Sclerotiorin: a Novel Azaphilone with Demonstrated Membrane Targeting and DNA Binding Activity Against Methicillin-Resistant Staphylococcus Aureus

Microbiol. Biotechnol. Lett. (2020), 48(4), 429–438 http://dx.doi.org/10.48022/mbl.2002.02006 pISSN 1598-642X eISSN 2234-7305 Microbiology and Biotechnology Letters

Sclerotiorin: a Novel Azaphilone with Demonstrated Membrane Targeting and DNA Binding Activity against Methicillin-Resistant Staphylococcus aureus

Chakradhar Dasagrandhi1*, Anup Pandith2,3, and Khalid Imran1 1Department of Microbiology, Krupanidhi Degree College, Varthur (Hobli), Off Sarjapur Road, Carmelaram Post, Bangalore 560035, India 2Department for Management of Science and Technology Development, Ton Duc Thang University, Ho Chi Minh City, Vietnam 3Faculty of Applied Science, Ton Duc Thang University, Ho Chi Minh City, Vietnam

Received: February 13, 2020 / Revised: March 6, 2020 / Accepted: April 11, 2020

The emergence of multi-drug resistant, pathogenic methicillin-resistant Staphylococcus aureus (MRSA) is a threat to global health and has created a need for novel functional therapeutic agents. In this study, we evaluated the underlying mechanisms of the anti-MRSA effect of an azaphilone pigment, sclerotiorin (SCL) from Penicillium sclerotiorum. The antimicrobial activity of SCL was evaluated using agar disc diffusion, broth microdilution, time-kill assays and biophysical studies. SCL exhibits selective activity against Gram positive bacteria including MRSA (range, MIC = 128-1028 µg/ml) and exhibited rapid bactericidal action against MRSA with a > 4 log reduction in colony forming units within three hours of adminis- tration. Biophysical studies, using fluorescent probes and laser or electron microscopy, demonstrated a SCL dose-dependent alternation in membrane potential (62.6 ± 5.0.4% inhibition) and integrity (> 95 ± 2.3%), and the release of UV260 absorbing materials within 60 min (up to 3.2 fold increase, p < 0.01) of exposure. Further, SCL localized to the cytoplasm and hydrolyzed plasmid DNA. While in vitro checkerboard studies revealed that SCL potentiated the antimicrobial activity of topical antimicrobials such as polymixin, neomycin, and bacitracin (Fractional Inhibitory Concentration Index range, 0.26-0.37). Taken together these results suggest that SCL targets the membrane and DNA of MRSA to facilitate its anti- MRSA antimicrobial effect.

Keywords: Azaphilones; sclerotiorin, antimicrobial agents, MRSA, membrane action, synergy

Introduction breaks of several superbugs in the recent past in several countries emphasized once again the need for the devel- Staphylococcus aureus (S. aureus), a versatile opportu- opment of novel antimicrobial resources [2]. With the nistic pathogen has been implicated in many soft tis- continuous withdrawal of pharmaceutical companies sues, implant related diseases. Emergence of multi-drug from the antibiotic market and discovery process, there resistance in methicillin-resistant Staphylococcus is a great demand for novel antibiotic agents against S. aureus (MRSA) is a serious public health concern. aureus are necessary. Although several natural products Further development of more virulent forms of S. of plant, and microbial origin have been reported against aureus causes increased healthcare burden [1]. The out- MRSA [3], relatively very few molecules are currently under clinical study. Hence there is tremendous oppor- *Corresponding author tunity for novel natural products with diverse mecha- Tel: +91-9740696123 nism of action to expand the drug arsenal and to E-mail: [email protected] © 2020, The Korean Society for Microbiology and Biotechnology contravene the rise of multidrug resistant MRSA.

