Possible mechanism of antifungal phenazine-1-carboxamide from Pseudomonas sp. against dimorphic fungi Benjaminiella poitrasii and human pathogen Candida albicans.

Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Possible mechanism of antifungal phenazine-1-carboxamide from Pseudomonas sp. against dimorphic fungi Benjaminiella poitrasii and human pathogen Candida albicans S.G. Tupe1, R.R. Kulkarni2, F. Shirazi1, D.G. Sant1, S.P. Joshi2 and M.V. Deshpande1 1 Biochemical Sciences Division, CSIR-National Chemical Laboratory, Pune, India 2 Organic Chemistry Division, CSIR-National Chemical Laboratory, Pune, India

Keywords apoptosis, Candida albicans, dimorphism, phenazines, Pseudomonas sp., reactive oxygen species. Correspondence Mukund V. Deshpande, Biochemical Sciences Division, National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411 008, India. E-mail: [email protected] 2014/1465: received 18 July 2014, revised 2 October 2014 and accepted 21 October 2014 doi:10.1111/jam.12675

Abstract Aim: Investigation of antifungal mechanism of phenazine 1-carboxamide (PC) produced by a Pseudomonas strain MCC2142. Methods and Results: An antifungal metabolite produced by a Pseudomonas was purified and identified as PC. Human pathogenic fungi such as Candida albicans, Candida glabrata, Cryptococcus neoformans, Fusarium oxysporum, Aspergillus fumigatus and Aspergillus niger were found to be inhibited by PC (MIC90 32–64 lg ml1). Addition of PC (20 lg ml1) during yeast (Y)–hypha (H) transitions inhibited germ tube formation by >90% and >99% in C. albicans National Collection of Industrial Microorganisms (NCIM) 3471 and nonpathogenic model Benjaminiella poitrasii, respectively. After exposure to PC (20 lg ml1), 75–80% yeast cells of B. poitrasii and C. albicans NCIM 3471 showed rhodamine 123 fluorescence indicating high intracellular reactive oxygen species (ROS) production. ROS further led to hyperpolarization of mitochondrial membrane, subsequently induction of apoptosis as evident by externalization of phosphatidylserine, DNA fragmentation, chromatin condensation and finally death in B. poitrasii. In C. albicans NCIM 3471, PC (20 lg ml1) induced apoptosis. Conclusions: The antifungal effect of PC in B. poitrasii and C. albicans may be due to ROS-mediated apoptotic death. Significance and Impact of the Study: Inhibition of Y–H transition of B. poitrasii and C. albicans by PC indicates that it may prove useful in the control of dimorphic human pathogens.

Introduction In recent years, increase in the morbidity and mortality due to invasive fungal infections is posing a serious challenge to overcome. The major fungi infecting humans include Candida sp. (Candida albicans, glabrata and parapsilosis) accounting for ~75% of all fungal infections (Tupe and Deshpande 2013). Worldwide-reported attributable mortality for candidemias ranges from 5 to 71%, with crude mortality as high as 81% (Horn et al. 2009) and this rate exceeds that of all Gram-negative bacterial septicemias, emphasizing the medical importance of

fungal diseases. Main reasons for escalation in invasive fungal infections include increase in the number of immunocompromised hosts such as patients with AIDS, autoimmune diseases, burns, radiotherapy, chemotherapy, transplantation and development of resistance against currently used antifungals. Secondly, drawbacks of existing drugs such as acute and chronic side effects, less clinical efficiency, effect on nontargeted cells have aggravated the situation and necessitate the need of search for new antifungal agents (Chaudhary et al. 2013). Many soil-borne, nonpathogenic micro-organisms have the ability to antagonize bacterial and fungal pathogens by

Journal of Applied Microbiology 118, 39--48 © 2014 The Society for Applied Microbiology

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Mechanism of antifungal phenazine

S.G. Tupe et al.

