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Research in Microbiology xx (2014) 1e9 www.elsevier.com/locate/resmic
Ocimum sanctum (L.) essential oil and its lead molecules induce apoptosis in Candida albicans Amber Khan1, Aijaz Ahmad2, Luqman Ahmad Khan, Nikhat Manzoor*
Q1
Medical Mycology Lab, Department of Biosciences, Jamia Millia Islamia, New Delhi 110025, India Received 1 October 2013; accepted 9 May 2014
Abstract Manipulation of endogenous responses during programmed cell death (PCD) in fungi can lead to development of effective therapeutic strategies. In the present study, we evaluated the physiology of cell death in Candida albicans in response to Ocimum sanctum essential oil (OSEO) and its two major constituents e methyl chavicol (MET CHAV) and linalool (LIN) at varying inhibitory concentrations. Apoptotic cell death was studied on the basis of externalization of membrane phosphatidylserine (PS) revealed by annexin-V-FITC labeling, morphological alterations revealed by transmission electron microscopy and DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Exposure of fungal cells to MIC/4 of OSEO, MET CHAV and LIN resulted in morphological features characteristic of apoptosis, while necrosis was observed at higher concentrations. Necrotic cells displayed reduced TUNEL staining and an inability to exclude propidium iodide. In addition, they lacked a defined nucleus and an intact external morphology. Exposed cells were TUNEL-positive, showed chromatin condensation and margination, nuclear envelope separation, nuclear fragmentation, cytoplasmic shrinkage and plasma membrane blebbing. A dose-dependent decrease in cytochrome c oxidase activity was observed with each compound, but the decrease was not comparable to that elicited by H2O2, eliminating the primary involvement of cytochrome c release in apoptosis thus induced. Previously reported data revealed induction of apoptosis at low concentrations as a result of oxidative insult. Studies aimed at identifying other mitochondrial factors activated during this course to mediate apoptosis will further elucidate the mechanism of antifungal action of these natural products. © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Ocimum sanctum; Apoptosis; Methyl chavicol; Linalool; Candida albicans
1. Introduction Candida albicans is associated with a number of clinical conditions ranging from irritating superficial infections to lifethreatening systemic disease in immunocompromised patients [24]. Candidemia is the fourth most common bloodstream infection among hospitalized patients [33]. Several distinct * Corresponding author. Tel.: þ91 11 2698 1717x3410; fax: þ91 11 2698 0229. E-mail address: [email protected] (N. Manzoor). 1 Present address: Department of Pharmacy and Pharmacology, Faculty of Health Sciences, Medical School, University of Witwatersrand, Johannesburg, South Africa. 2 Present address: Department of Pharmaceutical Sciences, Tshwane University of Technology, Arcadia Campus, Pretoria, SA-0001, South Africa.
classes of antifungal drugs are currently available for the treatment of clinical infections [19,25]. Most of these drugs affect the cell membrane or cell wall integrity through inhibition of either ergosterol biosynthesis or b-glucan/chitin synthesis, respectively. A further complication to this limited spectrum of drugs is the acquisition of resistance by the pathogenic fungi observed in clinical settings [11,12,23]. AmB, while fungicidal, has severe and potentially lethal side effects. The azoles are generally fungistatic and hence their prolonged administration leads to resistance in Candida species. The development of more effective antifungal therapies is therefore of paramount importance. There are only a few studies that explored the relationship between antifungal drugs and cell death [4,29]. Yeasts display stereotypical patterns of cell death when responding to environmental insults or
http://dx.doi.org/10.1016/j.resmic.2014.05.031 0923-2508/© 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Please cite this article in press as: Khan A, et al., Ocimum sanctum (L.) essential oil and its lead molecules induce apoptosis in Candida albicans, Research in Microbiology (2014), http://dx.doi.org/10.1016/j.resmic.2014.05.031
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endogenous signals [27]. A specific series of morphological and biochemical changes set apoptotic cell death apart from necrosis [13,34]. While the novel concept of programmed necrosis has just emerged in yeast [8], recent studies have revealed the existence of programmed cell death in C. albicans in response to cytotoxic agents [4]. An understanding of the mechanistic basis of cell death in yeast may provide new developments for effective antifungal therapies. Innumerable studies have evaluated the antifungal efficacies of plant essential oils and their lead molecules against various pathogenic drug-susceptible and drug-resistant fungi [1,28]. Ocimum sanctum (L.) essential oil (OSEO) is known for its broad spectrum antifungal activities [6,9,10,15,16,20]. Its major constituents, methyl chavicol (MET CHAV) and linalool (LIN), are also known to have anticandidal activity [14,30e32]. MICs of OSEO, MET CHAV and LIN against C. albicans were found to be 0.025%v/v, 0.025%v/v and 0.031% v/v, respectively [14]. OSEO showed mild hemolytic activity, causing only 5.1% hemolysis of human RBCs at very high concentrations (80 MIC), while the same amount of hemolysis could be achieved by AmB at concentrations as low as MIC/4 [15]. In addition, OSEO has been shown to attenuate the expression of virulence-associated genes as well as pathogenicity in C. albicans [18]. The present work is hence an extension of our previous study in which we showed that OSEO inhibits ergosterol biosynthesis and causes membrane damage in Candida cells [14]. The mechanism of antifungal action of OSEO, MET CHAV and LIN has now been explored at varying inhibitory concentrations and the physiology of cell death has been studied on the basis of externalization of membrane phosphatidylserine (PS), revealed by annexin V affinity labeling, morphological alterations characteristic of cell death revealed by transmission electron microscopy (TEM) and DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Cytochrome c oxidase activity was estimated so as to study the role of mitochondria in mediating cell death under the influence of these natural antifungal products.