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Fungal azaphilones are fungal pigments with wide health functional benefits. The biological activities of fungal azaphilones have been extensively reviewed [4]. Fungal azaphilones are known to possess inhibitory effect against nematodes, monoamine oxidases, cholesterol acetyl transferases, HIV replication, free radicals, gp120−CD4 binding, heat shock protein 90 (Hsp90), and protein-protein interactions [5−11]. Sclerotiorin (SCL), a azaphilone pigment produced by Penicillium species was reported to exhibit endothelin receptor binding [12]), anti lipoxygenase, aldose reductase [13], anti cholesterol [14], tumor suppressive [15], anti-HIV [16], antifungal [17], anti-mycobacterial [18] properties. The food compatible SCL analogs have been reported earlier [19]. Fig. 1. Sclerotiorin (SCL) structure. The 1NMR spectrum Although SCL was reported to be antibacterial, details obtained in CDCl3 at 125 MHZ for the TLC pure fraction. The pro- on its mechanism of action was lacking. ton peaks of NMR spectrum were attributable to the groups (a-l) SCL has been shown to exhibit some antimicrobial shown in the structure. Pure SCL crystal was shown in the inset. activities. However, a systematic study on the antimicrobial action of SCL has not been documented. (Methicillin resistant) and ATCC 29213 (methicillin- The present study which was carried out in order to sensitive) were procured from American Type Culture address the potential use of novel non-antibiotic natural Collection (USA). SA1199 and SA1199B are clinical product alternatives against MRSA infections, we evalu- strains obtained as gift by Dr. G.W. Kaatz (Wayne State ated the anti-microbial activity of SCL isolated from University, USA). All other strain information is pro- Penicillium sclerotiorum and evaluated mechanism of vided in the Table 1. The cultures were stored at -80℃ in action against methicillin-sensitive S. aureus (MSSA) TSB stocks (50% v/v glycerol). The test bacteria grown and methicillin-resistant S. aureus (MRSA). either TSB or NB for overnight (12 h) were used for the experiments. Materials and Methods Agar disc diffusion assay Chemicals and reagents The procedure for evaluation of anti-microbial potency Antibiotics (nisin, polymixin, neomycin, bacitracin), of SCL was performed as per the recommendations by ethidium bromide (EtBr), propidium iodide (PI), 4′,6- Clinical Laboratory Standards Institute [20]. Briefly, diamidino-2-phenylindole (DAPI), and dimethyl sulfox- test microorganisms (0.1 ml, 105 colony forming units ide (DMSO) were purchased from Sigma-Aldrich (USA). (cfu)/ml approx.) were inoculated to MH agar plate Thin layer chromatography (TLC) plates (Silica gel (20 ml). SCL stock solutions were prepared in sterile 60 F254) were purchased from Merck (Germany). DMSO (1 mg SCL in 0.1 ml DMSO) and 20 µl aliquot GenEluteTM Plasmid Mini Prep Kit was purchased from (100 µg) was loaded to sterile filter paper disk (6-mm Sigma-Aldrich. SCL was purified (>95% purity, Fig. 1) dia). The filter paper discs were placed equidistantly on from the culture broths of Penicillium sclerotiorum and the pre seeded agar. The inoculated plates were incu- identified by proton nuclear magnetic resonance as bated (37℃, 24 h) and the size of inhibitory zones around described previously [13]. the test and control disks was determined.

Media, cultures, and growth conditions Minimum inhibitory concentration (MIC) Tryptic soy broth (TSB), nutrient broth (NB), and A twofold serial dilution of SCL (range, 0.5−500 µg) or mueller hinton broth (MBH) were purchased from nisin (250−0.1 µg) were prepared in microtiter plates Becton-Dickinson (USA). S. aureus strains, ATCC 35139 (MP) containing 0.1 ml of MHB and test bacterial

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(106 cfu/ml approx.). The plates were incubated sta- 10 min. The DioC2 (3) labeled cells in PBS were either tionary 35℃ for 24−48 h. The bacterial growth was untreated or treated with SCL (31.2−125 µg/ml) for determined by measuring the optical density (OD) of cul- 5 min. The fluorescence intensity of DioC2 (3) of ture at 610 nm using a plate reader (Tecan M200 untreated and SCL treated cells was measured using Infinite Pro, Switzerland). MIC is defined as the lowest BD FACS flow cytometer (ex: 488) using argon laser. dilution of the test agent that inhibited >90% of the cell Nisin (62.5 µg/ml) served as positive control. growth.

