fphar-08-00301 May25,2017 Time:12:14 #1 ORIGINALRESEARCH published:29May2017 doi:10.3389/fphar.2017.00301 An Antifungal Mechanism of Protolichesterinic Acid from the Lichen Usnea albopunctata Lies in D the Accumulation of Intracellular ROS and Mitochondria-Mediated Cell E Death Due to Apoptosis in Candida T tropicalis C S.N.Kumar*andC.Mohandas DivisionofCropProtection,CentralTubAerCropsResearchInstitute,Sreekariyam,India Candida species causes superficial and life-threatening systemic infections and are R difficulttotreatduetotheresistanceoftheseorganismtovariousclinicallyuseddrugs. Protolichesterinic acid is a well-known lichen compound. Although the antibacterial T activity of protolichesterinic acid has been reported earlier, the antifungal property and Editedby: itsmechanismofactionarestilllargelyunidentified.Thegoalofthepresentinvestigation NicolaClementi, Eis to explore the anticandidal activity and mechanism of action of protolichesterinic Vita-SaluteSanRaffaeleUniversity, Italy acid, especially against Candida tropicalis. The Minimum Inhibitory Concentration *CorrespoRndence: (MIC) value was established through microdilution techniques against four Candida S.N.Kumar species and out of four species tested, C. tropicalis showed a significant effect [email protected]; (MIC: 2 µg/ml). In the morphological interference assay, we observed the enhanced [email protected] inhibition of hyphae when the cells were treated with protolichesterinic acid. Time- Specialtysection: kill assay demonstrated that the maximum rate of killing was recorded between 2 Thisarticlewassubmittedto ExperimentalPharmacologyandDrug and 6 h. C. tropicalis exposed to protolichesterinic acid exhibited an increased ROS Discovery, production, which is one of the key factors of fungal death. The rise in ROS was due asectionofthejournal tothedysfunctionofmitochondriacausedbyprotolichesterinicacid.Weconfirmedthat FrontiersinPharmacology protolichesterinicacid-induceddysfunctionofmitochondriainC.tropicalis.Thedamage Received:02March2017 Accepted:10May2017 of cell membrane due to protolichesterinic acid treatment was confirmed by the influx Published:29May2017 of propidium iodide and was further confirmed by the release of potassium ions. The Citation: treatment of protolichesterinic acid also triggered calcium ion signaling. Moreover, it Kumar SNandMohandas C(2017) AnAntifungalMechanism commenced apoptosis which is clearly evidenced by Annexin V and propidium iodide ofProtolichesterinicAcidfrom staining. Interestingly protolichesterinic acid recorded excellent immunomodulatory theLichenUsneaalbopunctataLies property when tested against lymphocytes. Finally protolichesterinic acid showed low intheAccumulationofIntracellular ROSandMitochondria-MediatedCell toxicity toward a normal human cell line Foreskin (FS) normal fibroblast. In in vivo DeathDuetoApoptosisinCandida test, protolichesterinic acid significantly enhanced the survival of C. tropicalis infected tropicalis.Front.Pharmacol.8:301. doi:10.3389/fphar.2017.00301 Caenorhabditis elegans. This investigation proposes that the protolichesterinic acid FrontiersinPharmacology|www.frontiersin.org 1 May2017|Volume8|Article301 fphar-08-00301 May25,2017 Time:12:14 #2 KumarandMohandas MechanismofProtolichesterinicAcidinCandidatropicalis induces apoptosis in C. tropicalis via the enhanced accumulation of intracellular ROS and mitochondrial damage, which leads fungal cell death via apoptosis. Our work revealedanewkeyaspectofmechanismsofactionofprotolichesterinicacidinCandida species. This article is the first study on the antifungal and mechanism of action of protolichesterinicacidinCandidaspecies. Keywords:protolichesterinicacid,Candida,mechanismofaction,apoptosis,ROS INTRODUCTION antifungals from natural sources to fight against deadly fungal diseases. The occurrence of infections due to pathogenic fungi has been Candida spp. are normal commensal microbes of humans, gradually growing as assessed to occur in more than a billion commonly found in the oral, gastrointestinal, urinary, and humans every year worldwide (Lee and Lee, 2014). Diseases vaginalmucosa(Alvesetal.,2014),andalsDoproducessuperficial caused by pathogenic Candida spp. and other fungi continue and severe systemic infections. Due to this, Candida spp. are to embody a noteworthy health burden, especially to human alsoknownasopportunisticfungalpathogens.Candidagenusis beings. The Candida species represent second most numerous most commonly recovered from hospital fungal infections and E agents causing fungal infection worldwide (Brown et al., 2012). is known as candidaemia (Alves et al., 2014). Candida spp. are Here some Candida spp. is highly resistant to various clinically presentinapproximately50%ofhumanpopulationandaround usedazolesandpolyenesdrugs.Moreover,Candidaspeciesisthe 80% of women haTve suffered from Candida infections, with fouthmostcommonsourceofhospital-acquiredsystemicfungal around 5% of these infections are recurring (de Castro et al., infections with more than 50% mortality rates (Hwang et al., 2013).Throughafailureofhumandefensesystems,Candidacan C 2014). It is extensively believed that more than 70% of all the causesevereinfectionsinalmostanypartofthebody,including fungal infections in humans are caused by the Candida species skin,nails,urogenital,orcanbesystemic(Wangetal.,2014). viz. Candida albicans, Candida tropicalis, Candida glabrata, Wide