chemical shift of H-18 (0.91 ppm) in compound 1, further verified its proximity to the Δ ¹⁵ double bond. HMBC correlations from the H-11 and H-14 methylene protons to carbons C-10 and C-12, and C-13 and C-15, respectively, COSY correlations to the group of olefinic protons at δ _H 5.34 ppm, and proton chemical shifts (2.77 ppm) for both methylenes were indicative of a doubly allylic position of this group and completed the determination of this polyunsaturated chain. As mentioned above, the carbon chemical shift observed for C-8, C-11, C-14, and C-17 (26.4, 25.0, 25.0, and 19.9 ppm, respectively) was indicative of a Z configuration in all three of the bonds of the olefinic system. This linear nonconjugated polyunsaturated chain is the same as that displayed by linolenic acid [11]. Compound 2 is thus linolenyl sulphate [12].

Compound 3 was isolated as a white amorphous solid. Its molecular formula was determined to be C₁₈H₃₆O₄S from the HRESIMS (m/z 366.2669, [M + NH₄]⁺, calcd. for C₁₈H₄₀NO₄S⁺, 366.2673) and ¹³C NMR spectra, containing one unsaturation less than compound 1. The ¹H NMR shifts showed the presence of two olefinic protons, one oxygenated methylene, two allylic methylenes, one aliphatic triplet methyl group, and twelve aliphatic methylenes. This linear monounsaturated chain is the same as that displayed by oleyl sulfate. The proton and carbon chemical shifts observed in the NMR spectra of this compound matched those described in the literature for that compound [10], [13].

The initial antifungal activity detected in the extract was successfully confirmed in the natural sulphates isolated. These were tested against a panel of 6 fungal parasitic strains, namely C. glabrata, C. krusei, C. parapsilosis, C. tropicalis, C. albicans, and A. fumigatus ([Table 2]), with MIC values ranging between 2 and 64 µg/mL. Cytotoxic activity of all of the compounds isolated was also tested against the THLE-2 epitelial human cell line, displaying ED₅₀ values greater than 20 µg/mL.

Although there is a previous report of other sulphated long-chain alcohols, 1-heptadecanyl sulfate and 1-octadecanyl sulfate [14], isolated from the Mediterranean tunicate Sidnyum turbinatum that exhibited in vitro antiproliferative activity against the WEHI 164 cell line, this article constitutes the first report of compounds belonging to this structural class isolated from microbial sources and their antifungal activity, as well as the first report of compounds isolated from fungi of the genus Chaetopsina. Compounds 1–3 have been previously described as synthetic products and two of them, 1 and 3, present the ability to produce the allosteric inhibition of SLO and 15-HLO, a potential target for therapies against cancer, asthma, and arthrosclerosis [9], [10] that might also be involved in their antifungal activity via inhibition of fungal lipooxygenase. On the other hand, although not directly related to the biological activity herein described, compound 2 was previously described as salt of the pro-drugs of omega-3 polyunsaturated alcohols and their use for the treatment of elevated triglyceride levels [12].

In conclusion, three long-chain alkenyl sulphates, linoleyl sulphate (1), linolenyl sulphate (2), and oleyl sulphate (3), two of them (1 and 3) displaying significant antifungal activity, have been isolated from fermentation broths of Chaetopsina sp. The instability observed for compound 2 when recording its NMR spectra in CD₃OD might be claimed as a possible explanation for the weaker antifungal activity observed for this molecule. Given the previous bioactivity described for some of these compounds as inhibitors of SLO and 15-HLO, the antifungal activity herein described might be due to inhibition of fungal lipoxygenase. Additionally, this paper confirms the potential of microbial natural products as a source of bioactive molecules and potential new drugs.

Materials and Methods

General experimental procedures

IR spectra were measured with a JASCO FT/IR-4100 spectrometer equipped with a PIKE MIRacle single reflection ATR accessory. NMR spectra were recorded on a Bruker Avance III spectrometer (500 and 125 MHz for ¹H and ¹³C NMR, respectively) equipped with a 1.7-mm TCI MicroCryoProbe using the signal of the residual solvent as the internal reference (δ _H 3.31 and δ _C 49.0 ppm for CD₃OD, and δ _H 2.50 and δ _C 39.5 ppm for DMSO-d ₆). LC-UV-MS analysis was performed on an Agilent 1100 single quadrupole LC-MS system using a Zorbax SB-C8 column (2.1 × 30 mm, 5 µm, flow rate 0.3 mL/min, 40 °C) [15]. HRESIMS spectra were acquired using a Bruker maXis QTOF mass spectrometer coupled to the same HPLC system as described above. Flash chromatography was performed with a CombiFlash Teledyne ISCO Rf400x. Semipreparative HPLC was done using a GILSON GX-281 322H2 coupled to a UV-VIS detector and an automatic fraction collector. All solvents employed for isolation were of HPLC grade.