overnight in 2.5% glutaraldehyde in phosphate/magnesium buffer (40 mM K2HPO4/KH2PO4, pH 6.5e0.5 mM MgCl2). Cells were washed twice for 15 min in 0.1 M sodium phosphate buffer (pH 6.0) and post-fixed for 2 h in 2% osmium tetroxide. Cells were washed twice for 15 min in distilled water and then en bloc stained with 1% uranyl acetate (aqueous) for 30 min. After two further washes, cells were dehydrated in 95% and 100% ethanol. Cells were exposed to propylene oxide for 2 10 min and infiltrated for 1 h in 1:1 propylene/epoxy embedding material (Epon) mixture and then overnight in fresh Epon. After polymerization for 48 h at 60 C, ultrathin sections were cut using a microtome (Leica EM UC6) and transferred to a copper grid. Samples were stained with uranyl acetate (saturated solution of uranyl acetate in 50% alcohol) followed by lead citrate. Samples were washed three times in Milli-Q (MQ) water and dried by touching Whatman filter paper. Sections were examined with a Jeol (Japan) JEM-2100F transmission electron microscope at 120 kV. 2.3. Protoplast preparation The cell wall was digested with lyticase (1 mg/g cells) in different washing steps in protoplast buffers (pH 7.4) containing 1 M sorbitol, DTT (buffer I e 30 mM; buffer II e 1 mM; buffer III e 0 mM), 50 mM tris base and 10 mM MgCl2. Briefly, cells were harvested and washed thrice for 5 min each with buffer I (3 mL/g cells). Cells were then incubated in buffer II (5 mL/g cells; supplemented with lyticase) for 2 h at 25 C. After removing buffer II by centrifugation, cells were incubated with buffer III (5 mL/g cells) for 15 min and then again centrifuged to remove buffer III. Protoplasts were finally washed once with PBS and re-suspended in the same. 2.4. Annexin V-FITC labeling
C. albicans ATCC 90028 cells used in the present study were grown and maintained on YPD medium. MET CHAV and LIN were purchased from SigmaeAldrich (Germany); Amphotericin B (AmB) from Himedia (India), and H2O2 from Merck (India). Reagents, organic solvents and salts were of analytical grade and procured from Merck (India) and SRL (India). Midlog phase cells were harvested and exposed (A595 ¼ 0.1) to different concentrations of test drugs (MIC/4, MIC/2, MIC, 4MIC), AmB (2 mg/mL), and H2O2 (2.5 mM) for 1 h.
The externalization of phosphatidylserine (PS) was studied using annexin V-FITC labeling by a APOAF kit (SIGMA) in yeast protoplasts. Propidium iodide (PI) was used to differentially label dead cells. The protoplasts were resuspended in binding buffer and incubated with annexin V-FITC conjugate and PI solution. The results were quantified using a FACSCalibur flow cytometer (Becton Dickinson Biosciences) and analyzed using Cell Quest Pro software (Becton Dickinson). Filter-FL1 revealed FITC fluorescence, whereas FL2 revealed PI fluorescence. Cell populations in the R1 quadrant corresponded to red fluorescence (PIþ; Debris); in the R2 quadrant they corresponded to green and red fluorescence (FITCþ, PIþ; necrosis); in the R3 quadrant they corresponded to neither fluorescence (FITC, PI; alive) and in the R4 quadrant they corresponded to green fluorescence (FITCþ; apoptosis).
2.2. Transmission electron microscopy (TEM)
2.5. TUNEL assay (fluorometric)
The morphology of Candida cells was analyzed using TEM following the standard protocol. Cells (1 108) were fixed
A terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed in order to
2. Materials and methods 2.1. Strains and growth conditions
Please cite this article in press as: Khan A, et al., Ocimum sanctum (L.) essential oil and its lead molecules induce apoptosis in Candida albicans, Research in Microbiology (2014), http://dx.doi.org/10.1016/j.resmic.2014.05.031
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66 67 68 69 70 71 72 73 74 75 76 3. Results 77 78 3.1. TEM 79 80 Fig. 1 shows the effect of OSEO and its two major com81 82 ponents, LIN and MET CHAV, on the morphology of the 83 Candida cells using TEM. The yeast cells were incubated with 84 the test compounds at their respective MICs for 1 h. Electron 2.6. TUNEL assay (colorimetric) 85 microscopic investigation of cells incubated with the test 86 compounds revealed chromatin condensation along the nuProtoplasts were studied further for DNA fragmentation 87 clear envelope typical for apoptosis (margination), with intact with dUTP-biotin labeling using TUNEL-DAB system 88 cellular morphology, plasma membrane and cell wall. On the (DeadEnd colorimetric TUNEL system, PROMEGA) accord89 90 other hand, nuclei of untreated cells (control) were homogeing to the manufacturer's instructions. DAB staining (TUNEL 91 neous in shape and density. In addition, some cells showed positive cells) was observed under an inverted microscope 92 cellular shrinkage and extensive plasma membrane folds (Motic, China). This procedure stains the apoptotic nuclei dark 93 (Fig. 1: LIN-exposed cell). This could be membrane blebbing, brown. 94 a characteristic marker of apoptosis [22] observed as tiny 95 vesicles in yeast cells. Invagination of the plasma-membrane 2.7. Cytochrome c oxidase activity 96 and appearance of tubular structures were also evident and 97 98 indicated onset of membrane blebbing. At very high concenProtoplasts exposed to varying concentrations (MIC/4, 99 trations of test compounds (above 4 MIC), most of the intraMIC/2 and MIC) of test entities were assayed for cytochrome 100 cellular structures were destroyed (data not shown), indicating c oxidase activity using CYTOCOX1 kit (SIGMA) according 101 necrotic cell death. This corroborates well with the TUNEL to the manufacturer's instructions. Cytochrome c oxidase 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 Fig. 1. Representative transmission electron micrographs of C. albicans ATCC 90028 cells exposed to OSEO, MET CHAV and LIN at their respective MICs. 129 Morphological changes and intracellular damages in exposed cells are shown with yellow arrows. Abbreviation ‘cw’ refers to cell wall; ‘pm’, plasma membrane; ‘m’, mitochondria; and ‘n’, nucleus. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Q2 130
confirm apoptotic cell death in Candida induced by OSEO and its lead molecules. After treatment with test compounds, Candida cells were washed twice in PBS and fixed with 4% paraformaldehyde in PBS (pH 7.4) for 1 h at 20 C. Protoplasts were then prepared as described above. They were rinsed twice with PBS and incubated with permeabilization solution for 2 min on ice. The cells were again rinsed in PBS and labeled, using 50 ml of a 9:1 solution of the label and enzyme solutions from an in situ cell death detection kit, fluorescein (Roche Applied Sciences, Mannheim, Germany), with appropriate controls labeled only with the label solution. The protoplasts were incubated for 1 h at 37 C in a humidified atmosphere in the dark, rinsed in PBS, and examined with a Nikon Eclipse-80i fluorescence microscope (Japan) with a detection range of 515e565 nm.
