Research Article Effects of Chitosan on Candida albicans: Conditions for Its Antifungal Activity

16 Pages·2015·2.97 MB·English

HindawiPublishingCorporation
BioMedResearchInternational
Volume2013,ArticleID527549,15pages
http://dx.doi.org/10.1155/2013/527549
Research Article
Candida albicans
Effects of Chitosan on : Conditions for
Its Antifungal Activity
AntonioPeña,NormaSilviaSánchez,andMarthaCalahorra
DepartamentodeGene´ticaMolecular,InstitutodeFisiolog´ıaCelular,UniversidadNacionalAuto´nomadeMe´xico,
CircuitoExteriors/n,CiudadUniversitaria,04510Me´xico,DF,Mexico
CorrespondenceshouldbeaddressedtoAntonioPen˜a;[email protected]
Received18April2013;Accepted19May2013
AcademicEditor:IsabelSa´-Correia
Copyright©2013AntonioPen˜aetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,
whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Theeffectsoflowmolecularweight(96.5KDa)chitosanonthepathogenicyeastCandidaalbicanswerestudied.Lowconcentrations
ofchitosan,around2.5to10𝜇g⋅mL−1produced(a)aneffluxofK+andstimulationofextracellularacidification,(b)aninhibition
+ 2+
ofRb uptake,(c)anincreasedtransmembranepotentialdifferenceofthecells,and(d)anincreaseduptakeofCa .Itisproposed
thattheseeffectsareduetoadecreaseofthenegativesurfacechargeofthecellsresultingfromastrongbindingofthepolymer
+
tothecells.Athigherconcentrations,besidestheeffluxofK ,itproduced(a)alargeeffluxofphosphatesandmaterialabsorbing
2+
at260nm,(b)adecreaseduptakeofCa ,(c)aninhibitionoffermentationandrespiration,and(d)theinhibitionofgrowth.The
effectsdependonthemediumusedandtheamountofcells,butinYPDhighconcentrationscloseto1mg⋅mL−1arerequiredto
producethedisruptionofthecellmembrane,theeffluxofprotein,andthegrowthinhibition.Besidesthefindingsatlowchitosan
concentrations,thisworkprovidesaninsightoftheconditionsrequiredforchitosantoactasafungistaticorantifungalandproposes
amethodforthepermeabilizationofyeastcells.
1.Introduction with the negatively charged membrane and alter bacterial
surfacemorphology,whicheitherincreasesmembraneper-
Chitosan is an N-deacetylated derivative of chitin, mostly meability, causing leakage of intracellular substances, or
extractedfromexoskeletonsofcrustacea;itisbiodegradable, decreases membrane functions, preventing nutrient trans-
biocompatible,andwithlowtoxicity,whichmakesituseful port[8].Chitosaneffectshavenotbeenfullystudied,andits
andconvenientinmanydifferentpracticalapplications,such directutilizationasanantimicrobialagentislimitedbyitslow
as those in water and waste treatment, food and beverages, solubilityatneutralpH[2].
cosmetics and toiletries, industrial processes, textile, paper
Severalothercationiccompoundsareknowntoproduce
and agriculture industry, biopharmaceutics, biotechnology, + +
aneffluxofK inyeast[9].Inplants,theeffluxofions(K ,
and medical uses, mostly based on its antimicrobial and + 2+ − +
H , Ca , and Cl ) has also been observed [10]. Also a K
antifungalpropertiesamongothers[1,2].
leakagehasbeenidentifiedinGlycinemax[11]andinfungi,
Antimicrobialpropertiesofcationicpolymershavebeen
likeSphaeropsissapineaandTrichodermaharzianum.These
knownforalongtime;thefirststudiesrevealedtheperme-
fluxes may alter the ionic balance needed to maintain the
abilization of yeast cells as their main effects [3]. Later on,
principalfunctionsoftheorganisms[12].
cytochrome c and diethylaminoethyl-dextran were used to
permeabilize the yeast cell membrane [4, 5]. Some studies It has been suggested that at a pH below 6.3, chitosan
have demonstrated the disruption of bacterial membranes, molecules become positively charged by protonation of the
both by chitosan itself and by some derivatives [6]. These amino groups that interact with the negative charges of the
effects have also been documented using three Candida cellsurface.Itwasalsofoundthatblockingtheaminogroups
species [7]. In bacteria, polycationic chitosan can interact preventstheantimycoticpropertiesofchitosan[13].
2 BioMedResearchInternational
Considering that Candida albicans is an opportunistic 2.3. Efflux of Molecules at Higher Chitosan Concentrations.
pathogenandinfectionswiththisfungusareaseriousprob- Yeastcellmembranepermeabilizationbychitosanwasexam-
lem,particularlyinimmune-compromisedpatients,experi- inedbymeasuringtheeffluxofmaterialabsorbingat260nm,
+
mentsreportedwereperformedwiththisyeast,butalsowith K , and phosphate. Effluxes were measured by incubating
itscloselyrelatedspecies,Candidadubliniensis[14],aswellas 50mg (12.5mg⋅mL−1) of starved cells in 10mM MES-TEA,
withSaccharomycescerevisiae. pH6.0,and20mMglucose,for10minat30∘Cinafinalvol-
Thispaperdescribestheeffectsofchitosan:alargeefflux umeof4.0mL.Afterincubationwithvariableconcentrations
+
of K and other small molecules, a remarkable increase of of chitosan, the cells were centrifuged, and the supernatant
Ca2+uptake,andtheinhibitionofrespiration,fermentation, was used to measure K+ with a flame photometer, material
andviability.However,incomplexmedium,highconcentra- absorbingat260nm(nucleotides)withaspectrophotometer,
tionsofchitosanarerequiredtoinhibitgrowth.Aproposalof and inorganic phosphate by the method of Fiske and Sub-
themechanismbywhichchitosanmayactasanantifungal, barow [16]. Total K+ of the cells was extracted by boiling a
as well as its possible use to study enzymes and metabolic similarsuspensionofcellsinawaterbathfor20min.Total
functions in situ, and compartmentalization of solutes are inorganic phosphate or material absorbing at 260nm was
included. obtained by a 20min extraction in ice of a similar amount
ofcellswith30%trichloroaceticacid.