Release of UV260 nm absorbing materials Time kill kinetics The release of UV260 nm absorbing material by S. Viable counts of S. aureus (ATCC 35139 and ATCC aureus in presence of SCL was quantified according to 29213) in presence of SCL were determined by time kill the method previously described [23] with slight modifi- kinetic study according to the method of [21] with some cations. Briefly, S. aureus cells (OD610 nm 0.1) were modifications. Briefly, MHB (10 ml) adjusted with SCL incubated with SCL (62.5, 125 and 250 µg/ml). At 0, 30, (31.2, 62.5, and 125 µg/ml) or Nisin (64 µg/ml) and inocu- 60, 120, 240 min after incubation, a 0.1 ml aliquot from lated with S. aureus (2 × 105 cfu/ml approx.). An aliquot each treatment was filtered (0.2 µm) and the D of the fil- of suspension (0.1 ml) was sampled at 0, 3, 6, 9 and 24 h, trate was determined 260 nm. Background OD serial diluted, spread on MHA (0.1 ml), and incubated at imparted by SCL at respective concentration was sub- 35℃ for 24−48 h. The viable colonies of S. aureus after tracted from the final values. 48 h were compared between control and test samples. Scanning electron microscopy (SEM) Determination of membrane integrity SEM analysis was carried out according to the method The integrity of S. aureus after treatment with SCL of [24]. Briefly, S. aureus (105 cfu/ml) in PBS was treated was determined using fluorescence assisted cell sorting with SCL (128 µg/ml) at 32℃ 60 min. After brief centrif- (FACS) analysis by PI staining and EtBr uptake assays. ugation for 5 min (8000 ×g), the cell pellet was reconsti- The samples were prepared according to the method [22] tuted with PBS and an aliquot (0.1 ml) was placed on with minor modifications. Briefly, the test bacteria (105 cellulose acetate filter (0.2 µm) and fixed with glutaral- cfu/ml) in two sets of PBS (pH 7.4, 0.1 ml) were incubated dehyde (4% v/v) for 3 h, rinsed with distilled water, and with SCL (0−128 µg/ml) at 32℃ for 60 min. One set of dehydrated with 50, 70, 80, 90, 95, and 100% (v/v) test bacteria were incubated with PI (20 µg/ml) for graded ethanol. The samples were sputter coated with 10 min at 32℃ and the samples were analyzed by flow gold and observed under SEM at 10 KV (JOEL, JSM- cytometer (BD FACS Aria III, USA). Further, for real 6390LV, Japan). time monitoring of membrane integrity, the second set of SCL treated cell suspension (0.1 ml) was incubated with Cell penetrating ability of SCL EtBr (5 µM) in a black bottom MP for 5 min and the S. aureus (105 cfu/ml) was incubated with sub MIC of EtBr fluorescence was recorded (ex; 360 nm, emi; 616 SCL (15.6 and 31.2 µg/ml) at 32℃ for 2 h. Those without nm) using MP reader (Tecan Infinite pro, Model 814). SCL served as negative controls. The bacteria were Control experiments were conducted without the addi- washed with PBS, harvested by centrifugation (3000 ×g/ tion of SCL or with the addition of nisin (128 µg). 5 min) and the reconstituted pellet in PBS was incubated with lysostaphin (10 U) for 10 min. The cell sus- Determination of membrane potential pension was filtered (0.2 µm) and the fluorescence of The membrane potential of MRSA was determined lysate was determined (ex: 312 nm, emi: 418 nm). using Bac LightTM bacterial membrane potential kit (Life Technologies, USA) using 3,3′-diethyloxacarbocy- DNA binding assay anine iodide (DioC2 (3)) as per manufacturer’s instruc- The interaction of SCL with plasmid DNA was deter- tions. Briefly ATCC 35139 cells (OD 0.1) suspended in mined by agarose gel electrophoresis. In brief, 200 ng of PBS were labeled with 5 µM of DioC2 (3) at 28℃ for plasmid DNA isolated from ATCC 35139 using Gen