Strain and fermentation

The producing fungi Chaetopsina sp. (CF-255912) was isolated from leaf litter of the endemic tree B. tawa (A.Cunn.) Kirk (Lauraceae) collected in Basil Hewett Reserve, New Zealand, using a method for plating of washed particles of plant litter [16]. Frozen stock cultures in 10 % glycerol (− 80 °C) are maintained in the collection of Fundación MEDINA under the accession number CF-255912. Total genomic DNA was extracted from mycelia grown on YM agar. The 28S rDNA fragments, containing D1−D2 regions, were PCR amplified, and sequence alignments and phylogenetic analyses were performed as previously described [17]. The fungal strain was initially grown in ten different culture media, according to a strategy and protocols for fermentation of fungi on nutritional arrays previously described [18], and only cultures in STP medium displayed antifungal activity. To scale up the fermentation of this bioactive extract to 1 L, 12 mycelial discs were used to inoculate 50 mL of SMYA (Difco neopeptone 10 g, maltose 40 g, Difco yeast extract 10 g, agar 4 g, distilled H₂O 1 L). After 7 days incubation at 22 °C and 220 rpm, 0.3 mL aliquots of this culture were used to inoculate the STP medium [sucrose 75 g, tomato paste 10 g, malt extract 5 g, soy flour 1 g, (NH₄)₃SO₄ 1 g, KH₃PO₄ 9 g; 1 L distilled H₂O] distributed among 100 × 10 mL in 40 mL TPP tubes. The TPP were incubated statically at 22 °C, 70 % relative humidity for 21 days (Fig. 2 S, Supporting Information).

Extraction and isolation

TTP slants were pooled and triturated with 1 L of water. The resulting mixture was extracted with acetone (1 L) under magnetic continuous stirring for 2 h at room temperature. The solid material was separated by filtration (Büchner), and acetone from the supernatant (2 L) was concentrated under a stream of nitrogen until a final volume of 1 L. This solution was loaded with a continuous 1 : 1 water dilution onto a column packed with SP207ss resin (brominated styrenic polymer, 65 g) previously equilibrated with water. The column was washed with water (1 L) and afterwards eluted at 10 mL/min using a stepped gradient from 10 % to 100 % acetone in water for 30 min with a final 100 % acetone wash step of 12 min, collecting 19 fractions of 20 mL. 1400 µL of DMSO were added to fractions and all the acetone and water were evaporated on a centrifugal evaporator until generating a solution in 1400 µL (100 % DMSO) of each fraction. Fractions were subjected to antifungal testing, and fractions 14 to 16, containing the active compounds (eluted with 60 % acetone in water), were pooled and further purified by reversed-phase semipreparative HPLC (Agilent Zorbax RX-C8, 9.4 × 250 mm, 5 µm; 3.6 mL/min, UV detection at 210 nm), making repeated injections, with a linear gradient from 25 % to 55 % acetonitrile in water over 36 min where 1 (29 mg) was eluted at 23 min, 2 (2.8 mg) at 20 min, and 3 (130.6 mg) at 25 min.

Linoleyl sulfate ( 1 ): White amorphous solid; UV (DAD) λ _max end absortion; IR (ATR) ν _max 3395, 2926, 1650, 1463, 1243, 1213, 1023, 805 cm⁻¹; for ¹H and ¹³C NMR data see [Table 1]; HRESIMS m/z 347.2240 [M + H]⁺ (calcd. for C₁₈H₃₅O₄S⁺, 347.2251); 364.2511 [M + NH₄]⁺ (calcd. for C₁₈H₃₈NO₄S⁺, 364.2516).

Linolenyl sulfate ( 2 ): White amorphous solid; UV (DAD) λ _max end absortion; for ¹H and ¹³C NMR data see [Table 1]; HRESIMS m/z 345.2091 [M + H]⁺ (calcd. for C₁₈H₃₃O₄S⁺, 345.2094); 362.2357 [M + NH₄]⁺ (calcd. for C₁₈H₃₆NO₄S⁺, 362.2360).

Oleyl sulfate ( 3 ): White amorphous solid; UV (DAD) λ _max end absortion; IR (ATR) ν _max 3420, 2922, 2853, 1645, 1464, 1213, 1062, 1020, 983, 808 cm⁻¹; for ¹H NMR (500 MHz, CD₃OD) δ 5.33 (m, 2H, CH=CH), 4.00 (t, J = 6.6 Hz, CH ₂O), 2.01 (m, 4H, CH ₂CH=CHCH ₂), 1.66 (m, 2H, CH ₂CH₂O), 1.40 (m, 2H, CH ₂CH₂CH₂O), 1.29 and 1.32 [2bs, 20H total, (CH₂)₆ and (CH₂)₄], 0.90 (t, J = 6.7 Hz, CH ₃). ¹³C NMR (125 MHz, CD₃OD) δ 130.80, 69.12, 33.03, 30.84, 30.81, 30.57, 30.44, 30.42, 30.37, 30.30, 28.13, 28.10, 26.88, 23.71, 14.44; HRESIMS m/z 366.2669 [M + NH₄]⁺ (calcd. for C₁₈H₄₀NO₄S⁺, 366.2673).

