Antimicrobial constituents of Hypocrea virens , an endophyte of the mangrove-associate plant Premna serratifolia L .

Emergence of multidrug-resistant pathogenic microorganisms has prompted a worldwide search for new antibiotics from various sources. Endophytic fungi from unique habitats are considered as potential sources of novel bioactive compounds. Sri Lankan mangrove ecosystem is such a distinctive and unexploited resource for the discovery of structurally diverse and biologically active metabolites including antimicrobials. Nine endophytic fungi were isolated from the leaves and twigs of Premna serratifolia L. from a mangrove habitat in the Negombo lagoon and the antimicrobial activities of their laboratory cultures were evaluated. The most promising antimicrobial activity was exhibited by the endophytic fungus Hypocrea virens. Bioassay guided fractionation of the organic extract of this fungus led to the isolation of two known metabolites; the antimicrobial epidithiodioxopiperazine, gliotoxin (1), and the closely related but less active bisdethiobis(methylthio)gliotxin (2). The chemical structures of the two compounds were determined by spectroscopy and confirmed by comparison of mass and nuclear magnetic resonance (NMR) spectral data with the reported values for these molecules. The minimum inhibitory concentration (MIC) values obtained for gliotoxin (1) in the current study are 0.13 μg mL for Bacillus subtilis, 16 μg mL for Staphylococcus aureus, 32 μg mL for Methicillin Resistant Staphylococcus aureus (MRSA) and Escherichia coli, 64 μg mL for Pseudomonas aeruginosa and Candida albicans fungus. This is the first study to report the isolation of endophytic fungi from P. serratifolia and their antimicrobial activities.


INTRODUCTION
The prevalence of antibiotic resistance, specially multidrug resistance among pathogenic bacteria is a serious human health concern (Levy & Marshall, 2004).According to Rice (2008), this resistance has increased in both Gram-positive and Gram-negative bacteria and they effectively escape the effects of current antibiotics.As more and more bacteria develop resistance, it has become crucial that new antibiotics with novel modes of action are introduced to replace the older ones, which have become ineffective.Natural products, specially the ones produced by microbes, are still a promising platform for such discovery (Butler et al., 2014).Strobel and Daisy (2003) have reported that plants from distinct environmental settings have a high possibility of harbouring endophyitc fungi that produce novel bioactive secondary metabolites.Li et al. (2009), Pang et al. (2008) and Zhou et al. (2014) have stated that endophytic fungi isolated from mangrove plants are a well-established source for structurally diverse and biologically active secondary metabolites.However up to now, the mangrove habitats in Sri Lanka have not been explored for endophytic fungi and their bioactive secondary metabolites.The high biodiversity and unique environment conditions of mangrove habitats such as sharp variation in moisture; temperature and salt concentration; high and low tides of water; anaerobic soil and intense competition among microorganisms; insects and herbivores etc., predestine the mangrove flora of Sri Lanka as promising sources for the isolation of endophytic fungi with bioactive properties (Bandaranayake, 2002;Debbab et al., 2013).
In a programme designed to investigate the antimicrobial secondary metabolites of endophytic fungi from distinct environmental settings of Sri Lanka, the isolation and characterisation of helvolic acid from a Xylaria sp.endophytic in the endemic orchid Anoectochilus setaceus from a rainforest, equisetin from an endophytic Fusarium sp. from an arid zone invasive cactus, and solanioic acid, a novel degraded steroid with potent antibacterial activity from an endophytic fungus of Cyperus rotundus was reported by us earlier (Ratnaweera et al., 2014;2015a;2015b).Our investigations on the antimicrobial constituents of a laboratory culture of the endophytic fungus Hypocrea virens inhabiting the mangrove-associate plant Premna serratifolia are now reported.This article describes the isolation of the endophytic fungi from P. serratifolia and the antimicrobial properties of the organic extracts of their laboratory cultures of which a preliminary account was also presented (Ratnaweera et al., 2013).A bioassayguided isolation and structure elucidation by nuclear magnetic resonance (NMR), low-resolution mass spectra (LRMS) of gliotoxin and bisdethiobis(methylthio)gliotoxin, and the antimicrobial constituents present in the organic extracts of Hypocrea virens are also reported.

