Identification of a new antifungal oligoacetal derivative produced by Streptomyces toxytricini against Candida albicans

Abstract Thirty actinomycete isolates were isolated from soil and tested against Candida albicans in vitro. The active isolate was identified by 16s-rRNA gene sequencing method as Streptomyces toxytricini. The antifungal compound was extracted with ethyl acetate followed by diethyl ether. Both HPLC and GC–MS analysis confirmed presence of one pure compound in the diethyl ether extract. The compound is a yellow liquid has a maximum absorbance at 240 nm in methanol. The chemical structure was elucidated by 1D and 2D-NMR and IR analyses. The elucidated molecular formula was C36H54O14. The compound is a polyacetal tricyclononane derivative, composed of a tricyclononane ring attached from the carbon atom number four with an oligo-acetal chain (six acetal groups in chain) and from the carbon atom number seven with a methoxy carbonyl benzene-1,3-dicarboxylic acid. The purposed name is: 4- {[tricycle(3.2.1.11,3)non-8-yl] methoxy carbonyl benzene-1,3-dicarboxylic acid} (2,4,5,6,7,8,9 heptaoxa, 3-ethoxy, 5,6,7,9-tetramethyl unidecane). Graphical abstract


Introduction
Candida albicans is known to be a member of natural flora in healthy humans. As a matter of fact, immunodeficiency could result from the increased use of immunosuppressive agents in ABSTRACT Thirty actinomycete isolates were isolated from soil and tested against Candida albicans in vitro. The active isolate was identified by 16s-rRNA gene sequencing method as Streptomyces toxytricini. The antifungal compound was extracted with ethyl acetate followed by diethyl ether. Both HPLC and GC-MS analysis confirmed presence of one pure compound in the diethyl ether extract. The compound is a yellow liquid has a maximum absorbance at 240 nm in methanol. The chemical structure was elucidated by 1D and 2D-NMR and IR analyses. The elucidated molecular formula was C 36 H 54 O 14 . The compound is a polyacetal tricyclononane derivative, composed of a tricyclononane ring attached from the carbon atom number four with an oligo-acetal chain (six acetal groups in chain) and from the carbon atom number seven with a methoxy carbonyl benzene-1,3dicarboxylic acid. The purposed name is: 4-{[tricycle(3.2.1.1 1,3 )non-8-yl] methoxy carbonyl benzene-1,3-dicarboxylic acid} (2,4,5,6,7,8,9 heptaoxa, 3-ethoxy, 5,6,7,9-tetramethyl unidecane). organ transplantations, aggressive anticancer chemotherapy, virus infection, antibiotic overuse and several medications intake; it has led to a substantial increase in the occurrence of serious fungal infections (Lamagni et al. 2001). Among these fungi, Candida sp. was more prominent. The overgrowth of C. albicans has caused pathogenic symptoms, such as oral, intestinal and vaginal candidiasis (Ferrer 2000;Yuuki et al. 2005;Enoch et al. 2006;Gary et al. 2011).
The most economically and biotechnologically valuable prokaryotes, actinomycetes, are able to produce a wide range of bioactive secondary metabolites (such as antibiotics, antitumor agents, immunosuppressive agents and enzymes) which possess antibacterial, antifungal, neuritogenic, anticancer, antialgal, antimalarial and antiinflammatory activities. Therefore, the secondary metabolite that possesses antifungal activity from actinomycetes can inhibit C. albicans (Valli et al. 2012).
The prevalence of systemic fungal infections has increased significantly during the past decade (Nesrin et al. 2007). So, guidelines for treatment of Candida infections have always emphasised the need for early implementation of antifungal therapy (Frank & Donna 2004). Furthermore, discovery of new antimicrobial compounds are required due to the fact that micro-organisms can acquire resistance against the used antimicrobial compounds. This work aimed to identify a new antimicrobial compound from an actinomycete isolate against C. albicans.

Isolation of actinomycetes
Thirty actinomycete isolates were isolated and only two had antimicrobial activity against C. albicans. The most active actinomycete isolate that showed a larger inhibition zone (13 mm diameter) was selected for identification and production of the antifungal compound.

Test for volatile antifungal compounds
The C. albicans wasn't inhibited by the volatile compounds produced by the selected actinomycete isolate, although the supernatant of this actinomycete showed an inhibition zone. This reflects the non-volatile nature of the antifungal compound.

Identification of the actinomycete
According to BLAST analysis, the resulting sequence had high similarity with the 16S ribosomal RNA gene sequence of Streptomyces toxytricini strain HBuM174624 (GenBank accession no. Eu841711.1). The sequence BLAST analysis showed 96% similarity to S. toxytridni, which was reported in previous research to produce a specific inhibitor of pancreatic lipase named lipstatin (Weibel et al. 1987;umesh & Dubey 2012). However, no previous research articles reported production of antifungal compounds from S. toxytridni.