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elute plasmid isolation kit (Sigma-Aldrich) was incu- Table 1. Anti-bacterial activity of Sclerotiorin against test bated with SCL (0−256 µg/ml) in binding buffer (Tris pathogens. ℃ ZOIa MICb Hcl, pH 8.4) at 28 for 30 min. The DNA migration was *Bacterial strain analyzed by EtBr staining on 1% agarose by gel electro- (mm) (μg/ml) phoresis. The degree of DNA condensation was deter- Gram-Positive pathogens mined using EtBr displacement assay. Plasmid DNA Bacillus subtilis (ATCC 6051) 12 125 (10 µg/ml) was incubated with EtBr for 1 h at 28℃. The Listeria monocytogens (ATCC 19111) 10 1000 EtBr-DNA complex was incubated with sub MIC of SCL Enterobacter clocae (ATCC 13048) 09 1000 (15.6, 31.2 µg/ml) and the fluorescent intensity of the Staphylococcus aureus complex was determined (exi: 520 nm; emi: 590 nm). Methicillin-sensitive SA1911 & 1911 B 11 250 Interaction of SCL with topical antibiotics ATCC 29213 10 125 The interaction of SCL with antibiotics was deter- Methicillin-resistant mined by checkerboard method [25]. Twofold serial dilu- ATCC 35139 12 125 tions of antibiotics (bacitracin, polymixin, neomycin) and 01ST001 12 125 SCL (0−64 µg/ml) were prepared in 0.1 ml of MBH in MP Gram-negative bacteria 5 containing the test bacteria (2 × 10 cfu/ml). The plates Escherichia coli (ATCC 8739) 07 ND were incubated at 37℃ for 24−48 h and OD610 of the cell Salmonella typhi (KCTC 2515) nil ND suspension was determined using MP reader. The frac- Yeast pathogens tional inhibitory concentration index (FICI) was deter- Candida albicans (KCTC 11282) nil ND mined by the following formula: Cyrptococcus neoformans(KCCM 50564) nil ND *ATCC: American Type Culture Collection center, KCTC: Korean FICI = ------FIC A- FIC B Collection for Type Culture (Daejeon, South Korea), KCCM: Korean Culture of Center of Microorganism (Sudaemun, Seoul). aZOI: Zone b where FIC A = MIC of SCL in combination with anti- of inhibition, MIC: minimum inhibitory concentration. biotic/MIC of SCL alone, FIC B = MIC of antibiotic in combination with SCL/MIC of antibiotic alone). The interaction can be synergy (FICI < 0.5), Indifference (FICI = 4) or antagonism (FICI > 4).

Statistical analysis A two tailed student’s t test using Graph Pad Prism (GraphPad Software, Version 5.00, USA) was performed.

Results

SCL exhibits anti-staphylococcal activity It is evident that SCL exhibited narrow spectrum activity with MIC range of 128−1028 µg/ml against Gram positive bacteria including S. aureus (Table 1). SCL exhibited dose dependent inhibitory effect achiev- Fig. 2. Time kill kinetic analysis of MRSA (ATCC 35139) in the ing complete suppression (> 90%) of bacterial growth at presence of increasing concentration of sclerotiorin (31.2- 125 µg/ml up to 24 h (Fig. S1). Further from Table S1 it 125 μg/ml). Mean ± SD (n = 4) value was represented. is evident that S. aureus strains (including clinical isolates of MRSA) exhibited susceptibility (MIC range, However, Gram-negative and yeast strains were found 125−250 µg/ml; MBC range, 125−500 µg/ml) to SCL. to be resistant to SCL toxicity. In time kill studies using

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Fig. 3. Confocal laser scanning microscopic (CLSM) images of MRSA either untreated (A), treated with DMSO (B), and sclerotiorin (SCL) at 31.2-125 μg/ml (C-E) or nisin at 62.5 μg/ml (F). S. aureus (ATCC 35139) cells were exposed to SCL or DMSO (negative control) were subsequently washed with saline, stained with DAPI/PI and subjected to CLSM. Cells with red color indicate dead cells. Each experiment was repeated twice with 5 random filed (Scale: 20 μm).