Collection of plant material, isolation of endophytic fungi and investigation of antimicrobial activity
Healthy leaves and twigs of P. serratifolia were collected from the Kadol Kale mangrove forest in the Negombo Lagoon Sri Lanka (7 o 11'41'' -7 o 11'54'' N and 79 o 50'32'' -79 o 50'48'' E) in March 2013 and brought to the laboratory in tightly sealed polythene bags under humid conditions.The plant material was identified and confirmed using A Revised Handbook of the Flora of Ceylon (Dassanayake & Fosberg, 1985).For surface sterilisation, the leaves and twigs were first washed thoroughly in running tap water for 10 min, then successively immersed in 70 % ethanol for 1 min, 5.25 % sodium hypochlorite for 3 min and 70 % ethanol for 30 s (Ratnaweera et al., 2014).Finally, the surface sterilised plant parts were washed with sterilised distilled water and allowed to dry in a laminar flow cabinet.Small pieces were cut from these surface sterilised specimens and placed on sterile potato dextrose agar (PDA) dishes.A few drops of sterilised distilled water used for the final washing was also spread on the sterile PDA plates to confirm the sterilisation process by the absence of any fungal growth.The endophytic fungi, which emerged from the tissues were transferred on to new PDA dishes and sequential sub culturing was done until pure cultures were obtained.Each fungus was grown on 5 PDA dishes and after 14 -21 days, depending on the growth of the fungus.The mycelium together with the medium was cut into small pieces and dipped in 200 mL of ethyl acetate.After leaving for 24 hrs, each ethyl acetate extract was filtered and evaporated under reduced pressure at 40 o C using a rotary evaporator (BUCHI-R-200).The residues obtained (crude ethyl acetate extracts) were tested for antimicrobial activity against Gram-positive Bacillus subtilis (UBC 344), Staphylococcus aureus (ATCC 43300), Methicillin Resistant Staphylococcus aureus (MRSA, ATCC 33591), Gram-negative Escherichia coli (UBC 8161), Pseudomonas aeruginosa (ATCC 27853) and pathogenic fungus Candida albicans (ATCC 90028) at 200 µg per disc using agar disc diffusion method according to the National Committee for Clinical Laboratory Standards (NCCLS, 2003).The bioassay was carried out in triplicate to confirm the activity.polymyxin B (30 µg/disc) for P. aeruginosa, E. coli and B. subtilis, rifamycin (10 µg/disc) for S. aureus, MRSA and amphotericin B (20 µg/disc) for C. albicans were used as positive controls, while methanol was used as the negative control.The results obtained were statistically analysed by one-way ANOVA using Minitab.

Large scale culturing and extraction of endophytic fungi
The endophytic fungus, which showed the most promising antimicrobial activity was cultured on PDA medium (200 medium sized Petri dishes -100 × 20 mm) for 15 days at room temperature.At the end of the incubation period, the fungus together with the medium were cut into small pieces and immersed in 1 L of ethyl acetate for 48 hrs and filtered through filter paper (Whatman No.1).This extraction and filtration process was repeated thrice.The filtrates were combined and the organic solvent was evaporated to dryness under reduced pressure at room temperature.The crude extract obtained was weighed and tested for antimicrobial activity at 50 µg per disc using agar disc diffusion method to confirm the presence of antimicrobial activity.

Identification of the endophytic fungi exhibiting antimicrobial activity
First, the colony morphological features of the endophytic fungus was recorded.Then fungal DNA was extracted using a published protocol (Kariyawasam et al., 2012).The extracted DNA was subjected to polymerase chain

Isolation and structure elucidation of the bioactive components
To isolate the principal bioactive component(s) from the complex mixture of crude ethyl acetate extract, a series of bioassay guided chromatographic techniques were performed.The initial fractionation of the crude extract (570 mg) by size exclusion column chromatography on Sephadex LH-20 (3 × 115 cm; methanol as the eluent) resulted in seven fractions A-G, based on thin layer chromatographic (TLC) analysis.These fractions were tested for antimicrobial activity against MRSA and B. subtilis using a bioautographic TLC assay (Choma & Grzelak, 2011).The most active fraction E (22 mg) was next chromatographed on normal phase silica (2 × 30 cm column) with step-gradient elution (hexane: ethyl acetate 46:54 to ethyl acetate followed by ethyl acetate/methanol mixtures up to 1:1 ratio) to obtain the main active component 1.A second bioactive fraction D (60 mg) from Sephadex LH-20 chromatography was also chromatographed on normal phase silica (2 × 30 cm column) with step-gradient elution (hexane:ethyl acetate 35:65 to ethyl acetate followed by ethyl acetate/methanol mixture up to 1:1 ratio) to obtain a less bioactive component 2.
The structure elucidation of the isolated compounds 1 and 2 was done using NMR and mass spectral data. 1 H, 13 C and 2D NMR spectral datasets in CDCl 3 were obtained using a Bruker AVANCE 600-MHz spectrometer with a 5 mm cryoprobe, while the electrospray ionisation mass spectral (ESIMS) data were obtained using Bruker Esquire-LC electrospray spectrometer.