producing different antibiotics, enzymes and volatiles. Several clinically used antifungal drugs are derived from different micro-organisms. For instance, polyenes amphotericin B and nystatin are produced by Streptomyces nodosus and S. noursei, respectively (Andriole 1999). Echinocandins such as Caspofungin, Micafungin and Anidulafungin are synthetic modifications of lipopeptides originally derived from fermentation broths of various fungi including Papularia sphaerosperma, Aspergillus rugulovalvus, Aspergillus aculeatus and Zalerion arboricola (Denning 2002). Nikkomycin and polyoxins A–L were discovered from fermentation broths of Streptomyces tendae T€ u 901 and Streptomyces cacaoi var. asoensis, respectively (Chaudhary et al. 2013). Pyrrolnitrin produced by Pseudomonas cepacia and other Pseudomonas sp. is being marketed in Japan as topical antifungal medication (Barrett 2002). Pseudomonas sp., in general, were reported to produce extracellularly different secondary metabolites such as 2,4-diacetylphloroglucinol, phenazines, oomycin A, pyoluteorin, pyrrolnitrin, viscosinamide, pantocin A and B, butyrolactones, which showed antifungal activity (Raaijmakers et al. 2002). Many of these antibiotics produced by pseudomonads have broad-spectrum activity. For instance, 2,4-diacetylphloroglucinol is antifungal, antibacterial, anthelminthic (Keel et al. 1992). During our search for bacterial antifungal metabolites, one of the isolated Pseudomonas sp. was identified to inhibit growth of different plant and human pathogenic fungi (unpublished results). Most of the human pathogenic fungi such as Blastomyces dermatitidis, Candida albicans, Coccidioides immitis, Histoplasma capsulatum, Paracoccidioides brasiliensis and others are dimorphic, that is they change their morphology reversibly between yeast (Y) and hyphal (H) form for the survival and proliferation in the host. The change is usually from saprophytic to pathogenic form. Therefore, Y–H reversible transition inhibition can be one of the target mechanisms to identify potent antifungal compounds (Ghormade and Deshpande 2000; Ghormade et al. 2012). A nonpathogenic dimorphic zygomycetous fungus Benjaminiella poitrasii has been established as a model for antifungal susceptibility testing (Ghormade et al. 2012) and Y–H transition inhibition screening of different classes of antifungal agents such as 1,2,3-triazolyl-linked uridine derivatives (Chaudhary et al. 2009), 2-amino-5-oxo-4-phenyl-5, 6, 7, 8-tetrahydroquinoline-3carbonitrile and its analogues (Gholap et al. 2007), 1,2,3 triazole-linked b-lactam bile acid conjugates and bile acid dimers linked with triazole and bis b-lactam (Joshi et al. 2013). In this study, investigations on bioassay-guided isolation and characterization of the active compound from Pseudomonas sp., its antifungal activity, mode of action studies in B. poitrasii and validation in C. albicans are presented. 40

Materials and methods Micro-organisms and growth conditions Pseudomonas sp. was one of the isolates from garden soil collected from Mahabaleshwar, India, and maintained on nutrient agar. The strain has been deposited in Microbial Culture Collection (MCC), National Centre for Cell Science (Pune, India) with accession number Pseudomonas sp. MCC 2142. Fungal strains used in the study were procured from National Collection of Industrial Microorganisms (NCIM), National Chemical Laboratory (Pune, India). B. poitrasii NCIM 1240, C. albicans NCIM 3471, C. albicans NCIM 3557, C. glabrata NCIM 3237 and C. neoformans NCIM 3542 were maintained on YPG (yeast extract, 03%; peptone, 05%; glucose, 1%; Himedia, India) agar (2%) slants, whereas A. fumigatus NCIM 902, A. niger NCIM 628 and F. oxysporum NCIM 1043 were maintained on potato dextrose (PD) agar (Himedia, India) plates. B. poitrasii grows in yeast form in YPG broth at 37°C, whereas yeast extract-peptone (YP; without glucose) broth and 28°C favours filamentous form (Khale et al. 1992). B. poitrasii sporangiospores were obtained from a 7-day (7-d)-old culture grown on YPG agar slants. The spores were inoculated in 50 ml YPG broth and incubated at 37°C for 24 h to obtain yeast (Y) cells, which were washed with distilled water and used as an inoculum (16 9 105 cells ml1) for the experiments, unless otherwise mentioned. Isolation of antifungal metabolite from Pseudomonas sp The Pseudomonas sp. MCC 2142 was inoculated in a broth containing (g l1) the following: soya bean meal, 100; mannitol, 15; yeast extract, 100; soluble starch, 50; and incubated at 28°C for 96 h under shaking conditions (180 rev min1). The cells were harvested by centrifugation at 10 000 g for 15 min, and the supernatant (3 l) obtained was evaporated at 50°C till dryness to yield yellow brown residue 205 g. The residue was extracted successively with acetone and ethyl acetate. The solvents were evaporated to yield acetone extract 13 g (A), ethyl acetate extract 02 g (B) and residue (C). The harvested cells were washed with water and suspended in 200 ml acetone for 12 h with occasional stirring, then centrifuged at 10 000 g for 15 min, and the acetone solubles were decanted. Acetone was evaporated, and the yellow extract obtained was successively extracted with chloroform and ethyl acetate, which on solvent evaporation gave chloroform soluble 045 g (D), ethyl acetate solubles 02 g (E) and residue 07 g (F).