activity was measured using a spectrophotometer (Systronics, India) at 550 nm by estimating the decrease in absorbance of ferrocytochrome c due to its oxidation to ferricytochrome c by the enzyme. Enzyme activity was defined as the amount of ferrocytochrome c oxidized in mmoles per minute. Each experiment was performed twice and in triplicate. Results obtained were expressed in terms of mean ± standard error (SEM).
Please cite this article in press as: Khan A, et al., Ocimum sanctum (L.) essential oil and its lead molecules induce apoptosis in Candida albicans, Research in Microbiology (2014), http://dx.doi.org/10.1016/j.resmic.2014.05.031
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reaction data shown in Fig. 4 where the number of TUNELFITC-positive cells decreased with increasing concentrations of the test compounds. 3.2. Annexin V-FITC affinity labeling Apoptotic cell death is accompanied by a change in plasma membrane structure by surface exposure of PS, while membrane integrity remains unchallenged. Surface-exposed PS can be detected by its affinity for annexin V, a phospholipid binding protein. The extent of PS externalization in C. albicans was monitored by flow cytometry using Annexin V-FITC labeling. The cytogram in Fig. 2 shows bivariate annexin V/PI analysis of the unexposed and exposed cell suspension. Viable cells are negative for both PI and annexin V (R3), apoptotic cells are PI-negative and annexin V-positive (R4),
while necrotic cells are positive for both PI and annexin V (R2). Exposure to MIC/4 of OSEO, MET CHAV and LIN resulted in 9e11% apoptotic cell death, whereas in controls, apoptotic cells represented only 2% of the cell population (Fig. 3). Cell death was dose-dependent and exposure to increasing concentrations of test compounds resulted in a concomitant increase in the percentage of dead cells. Exposure to MIC/2 of test compounds caused 22e23% cell death. Moreover, at sub-MICs, the exposed cells showed an apoptotic rather than a necrotic mode of cell death. Incubation of OSEO, MET CHAV and LIN at their respective MIC values resulted in 50%, 46% and 51% cell death, respectively, whereas incubation at higher concentrations (4MIC) caused an increase in the number of dead cells with a value of 94%, 74% and 85%, respectively. Cells incubated with 2.5 mM H2O2 showed 26% cell death (including 66% of the total oil. LIN causes 13e77% GSH depletion, while MET CHAV results in 18e73.6% GSH depletion in Candida cells exposed to a 10e100 mg/mL test concentration. In addition, both constituents at the same concentrations compromise anti-oxidant defense armory and cause severe membrane oxidation in Candida. LIN exhibited higher destruction of lipids (2.8e4.9-fold relative to control) than MET CHAV (2.7e3.8-fold) [17]. Lipid peroxidation (LPO) induced by test compounds may result in part from the suppression of ergosterol biosynthesis by test entities [14], which could otherwise physically protect lipids from oxidation. In the present study, the apoptosis induced at lower concentrations of test entities overall could be justified in the light of MET CHAV- and
LIN-induced GSH depletion, which is a well-established initiator of apoptosis [35], as well as attenuation of the anti-oxidant defense system (unpublished data), resulting in an outburst of ROS. Complete ergosterol depletion and membrane disintegration at high concentrations, on the other hand, explains necrotic cell death observed in the present study. In H2O2 and AmB treated cells the triggers of apoptosis are induction of oxidative stress and accumulation of ROS [27]. These triggers may involve mitochondrial routes of PCD. Apoptosis operates via two major classical pathways e extrinsic and intrinsic. The extrinsic pathways are triggered only by external means like pheromone signaling and quorum sensing. This includes signaling via ‘Ste’ proteins in the yeast cells. The intrinsic pathway, which has significance in the present study (because of the lipophilic nature of test entities, and their reported effects on the intracellular pool), is mediated by the mitochondria, where mitochondrial membranes are sensitized by the pro-apoptotic proteins encoded by the Bcl2 family genes (BAD, BAX, BID) and cytochrome c is released into the cytoplasm. In the mammalian system, cytochrome c activates the apoptosis-activating factor 1(Apaf1) which activates caspase 9 required to activate caspases 3, 6, and 7. However, in the case of Candida, the only caspase identified as executing apoptosis is a metacaspase known as Yca1p [7]. The induction of oxidative stress followed by apoptosis in Candida cells by low concentrations of OSEO and its compounds, therefore, is likely to involve the mitochondrial routes, probably via the release of cytochrome c. This is observed most commonly with such forms of cell death in yeast. Furthermore, the release of Aifp or Nuc1p (mammalian ENDOG homolog) and the involvement of Nde1p or Ndi1p could also be studied in order to detail the sequence of actions. In the present study we evaluated the release of cytochrome c as a mechanism of cell death induced by test entities. A concomitant release of cytochrome c from the mitochondria directly correlates with a decrease in cytochrome c oxidase activity. In the present study, the decrease in enzyme activity was strongly shown by H2O2 (93%), indicating that the concomitant release of cytochrome c is required to elicit apoptosis, while the test compounds did not cause a substantial