S. aureus (Fig. 2), the untreated ells exhibited exponen- 4 log cfu/ml. Whereas SCL at 64 µg/ml (0.5 ×MIC) tial growth reaching >10 log cfu in 24 h. Cells treated caused a 3 log reduction within 6 h but failed to control with SCL (125 µg/ml) reduced viable counts MRSA by till 24 h. Similar results were also observed in MSSA

Fig. 4. Sclerotiorin (SCL) induced cell membrane integrity damage in MRSA. Flowcytometry dot plots (A) and percentage of MRSA cells (ATCC35139) showing enhanced propidium iodide fluorescence (B) in presence of SCL (32-128 μg/ml). Nisin (128 μg) served as positive control. Mean ± SD (n = 4) value was represented. Student’s t test: ***p < 0.001, **p < 0.01.

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uptake was evident in presence of SCL (64−256 µg/ml) resulting in maximum cell damage by MIC of SCL (p < 0.001, 99.6 ± 0.2%) and this effect was comparable to nisin treatment (p < 0.001, 99.4 ± 0.2%). In time dependent assay, cells treated with SCL (31.2 µg/ml) exhibited PI fluorescence which was not significantly dif- ferent from controls (Fig. 5). However cell treated with SCL (62.5 µg/ml) exhibited increased PI fluorescence (2.3 ± 1.1 fold) until 120 min. Further increase in SCL (125 µg/ml) rapidly increased PI fluorescence (p <0.01, 3.1 ± 1.3 fold) within 60 min and then slightly decreased (Fig. 3B). Fig. 5. Time dependent uptake of ethidium bromide facilitated by sclerotiorin (SCL) in MRSA. EtBr uptake by ATCC Localization of SCL in MRSA 35139 cells in the presence of SCL was quantified by fluores- S. aureus cells treated with sub MIC of SCL (15.6−31.2 cent spectrophotometer. Nisin served as positive control). Mean ± SD (n = 4) value was represented. Student’s t test: µg/ml) exhibited green fluorescence (Fig. 6). Further we ***p < 0.001, **p < 0.01 quantified the intracellular abundance of SCL by measuring its fluorescence by fluorescent spectrophotome- (data not shown). ter. From Fig. 6B, it is evident that the fluorescent intensity of cell lysate of S. aureus treated with SCL SCL causes membrane integrity damage (15.6−31.2 µg/ml) was significantly high (240 ± 5.2 AU, The confocal laser scanning microscopy (CLSM) study 1.6 fold, p < 0.05) compared to the control (161 ± 2.4). using DAPI (Blue) and PI (red) suggested that untreated cells stained blue, whereas SCL treated cells exhibited SCL induces membrane damage in S. aureus red fluorescence (Fig. 3). Further FACS analysis (Fig. From flow cytometric dot plots (Fig. 7A), it is evident 4A, B), showed that only 8.7 ± 0.3% cells exhibited PI flu- that SCL (62.5−125 µg/ml) resulted in increased propor- orescence. However, a dose dependant increase in PI tion of red fluorescence of MRSA cells. Further when

Fig. 6. Sclerotiorin (SCL) uptake by individual MRSA cells. Bright field (BF, left), fluorescent (UV, middle) and merge (right) images of ATCC 35139 (A) either untreated (-SCL) or treated with 31.2 μg/ml SCL (+SCL). Scale bar: 20 μm. Fluorescent intensity of cell lysates of ATCC 35139 cells (B) incubated with SCL (16-32 μg/ml) incubated for 40 min. (n = 6, **p < 0.01).

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Fig. 7. Membrane potential alterations induced by sclerotiorin (SCL) in MRSA. Comparison of flow cytometric profiles of untreated (control), positive control (nisin), and SCL treated ATCC 35139 cells (A). Mean fluorescence intensity of DioC2 (3) labeled S. aureus cells either untreated or treated with 31.2-125 μg/ml of SCL (B). (n = 3, **p < 0.01, ***p < 0.001). compared to controls (Fig. 7B) there was a statistically absorbing values of untreated cells slightly increased significant reduction (p < 0.01) in the mean fluorescence from 0−240 min. sub MIC of SCL (31.2−62.5 µg/ml) intensity value of cells incubated with SCL which was exhibited no considerable increase (0.6 fold) increase in comparable to the positive control (Nisin, 62.5 µg). UV260. In presence of SCL (125−250 µg/ml), a rapid Further from Fig. 8A, it is evident that the UV260 increase (p < 0.01, 1.7−3.2 fold) was evident with in