spread used of various immunosuppressive drugs, the A and Candida parapsilosis (Hwang et al., 2014). Investigation useofbroad-spectrumantibioticstotreatvariousinfectionsare on the development of novel antifungal agents over the past responsible for an increasing number of immunocompromised few decades has resulted in only a few drugs, which are being peoplesandthisinternleadstovariousopportunisticinfections R clinically used (Bhattacharya et al., 2015). Five major groups of worldwide. A debilitated or reduced immune system offers compounds are used to treat various the fungal infections and favorable situations for infectious diseases caused by various thisincludesazoles,polyenes,fluoropyrimiTdines,echinocandins, pathogenic microorganisms. AIDS due to HIV (human and allylamines (Bhattacharya et al., 2015). But, most of the immunodeficiency virus) is one of the main causative features compoundsusedtotreatpathogenicfungiareusuallysynthetic for the rising figure of peoples with various deadly fungal E innature.Butafewdrugstotreatfungalinfectionslikepolyenes infections, especially by Candida spp. (Nissapatorn et al., 2003; andechinocandinsarepurelynaturalcompounds,whichisolated Kothavadeetal.,2010).Thewidespreaduseofantifungaldrugs from microbial sourceRs. Fluconazole which comes under azole for prophylaxis in AIDS patients became one of the foremost family, is the backbone in treating various infections caused by reason of establishment of pathogenic Candida-non-albicans Candida species due to the great efficacy, reduced toxicity and species (CNA) and increased resistance to various antifungals inexpensive (Liu et al., 2016). However, with the widespread (Kothavade et al., 2010). Surprisingly, in India, C. tropicalis, an usageoffluconazole,theoccurrenceofdrugresistancetomany importantCNA,isoneofthemajorcauseofseverenosocomial other azole derivatives emerged (Liu et al., 2016). Moreover, candidaemiainmanypatients(Kothavadeetal.,2010). prolonged use of antifungals other than fluconazole in treating Medicinal plants are well-known natural sources for the infections caused by Candida species has led to the emergence treatmentofvariousdiseasessinceancienttimes(Srivastavaetal., of resistance to almost all drugs used clinically. This leads 2013). Lichens are among the most mesmerizing organisms on to drug resistance in clinical isolates of Candida species ourplanet.Lichensareawell-known,self-supporting,mutualistic mostly resulted through cross-resistance to many other drugs, association between fungi (mycobiont) and algae (photobionts) aphenomenontermedasmultidrugresistance(MDR).MDRis which form a unique symbiotic structure, known as lichen a serious problem during the treatment of opportunistic fungal thallus (Suzuki et al., 2016). Lichen can adapt to grow in many infectionswhichposessevereconcerngiventhelimitednumber adverse environmental conditions. Interestingly, there are no of clinically useful antifungal drugs available in the market. reportsonthedestructivemicrobialinfectionsinlichensandthis Despite the accessibility of various other classes of antifungals, clearlyindicatedthepresenceofmanyantimicrobialcompounds. inherent toxicity, and development of multi drug resistance The ability of lichen to produce a wide range of biologically continuemajorproblemsinmanagingvariousdeadlypathogenic active metabolites help to explain the fact that lichens have fungi and the availability of novel antifungal drugs to over also been traditionally used as sources of human medicines, comes these situations are still limited (Sharma et al., 2015). pigments, perfumes etc. (Suzuki et al., 2016). Various chemical These problems point to an urgent and unmet need for the compounds have consistently been associated with lichens, development of novel antifungal agents or searching for new and these compounds may have pharmaceutically important FrontiersinPharmacology|www.frontiersin.org 2 May2017|Volume8|Article301 fphar-08-00301 May25,2017 Time:12:14 #3 KumarandMohandas MechanismofProtolichesterinicAcidinCandidatropicalis DivisionofCTCRIforfuturereference.Finelydrygroundthalli ofU.albopunctata(50g)wereextractedusingacetone:methanol (1:1)inaSoxhletapparatus.Thefinalextractswerefilteredand thenconcentratedunderreducedpressureinarotaryevaporator at 40◦C. The dried extract was stored at −20◦C for future FIGURE1|Chemicalstructureofprotolichesterinicacidisolatedfrom U.albopunctata.Thechemicalstructureofprotolichesterinicacidwas experiments. The dried extract of the lichen U. albopunctata confirmedbasedontheNMRandMSspectralanalyses. (1 g) was dissolved in ethyl acetate. After filtration, the filtrate was fractioned using silica gel column chromatography (silica gel 60, 230–400 mesh). The silica gel column was eluted properties. Interestingly, most of the lichen compounds are with methanol-ethyl acetate solvent gradient (10: 1 and 5: 1) phenolicinnature(Suzukietal.,2016). which yield five major fractions. The fourth fraction yielded Protolichesterinic acid (Figure 1), a higher aliphatic acid pure protolichesterinic acid (60 mg). The chemical structure is a typical lichen compound exhibiting a wide range of of protolichesterinic acid was confirmed based on the detailed pharmaceutically interesting properties. In spite of the spectralanalyses(NMRandHRMS)(KumDaretal.,2014). pharmacological effects that have already been recognized, variousreportshaveshownthatprotolichesterinicacidexhibits