S.G. Tupe et al.

The active fraction D was separated by column chromatography using silica gel 200–300 mesh as stationary phase and methanol: chloroform (5 : 95) as eluting system into 10 fractions. Fractions 2–4 which showed presence of antifungal activity were combined and purified by preparative thin-layer chromatography (TLC) to isolate the compound. It was purified further by crystallization from acetone. Structure elucidation of the compound € Melting point was recorded using a BUCHI B 540 appara€ tus (BUCHI Labortechnik AG, Flawil, Switzerland). An IR spectrum was recorded in chloroform with a Perkin–Elmer FT-IR spectrometer (Perkin–Elmer Life and Analytical Sciences, Shelton, CT). Nuclear magnetic resonance (NMR) spectra of the purified compound were recorded on a Bruker Ultrashield 500 MHz for protons and Bruker Ultrashield 125 MHz for carbon-13. Spectra were measured in deuterated chloroform (CDCl3) at room temperature. Mass spectrum was recorded with electronspray ionization mass spectrometer, ESI-MS (API-QSTAR-PULSAR, Applied Biosystems, Framingham, MA), in positive ion mode. Screening for antifungal activity In vitro antifungal activity of extracts and fractions was evaluated against B. poitrasii by disc diffusion assay. Benjaminiella poitrasii yeast cells (01 ml; 1 9 106 CFU ml1) were spread on 1% YPG agar plates. Whatman filter paper No. 1 discs (5 mm) impregnated with the fractions/extracts were placed on the plates, and the plates were incubated at 37°C for 24 h. The diameter of the zone of inhibition was measured. The purified compound was evaluated for antifungal susceptibility testing against all the cultures under study by Clinical and Laboratory Standards Institute’s broth microdilution methods M27A3 (for yeasts) and M38-A2 (for filamentous fungi) (Clinical and Laboratory Standards Institute 2008a, b). The medium used for the assay was Roswell Park Memorial Institute (RPMI 1640) broth (with glutamine and phenol red, but without bicarbonate) supplemented with 2% glucose and buffered with 0165 mol l1 3-(N-morpholino) propanesulphonic acid. Purified compound was dissolved in dimethyl sulphoxide to get 1009 final concentration. The stock is then diluted 1 : 50 in RPMI 1640 broth, and 200 ll from this is added in the first row of a 96-well microtiter plate. The compound is then serially diluted twofold in successive wells to get a range of 256– 2 lg ml1. Freshly grown yeast cells (2 9 104 CFU ml1) of B. poitrasii, C. albicans NCIM 3471, C. albicans NCIM 3557, C. glabrata NCIM 3237 and C. neoformans NCIM 3542 were suspended in RPMI 1640 broth and inoculated (100 ll) in the wells (in triplicate, that is wells of three