Fig. 8. Membrane permeability induced by sclerotiorin (SCL) in MRSA. Leakage of UV260 nm absorbing materials from S. aureus incubated with SCL (A). Scanning electron microscopy (SEM) analysis of cell morphology of ATCC 35139 cells either untreated (B) or treated with SCL at 128 μg/ml (C)

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Fig. 9. Interaction of sclerotiorin (SCL) with plasmid DNA. Electrophoretic mobility of plasmid DNA of ATCC 35139 either untreated (Lane 1) or treated with 15.6-128 μg/ml of SCL (A). Displacement of EtBr from the EtBr-plasmid DNA complex by SCL (B). Mean ± SD (n = 4) value was represented.

60 min and remained stable thereafter. In addition, the SCL and antibiotic combinations were ineffective SEM studies indicated that the untreated S. aureus cells against Gram negative bacteria. exhibited smooth cell surface (Fig. 8B). In contrast, SCL treated cells exhibited surface blebs with altered mem- Discussion brane integrity (Fig. 8C). SCL was reported to anti-staphylococcal [26]. However SCL interact with MRSA plasmid DNA the mechanism of anti-staphylococcal action of SCL has At SCL/DNA ratio (0.01 w/w), a fraction of the plasmid not been reported and hence we investigated the anti- DNA is less mobile than plasmid DNA alone. Above staphylococcal mechanism of action of SCL was investi- SCL/DNA ratio of 0.04, retardation of the plasmid DNA gated. In the present study, we found that SCL was mobility is completely inhibited (Fig. 9A). Additionally, Gram positive specific especially against S. aureus. The SCL dose dependently (31.2−125 µg/ml) displaced EtBr- MIC (125−250 µg/ml) of SCL of this study is comparable DNA complex and resulted in decrease in EtBr fluores- to the MIC of some azaphilones (MIC range, 15−250 µg/ cence in a time dependent manner (Fig. 9B). ml) previously reported against S. aureus [27, 28, 16]. The bactericidal activity exhibited by SCL of the present SCL potentiates topical antimicrobial efficacy study (> 4 log cfu reduction) is in contrast with the bacte- From Table 2, it is evident that sub MIC (31.2 µg/ml) riostatic property reported earlier for SCL [27]. In our of SCL exhibited synergy with bacitracin and neomycin study SCL exhibited narrow spectrum activity against (FICI range, 0.26−0.37) and an additive effect with poly- Gram positive bacteria. The apparent lack of activity of mixin against MRSA and MSSA (FICI = 0.5). However SCL against Gram-negative bacteria may be due to

Table 2. Synergy of Sclerotiorin (SCL) with topical antibiotics on Gram positive and Gram negative bacteria. bMIC (μg/ml) MIC (μg/ml) MIC (μg/ml) aStrain SCL(a) PMX(b) (a) + (b) cFICI BAC(c) (a) + (c) FICI NEO(d) (a) + (d) FICI ATCC35139 125 128 32+128 1.25 256 32+16 0.31 1028 32+64 0.26 ATCC29213 125 256 32+128 0.5 128 32+8 0.31 4 32+0.5 0.37 ATCC8739 >125 256 32+256 1.25 512 32+512 1.25 8 32+4 0.75 KCTC2515 >125 128 32+128 1.25 512 32+512 1.25 2 32+2 1.25 aATCC 35139 and 29213 are susceptible for the synergistic combinations of Sclerotiorin (SCL) with bacitracin (BAC) and neomycin (NEO). bMinimum Inhibitory concentration, cFICI; Fractional Inhibitory Concentration Index. Potential synergistic interaction are indicated in bold interface (FICI < 0.5).