Preparation of Protolichesterinic Acid several interesting biological properties such as antimicrobial, and the Fungal StraEins antitumor, and antioxidant activities (Ingolfsdottir et al., 1997; Astocksolutionofprotolichesterinicacid(50mg/ml)dissolved Turketal.,2003;Mitrovic´etal.,2011;Russoetal.,2012).Allthese in0.5%dimethylsulfoxide(DMSO)wasusedforallassays.The properties were suggesting that this compound is important T CandidastrainsCandidaalbicansMTCC277,Candidatropicalis classes of perfect natural bioactive compound. Even though MTCC 230, Candida glabrata MTCC 3019, and Candida the antibacterial and anticancer properties of this compound parapsiloCsis MTCC 6510 were used for antifungal studies. All have been explored extensively and data about the antifungal the test fungi were obtained from MTCC (Microbial Type activityespeciallyagainstCandidaspecieslacksintheliterature. Culture Collection Centre), Institute of Microbial Technology The reported biological properties of protolichesterinic acid A(IMTECH, Chandigarh, India). The test fungi were preserved clearlyhighlightedtheimportanceofthiscompoundasleading on potato dextrose agar (PDA) slants. The fungal cells were bioactivecompoundwithpharmacologicalandmedicalpotential cultured in potato dextrose broth (PDB) with aeration at 37◦C inthedevelopmentofnoveldrugformulationsinnearbyfuture R under agitation at 150 rpm. Initially, the fungal growth was (Nguyenetal.,2014;Bessadóttiretal.,2015). monitored by measuring the OD at 600 nm with a microtiter Recentlywehavereportedthepurificationandantimicrobial ELISA plate reader. After incubation, the Candida cells were property of protolichesterinic acid fromTUsnea albopunctata harvested by centrifugation at 5000 × g for 5 min at 4◦C and (Kumar et al., 2014). We also reported the preliminary washedtwicein10mlof0.1Mphosphate-bufferedsaline(PBS; anticandidalactivityofprotolichesterinicacidagainstC.albicans E pH7).Cellpelletsthusformedwereresuspendedin10mlPDB and C. tropicalis in the above cited paper. In the present and the cell density was adjusted to 1 × 106 cells/ml using a investigation, we studied the detailed antifungal properties hemocytometer. of protolichesterinic Racid by metabolic and, morphological analyses to confirm its mechanism of action in Candida cells, Determination of Minimum Inhibitory especiallyC.tropicalis.Here,weexaminedboththeCandidacell Concentration (MIC) of Protolichesterinic proliferationandtheproductionofintracellularreactiveoxygen species(ROS)byprotolichesterinicacidasROSplayavitalrole Acid incelldeathduetoapoptosis.Besides,wealsoobservedapoptotic Minimum inhibitory concentrations (MICs) were done by the topographies caused by protolichesterinic acid, which includes microdilution method conferring to the reference of CLSI mitochondrial membrane depolarization and externalization of (ClinicalandLaboratoryStandardsInstitute[CLSI],2005)using phosphatidylserine and calcium signals. Further, we also tested multi-wellELISAplatesalteredaccordingtothecommendations the toxicity profile of this compound against Foreskin (FS) that have been mentioned for the more efficient determination normalfibroblastcellline.Finally,weshowedprotolichesterinic of antimicrobial potential of natural compounds (Cos et al., acid greatly prolonged the survival of C. tropicalis infected 2006). In brief, 180 µl of PDB was dispensed aseptically to C.elegans. the wells of a 96-well microdilution plates. After that, 10 µl each of the serial dilutions of protolichesterinic acid and amphotericin B (AmpB) (reference antimycotic drug) were MATERIALS AND METHODS prepared in the PDB (1 to 2000 µg/ml). About 10 µl of Candidasuspensionatadensityof106 CFU/mlwasinoculated Extraction and Isolation of in each well and the plates were incubated for 48 h at 37◦C. Protolichesterinic Acid The Candidal growth was measured by taking the absorbance Usnea albopunctata were collected from Trivandrum, Kerala, at 600 nm using a microtiter plate reader. Dimethyl sulfoxide India and were identified through morphological analysis and and sterile water (deionized) were used as a blank control a voucher specimen has been deposited in the Crop Protection which did not inhibit the growth of Candida species tested. FrontiersinPharmacology|www.frontiersin.org 3 May2017|Volume8|Article301 fphar-08-00301 May25,2017 Time:12:14 #4 KumarandMohandas MechanismofProtolichesterinicAcidinCandidatropicalis Minimum inhibitory concentration was defined as the lowest (0.5× MIC, MIC, and 2× MIC) and amphotericin B (MIC). concentrationsofanyagentthatinhibitedthegrowthofthetest Control cells were incubated with DMSO and sterile deionized microbesby≥90%comparedwiththatofthecontrol.Minimum water.About10µlsamplesfromeachtreatmentwerecollected inhibitoryconcentrationswereattainedfromthreeindependent at 0, 1, 2, 4, 6, 12, 24, and 48 h and serially diluted in PBS, tests that performed in triplicate (Wonyoung and Dong Gun, and 50 µl aliquots were gently plated on the surface of PDA 2014). plates.TheviableCandidacolonieswerecountedafterincubation at 37◦C for 48 h. All of the experiments were performed in Minimum Fungicidal Concentration triplicate. (MFC) of Protolichesterinic Acid After the incubation period, the number of viable Todeterminetheminimumfungicidalconcentrations(MFCs)of Candida cells was counted and expressed in log CFU/ml. protolichesterinic acid and amphotericin B, an