Please cite this article in press as: Khan A, et al., Ocimum sanctum (L.) essential oil and its lead molecules induce apoptosis in Candida albicans, Research in Microbiology (2014), http://dx.doi.org/10.1016/j.resmic.2014.05.031
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decrease in enzyme activity. The effect of test compounds on cytochrome c oxidase activity is therefore speculated to be secondary to their antifungal activity. In conclusion, the primary involvement of cytochrome c might be excluded from the apoptosis-inducing mechanisms of OSEO and its compounds in C. albicans. Cytochrome c is present as loosely and tightly bound pools attached to the inner membrane by its association with cardiolipin. Lipid peroxidation (particularly of cardiolipin) disrupts binding of cytochrome c to the inner membrane. Once cytochrome c is dissociated, permeabilization of the outer mitochondrial membrane by Bax is required to permit the release of cytochrome c into the extramitochondrial environment. This two-step process is crucial for triggering cytochrome c release into cytosol [26]. Although test compounds elicit forceful oxidative insult [17], the release of cytochrome c into the cytoplasm does not occur. There is a possibility of involvement of other mitochondrial factors in activating caspases; probably, the ROS so generated could also be directly activating Yca1p to execute apoptosis [7]. At high concentrations, the evidence of necrosis signifies lethal, rapid and irreversible antifungal action of the compounds tested. This might originate from their damaging effects on the structural and functional integrity of membranes already investigated and reported in our previous studies [14]. To the best of our knowledge, the present study is the first to demonstrate apoptosis-inducing abilities for a plant essential oil in C. albicans. The role of mitochondria in mediating cell death should be further investigated by studying Nuc1p and Aif1p release. Characterization of the mechanistic switches that regulate active death processes in C. albicans may lead to development of novel antifungal agents that switch on endogenous cell suicide mechanisms. Acknowledgments This study was supported by Indian Council of Medical Research (India), Grant No. 59/24/2008/BMS/TRM [200804780] to Dr. NM and Dr. LAK. Dr. AK is a recipient of the Claude Leon Foundation (Cape Town) postdoctoral fellowship and expresses gratitude to the Foundation for the bursary. The authors wish to thank the DST-FIST for flourescence microscope facilities at the Department of Biosciences, Jamia Millia Islamia, New Delhi and Prof. Jawaid Khan for his kind assistance. References [1] Ahmad A, Khan A, Akhtar F, Yousuf S, Xess I, Khan LA, et al. Fungicidal activity of thymol and carvacrol by disrupting ergosterol biosynthesis and membrane integrity against Candida. Eur J Clin Microbiol Infect Dis 2011;30(1):41e50. [2] Al-Dhaheri RS, Douglas LJ. Absence of amphotericin B-tolerant persister cells in biofilms of some Candida species. Antimicrobial Agents Chemother 2008;52(5):1884e7. [3] Al-Dhaheri RS, Douglas LJ. Apoptosis in Candida biofilms exposed to amphotericin B. J Med Microbiol 2010;59(2):149e57. [4] Almeida B, Silva A, Mesquita A, Sampaio-Marques B, Rodrigues F, Ludovico P. Drug-induced apoptosis in yeast. Biochim Biophys Acta 2008;1783(7):1436e48.
[5] Arana DM, Nombela C, Pla J. Fluconazole at subinhibitory concentrations induces the oxidative- and nitrosative-responsive genes TRR1, GRE2 and YHB1, and enhances the resistance of Candida albicans to phagocytes. J Antimicrob Chemother 2010;65(1):54e62. [6] Bansod S, Rai M. Antifungal activity of essential oils from Indian medicinal plants against human pathogenic Aspergillus fumigatus and A. niger. World J Med Sci 2008;3(2):81e8. [7] Cao Y, Huang S, Dai B, Zhu Z, Lu H, Dong L, et al. Candida albicans cells lacking CaMCA1 encoded metacaspase show resistance to oxidative stress induced death and change in energy metabolism. Fungal Genet Biol 2009;46(2):183e9. [8] Carmona-Gutierrez D, Eisenberg T, Buttner S, Meisinger C, Kroemer G, Madeo F. Apoptosis in yeast: triggers, pathways, subroutines. Cell Death Differ 2010;17:763e73. [9] Devkatte AN, Zore GB, Karuppayil SM. Potential of plant oils as inhibitors of Candida albicans growth. FEMS Yeast Res 2005;5:867e73. [10] Dharmagadda VSS, Tandon M, Vasudevan P. Biocidal activity of the essential oils of Lantana camara, Ocimum sanctum and Tagetes patula. J Sci Ind Res 2005;64:53e6. [11] Gulshan K, Moye-Rowley WC. Multidrug resistance in fungi. Eukaryot Cell 2007;6:1933e42. [12] Kanafani Z, Perfect J. Antimicrobial resistance: resistance to antifungal agents: mechanisms and clinical impact. Clin Infect Dis 2008;46:120e8. [13] Kerr JFR, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972;26:239e57. [14] Khan A, Ahmad A, Akhtar F, Yousuf S, Xess I, Khan LA, et al. Ocimum sanctum essential oil and its active principles exert their antifungal activity by disrupting ergosterol biosynthesis and membrane integrity. Res Microbiol 2010c;161:816e23. [15] Khan A, Ahmad A, Xess I, Khan LA, Manzoor N. Anticandidal effect of Ocimum sanctum essential oil and its synergy with fluconazole and ketoconazole. Phytomedicine 2010b;17:921e5. [16] Khan A, Ahmad A, Manzoor N, Khan LA. Antifungal activities of Ocimum sanctum essential oil and its lead molecules. Nat Prod Commun 2010a;5(2):345e9. [17] Khan A, Ahmad A, Akhtar F, Yousuf S, Xess I, Khan LA, et al. Induction of oxidative stress as a possible mechanism