columns for each culture) of the plate. In case of filamentous fungi A. fumigatus NCIM 902, A. niger NCIM 628 and F. oxysporum NCIM 1043, 100 ll from spores (2 9 105 spores ml1) suspension in RPMI 1640 broth was added in the wells. The microtiter plates were incubated for 24 h for yeasts and 48 h for filamentous fungi. Growth was checked visibly and by measuring absorbance at 600 nm using microtiter plate reader (xMark Microplate Absorbance Spectrophotometer, Bio-Rad, Hercules, CA). The minimum inhibitory concentration (MIC) was defined as the lowest concentration exhibiting >90% inhibition of visible growth compared to growth of the control. Effect of phenazine-1-carboxamide (PC) on yeast (Y)— hypha (H) and H–Y transition The Y–H transition experiment was carried out using B. poitrasii, as described earlier (Khale et al. 1992). The yeast cells (16 9 105 cells ml1) were inoculated in YP broth (with different concentrations of PC up to 50 lg ml1) and incubated at 28°C for 6 h. After incubation, using haemocytometer grid, microscopically single or budding cells were counted as one yeast morphological unit; cells with one or more germ tubes were counted as one hyphal morphological unit. Minimum 300 cells were counted, and percentage of germ-tube-forming cells was calculated. Similarly, H–Y transition was studied by inoculating hyphal cells in YPG (G, 05%) with incubation for 20 h at 37°C. The number of yeast cells formed was obtained by subtracting the number of hyphal cells remaining from total number of cells counted and used to determine % budding. For validation in pathogen, overnight grown C. albicans NCIM 3471 cells (1 9 106 yeast cells ml1) were inoculated in YPG broth containing 15% foetal bovine serum (with different concentrations upto 50 lg PC ml1) and incubated at 37°C for 6 h (Nantel et al. 2002), and the percentage of cells forming germ tubes were determined as described for B. poitrasii. Detection of intracellular reactive oxygen species (ROS) production Amount of ROS was measured by fluorimetric assay with 20 ,70 -dichlorofluorescein diacetate (DCFH-DA) as described by Kobayashi et al. (2002). After incubation of the cells with different concentrations of PC at 37°C for 120 min, 10 lmol l1 DCFH-DA in phosphate-buffered saline (PBS) was added. The fluorescence intensities (excitation 485 nm and emission 538 nm) of the resuspended cells were measured with a spectrofluorometer (LS 50B fluorescence Spectrometer, PerkinElmer, Waltham, MA) after 30 min.


41


S.G. Tupe et al.

ROS generation in B. poitrasii and C. albicans NCIM 3471 was assessed by di-hydrorhodamine 123 (DHR 123) staining. The cells treated with 5–50 lg ml1 PC were spiked with DHR123 (5 lg ml1 from a 25 mg ml1 stock solution in ethanol) and incubated further for 30 min. Cells were then harvested and directly viewed using epifluorescence microscope (Leitz Laborlux S, Wetzlar, Germany). Effect of PC on mitochondrial membrane potential (MMP) Benjaminiella poitrasii yeast cells (1 9 107 CFU ml1) were suspended in phosphate buffer (pH 70) containing different concentrations of PC upto 50 lg ml1 and kept at 37°C for 120 min. After 120 min. Rh 123, at 2 lmol l1 final concentration, was added and further incubated for 30 min. The cells were then harvested, washed and suspended in phosphate buffer (pH 70). The fluorescence intensities of the control and treated samples were taken using spectrofluorometer (Excitation 480 nm and emission 525 nm) (LS 50B fluorescence Spectrometer, PerkinElmer, Waltham, MA). Membrane permeabilization assay Membrane permeabilization assay using propidium iodide (PI) was carried out as described previously (Maurya et al. 2011). PC concentration used was in the range 0–50 lg ml1.