aliquot (10 µl) The results were scrutinized and represented graphically, taken from each well that recorded total inhibition of fungal that is a fungal death curve vs. function of time. Data growth was plated onto PDA plates. The presence of fungal analysis for the protolichesterinic acid was measured as colonies was monitored after 24 h of incubation at 37◦C. MFC showing fungicidal activity when there wD as a decrease greater wasdefinedasthelowestconcentrationofprotolichesterinicacid than or equal to 3 log10 CFU/ml of the initial inoculum, and amphotericin B that produced a total reduction in CFU resulting in the reduction of 99.99% or more CFU/ml in 48 h (99.99%inhibitioncomparedtonon-treatedmicrobialgrowth). compared with the initial iEnoculum. Fungistatic activity was Theexperimentwasrepeatedforthreetimes. considered as the decrease in growth lower than 99.99% or <3 log in CFU/ml from the original inoculum (Leite et al., 10 Agar DisK Diffusion Assay 2015). T Hundred microliter of overnight Candida cultures were spread Mechanism Action on Protolichesterinic over PDA plates. Six millimeter sterile filter paper disks (Hi- C Media)containingMICconcentrationofprotolichesterinicacid Acid on Candida andamphotericinBwereplacedontheagarplates.Filterpaper Out of four Candida species tested, C. tropicalis recorded the disks incorporated with 10 µl of DMSO served as negativeAbestactivityandthusthisstrainwasselectedforfurtherdetailed control.Plateswereincubatedat37◦Cfor24h.Afterincubation, studiesincludingmechanismofaction. thediameterofzoneofinhibitionwasenvisagedaroundthedisks containingtestsubstances.TheantimicrobialactivityRofthetest Detection of Reactive Oxygen Species compoundswasdeterminedbymeasuringthezoneofinhibition (ROS) in C. tropicalis after Treatment (diameter)andexpressedinmm.Theexperimentwasrepeated forthreetimes. T with Protolichesterinic Acid 5-(and-6)-chloromethyl-2(cid:48),7(cid:48)-dichlorodihydrofluorescein Cell Viability Assay diacetate acetyl ester (CMH DCFDA) effortlessly diffuses 2 E Antifungalactivityofprotolichesterinicacidwastestedwiththe through the microbial cell membrane, and then it is freely colony-formingunit(CFU)assay.Candidaspeciesweretreated hydrolyzedbyintracellularesterasestoproducenon-fluorescent with different concenRtrations of protolichesterinic acid (0.5× dichlorofluorescein (DCFH), which is then very quickly MIC,MIC,and2×MIC)andincubatedfor8hat37◦C.About oxidizedtoformextremelyfluorescent2(cid:48),7(cid:48)-dichlorofluorescein 100 µl samples from each treatment were collected at 0, 2, 4, (DCF) by various intracellular oxidative stressors (da Silva 6, and 8 h and cells were washed with PBS and diluted serially. et al., 2014). The DCF fluorescence intensity thus formed is DilutionswerespreadonPDAplatesindividuallyandincubated directly proportional to the sum of intracellular ROS formed for24hat37◦C.Tomeasurethecellviability,CFUswerecounted by the action of any test compounds (da Silva et al., 2014). byeye.Theexperimentwasrepeatedtimetimes. For revealing of ROS produced over a 24 h culture period, when treated with protolichesterinic acid, C. tropicalis was Hyphal Induction Assay incubated with 20 µM CMH DCFDA at 37◦C for 2 h in 2 Candidahyphaewereinducedasreportedpreviously(Yuetal., the dark. Then, the C. tropicalis cells were collected, washed 2014), except C. glabrata (As this species does not form any three times thoroughly with PBS buffer, resuspended in PBS, hyphae). Briefly, 24 h Candida cells were washed thoroughly and immediately examined using a confocal microscopy (BD withPBSandsuspendedinRPMI-1640mediumcomprisingthe Pathways). The fluorescence was further measured using a protolichesterinic acid with the specified concentrations (0.5× spectrofluorophotometer (Shimadzu RF-5301PC, Shimadzu, MIC, MIC, and 2× MIC). After that, the tubes were incubated Kyoto, Japan) at an excitation wavelength of 495 nm and an inashakingincubatorfor4hat37◦C.Thepercentageofhyphal emission wavelength of 525 nm. 2.5 mM H2O2 was used as inductionwascalculatedasthepreviouslyreportedmethod(Yu positivecontrol. etal.,2015). N-Acetyl Cysteine (NAC) Assay Time-Kill Kinetic Analysis ForROSquenching,NAC(Sigma–Aldrich,St.Louis,MO,United Exponential-phase Candida cells (1 × 106 cells/ml) were States) was added to the C. tropicalis culture (1 × 106 cells/ml) incubatedwithdifferentconcentrationofprotolichesterinicacid toattainafinalconcentrationof5mmol/L.C.tropicalisculture FrontiersinPharmacology|www.frontiersin.org 4 May2017|Volume8|Article301 fphar-08-00301 May25,2017 Time:12:14 #5 KumarandMohandas MechanismofProtolichesterinicAcidinCandidatropicalis was then treated with protolichesterinic acid and amphotericin without the compounds and with sonication, respectively (Yun B followed by the addition of NAC. The concentrations of etal.,2015). NAC were determined to reduce the growth inhibition of C. tropicalis. Colony count (CFU/ml) was monitored after 4 h Determination of Changes in exposure to the protolichesterinic acid and amphotericin B. Mitochondrial Membrane Potential For measuring the colony count, 100 µl of the C. tropicalis The fluorescent dye rhodamine 123 or RHO123 (2-[6-amino- were collected, thoroughly washed with PBS buffer. This was 3-imino-3Hxanthen-9-yl] benzoic acid methyl ester) was used then serially diluted in PBS buffer. All dilutions were plated to study the mitochondrial membrane potential ((cid:49)(cid:57)m) of onto PDA plates, and incubated at 37◦C for 18 h. Serial C. tropicalis after the treatment of test compound. RHO123 dilutions that produced between 10 and 100 fungal colonies is a positively charged dye that usually enters any cells were counted, and the CFU/ml was stated as Candida survival by diffusion. The RHO123 accumulation and retention in (expressed in percentage) using the formula: [(CFU/ml of cells mainly depend on the (cid:49)(cid:57)m value in response to the C. tropicalis treated with test compound)/(CFU/ml of non- result of protolichesterinic acid treatment on mitochondrial treated control) × 100] (Hwang et al., 2014). The results were transmembrane. Briefly, C. tropicalis (1 ×D 106 cells/ml) in the express as mean ± standard deviation for three independent mid-logphasewerecentrifugedandresuspendedinPBS(pH7.4) tests. andincubatedfor15minat37◦Cwithrhodamine123(10mM). The cells were again washedEwith PBS and treated with 0.5× Determination of Membrane MIC,MIC,and2×MICconcentrationofprotolichesterinicacid Permeabilization in C. tropicalis for 2 h at 37◦C. 2.5 mM H O was used as positive control. T 2 2 To investigate the alteration of membrane permeabilization The microscopic examination of the delivery of rhodamine under protolichesterinic acid treatment, cell membrane 123 in the C. tropicalis cells was achieved through confocal impermeable fluorescent dye propidium iodide (PI) was laserscanCningmicroscopy(BDPathways).Thefluorescencewas used. C. tropicalis was treated with the concentrations of further measured using a spectrofluorophotometer (Shimadzu 0 (control), 0.5× MIC, MIC, and 2× MIC concentration RF-5301PC,Shimadzu,Kyoto,Japan)atanexcitationwavelength of protolichesterinic acid in PDB with 1 × 106 CandidaAof 511 nm and an emission wavelength of 534 nm. Untreated cells/ml for 4 h. The amphotericin B treatment severed as C.tropicaliscellswereusedascontrols. the control. 2.5 mM H O was used as the positive control. 2 2 After incubation at 37◦C for 3 h, the Candida Rcells were Detection of Phosphatidylserine stainedwith5µg/mlofPIat37◦Cfor30mininthedark.The Externalization through Annexin-V Candida cell membrane permeabilization was directly analyzed T Staining by confocal microscopy (BD Pathways). The fluorescence ProtoplastPreparation was further measured using a spectrofluorophotometer The C. tropicalis cell wall was digested with lyticase (1 mg/g (Shimadzu RF-5301PC, ShiEmadzu, Kyoto, Japan) at cells) in different washing steps in protoplast buffers (pH an excitation wavelength of 535 nm and an emission 7.4) containing 1 M sorbitol, DTT (buffer 1–30 mM; buffer wavelength of 617 nm. The test was performed trice (Li R 2–1 mM; buffer 3–0 mM), 50 mM tris base and 10 mM etal.,2015). MgCl . Briefly, cells were harvested and washed thrice for 2 Determination of the Release of 5 min each with buffer 1 (3 ml/g cells). Cells were then incubated in buffer 2 (5 ml/g cells; supplemented with lyticase) Potassium Ions after Protolichesterinic for 2 h at 37◦C. After removing buffer 2 by centrifugation, Acid Treatment cells were incubated with buffer 3 (5 ml/g cells) for 15 min The variation in the ion concentration due to the treatment and then again centrifuged to remove buffer 3. Protoplasts of protolichesterinic acid was determined by the release of were finally washed once with PBS and re-suspended in the potassium ions. C. tropicalis cells (OD approximately 1) were same. centrifuged at 5000 rpm for 5 min and the pellet thus obtained was resuspended in 5 ml PBS and treated with Annexin-VStaining the protolichesterinic acid (0.5× MIC, MIC, and 2× MIC). Phosphatidylserine externalization due to apoptosis after Amphotericin B serves as the drug control. After incubation protolichesterinicacidtreatmentwasstudiedbyFITC-Annexin for 5-min intervals at 37◦C, the cells were centrifuged at Vapoptosisdetectionkit(Sigma–Aldrich).Forthisexperiment, 13,000rpmfor10min.Thesupernatantwasmeasuredusingan the protoplasts were incubated with 0.5× MIC, MIC, and 2× ion-selective electrode (ISE) meter (Orion Star A214, Thermo MICconcentrationofprotolichesterinicacidfor2hat37◦Cand Scientific, Singapore). The cells were sonicated to determine incubatedfor15–20mininanannexinbindingbuffercontaining 100% potassium release. The percentage of potassium release 5 µl of FITC-Annexin-V/ml and 5 µl of PI. After this the cells caused by the compounds was calculated as follows: potassium were then analyzed by confocal laser scanning microscopy. release (%) = 100 × ([K+]-[K+] )/([K+]-[K+] ), where [K+] 2.5 mM H O was used as positive control. The experiment 0 t 0 2 2 represents the potassium release achieved after addition of the wasperformedthreetimes,andtheresultswereshownasmean compoundsand[K+] and[K+] representthepotassiumrelease values±SDs. 0 t FrontiersinPharmacology|www.frontiersin.org 5 May2017|Volume8|Article301 fphar-08-00301 May25,2017 Time:12:14 #6 KumarandMohandas MechanismofProtolichesterinicAcidinCandidatropicalis Studying the Cytosolic and In Vitro Antifungal Property of Mitochondrial Ca2+ Level Up on the Protolichesterinic Acid in C. elegans Treat of Protolichesterinic Acid Infected with C. tropicalis ToexaminecytosolicandmitochondrialCa2+levels,Fura-2AM Caenorhabditis elegans – C. tropicalis assay was performed (Molecular Probes) and Rhod-2AM (Molecular Probes) were according to previously reported method (Chang et al., 