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absence of specific target sites for SCL. The lipopolysac- observations are in line with the earlier findings of [34] charides of E. coli were reported to prevent the entry of that the agents that selectively collapse proton motive hydrophobic drugs [29]. force (PMF) can kill bacteria. The intracellular localiza- Our study using CLSM and flow cytometry suggested tion of SCL further can access plasmid DNA and inhibit that SCL could dose dependently alter the cell mem- replication and transcription of plasmid DNA. The brane integrity. PI can’t enter the intact cell mem- potential of membrane targeting compounds to alter branes. In the present study SCL facilitated the entry of PMF, metabolic activity, and intracellular function has small molecules such as PI and EtBr by loss of mem- been reported [34, 35]. Further, the membrane integrity brane integrity mechanisms. This loss in membrane loss caused by SCL at sub MIC concentration was integrity may further result in temporary pore forma- thought be responsible for neomycin and bacitracin tion in the cell membrane leading to altered membrane uptake which augmented the toxicity against S. aureus. potential, membrane damage and release of large mole- The membrane targeting and DNA targeting action of cules such as DNA as evident from depolarization, SEM SCL is encouraging. Owing to the health benefits of SCL and UV260 nm releasing experiments. SCL exhibits auto as antioxidant, anti-inflammatory and antimicrobial, it fluorescence (exi, 358 nm; emi 512 nm). Using CLSM can be used in topical preparations both as preservative and fluorescent spectrophotometer, we found that SCL and antimicrobial. However a thorough investigation on fluorescence was internalized in the cell cytoplasm of S. the development of structural analogs to and to test its aureus. In support of our observation, epicocconone with efficacy In-vivo is required inorder to develop SCL in to intrinsic fluorescence was reported to be cell permeable an effective therapeutic agent. and stain the cytoplasmic components [30, 31]. This made us to look for extra cellular targets for SCL and we Acknowledgment confirmed the affinity of SCL to bind plasmid DNA and compete for EtBr in EtBr-DNA complex. Sub MIC of CD would like to thank the Management, Krupanidhi Group of Insti- SCL (15.6−31.2 µg/ml) also potentiated the efficacy of tutions (KGI) and Krupanidhi-Research Incubation Center (K-RIC, KGI) for constant support and encouragement. bacitracin and neomycin and these results in agreement with the synergistic action of SCL (20 µM) with rifampin Conflict of Interest against mycobacterium [18]. S. aureus acquires multidrug resistance due to thick The authors have no financial conflicts of interest to declare. cell wall, antibiotic modifying enzymes, efflux pumps, and plasmid encoded genes. From our experiments, we References deduce certain understanding of mechanism of action of SCL against MRSA. The negative charges of SCL help 1. Stewart CM, Cole MB, Legan JD, Slade L, Vandeven MH, Schaffner in preliminary interaction with the cell membrane and DW. 2002. Staphylococcus aureus growth boundaries: moving establish an electrostatic interaction with positively mechanistic predictive mode is based on solute-specific effects. Appl. Environ. Microbiol. 68: 1864-1871. charged cell components. SCL with its polyene side 2. Tommasi R, Brown DG, Walkup GK, Manchester JI, Miller AA. chain attached to isochromane ring increase the hydro- 2015. ESKAPEing the labyrinth of antibacterial discovery. Nat. phobicity of SCL (log P = 1.8) and increase its tendency Rev. Drug. Disc. 14: 529-542. to partition in to the cell membrane and drives the pene- 3. Friedman M. 2015. Antibiotic-resistant bacteria: prevalence in tration of SCL. An electrochemical gradient know as food and inactivation by food compatible compounds and plant proton motive force (PMF) across the cell membrane is extracts. J. Agric. Food. Chem. 63: 3805-3822. 4. Osmanova N, Schultz W, Ayoub N. 2010. Azaphilones a class of maintained by the bacteria [32]. Although SCL is known fungal metabolites with diverse biological activities. Phytochem. to interact with transmembrane proteins (PknG) via Rev. 9: 315-342. hydrogen interactions [14, 33], our biophysical studies 5. Dong J, Zhou Y, Li R, Zhou W, Li L, Zhu Y, et al. 2006. New nemati- indicated that rapid perturbation of membrane integrity cidal azaphilones from the aquatic fungus Pseudohalonectria allows slow release of biomolecules resulting in permea- adversaria YMF1.01019. FEMS. Microbiol. Lett. 264: 65-69. bilization of cell membrane and loss of viability. Our 6. Yoshida E, Fujimoto H, Yamazaki M. 1996. Isolation of three new

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http://dx.doi.org/10.48022/mbl.2002.02006