Extraction and purification of the antifungal compound
The supernatant was extracted with ethyl acetate and methylene chloride. Both extracts were tested in the inhibition zone test against C. albicans. Only the ethyl acetate extract showed an inhibition zone against C. albicans (12 mm diameter), while the methylene chloride didn't display any inhibition zone. Therefore, the ethyl acetate extract was evaporated and the remaining residue was subsequently extracted with different solvents with increased polarity. Only the diethyl ether extract showed an inhibition zone of 12-mm diameter. This extract was analysed by HPLC and GC/MS to identify the number of entire compounds that was present.

HPLC and GC/MS analysis
The HPLC analysis of the diethyl ether extract was performed using two different stationary phases (reversed and normal phases). These two columns were used to enable elution of the compounds that may have varied polarity. The normal phase may entrap the polar compounds and elute the polar one, while the reversed phase may entrap the non-polar compounds and elute the more polar one. The analysis by these two columns resulted in the presence of one pure compound ( Figure S1). The compound has a retention time 5.1 and 4.5 min for each column, respectively. This compound is a yellow liquid that has a strong characteristic actinomycete odor.
The GC/MS analysis was performed to confirm the HPLC result as well as to obtain the mass spectrum. The analysis also showed one pure compound ( Figure S2). The obtained mass spectrum showed fragments of m/z 55 (the base ion), 61, 70, 119, 165 and 207 ( Figure S3). Although several natural compounds are usually produced in microbial culture and require several purification techniques to obtain one pure compound. One pure compound was obtained by these simple extraction and purification methods. The purity of this compound was confirmed by both the HPLC and GC/MS analyses' results.
The COSY data interpretation showed presence of two groups of the methyl group δ H 1.28 (d, 5.9) (one of them was correlated with the δ H 5.14 and the second with δ H 4.95), two groups of the methyl group δ H 1.33 (d, 5.7) (one of them was correlated with δ H 5.23 and the second with δ H 5.38) and two groups of the methyl group δ H 1.18 (t, 7) (one of them was correlated with δ H 3.59 and the second with δ H 3.48, while there was only one methyl group at δ H 1.28 (t, 7), which is correlated with δ H 3.59. Therefore, the total number of the methyl groups in this compound was seven ( Figure S8).
The HMBC data interpretation matched the elucidated fragments and confirmed the overall elucidated structure (Figures S9 and S10). The carbonyl group was correlated with the protons of the phenyl ring as well as with the proton δ H 4.21 (CH 2 , δ C 69). This result confirmed the attachment of one carbonyl group with the carbon atom δ C133 of the ring from one side and with δ C69 from another side through ester bond. Also, this result confirmed the presence of two carboxyl groups directly attached with the phenyl ring, where there are two hydroxyl signals corresponding for these two carboxyl groups. For this reason, it was clearly appeared that the phenyl ring attached with three carbonyl groups.
The mass fragments pattern obtained from the GC/MS analysis matched the elucidated structure, where the m/z 61 fragments is corresponding to methyl acetal moiety of the four moieties after combination with a hydrogen radical (CH 3 CHOO + H˙), the m/z 119 fragment is the carboxy-phenyl ion (75 + 44), m/z 165 is the di-carboxy-phenyl ion (75 + (2 × 45), the m/z 207 is the tri-carboxy-phenyl ion (75 + (3 × 44), while the other fragments can be obtained from the fragmentation of the tricyclononane moiety. Absence of the molecular ion moiety is due to the sensitivity of the molecule toward the fragmentation, especially the poly-acetal moiety of this molecule.
S. toxytridni was previously used for production of lipstatain, which is a derivative of a tetra-membered ring lactone (Weibel et al. 1987;umesh & Dubey 2012). However, there aren't any reports of antimicrobial compounds produced by this actinomycete. Other new actinomycete isolates have been recently isolated. Awad et al. (2014) identified a new chitinase-producing actinomycete strain named Streptomyces glauciniger WICC-A03 that has antifungal activity against several phytopathogens. Shaaban et al. (2014) isolated an actinomycete isolate (Streptomyces sp. RM-14-6) that produces the sesquiterpene isopterocarpolone. The chemical structure of the produced sesquiterpene was identified by 1D and 2D-NMR spectroscopy and HR-ESI mass spectrometry.

Isolation of actinomycetes
Six soil samples were collected from different cultivated soils in 6th October City, Egypt. The isolation was performed on casein starch agar medium. Each obtained actinomycete isolate was purified on the same medium.

Isolation of C. albicans
Swabs from six women with vaginal candidiasis (with permission from the patients at Kaser EL-Ainy hospital, College of Medicine, Cairo university, Egypt) were taken and suspended in 3 mL of sterilized saline solution. Taking swabs for the detection of Candida spp. from human subjects is a routine work in this hospital. A loop-full from this solution was streaked on chromogenic agar medium (Oxoid, England), which is selective for isolation of C. albicans (Murray et al. 2005). The isolate that showed the characteristic green colonies were picked, microscopically examined and maintained on potato dextrose agar (PDA) slants (Oxoid, England).