Antimicrobial activity of compounds 1 and 2
The minimum inhibitory concentrations (MICs) of the major bioactive compound 1 was tested for antimicrobial activities against three Gram-positive bacteria, B. subtilis (UBC 344), S. aureus (ATCC 43300) and MRSA (ATCC 33591), two Gram-negative bacteria, E. coli (UBC 8161), P. aeruginosa (ATCC 27853) and a pathogenic fungus C. albicans (ATCC 90028) using broth microdilution method according to NCCLS with modification using Mueller Hinton broth as the medium (NCCLS, 2002).MIC assay was done in triplicate and the mean was taken to calculate the MIC value.The less bioactive compound 2 was checked for bioactivity against the above microorganisms at 200 µg per disc using agar disc diffusion method (NCCLS, 2003).Agar disc diffusion assay was also carried out in triplicate and the mean inhibition zone was taken.The commercial antimicrobial agents polymyxin B, rifamycin and amphotericin were used as positive controls and methanol was used as the negative control.

Isolation and antimicrobial activities of the endophytic fungi, and identification of the active fungal strain
In the current study nine endophytic fungi, seven from the leaves and two from the twigs were isolated from P. serratifolia.The antibacterial activities of the crude fungal extracts are given in Table 1.The extract WM-1.6 originating from the twigs showed the most promising antibacterial activities (ANOVA, p < 0.0001) being active against Gram-positive B. subtilis, S. aureus, MRSA and Gram-negative E. coli.The extracts WM-1.1 and WM-1.4 showed selective activities against B. subtilis, while WM-1.3 and WM-2.3 showed weak inhibitions of P. aeruginosa.Although the inhibition zones against P. aeruginosa were considerable, the zones were not completely clear of the bacterium, which showed that the extracts did not completely kill the organism at 200 µg per disc.Of the nine extracts, four were inactive against all test organisms while none were active against C. albicans.
Fungal mycelium, which led to the extract WM-1.6 was light green and white, and concentric rings were observed in the culture.After 13 -15 days, it started to secrete a light yellow pigment to the medium.According to DNA sequence data and blast results obtained, this fungus showed 100 % identity to the previously isolated Hypocrea virens (Syn.Trichoderma virens) species (ex: accession numbers, EF596954.1,JX492998.1,JX174053.1)(http://www.ncbi.nlm.nih.gov/pubmed, 17/12/2014).Therefore on the basis of its 18S ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence and 28S ribosomal RNA gene, partial sequence, the WM-1.6 active fungus isolated in the current study was assigned to H. virens species.

Isolation and structure elucidation of the active compounds 1 and 2
Size exclusion chromatography of the crude extract (570 mg) followed by silica gel chromatography led to the isolation of 7 mg of the main active compound 1 as a white crystalline compound.This gave a [M+Na] + ion with m/z 349 in the low-resolution electrospray ionisation mass spectrum consistent with a molecular weight of 326 daltons and a molecular formula of C 13 H 14 N 2 O 4 S 2 .
Analysis of 1 H (Figure 1) and 13 C NMR data as well as 2D NMR (COSY, HSQC, HMBC, TROESY) spectral data in CDCl 3 revealed that the structure of the active compound 1 (Figure 3a) matches that of the known epidi thiodioxopiperazine, gliotoxin (Kaouadji, 1990).The only significant differences were the appearance of two additional methyl singlets at 2.22 ppm (12-Me) and 2.24 ppm (11-Me) in the 1 H NMR spectrum (Figure 2) and two additional signals at 13.3 and 14.6 ppm in the 13 C NMR spectrum of the compound 2.These differences could readily be explained by reductive methylation of the disulfur-bridge of gliotoxin resulting in two additional S-methyl groups leading to the known fungal metabolite bisdethiobis(methylthio)gliotoxin (Figure 3b).Comparison of 1 H and 13 C NMR data of compound 2 with the reported values of bisdethiobis(methylthio)gliotoxin confirmed this deduction (Lee et al., 2001).A comparison of 13 C NMR values obtained in the present study for gliotoxin (1) and bisdethiobis(methylthio)gliotoxin ( 2) with already reported data is given in Table 2.