of the antifungal action of three phenylpropanoids. FEMS Yeast Res 2011;11(1):114e22. [18] Khan A, Ahmad A, Xess I, Khan LA, Manzoor N. Ocimum sanctum essential oil inhibits virulence attributes in Candida albicans. Phytomedicine; 2013. http://dx.doi.org/10.1016/j.phymed.2013.10.028. [19] Kontoyiannis DP, Lewis RE. Antifungal drug resistance of pathogenic fungi. Lancet 2002;359:1135e44. [20] Kumar A, Shukla R, Singh P, Dubey NK. Chemical composition, antifungal and antiaflatoxigenic activities of Ocimum sanctum L. essential oil and its safety assessment as plant based antimicrobial. Food Chem Toxicol 2010;48(2):539e43. [21] Madeo F, Carmona-Gutierrez D, Ring J, Bu¨ttner S, Eisenberg T, Kroemer G. Caspase-dependent and caspase-independent cell death pathways in yeast. Biochem Biophys Res Commun 2009;382(2): 227e31. [22] Madeo F, Frohlich E, Ligr M, Grey M, Sigrist SJ, Wolf DH, et al. Oxygen stress: a regulator of apoptosis in yeast. J Cell Biol 1999;145:757e67. [23] Morschh€auser J. Regulation of multidrug resistance in pathogenic fungi. Fungal Genet Biol 2010;47:94e106. [24] Odds FC. Candida and candidiasis: a review and bibliography. London, UK: Bailliere Tindall; 1988. p. 67. [25] Odds FC, Brown AJ, Gow NA. Antifungal agents: mechanisms of action. Trends Microbiol 2003;11(6):272e9. [26] Ott M, Robertson JD, Gogvadze V, Zhivotovsky B, Orrenius S. Cytochrome c release from mitochondria proceeds by a two-step process. Proc Natl Acad Sci U S A 2002;99(3):1259e63. [27] Phillips AJ, Sudbery I, Ramsdale M. Apoptosis induced by environmental stresses and amphotericin B in Candida albicans. Proc Natl Acad Sci U S A 2003;100:14327e32. [28] Pinto E, Hrimpeng K, Lopes G, Vaz S, Gonçalves MJ, Cavaleiro C, et al. Antifungal activity of Ferulago capillaris essential oil against Candida,
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essential oils and their major components. Mycopathologia 2005; 159(3):339e45. [33] Wisplinghoff HT, Bischoff T, Tallent SM, Seifert H, Wenzel RP, Edmond MB. Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study. Clin Infect Dis 2004;39:309e17. [34] Wyllie AH, Kerr JFR, Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol 1980;68:251e306. [35] Zhu J, Krom BP, Sanglard D, Intapa C, Dawson CC, Peters BM, et al. Farnesol-induced apoptosis in Candida albicans is mediated by Cdr1-p extrusion and depletion of intracellular glutathione. PLoS One 2011; 6(12):e28830.
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MODEL
Research in Microbiology xx (2014) 1e9 www.elsevier.com/locate/resmic
Ocimum sanctum (L.) essential oil and its lead molecules induce apoptosis in Candida albicans Amber Khan1, Aijaz Ahmad2, Luqman Ahmad Khan, Nikhat Manzoor*
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Medical Mycology Lab, Department of Biosciences, Jamia Millia Islamia, New Delhi 110025, India Received 1 October 2013; accepted 9 May 2014
Abstract Manipulation of endogenous responses during programmed cell death (PCD) in fungi can lead to development of effective therapeutic strategies. In the present study, we evaluated the physiology of cell death in Candida albicans in response to Ocimum sanctum essential oil (OSEO) and its two major constituents e methyl chavicol (MET CHAV) and linalool (LIN) at varying inhibitory concentrations. Apoptotic cell death was studied on the basis of externalization of membrane phosphatidylserine (PS) revealed by annexin-V-FITC labeling, morphological alterations revealed by transmission electron microscopy and DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Exposure of fungal cells to MIC/4 of OSEO, MET CHAV and LIN resulted in morphological features characteristic of apoptosis, while necrosis was observed at higher concentrations. Necrotic cells displayed reduced TUNEL staining and an inability to exclude propidium iodide. In addition, they lacked a defined nucleus and an intact external morphology. Exposed cells were TUNEL-positive, showed chromatin condensation and margination, nuclear envelope separation, nuclear fragmentation, cytoplasmic shrinkage and plasma membrane blebbing. A dose-dependent decrease in cytochrome c oxidase activity was observed with each compound, but the decrease was not comparable to that elicited by H2O2, eliminating the primary involvement of cytochrome c release in apoptosis thus induced. Previously reported data revealed induction of apoptosis at low concentrations as a result of oxidative insult. Studies aimed at identifying other mitochondrial factors activated during this course to mediate apoptosis will further elucidate the mechanism of antifungal action of these natural products. © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Ocimum sanctum; Apoptosis; Methyl chavicol; Linalool; Candida albicans
1. Introduction Candida albicans is associated with a number of clinical conditions ranging from irritating superficial infections to lifethreatening systemic disease in immunocompromised patients [24]. Candidemia is the fourth most common bloodstream infection among hospitalized patients [33]. Several distinct * Corresponding author. Tel.: þ91 11 2698 1717x3410; fax: þ91 11 2698 0229. E-mail address: [email protected] (N. Manzoor). 1 Present address: Department of Pharmacy and Pharmacology, Faculty of Health Sciences, Medical School, University of Witwatersrand, Johannesburg, South Africa. 2 Present address: Department of Pharmaceutical Sciences, Tshwane University of Technology, Arcadia Campus, Pretoria, SA-0001, South Africa.