B. poitrasii cells (16 9 105 cells ml1) were inoculated in YPG (05% G) broth with (5–50 lg ml1) and without PC and incubated for 200 min at 37°C under shaking condition (180 rev min1). The cells were harvested and washed twice with potassium phosphate buffer (PB, 200 mmol l1, pH 58) containing KCl (06 mol l1). The protoplasts of yeast cells were obtained using lysing enzyme mixture as described by Chitnis and Deshpande (2002). The integrity of the isolated protoplasts was checked by vital staining using 01% (w/v) eosin in a PBK buffer. The protoplasts were analysed for apoptosis markers, viz. phosphatidylserine externalization, DNA strand breaks and chromatin fragmentation by annexin V, TUNEL and diaminophenylindole (DAPI) staining, respectively, as described by Madeo et al. (1997, 1999). After staining, the cells were viewed using epifluorescence microscope, and percentage of annexin V-, TUNEL- and DAPI-positive cells were determined by counting at least 300 cells from three independent experiments. Haemolysis assay The toxicity of PC was checked by the red blood cell (RBC) lysis assay as described by Khan and Ahmad (2011). The concentrations of PC tested were in the range of 4-512 lg ml1. Results Isolation and physical characterization of PC

Superoxide dismutase (SOD, EC 1.15.1.1) activity assay Hyphal and the yeast form cells were collected on Whatman filter paper No.1 and washed with 085% saline, followed by extraction buffer containing 250 mmol l1 sucrose, 12 mmol l1 Tris-HCl and 01 mmol l1 DTT, pH 74. Yeast and hyphal cells of B. poitrasii (yeast 1 g 5 ml1; hyphal 1 g 2 ml1 of extraction buffer) were disrupted using glass beads in Braun’s homogenizer. The samples were centrifuged at 12 000 g for 10 min at 4°C to obtain cell extracts for the estimation of enzyme activities. The SOD activity in B. poitrasii was estimated by nitroblue tetrazolium (NBT) assay as described by Kono (1978). Protein was estimated using crystalline bovine serum albumin as a standard according to Lowry et al. (1951). Detection of apoptosis markers Synchronized yeast cells for apoptosis experiments were obtained by filtration of inoculum yeasts cells through G1 filter (Jinsel, India). Inoculation in fresh 1% YPG and filtration of cells were repeated five times. Actively growing 42

The isolate Pseudomonas sp. MCC 2142 produced extracellularly yellowish green pigment in soya bean meal broth after prolonged incubation (28°C for 4 days). Supernatant of the soya bean meal broth showed inhibition of B. poitrasii growth in disc diffusion assay. Through bioactivity-guided isolation, a pure compound was obtained. The compound was pale yellow crystalline needles with a melting point 245°C and was extensively characterized by ESI-MS, IR, 1H-NMR and 13C NMR. ESI-MS spectrum showed [M + H]+ peak at 224 (base peak) and [M + Na]+ peak at 246, corresponding to molecular formula C13H9N3O (molecular weight 223) with 11 indices of hydrogen deficiency. Peak at m/z 207 due to loss of NH3 [M + 1–17] suggested a fragmentation typical of amide-containing compound (Fig. S1). This was supported by absorption peaks at 3367, 2927, 1648 and 1215 cm1 in IR spectrum. 1H-NMR revealed presence of three methane at d 897 (dd, J = 70; 13 Hz), 854 (dd, J = 87; 13 Hz) and 802 (dd, J = 70; 87 Hz), corresponding to 1,2-3 trisubstituted benzene ring. 1H-NMR


S.G. Tupe et al.