2012). used, respectively. C. tropicalis cells (1 × 106 cells/ml) were Briefly, nematodes were transferred on 48 h old C. tropicalis incubatedwithprotolichesterinicacid(0.5×MIC,MIC,and2× lawns [on Brain Heart Infusion agar (BHI) plates] for 2 h. The MIC)andamphotericinBat37◦Cfor4h.Ca2+levelwasstudied nematodeswerewashedofftheplateswithscreenmedium[30% according to the previously reported method (Choi and Lee, BHI broth in M9 buffer containing kanamycin (100 µg/ml), 2015).2.5mMH O wasusedaspositivecontrol. ampicillin (200 µg/ml), and streptomycin (200 µg/ml)]. The 2 2 screen buffer containing nematodes were re-suspended at a Protolichesterinic Acid Toxicity to densityoftwonematodes/µlscreenmedium.Thesuspensionof preinfectednematodes(20µl)wasaddedtowellsofELISAplates. Leukocytes D 80 µl of screen medium containing the various concentration Thetoxicityofprotolichesterinicacidtoleukocyteswasevaluated of protolichesterinic acid (0.5× MIC, MIC, and 2× MIC) was using MTT assay. Briefly, the blood was collected in a sterile dispensedintotheindicatedwell.AmphotericinBservesasthe heparinized tube under sterile environment. Blood was diluted positive control. The nematoEdes’ survival in all treatment was with a double volume of PBS and mixed well. Using a 10 ml checkedeachdayuntil5days(Changetal.,2015).Thenematode syringe with needle attached, 3 ml Ficoll was transferred to a survival rates wereTcalculated by counting the live and dead cleansterile50mlvial.Thebloodwasdilutedontothegradient nematodes based on shape (live nematodes usually appear as withoutmixingusingasterilepipette.Afterthisitwascentrifuged sinusoidalwhilethedeadnematodesappearrodshape). for 10 min at 3000 rpm. Following centrifugation, the test tube C wascarefullyremovedandkeptinsidethelaminar.Usingasterile Statistical Analysis Pasteur pipette, it was removed gradually and the upper layer of the solution was discarded down till within ∼1 cm of theAIn vitro susceptibility tests were repeated at least thrice on interphase.Afterthat,middlelayerwascarefullyremoveddown differentdays.Alltheresultswerepresentedasmeans±standard till∼0.5cmnowleavingalittlematerialthere.Anequalvolume deviations. Geometric means were used to compare the MIC ofPBSwasaddedandcentrifugedfor5minat3000rRpm.After results. Significant differences between the treatments were thatthesupernatantsolutionwasremovedcarefullyandthepellet establishedusingone-wayANOVAtest(P<0.05). wasdilutedusingRPMImedium.Thecellswerecountedusinga hemocytometer.About1×105 cells/mlfoTrculturewasdiluted and MTT experiment was performed as reported previously RESULTS (Aravind et al., 2014). Proliferation Rate was calculated using E formulaT/C×100. Protolichesterinic Acid Recorded Strong Antifungal Effect Foreskin (FS) NRormal Fibroblast Cell Although previous studies conducted by various researchers Proliferation Inhibition Using the MTT on protolichesterinic acid have mainly focused on studying Test the preliminary antimicrobial property, there is a shortage CultivationofFSNormalFibroblastCell of information on the antifungal effects especially against FS cells were cultivated under standard culture conditions in Candida species of this compound in literature. The foremost minimalessentialmediumwithEarle’ssalts.FSnormalfibroblast emphasis of the current investigation was to elucidate the growthwasanalyzedonthebasisoftheabilityoflivingFScells antifungal activity and preliminary mechanism of action of to reduce the yellow color of MTT dye (Sigma–Aldrich) to the protolichesterinic acid in Candida. Our early results clearly purple formazan product. FS cells were plated in 96-well plates indicated that protolichesterinic acid has significant antifungal (3000 cells/well), and protolichesterinic acid (1–200 µg/ml) activity against Candida species, especially to C. tropicalis dissolved in 0.1% DMSO were added to each well, followed and therefore, we further conducted detailed experiments to by incubation for 24 h under standard cultivation conditions. determineitsmechanismofactiononC.tropicalis. Afterward, the plates were centrifuged and the medium was Theresultsofthesensitivitytestofprotolichesterinicacidand replacedwithfreshmedium(150µl)containing0.5mg/mlMTT. thestandarddrug(amphotericinB)againsttheCandidaspecies Three hours later, the MTT formazan product was dissolved in are presented in Table 1. The MIC values of protolichesterinic 150µlDMSOandtheabsorbancewasmeasuredusinganELISA acid ranged from 2 to 16 µg/m1, whereas the MFC values plate reader. The effects of the test compounds were quantified range from 4 to 32 µg/ml. But the antifungal activity of as the percentage of control absorbance of the reduced dye at protolichesterinicacidwasinferiorwhencomparedtotheactivity 595 nm. Cisplatin was used as positive control. Experiments of amphotericin B (Table 1). Out of four Candida species were carried out in triplicate and expressed mean ± standard tested, C. tropicalis recorded best MIC value (2 µg/ml). Zone deviation. of inhibition obtained from agar disk diffusion experiment was FrontiersinPharmacology|www.frontiersin.org 6 May2017|Volume8|Article301 fphar-08-00301 May25,2017 Time:12:14 #7 KumarandMohandas MechanismofProtolichesterinicAcidinCandidatropicalis presentedinTable2andsignificantinhibitionofzonewasalso In the time kill curve experiment, significant killing kinetics observedforC.tropicalis(26mm). wasrecordedby2×MICconcentrations,followedbyMICand The