classes of antifungal drugs are currently available for the treatment of clinical infections [19,25]. Most of these drugs affect the cell membrane or cell wall integrity through inhibition of either ergosterol biosynthesis or b-glucan/chitin synthesis, respectively. A further complication to this limited spectrum of drugs is the acquisition of resistance by the pathogenic fungi observed in clinical settings [11,12,23]. AmB, while fungicidal, has severe and potentially lethal side effects. The azoles are generally fungistatic and hence their prolonged administration leads to resistance in Candida species. The development of more effective antifungal therapies is therefore of paramount importance. There are only a few studies that explored the relationship between antifungal drugs and cell death [4,29]. Yeasts display stereotypical patterns of cell death when responding to environmental insults or
http://dx.doi.org/10.1016/j.resmic.2014.05.031 0923-2508/© 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Please cite this article in press as: Khan A, et al., Ocimum sanctum (L.) essential oil and its lead molecules induce apoptosis in Candida albicans, Research in Microbiology (2014), http://dx.doi.org/10.1016/j.resmic.2014.05.031
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endogenous signals [27]. A specific series of morphological and biochemical changes set apoptotic cell death apart from necrosis [13,34]. While the novel concept of programmed necrosis has just emerged in yeast [8], recent studies have revealed the existence of programmed cell death in C. albicans in response to cytotoxic agents [4]. An understanding of the mechanistic basis of cell death in yeast may provide new developments for effective antifungal therapies. Innumerable studies have evaluated the antifungal efficacies of plant essential oils and their lead molecules against various pathogenic drug-susceptible and drug-resistant fungi [1,28]. Ocimum sanctum (L.) essential oil (OSEO) is known for its broad spectrum antifungal activities [6,9,10,15,16,20]. Its major constituents, methyl chavicol (MET CHAV) and linalool (LIN), are also known to have anticandidal activity [14,30e32]. MICs of OSEO, MET CHAV and LIN against C. albicans were found to be 0.025%v/v, 0.025%v/v and 0.031% v/v, respectively [14]. OSEO showed mild hemolytic activity, causing only 5.1% hemolysis of human RBCs at very high concentrations (80 MIC), while the same amount of hemolysis could be achieved by AmB at concentrations as low as MIC/4 [15]. In addition, OSEO has been shown to attenuate the expression of virulence-associated genes as well as pathogenicity in C. albicans [18]. The present work is hence an extension of our previous study in which we showed that OSEO inhibits ergosterol biosynthesis and causes membrane damage in Candida cells [14]. The mechanism of antifungal action of OSEO, MET CHAV and LIN has now been explored at varying inhibitory concentrations and the physiology of cell death has been studied on the basis of externalization of membrane phosphatidylserine (PS), revealed by annexin V affinity labeling, morphological alterations characteristic of cell death revealed by transmission electron microscopy (TEM) and DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Cytochrome c oxidase activity was estimated so as to study the role of mitochondria in mediating cell death under the influence of these natural antifungal products.
overnight in 2.5% glutaraldehyde in phosphate/magnesium buffer (40 mM K2HPO4/KH2PO4, pH 6.5e0.5 mM MgCl2). Cells were washed twice for 15 min in 0.1 M sodium phosphate buffer (pH 6.0) and post-fixed for 2 h in 2% osmium tetroxide. Cells were washed twice for 15 min in distilled water and then en bloc stained with 1% uranyl acetate (aqueous) for 30 min. After two further washes, cells were dehydrated in 95% and 100% ethanol. Cells were exposed to propylene oxide for 2 10 min and infiltrated for 1 h in 1:1 propylene/epoxy embedding material (Epon) mixture and then overnight in fresh Epon. After polymerization for 48 h at 60 C, ultrathin sections were cut using a microtome (Leica EM UC6) and transferred to a copper grid. Samples were stained with uranyl acetate (saturated solution of uranyl acetate in 50% alcohol) followed by lead citrate. Samples were washed three times in Milli-Q (MQ) water and dried by touching Whatman filter paper. Sections were examined with a Jeol (Japan) JEM-2100F transmission electron microscope at 120 kV. 2.3. Protoplast preparation The cell wall was digested with lyticase (1 mg/g cells) in different washing steps in protoplast buffers (pH 7.4) containing 1 M sorbitol, DTT (buffer I e 30 mM; buffer II e 1 mM; buffer III e 0 mM), 50 mM tris base and 10 mM MgCl2. Briefly, cells were harvested and washed thrice for 5 min each with buffer I (3 mL/g cells). Cells were then incubated in buffer II (5 mL/g cells; supplemented with lyticase) for 2 h at 25 C. After removing buffer II by centrifugation, cells were incubated with buffer III (5 mL/g cells) for 15 min and then again centrifuged to remove buffer III. Protoplasts were finally washed once with PBS and re-suspended in the same. 2.4. Annexin V-FITC labeling
C. albicans ATCC 90028 cells used in the present study were grown and maintained on YPD medium. MET CHAV and LIN were purchased from SigmaeAldrich (Germany); Amphotericin B (AmB) from Himedia (India), and H2O2 from Merck (India). Reagents, organic solvents and salts were of analytical grade and procured from Merck (India) and SRL (India). Midlog phase cells were harvested and exposed (A595 ¼ 0.1) to different concentrations of test drugs (MIC/4, MIC/2, MIC, 4MIC), AmB (2 mg/mL), and H2O2 (2.5 mM) for 1 h.
The externalization of phosphatidylserine (PS) was studied using annexin V-FITC labeling by a APOAF kit (SIGMA) in yeast protoplasts. Propidium iodide (PI) was used to differentially label dead cells. The protoplasts were resuspended in binding buffer and incubated with annexin V-FITC conjugate and PI solution. The results were quantified using a FACSCalibur flow cytometer (Becton Dickinson Biosciences) and analyzed using Cell Quest Pro software (Becton Dickinson). Filter-FL1 revealed FITC fluorescence, whereas FL2 revealed PI fluorescence. Cell populations in the R1 quadrant corresponded to red fluorescence (PIþ; Debris); in the R2 quadrant they corresponded to green and red fluorescence (FITCþ, PIþ; necrosis); in the R3 quadrant they corresponded to neither fluorescence (FITC, PI; alive) and in the R4 quadrant they corresponded to green fluorescence (FITCþ; apoptosis).