also exhibited four peaks in the range of d 834–797 (4H, m), indicating towards disubstituted ring (Fig. S2A). 13 C NMR revealed 13 resonances which included seven methine and six quaternary carbons. Peak at d 16593 was indicative of amide carboxyl group (Fig. S2B). By comparing the data with previous report (Chin-A-Woeng et al. 1998), the compound was identified as PC (Fig. S3). Antifungal activity of PC PC displayed broad-spectrum antifungal activity by inhibiting all the strains tested. As shown in Table 1, the MIC for C. albicans NCIM 3557 and A. fumigatus NCIM 902 was 32 lg ml1, whereas the MIC value was 64 lg ml1 for all other tested strains indicating strain-specific sensitivity to PC. Inhibition of Y–H reversible transition in Benjaminiella poitrasii by PC Dimorphic fungus, B. poitrasii, was used as a model to study the effect of PC on differentiation, viz. Y–H reversible transition. As mentioned in Table 2, the transition of yeast to hyphal form (germ tube formation, 538 23%) inoculated in YP broth without PC and incubated at 28°C for 6 h was considered as a control, while for H–Y transition, the hyphal cells were incubated in YP + 1% glucose broth at 37°C for 20 h to obtain yeast form (budding 45 3%). The addition of PC at 5– 15 lg ml1 concentration during Y–H transitions inhibited germ tube formation by 62–82% as compared to the control, while, similar treatment during H–Y transition resulted in 41–87% inhibition of budding. At 20 lg ml1 Table 1 The effect of phenazine-1-carboxamide on different yeast and filamentous fungi

Strain Yeasts Candida albicans National Collection of Industrial Microorganisms (NCIM) 3471 Candida albicans NCIM 3557 Candida glabrata NCIM 3237 Cryptococcus neoformans NCIM 3542 Filamentous fungi Aspergillus fumigatus NCIM 902 Aspergillus niger NCIM 628 Fusarium oxysporum NCIM 1043 Dimorphic model fungus Benjaminiella poitrasii NCIM 1240

MIC90 (lg ml1)*

64 32 64 64 32 64 64 64

*The antifungal assay was carried out using CLSI micro-broth dilution method as described under “Materials and Methods”.

Table 2 Effect of phenazine-1-carboxamide (PC) on morphological transition of Benjaminiella poitrasii and Candida albicans National Collection of Industrial Microorganisms 3471 Benjaminiella poitrasii % Germ tube formation Y–H

% Budding H–Y

No addition 5

538 23

45 35

201 19 (626%)

10

115 08 (786%)

15

95 05 (823%)

26 20 (418%) 14 15 (689%) 6 10 (867%) ND

PC (lg ml1)

20

ND

Candida albicans % Germ tube formation Y–H 96 16 573 36 (403%) 391 22 (592%) 255 18 (734%) 66 09 (931%)

ND, not detected; Values in parentheses denote % inhibition in comparison with control.

PC, transition (Y–H and H–Y) was not detected (Table 2). In case of C. albicans NCIM 3471, the Y–H transition was inhibited >90% with 20 lg ml1 PC (Table 2). Mechanism of action of PC in Benjaminiella poitrasii To understand mechanism of action of PC, its effect on intracellular ROS production, MMP, SOD activity and membrane integrity in B. poitrasii were studied. DCFH-DA is a ROS probe that undergoes deacetylation in the cell, followed by ROS-mediated oxidation to a fluorescent 20 ,70 -dichlorofluorescein. Addition of PC (5– 20 lg ml1) resulted in concentration-dependent increase in fluorescence indicating higher ROS production in B. poitrasii yeast cells. The relative intensity of fluorescence as measured by spectrofluorometry increased from 139 arbitrary units (A.U.) for cells without addition of PC to 251 A.U. with an addition of 20 lg ml1 of PC (Fig. 1). In another ROS detection assay, 75-80% yeast cells showed fluorescence with same concentration of PC due to conversion of DHR123 to rhodamine 123 (Rh123) indicating high intracellular ROS production (Fig. 2a I and II). Same observations were validated in case of C. albicans NCIM 3471. High intracellular ROS levels due to 20 lg ml1 PC were observed in DHR 123 staining of Candida cells (Fig. 2b I and II). For MMP assay, Rh123, a membrane-permeable lipophilic cationic fluorescent probe, was used. It selectively accumulated in mitochondria and distributed electrophoretically into the mitochondrial matrix in response to MMP (ΔΨm) as mentioned by Baracca et al. (2003). The presence of PC (20 lg ml1) triggered hyperpolarization of MMP with >2-fold increase in the relative fluorescence


43

S.G. Tupe et al.