antifungal activity of protolichesterinic acid was also 0.5×MICconcentrationofprotolichesterinicacid.Interestingly analyzed by CFU assay. Protolichesterinic acid treated Candida regrowthwasnotrecordedforprotolichesterinicacid. recorded significant cell survival percentages (Figure 2). Maximum inhibition was recorded at 4 h (Figure2). Here also Mechanism of Action of C.tropicalisrecordedsignificantinhibitionasevidencedbylow Protolichesterinic Acid on C. tropicalis cellsurvivalpercentages. Out of four Candida species tested, C. tropicalis recorded the bestactivityinthepreliminaryexperimentsandthusthisstrain Protolichesterinic Acid Strongly Inhibits wasselectedforfurtherdetailedstudiesincludingmechanismof the Formation of Hyphae in Candida action. Species Protolichesterinic Acid Increases Total Hyphal development is an important determinant for Candida Intracellular ROS Levels in CD. tropicalis species to effectively invade host tissues. So, we further tested the effect of the protolichesterinic acid on the inhibition of Candidatropicalistreatedwithprotolichesterinicacidexhibited hyphaldevelopment.Excitingly,protolichesterinicacidexhibited elevated ROS levels compared with untreated C. tropicalis. In stronger activity against the hyphal development of three the H2O2 (positive control)Etreated C. tropicalis, there was a Candida species tested (Figure 3). This result clearly indicated significant increase in fluorescence. As shown in Figures5A,B, thehighefficiencyoftheprotolichesterinicacidagainstCandida cells exposed to protolichesterinic acid showed a significant T hyphaldevelopment. raiseintheintracellularROSlevelwhencomparedtountreated control C. tropicalis cells. Our results clearly showed that Time-Kill Assay accumulCation of ROS induced by protolichesterinic acid was In the time-kill experiment, the outcome presented regarding increasedinadose-dependentmanner(Figures5A,B). the changes in the CFU/ml of viable Candida colonies In addition, changes in C. tropicalis viability after adding indicated that the protolichesterinic acid exhibited potentAthe ROS scavenger NAC were also studied. NAC (5 mM) did anticandidal property (Figure 4). From the figure, it is not record any adverse result on C. tropicalis. NAC enhances very clear that the net growth rates sharply decreased from the survival of the C. tropicalis from 46.3 to 84.25% for R 2 h onward and the significant reduction in the number protolichesterinicacid(Figure5C). of Candida cells was recorded between 2 and 6 h when compared with untreated controls at T37◦C. Interestingly, Loss of Membrane Integrity by the protolichesterinic acid showed significantly better activity Protolichesterinic Acid as Evidenced by than amphotericin B regarding the growth of the yeast Propidium Iodide Staining cells until 48 h incubation atE37◦C. In the time-kill assay, Propidium iodide has been reported to be a useful fluorescent protolichesterinicacidrecordedbestactivityagainstC.tropicalis dyeforstainingmembraneimpermeablenucleicacid.Propidium (Figure4). R iodideentersthecellswithcompromisedpermeabilityonly,and thenitbindstightlytothedoublestrandedDNA.Afterbindingit producesasignificantredfluorescencewhenexcitedat480nm. TABLE1|MinimumInhibitoryConcentration(MIC)ofprotolichesterinic We examined the propidium iodide uptake by Candida cells in acid. the presence of the protolichesterinic acid at 0.5×, 1×, and 2× Testfungi MIC/MFC(µg/ml) MICbyconfocalmicroscopy(Figure6).Hereprotolichesterinic acid disrupted the membrane of C. tropicalis as evidenced by Protolichesterinicacid AmphotericinB the staining of propidium iodide. As shown in Figure 6, cells treatedwithprotolichesterinicaciddisplayedanincreaseofred C.albicans 8/16 0.5/1 fluorescencelevelwhencomparedtothatofuntreatedcells.This C.tropicalis 2/4 1/1 resultindicatedthatthefungalcellmembranewasdamagedby C.glabrata 16/32 0.5/0.5 thetreatmentofprotolichesterinicacid. C.parapsilosis 16/32 1/1 Efflux of Potassium Ions TABLE2|Diskdiffusiondataofprotolichesterinicacid. Figure 7 clearly showed that the concentration of potassium Testfungi Zoneofinhibition(mm) ion outside of the C. tropicalis cell was significantly enhanced afterprotolichesterinicacidtreatment.Usuallytheconcentration Protolichesterinicacid AmphotericinB of potassium ions within the cell is high and for maintaining C.albicans 21±0.57 26±2.21 the cell homeostasis it must remain as such when there are no C.tropicalis 26±1.52 27±1.77 changesinthesurroundings.Ifanychangeslikecellmembrane C.glabrata 24±1.52 28±1.12 damage happen to the cells, the ions levels in the surroundings C.parapsilosis 22±1 28±0.53 get increased. C. tropicalis treated with protolichesterinic acid FrontiersinPharmacology|www.frontiersin.org 7 May2017|Volume8|Article301 fphar-08-00301 May25,2017 Time:12:14 #8 KumarandMohandas MechanismofProtolichesterinicAcidinCandidatropicalis D E T C A R T FIGURE2|Antifungaleffectofprotolichesterinicacid.Candidaspeciescellsweretreatedwith0.5×MIC,MIC,and2×MICconcentrationofprotolichesterinic acidandincubatedfor8h.After0,2,4,6,and8hthesampleswerecollected,seriallydiluted,spreadinginPDAagarplatesandcellcolonieswerecountedafter 24hincubationat37◦C. E andamphotericinBforR25minrecordedexcellentpotassiumions thatprotolichesterinicacidenhancestheintensityoffluorescent releasewhencomparedtonon-treatedcells.Basedontheresults, C. tropicalis cells in a dose-dependent manner. Therefore, we confirmed that