Production of the antimicrobial compounds by the actinomycetes isolates
A loop from a 5-day old slant of each actinomycete isolate was used in inoculation of 50 mL of the seed medium (casein-starch broth) in a 250-mL conical flask. The flasks were incubated at 28 °C for 24 h. The optical density (OD) was measured at 600 nm and then adjusted to OD 0.1 for each actinomycete broth. One millilitre from each broth was then used to inoculate the production medium (50 mL of casein-starch broth) (Singh et al. 2012). The flasks were incubated at 28 °C for five days on rotary shaker at 150 rpm.

Disc and well diffusion test
Disc diffusion and well diffusion methods were used to test the antimicrobial activity of both the actinomycete supernatant and the culture extracts against C. albicans. A suspension of the C. albicans was spread on PDA plates using a sterilised cotton swab. Ten millilitre of the actinomycete culture was centrifuged at 3000 rpm to separate the supernatant from cells. The supernatant was then sterilised through a 0.2 µm syringe filter. A sterile filter paper disk (5 mm diameter) was impregnated with supernatant then placed on inoculated plates or a well on inoculated plate was filled with 100 µL of the supernatant. The dishes were then incubated at 28 °C for three days.

Test for volatile antifungal compound
This test was done to indicate if the antifungal compound was volatile or nonvolatile. C. albicans was streaked on the PDA plate. The actinomycete isolate was streaked on casein starch agar medium plate. The covers of both Petri dishes were removed and the C. albicans plate was inverted on the actinomycete plate. Then, it was sealed by parafilm and incubated for three days.

Identification of the actinomycete
Molecular identification of the actinomycete isolate was carried out by combination of polymerase chain reaction (PCR) amplification of its ribosomal DNA genes corresponding to unique sequences within the internally transcribed spacer (ITS) (Isogai et al. 2010). Genomic DNA was isolated using GeneJet genomic DNA purification Kit (Thermo), Catalogue No. K0722. Primers 8F (5′-AGA GTT TGA TCC TGG CTC AG-3′) as a forward primer and u1492R (GGT TAC CTT GTT ACG ACT T) as a reverse primer were designed to target the conserved regions and amplify the hypervariable regions that can provide species-specific signature sequences within the ITS sequences. PCR reaction conditions were optimised. Agarose (1.5%) was used for resolving the PCR products. The PCR product was purified using GeneJET™ PCR Purification Kit (Thermo, k0702). Sequencing of the PCR product was performed using ABI 3730xl DNA sequencer. The nucleotide sequences were compared with sequence database using the BLASTN algorithms (Altschul et al. 1997).

Production of the antimicrobial compound by the selected actinomycete isolate
Fifty millilitre of the seed inoculum was used for inoculation of 4 L of the casein-starch broth in a 5-L bioreactor (Bioflow-3000). The temperature was adjusted to 28 °C, agitation 150 rpm, pH 7.

Extraction and purification of the antifungal compound
The actinomycete culture filtrate was subsequently extracted with equal volumes of methylene chloride (CH 2 Cl 2 ) and ethyl acetate (EtOAc). The obtained extracts were tested for antifungal activity against C. albicans. The active extract (the ethyl acetate extract) was evaporated to dryness using rotary evaporator and the remaining residue was subsequently extracted by different solvents with increasing polarity: n-hexane, benzene, diethyl ether and ethyl acetate, each obtained extract was tested for antifungal activity against C. albicans.

HPLC analysis
The wavelength scanning for the diethyl ether active extract was analysed by spectrophotometer (Beckman, Du640) to obtain lambda max. The extract was analysed by HPLC Agilent 1260, equipped with 1260 quaternary pump and 1260 DAD. Two columns were tested: (1) reversed phase, hypersil ODS, 30 cm × 4.6 mm × 4.6 µm and (2) normal phase using silica column with the same dimensions. The used mobile phases were methanol in both columns at a flow rate 1 mL min −1 . The eluted compounds were detected at 240 nm.

GC/MS analysis
The ether extract was analysed by HP-5890 GC equipped with HP-5972 mass spectrometer. The analysis was performed by using HP-5MS column 30 m × 0.25 mm id × 0.25 μm film thickness. Mobile phase: helium, flow rate 1 mL min −1 . Oven temperature start at 70 °C for 1 min, up to 280 °C at a rate of 15 °C min −1 The mass detector temperature was 300 °C. The fragmentation pattern in mass spectra was automatically compared with that in the GC-MS software (Wiley 7 N mass library).

IR analysis
The analysis was conducted using Affinity-1, Shimadzu FTIR spectrophotometer.
From literature, this actinomycete has been studied before for the production of a specific inhibitor of pancreatic lipase named lipstatin, which is a tetra-membered ring lactone derivative. Recently, there aren't any reports of antimicrobial compounds produced by this actinomycete.

Disclosure statement
No potential conflict of interest was reported by the authors.