700 600 500 400 300 200 100 0

0

5

10

20

50

Figure 3 Spectrofluorometric analysis of Rh123 fluorescence of yeast cells of Benjaminiella poitrasii treated with different concentrations of phenazine-1-carboxamide.

300

25

250 200 150 100 50 0

20 15 10 5 0

0

5

10

20

50

0

Figure 1 Spectrofluorometric analysis of 20 ,70 -dichlorofluorescein fluorescence in yeast cells of Benjaminiella poitrasii treated with different concentrations of phenazine-1-carboxamide.

(a1)

(a2)

(b1)

(b2)

5

10

15

20

50

PC concentration (µg ml–1)


44

800


SOD activity (U mg–1)

Fluorescence intensity (A. U.)

intensity in the B. poitrasii yeast cells as compared to the control (Fig. 3). Addition of PC to the cells resulted in dose-dependent increase in SOD activity with >15-fold increase for 20 lg ml1 (218 15 U mg1) as compared to the control without PC (14 001 U mg1) (Fig. 4). PI is a membrane-impermeable dye and has often been used as a probe for nonviable cells. PI staining of the PC-treated cells indicated no membrane damage upto 20 lg ml1 concentration, as only ~5% cells were PI positive (Table 3). However, at 50 lg ml1 PC, the proportion of PI positive cells increased to 35% (Table 3). ROS generation is one of the hallmarks of apoptosis. Therefore, to check whether PC induced apoptosis in B. poitrasii, the percentage of protoplasts (containing nuclei) after PC treatment that stained positive with

Fluorescence intensity (A. U.)


Figure 4 Superoxide dismutase (SOD) activity in yeast cells of Benjaminiella poitrasii treated with different concentrations of phenazine1-carboxamide.

Figure 2 Epifluorescence microscopy for DHR 123-stained yeast cells of (a) Benjaminiella poitrasii treated with phenazine-1carboxamide (20 lg ml1). I. Cells under bright field; II. Cells with fluorescence and (b) Candida albicans National Collection of Industrial Microorganisms 3471 treated with phenazine-1-carboxamide (20 lg ml1). I. Cells under bright field; II. Cells with fluorescence (magnification 409).


S.G. Tupe et al.


Table 3 Effect of phenazine-1-carboxamide (PC) on apoptosis of Benjaminiella poitrasii

DHR123

Necrotic protoplasts (%) PI

ND 80 100 400 800 350

ND 10 20 20 50 350

Apoptotic protoplasts (%) PC (lg ml1)

Annexin V-FITC

No addition 5 10 15 20 50

1 40 150 350 600 400

12 12 30 15 46 75

TUNEL ND 70 150 250 450 150

DAPI ND 40 80 380 700 300

08 06 15 15 15

11 11 20 105 50

10 06 55 110 50

03 08 06 10 30

ND, not detected. The values mentioned, average from two experiments, are obtained by counting at least 300 protoplasts in each experiment.

annexin V-FITC, terminal deoxynucleotidyl transferasemediated dUTP nick end labelling (TUNEL) and DAPI staining were measured (Table 3). Protoplasts without PC treatment showed low level of staining with annexin V-FITC, TUNEL and DAPI (99% inhibition of Y–H transition for B. poitrasii can be correlated with 40-60% induction of apoptosis (Tables 2 and 3). At higher concentrations of PC, increase in necrotic protoplasts was seen (Table 3). The antagonistic effects of most of the phenazines are due to their redox activity. Phenazines are thought to diffuse across the cell membrane and act as reducing agents. This results in uncoupling of oxidative phosphorylation and generation of toxic intracellular ROS which are harmful to the organism (Turner and Messenger 1986). In the present investigations, ROS production increased with increase in the concentration of PC (Fig. 1). MMP


45


S.G. Tupe et al.