Biological activity

Candida strains. Frozen stocks of C. albicans ATCC64124, C. glabrata ATCC2001, C. krusei ATCC6258, C. parapsilosis ATCC22019, and C. tropicalis ATCC75, were used to inoculate Sabouraud dextrose agar (SDA) plates for confluent growth. Plates were incubated for 24 h at 37 °C. The grown colonies were harvested from the SDA plates and suspended in RPMI-1640 modified medium. RPMI-1640 modified medium was prepared as follows: 20.8 g of RPMI powder (Sigma) were poured into a 2-L flask, together with 13.4 g of YNB, 1.8 L of milliQ water, 80 ml of Hepes 1 M, and 72 mL of glucose 50 %. The volume was adjusted to 2 L and filtered. The OD₆₆₀ was adjusted to 0.25 using RPMI-1640 modified as diluent and blank. According to each strain, dilutions from the adjusted inoculum were prepared at 1/10 (C. krusei and C. tropicalis) and 1/100 (C. albicans, C. glabrata, and C. parapsilosis) and kept on ice until used to inoculate 96-well assay plates.

For the assay, 90 µL of the 1/10 or 1/100 diluted inoculum were mixed with 10 µL of the different concentrations of a solution of pure compounds or extracts in 20 % DMSO in water. Twofold serial dilutions from 64 to 0.125 µg/mL were tested for each compound. Additionally, a curve of amphotericin B (Sigma, ~ 80 % HPLC) (8 points, ½ serial dilutions starting at 160 µg/mL) was included as an internal positive control in each assay plate. After dispensing the inoculum, plates were read in a multilabel plate reader (EnVision Perkin Elmer) at 612 nm for T₀ (zero time). Plates were then statically incubated at 37 °C for 20 h. After incubation, the plates were shaken in a DPC Micromix-5 and read again for T_f (final time). Percentage of growth inhibition was calculated using the following formula:

% Inhibition = [1 – [(T_fSample – T₀Sample) – (T_fBlank – T₀blank)/(T_fGrowth – T₀Growth) – (T_fBlank – T₀blank)] × 100.

Thereafter, 10 µL of resazurin dye 0.02 % (w/v) were added to the assay plates and they were incubated again for 2 h at 37 °C. After this second incubation, fluorescence was recorded using wavelength settings for resorufin (excitation 570 nm, emission 600 nm). The percentage of growth inhibition was calculated using the following normalization equations:

% Reduction = 100 × (fluorescence intensity of test agent – fluorescence intensity of untreated control)/(fluorescence intensity of reduced resazurin – fluorescent intensity of untreated control); and % Inhibition = 100 – % Reduction.

A. fumigatus. A method previously described was used to test antifungal activities against A. fumigatus [19].

Absorbance and fluorescence data were analyzed with Genedata Screener (Genedata AG). The MIC was defined as the concentration of antifungal compound that inhibited 90 % of growth of each fungal strain after overnight incubation. Assays were performed in triplicate and repeated two different days. In all experiments performed in this work, the RZ′ factor [20], [21] obtained was between 0.85 and 0.92.

MTT assay. The THLE-2 (ATCC CRL-10149) cell line was derived from primary normal liver cells by infection with SV40 large T antigen. The virus was generated by introducing a retroviral vector containing the of Bgl I-Hpa I fragment of the SV40T antigen into the amphotropic packaging cell line PA317. THLE-2 cells expressed phenotypic characteristics of normal adult liver epithelial cells. These were seeded at a concentration of 1 × 10⁴ cells/well in 200 µL culture medium and incubated at 37 °C in 5 % CO₂. After 24 h, when the monolayer was formed, the medium was replaced with a final volume of 195 µL, and 5 µL of compounds and controls were added to the plates. Methyl methanesulfonate (MMS) and DMSO were used as positive and negative controls, respectively. Four points of rotenone and doxorubicin with an initial concentration of 10 mM and dilution ½ were also used as controls. After the addition of compounds and controls, plates were incubated at 37 °C in 5 % CO₂ for 24 h. After this time, an MTT solution at 5 mg/mL in PBS 1× was diluted to 0.5 mg/mL in MEM without phenol red. The sample solution in wells was removed and 100 µL of MTT dye were added to each well. The plates were gently shaken and incubated for 3 h at 37 °C in 5 % CO₂. The supernatant was removed and 100 µL of DMSO 100 % were added. The plates were gently shaken to solubilize the formazan that originated, and absorbance was measured at a wavelength of 570 nm [22].

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgements

Financial support from the Junta de Andalucía through the Project RNM-7987 is fully acknowledged. The polarimeter, HPLC, IR, and NMR equipment, and plate reader used in this work were purchased via grants for scientific and technological infrastructures from the Ministerio de Ciencia e Innovación [Grants No. PCT-010000–2010–4 (NMR), INP-2011-0016-PCT-010000 ACT6 (polarimeter, HPLC, and IR), and PCT-01000-ACT7, 2011–13 (plate reader)].

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Correspondence

• References

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  CrossrefPubMedSearch in Google Scholar
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