2.2. Transmission electron microscopy (TEM)
2.5. TUNEL assay (fluorometric)
The morphology of Candida cells was analyzed using TEM following the standard protocol. Cells (1 108) were fixed
A terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed in order to
2. Materials and methods 2.1. Strains and growth conditions
Please cite this article in press as: Khan A, et al., Ocimum sanctum (L.) essential oil and its lead molecules induce apoptosis in Candida albicans, Research in Microbiology (2014), http://dx.doi.org/10.1016/j.resmic.2014.05.031
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66 67 68 69 70 71 72 73 74 75 76 3. Results 77 78 3.1. TEM 79 80 Fig. 1 shows the effect of OSEO and its two major com81 82 ponents, LIN and MET CHAV, on the morphology of the 83 Candida cells using TEM. The yeast cells were incubated with 84 the test compounds at their respective MICs for 1 h. Electron 2.6. TUNEL assay (colorimetric) 85 microscopic investigation of cells incubated with the test 86 compounds revealed chromatin condensation along the nuProtoplasts were studied further for DNA fragmentation 87 clear envelope typical for apoptosis (margination), with intact with dUTP-biotin labeling using TUNEL-DAB system 88 cellular morphology, plasma membrane and cell wall. On the (DeadEnd colorimetric TUNEL system, PROMEGA) accord89 90 other hand, nuclei of untreated cells (control) were homogeing to the manufacturer's instructions. DAB staining (TUNEL 91 neous in shape and density. In addition, some cells showed positive cells) was observed under an inverted microscope 92 cellular shrinkage and extensive plasma membrane folds (Motic, China). This procedure stains the apoptotic nuclei dark 93 (Fig. 1: LIN-exposed cell). This could be membrane blebbing, brown. 94 a characteristic marker of apoptosis [22] observed as tiny 95 vesicles in yeast cells. Invagination of the plasma-membrane 2.7. Cytochrome c oxidase activity 96 and appearance of tubular structures were also evident and 97 98 indicated onset of membrane blebbing. At very high concenProtoplasts exposed to varying concentrations (MIC/4, 99 trations of test compounds (above 4 MIC), most of the intraMIC/2 and MIC) of test entities were assayed for cytochrome 100 cellular structures were destroyed (data not shown), indicating c oxidase activity using CYTOCOX1 kit (SIGMA) according 101 necrotic cell death. This corroborates well with the TUNEL to the manufacturer's instructions. Cytochrome c oxidase 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 Fig. 1. Representative transmission electron micrographs of C. albicans ATCC 90028 cells exposed to OSEO, MET CHAV and LIN at their respective MICs. 129 Morphological changes and intracellular damages in exposed cells are shown with yellow arrows. Abbreviation ‘cw’ refers to cell wall; ‘pm’, plasma membrane; ‘m’, mitochondria; and ‘n’, nucleus. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Q2 130
confirm apoptotic cell death in Candida induced by OSEO and its lead molecules. After treatment with test compounds, Candida cells were washed twice in PBS and fixed with 4% paraformaldehyde in PBS (pH 7.4) for 1 h at 20 C. Protoplasts were then prepared as described above. They were rinsed twice with PBS and incubated with permeabilization solution for 2 min on ice. The cells were again rinsed in PBS and labeled, using 50 ml of a 9:1 solution of the label and enzyme solutions from an in situ cell death detection kit, fluorescein (Roche Applied Sciences, Mannheim, Germany), with appropriate controls labeled only with the label solution. The protoplasts were incubated for 1 h at 37 C in a humidified atmosphere in the dark, rinsed in PBS, and examined with a Nikon Eclipse-80i fluorescence microscope (Japan) with a detection range of 515e565 nm.
activity was measured using a spectrophotometer (Systronics, India) at 550 nm by estimating the decrease in absorbance of ferrocytochrome c due to its oxidation to ferricytochrome c by the enzyme. Enzyme activity was defined as the amount of ferrocytochrome c oxidized in mmoles per minute. Each experiment was performed twice and in triplicate. Results obtained were expressed in terms of mean ± standard error (SEM).
Please cite this article in press as: Khan A, et al., Ocimum sanctum (L.) essential oil and its lead molecules induce apoptosis in Candida albicans, Research in Microbiology (2014), http://dx.doi.org/10.1016/j.resmic.2014.05.031
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reaction data shown in Fig. 4 where the number of TUNELFITC-positive cells decreased with increasing concentrations of the test compounds. 3.2. Annexin V-FITC affinity labeling Apoptotic cell death is accompanied by a change in plasma membrane structure by surface exposure of PS, while membrane integrity remains unchallenged. Surface-exposed PS can be detected by its affinity for annexin V, a phospholipid binding protein. The extent of PS externalization in C. albicans was monitored by flow cytometry using Annexin V-FITC labeling. The cytogram in Fig. 2 shows bivariate annexin V/PI analysis of the unexposed and exposed cell suspension. Viable cells are negative for both PI and annexin V (R3), apoptotic cells are PI-negative and annexin V-positive (R4),
while necrotic cells are positive for both PI and annexin V (R2). Exposure to MIC/4 of OSEO, MET CHAV and LIN resulted in 9e11% apoptotic cell death, whereas in controls, apoptotic cells represented only 2% of the cell population (Fig. 3). Cell death was dose-dependent and exposure to increasing concentrations of test compounds resulted in a concomitant increase in the percentage of dead cells. Exposure to MIC/2 of test compounds caused 22e23% cell death. Moreover, at sub-MICs, the exposed cells showed an apoptotic rather than a necrotic mode of cell death. Incubation of OSEO, MET CHAV and LIN at their respective MIC values resulted in 50%, 46% and 51% cell death, respectively, whereas incubation at higher concentrations (4MIC) caused an increase in the number of dead cells with a value of 94%, 74% and 85%, respectively. Cells incubated with 2.5 mM H2O2 showed 26% cell death (including 66% of the total oil. LIN causes 13e77% GSH depletion, while MET CHAV results in 18e73.6% GSH depletion in Candida cells exposed to a 10e100 mg/mL test concentration. In addition, both constituents at the same concentrations compromise anti-oxidant defense armory and cause severe membrane oxidation in Candida. LIN exhibited higher destruction of lipids (2.8e4.9-fold relative to control) than MET CHAV (2.7e3.8-fold) [17]. Lipid peroxidation (LPO) induced by test compounds may result in part from the suppression of ergosterol biosynthesis by test entities [14], which could otherwise physically protect lipids from oxidation. In the present study, the apoptosis induced at lower concentrations of test entities overall could be justified in the light of MET CHAV- and