is an important factor that controls the level of mitochondrial ROS production. At higher MMP, more ROS production was seen. Concomitant to the elevated ROS production, hyperpolarization of MMP was observed by spectrofluorimetry at increasing concentrations of PC (Fig. 3). The elevated levels of SOD, another oxidative stress marker, with increasing PC concentration indicated its action through ROS production (Fig. 4). The role of ROS generation in antimicrobial action has been demonstrated for phenazines such as pyocyanin (Hassan and Fridowich 1980), 5-methylphenazine-1-carboxylic acid (Morales et al. 2010). A number of studies have demonstrated that accumulation of ROS within the cytoplasm plays a central role in yeast apoptotic cell death (Madeo et al. 1999; Zhang et al. 2007; Carmona-Gutierrez et al. 2010). Therefore, to find out whether PC-induced ROS production caused apoptosis, annexin V-FITC, TUNEL, DAPI staining of the protoplasts was carried out after PC treatment. Treatment with PC upto 20 lg ml1 concentrations led to a dose-dependent increase in annexin V-FITC-stained protoplasts. Similar results were obtained for TUNEL and DAPI staining indicating dose-dependent increase in DNA fragmentation and chromatin condensation at these concentrations (Table 3). At higher PC concentration (50 lg ml1), the percentage of TUNEL-positive protoplasts decreased with concomitant increase in PI-stained cells, indicating loss of membrane permeability and necrotic death. Thus, PC caused induction of apoptosis at lower concentrations and necrosis at high concentrations. Similar concentration-dependent effect was reported for S. cerevisiae cells treated with H2O2 and acetic acid (Madeo et al. 1999; Ludovico et al. 2001). In conclusion, ROS production, hyperpolarization of mitochondrial membrane and subsequent induction of apoptosis contribute to the antifungal activity of PC. The inhibitory effect of nonhaemolytic PC on the Y–H transition of B. poitrasii and C. albicans demonstrated in the present study indicates its potential in the control of dimorphic human pathogens. Acknowledgements Funding: SGT thanks Council of Industrial and Scientific Research, Government of India, for fellowship. Authors are thankful to Department of Biotechnology, Government of India, for research grants (BT/PR7442/MED/29/ 680/2012; BT/PR15003/BRB/10/893/2010). Conflict of interest No conflict of interest declared. 46

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Supporting Information Additional Supporting Information may be found in the online version of this article:

Figure S3 Structure of Phenazine 1-carboxamide (PC). Table S1 Nuclear magnetic resonance (NMR) signals from phenazine-1-carboxamide (A) 1H-NMR (B) 13CNMR.

Figure S1 ESI-MS spectra of compound 1 (PC). Figure S2 Spectroscopic studies of PC. (A) 1H-NMR, (B) 13C-NMR, (C) DEPT in CDCl3 (1H: 400 MHz, and 13 C: 100 MHz).

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Potentiation of antifungal activity of sesquiterpene dialdehydes against Candida albicans and two other fungi.

Liquid and vapour-phase antifungal activities of essential oils against Candida albicans and non-albicans Candida.

Evaluation of Antifungal Activity and Mechanism of Action of Citral against Candida albicans.

Possible mechanisms of the antifungal activity of fluconazole in combination with terbinafine against Candida albicans.

Investigating the antifungal activity and mechanism(s) of geraniol against Candida albicans strains.

Antifungal activity and pore-forming mechanism of astacidin 1 against Candida albicans.

Antifungal activity of essential oils from Iranian plants against fluconazole-resistant and fluconazole-susceptible Candida albicans.

NAD glutamate dehydrogenase ratio in the dimorphic behavior of Benjaminiella poitrasii and its morphological mutants.

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Antifungal activity of extracts and isolated compounds from Buchenavia tomentosa on Candida albicans and non-albicans.

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Oxidative stress responses in the human fungal pathogen, Candida albicans.

Azole Antifungal Resistance in Candida albicans and Emerging Non-albicans Candida Species.

Comparison of Antifungal Activity of 2% Chlorhexidine, Calcium Hydroxide, and Nanosilver gels against Candida Albicans.

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In Vitro and In Vivo Antifungal Activity of Lichochalcone-A against Candida albicans Biofilms.

The isolation and characterization of a calmodulin-encoding gene (CMD1) from the dimorphic fungus Candida albicans.