An Antifungal Mechanism of Protolichesterinic Acid from the Lichen Usnea albopunctata Lies in the

18 Pages·2017·6.73 MB·English

by  S. N. Kumar

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
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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
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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.
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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
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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
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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
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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
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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.
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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
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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
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