LIN-induced GSH depletion, which is a well-established initiator of apoptosis [35], as well as attenuation of the anti-oxidant defense system (unpublished data), resulting in an outburst of ROS. Complete ergosterol depletion and membrane disintegration at high concentrations, on the other hand, explains necrotic cell death observed in the present study. In H2O2 and AmB treated cells the triggers of apoptosis are induction of oxidative stress and accumulation of ROS [27]. These triggers may involve mitochondrial routes of PCD. Apoptosis operates via two major classical pathways e extrinsic and intrinsic. The extrinsic pathways are triggered only by external means like pheromone signaling and quorum sensing. This includes signaling via ‘Ste’ proteins in the yeast cells. The intrinsic pathway, which has significance in the present study (because of the lipophilic nature of test entities, and their reported effects on the intracellular pool), is mediated by the mitochondria, where mitochondrial membranes are sensitized by the pro-apoptotic proteins encoded by the Bcl2 family genes (BAD, BAX, BID) and cytochrome c is released into the cytoplasm. In the mammalian system, cytochrome c activates the apoptosis-activating factor 1(Apaf1) which activates caspase 9 required to activate caspases 3, 6, and 7. However, in the case of Candida, the only caspase identified as executing apoptosis is a metacaspase known as Yca1p [7]. The induction of oxidative stress followed by apoptosis in Candida cells by low concentrations of OSEO and its compounds, therefore, is likely to involve the mitochondrial routes, probably via the release of cytochrome c. This is observed most commonly with such forms of cell death in yeast. Furthermore, the release of Aifp or Nuc1p (mammalian ENDOG homolog) and the involvement of Nde1p or Ndi1p could also be studied in order to detail the sequence of actions. In the present study we evaluated the release of cytochrome c as a mechanism of cell death induced by test entities. A concomitant release of cytochrome c from the mitochondria directly correlates with a decrease in cytochrome c oxidase activity. In the present study, the decrease in enzyme activity was strongly shown by H2O2 (93%), indicating that the concomitant release of cytochrome c is required to elicit apoptosis, while the test compounds did not cause a substantial
Please cite this article in press as: Khan A, et al., Ocimum sanctum (L.) essential oil and its lead molecules induce apoptosis in Candida albicans, Research in Microbiology (2014), http://dx.doi.org/10.1016/j.resmic.2014.05.031
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decrease in enzyme activity. The effect of test compounds on cytochrome c oxidase activity is therefore speculated to be secondary to their antifungal activity. In conclusion, the primary involvement of cytochrome c might be excluded from the apoptosis-inducing mechanisms of OSEO and its compounds in C. albicans. Cytochrome c is present as loosely and tightly bound pools attached to the inner membrane by its association with cardiolipin. Lipid peroxidation (particularly of cardiolipin) disrupts binding of cytochrome c to the inner membrane. Once cytochrome c is dissociated, permeabilization of the outer mitochondrial membrane by Bax is required to permit the release of cytochrome c into the extramitochondrial environment. This two-step process is crucial for triggering cytochrome c release into cytosol [26]. Although test compounds elicit forceful oxidative insult [17], the release of cytochrome c into the cytoplasm does not occur. There is a possibility of involvement of other mitochondrial factors in activating caspases; probably, the ROS so generated could also be directly activating Yca1p to execute apoptosis [7]. At high concentrations, the evidence of necrosis signifies lethal, rapid and irreversible antifungal action of the compounds tested. This might originate from their damaging effects on the structural and functional integrity of membranes already investigated and reported in our previous studies [14]. To the best of our knowledge, the present study is the first to demonstrate apoptosis-inducing abilities for a plant essential oil in C. albicans. The role of mitochondria in mediating cell death should be further investigated by studying Nuc1p and Aif1p release. Characterization of the mechanistic switches that regulate active death processes in C. albicans may lead to development of novel antifungal agents that switch on endogenous cell suicide mechanisms. Acknowledgments This study was supported by Indian Council of Medical Research (India), Grant No. 59/24/2008/BMS/TRM [200804780] to Dr. NM and Dr. LAK. Dr. AK is a recipient of the Claude Leon Foundation (Cape Town) postdoctoral fellowship and expresses gratitude to the Foundation for the bursary. The authors wish to thank the DST-FIST for flourescence microscope facilities at the Department of Biosciences, Jamia Millia Islamia, New Delhi and Prof. Jawaid Khan for his kind assistance. References [1] Ahmad A, Khan A, Akhtar F, Yousuf S, Xess I, Khan LA, et al. Fungicidal activity of thymol and carvacrol by disrupting ergosterol biosynthesis and membrane integrity against Candida. Eur J Clin Microbiol Infect Dis 2011;30(1):41e50. [2] Al-Dhaheri RS, Douglas LJ. Absence of amphotericin B-tolerant persister cells in biofilms of some Candida species. Antimicrobial Agents Chemother 2008;52(5):1884e7. [3] Al-Dhaheri RS, Douglas LJ. Apoptosis in Candida biofilms exposed to amphotericin B. J Med Microbiol 2010;59(2):149e57. [4] Almeida B, Silva A, Mesquita A, Sampaio-Marques B, Rodrigues F, Ludovico P. Drug-induced apoptosis in yeast. Biochim Biophys Acta 2008;1783(7):1436e48.
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