protolichesterinic acid affects C. tropicalis protolichesterinic acid treated C. tropicalis were injured in cells by destructing the cell membrane, which intern cause the permeabilityofmitochondriabythecollapseof(cid:49)(cid:57)m,whichisa permeabilization of cell membrane, and leads to the increased significantstepinC.tropicaliscellsundergoingprogrammedcell concentrationofpotassiumionoutsidethecells. death(apoptosis). Protolichesterinic Acid Induces Collapse Phosphatidylserine Externalization by of Mitochondrial Membrane Potential Protolichesterinic Acid in Candida ((cid:49)(cid:57)m) in C. tropicalis To differentiate between apoptotic and necrotic Candida cell To further conclude whether the mitochondria-mediated death induced by protolichesterinic acid, Annexin V double pathway is associated with protolichesterinic acid induced stainingassayusingtheFITCAnnexinVandPIwasselected.As apoptosis in C. tropicalis, we studied the changes of (cid:49)(cid:57)m revealed in Figure 9, C. tropicalis exposed to protolichesterinic by using the membrane sensitive florescence dye rhodamine acid were considerably stained green fluorescence (positive 123 (Rho 123) that usually aggregates in mitochondria and FITC-Annexin V and negative PI) at the edge of the cell produces an intense green fluorescence. The depolarization after digestion of the cell wall, representing a very clear of mitochondrial membrane was significantly visible after externalization of phosphatidylserine. FITC-Annexin V stained treatmentwithH O (whichwasusedasthepositivecontrol).In C. tropicalis cells were not recorded in experiments conducted 2 2 agreementwiththis,protolichesterinicaciddisruptedthe(cid:49)(cid:57)m without protolichesterinic acid (control set). The exposure asevidencedbyanincreaseintheintensityofgreenfluorescence phosphatidylserinefromtheinnertotheouterleafletofplasma by Rho 123 (Figure 8). Moreover our results clearly showed membraneincellsisanearlymorphologicalmarkerofapoptosis. FrontiersinPharmacology|www.frontiersin.org 8 May2017|Volume8|Article301 fphar-08-00301 May25,2017 Time:12:14 #9 KumarandMohandas MechanismofProtolichesterinicAcidinCandidatropicalis D E T C A R T E R FIGURE3|InhibitionofCandidalhyphaewhentreatedwithprotolichesterinicacid. Protolichesterinicacid, AmpB.Differentlettersinthe superscriptwerestatisticallydifferentaccordingtoDuncan’smultiplerangetest(p<0.05). Therefore, these results suggest that protolichesterinic acid was higher than those in the control cells. The fluorescence induced early apoptosis in C. tropicalis cells, as shown by a intensity of Rhod-2AM was increased in cells treated with significantstaininginAnnexinV-positiveapoptoticcells.From protolichesterinic acid (Figure 10A). Free calcium levels were our results, we established that protolichesterinic acid induces also increased in the mitochondria of cells treated with theproductionandaccumulationofintracellularROS,andwhich protolichesterinic acid (Figure 10B), indicating that calcium finallyinducesapoptoticfeaturesinC.tropicalis. signalingwasinvolvedinfungalcelldeath. Influence on the Cytoplasmic Free Ca2+ Immunomodulatory Properties of Concentration Protolichesterinic Acid Intracellularcalciumlevelswereevaluatedusingtwomembrane- The proliferation of lymphocyte was evaluated to assess the permeable derivatives of the ratio metric calcium indicator, immunomodulatory properties of protolichesterinic acid. The Fura-2AM and Rhod-2AM to detect whether calcium signaling proliferation of lymphocyte was slightly improved in cells was involved in cell death. The concentration of cytoplasmic treated with protolichesterinic acid when compared to that of free Ca2+ in C. tropicalis cells treated protolichesterinic acid the cells treated without test compound (control). Lymphocyte FrontiersinPharmacology|www.frontiersin.org 9 May2017|Volume8|Article301 fphar-08-00301 May25,2017 Time:12:14 #10 KumarandMohandas MechanismofProtolichesterinicAcidinCandidatropicalis D E T C A R T E R FIGURE4|Time-killassayofprotolichesterinicacidagainstCandidaspecies.AnovernightcultureofCandidaspeciesweretreatedwith0.5×MIC,MIC, and2×MICofprotolichesterinicacidandincubatedat37◦C.SampleswerecollectedatindicatedtimepointsandplatedontoPDAplates.Plateswereincubated for48hpriortocountingthecolonyformingunits(CFU). Control, AmpB, 0.5×MIC, MIC, 2×MIC. proliferation index was recorded to be 1.19 for 10 µg/ml of C.tropicalis(Figure13).Protolichesterinicacid(0.5×MIC,MIC, protolichesterinicacidinthepresenceofPHA(Figure11). and 0.5× MIC) enhanced the survival of C. tropicalis infected nematodeswithcomparableefficacyasamphotericinBtreatment Protolichesterinic Acid Recorded no (Figure13). Toxicity toward Normal Cell Line Interestinglyprotolichesterinicacidrecordednotoxicitytoward DISCUSSION FSnormalfibroblastwhentestedupto200µg/ml(Figure12). As an opportunistic, deep-infective fungus, Candida species is The In Vivo Efficacy of Protolichesterinic an important infective microorganism for the patients mainly Acid with immune dysfunction [for e.g., chronic diabetes, malignant In vivo studies with C. elegans establish that protolichesterinic tumors, leukemia, organ transplantations, and acquired acidprovidedsubstantialprotectionforthewormsinfectedwith immunodeficiency syndrome (AIDS)]. Candida species are a FrontiersinPharmacology|www.frontiersin.org 10 May2017|Volume8|Article301
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