DISCUSSION AND CONCLUSION
Although mangrove-derived fungi are well established as a potential source of new bioactive compounds, there have been no reports [other than a preliminary study (Ratnaweera et al., 2013)] of endophytic fungi of mangrove and mangrove associated plants of Sri Lanka, and their secondary metabolites.Therefore initiating a programme to investigate the endophytic fungi and their bioactivities from Sri Lankan mangrove habitats becomes a meaningful effort.
This pioneering investigation on the production of antimicrobial constituents by endophytic fungi from the mangrove habitat of Sri Lanka has revealed that the mangrove associated plant P. serratifolia harbours several endophytic fungi, which are capable of producing antibacterial substances with selective activities.Our investigation further revealed that the fungus H. virens, which showed the most promising antimicrobial activity among the isolated endophytic fungi produces two known epidithiodioxopiperazine antibiotics, gliotoxin and bisdethiobis(methylthio)gliotoxin (Kaouadji, 1990;Lee et al., 2001), making this the first study to identify the antimicrobial constituents of an endophyte isolated from P. serratifolia.Gliotoxin was active against both Gram positive and Gram negative bacteria tested and the pathogenic fungus C. albicans with varying potencies, and was most active against B. subtilis with a MIC of 0.13 µg mL -1 .Bisdethiobis(methylthio)gliotoxin was less potent and was active only against B. subtilis at 200 µg per disc concentration.
H. virens was first reported from Indiana, USA from a decorticated wood, and it has been found to be the telemorph of T. virens according to molecular and morphological characters (Chaverri et al., 2001).H. virens has generally been found earlier from soil, decaying wood, on other fungi (such as basidiomycetes) and as opportunistic plant symbionts (Herman et al., 2004;Druzhinina et al., 2011).According to some previous reports H. virens and H. lixii have been isolated from several mangrove plants, Rhizophora apiculata, R. mucronata, Avicenna officialis and A. marina (Liu et al., 2011;Bhimba et al., 2012).However, to the best of our knowledge this is the first isolation of an endophytic H. virens from the plant P. serratifolia.Weindling and Emerson (1936)  and 1966, respectively (Weindling, 1941;Beecham et al., 1966).During several years there had been confusion about the nomenclature of the gliotoxinproducing fungus (Webster & Lomas, 1964) Antimicrobial activities of gliotoxin isolated in the current study also match with the previous reports (Taylor, 1971;Jordan & Cordiner, 1987).According to reported data of Brian and Hemming (1945), gliotoxin has shown MIC values of 3, 10 and 20 µg mL -1 against S. aureus (NCTC 3750), B. subtilis (NCTC 3610) and E. coli (NCTC 4144), respectively.Kaouadji (1990) has reported some antifungal activity of gliotoxin during its isolation against C. albicans and C. tropicalis, while Li et al. (2006) has reported 1 µg mL -1 activity of gliotoxin against MRSA and multidrug resistant S. aureus (MDRSA).It has been reported that the wide range of biological properties, such as antiviral, antibacterial and immunosuppressive activities of gliotoxin is a direct consequence of the reactivity of the epidithio bridge, which is capable of generating reactive oxygen species and engaging in protein conjugation (Waring et al., 1995).
Gliotoxin has attracted considerable attention mainly due to its antibacterial, antifungal and antiviral activities (Jordan & Cordiner, 1987).It has also shown selective toxicity to cells of the haematopoietic system (Mullbacher & Eichner, 1984;Mullbacher et al., 1985).However, their potential to be chemotherapeutic agents has diminished due to the discovery of in vivo toxicity.More recent work on this type of toxins has shown that they may be involved in aetiology of fungal diseases as well as design of novel and specific enzyme inhibitors (Waring & Beaver, 1996).Apart from the above, Anitha and Murugesan (2005) have mentioned gliotoxin as the first antibiotic to be used for plant disease control against pathogenic fungi.
According to Li et al. (2006), bisdethiobis(methylthio) gliotoxin have shown a 31.2 µg mL -1 MIC value against MRSA and MDRSA contradicting the results of this study.However, this compound has not shown any radical scavenging activity against DPPH (Li et al., 2006).The lower potency of bisdethiobis(methylthio)gliotoxin against the tested microorganisms compared to gliotoxin in the current study is in agreement with similar results obtained for anti-angiogenic activities of these two compounds, thus again confirming that the bioactivity is correlated with presence and absence of the disulfide bond in the compounds (Lee et al., 2001).
The investigation of endophytic fungi from terrestrial plants is a relatively new area of research in Sri Lanka.The results of the current investigation together with our recent findings of solanioic acid, a novel degraded steroid with potent antibacterial activity from an endophytic fungus of C. rotundus and a nortiterpenoid helvolic acid from an endophytic Xylaria from a unique rainforest setting, demonstrate the potential of Sri Lankan plant endophytes for producing novel drug leads (Ratnaweera et al., 2014;2015b).Thus, it is hoped that this study will encourage further investigations of endophytic fungi of distinctive ecological settings of Sri Lanka for fruitful findings for the welfare of humans.