2.MaterialsandMethods
2.4. Glucose-6-phosphate Dehydrogenase Activity. To exam-
2.1. Strains and Growth Conditions. Most studies were per- ine the yeast cell membrane permeability to proteins pro-
formed with C. albicans strain ATCC 10231, reported by duced by chitosan, as well as the integrity of the internal
the American Type Culture Collection as isolated from a enzymes, glucose-6-phosphate dehydrogenase activity was
patient with bronchomycosis. The S. cerevisiae strain was measuredbothinthesupernatantofthesamplesincubated
a commercial diploid strain isolated from a single colony with variable concentrations of chitosan, or with a simi-
(LaAzteca,SA,Me´xico).Someexperimentswereperformed lar incubation mixture with whole cells to which variable
with C. dubliniensisstrain CD 36. Besides, considering that concentrations of chitosan were added. The measurements
our results with the C. albicans strain 10231 might be due weredonein10mMMES-TEA,pH6.0,600𝜇Mglucose-6-
toitsparticularproperties,someexperimentswererepeated phosphate, 500𝜇M NADP+, and 12.5mg⋅mL−1 of cells in a
withthestrainATCC90028,whereindicated.Thisstrainis finalvolumeof2.0mL.Absorbancechangesweremeasured
reportedbytheATCCasisolatedfromhumanblood. at 340nm in an Olis converted DW 2a UV/VIS AMINCO
Cells were maintained in solid YPD medium (1% yeast spectrophotometer.
extract,2%bactopeptone,2%glucose,and2%agar),renew-
ingtheculturesonceamonth.Aloopholeofcellsfromthe 2.5. Plasma Membrane Potential Difference Estimation. The
solid medium was placed into 500mL of medium without plasma membrane potential difference was estimated as
∘
agar.Aftergrowthinanorbitalshakerat29 Cfor24hours, described before [17] following the fluorescence changes of
the cells were collected by centrifugation, washed twice DiSC3(3). Starved cells (12.5mg⋅mL−1) were added to the
with water, and suspended in water. Then they were either
spectrofluorometercellcontaining10mMMES-TEAbuffer,
used during the same day or fasted by suspending them in 20𝜇M CCCP, 10𝜇M BaCl2, and 200nM cyanine, in a final
250mL of water and aerating for 48h in the orbital shaker
volume of 2.0mL. Where indicated, either 10mM glucose
∘
at 250rpm, at 29 C. S. cerevisiae was starved 24h, but C.
or 10mM KCl, or variable amounts of chitosan was added.
albicans required 48h to reach almost the same starvation
Thefluorescencechangesat540–590nm,forexcitationand
state [15]. After collection and washing by centrifugation,
emission,respectively,werefollowedagainsttimeinanOlis
the cells were suspended in water to a ratio of 0.5g⋅mL−1 convertedSLMAMINCOspectrofluorometer,equippedwith
(wetweight,workingsuspension)andmaintainediniceuntil athermostatedcellholderat30∘Cundercontinuousstirring.
theiruseduringthesameday.Fromtheinitialexperiments,
it was found that the effects of different concentrations of
2.6. Zeta Potential. This parameter was obtained from the
chitosan varied depending on the amount of cells, in most
displacementvelocityofstarvedcellsunderanelectricfield
experiments,12.5mg⋅mL−1,wetweightwasused.
andmeasureddirectlyunderthemicroscopeinaZetaMeter
ZM-75 cell electrophotemeter, which allows calculation of
2.2.PotassiumEffluxandAcidification. Potassiumeffluxand the electrophoretic mobility. From this last parameter, the
acidification of the medium were followed by means of pH zeta potential can be obtained by the following Helmholtz-
andK+ selectiveelectrodesconnectedtoapHmeterandan Smoluchowskiequation:
𝑉
adequate acquisition system. Usually, 125mg wet weight of Zeta potential=EM 4𝜋 𝑡, (1)
starvedcells(12.5mg⋅mL−1)asincubatedin2mMMES-TEA, 𝐷𝑡
pH 6.0, and 20mM glucose, in a final volume of 10.0mL.
where
While following the tracings and at the indicated times,
(i)EMistheelectrophoreticmobility;
variableamountsofchitosanwereaddedasindicatedinthe
figures. Experiments were carried out in a chamber with (ii)𝑉𝑡istheviscosityoftheliquidmeasuredinpoises,at
∘
continuousmagneticstirring,maintainedat30 C. thetemperatureofthemeasurement;
BioMedResearchInternational 3
(iii)𝐷𝑡isthedielectricconstantofthesuspendingfluidat 10h,24h,and48handcentrifuged.Supernatantswereused
thetemperatureofthemeasurement; tomeasurechitinaseactivityusingChitinaseassaykitfrom
(iv)4𝜋=12.57. Sigma-Aldrich(Cat.numberCS0980-1KT)asrecommended
bythemanufacturers.
The medium (13mL final volume) contained 10mM
MES-TEAbuffer,2mMglucose,12.5mgofcells(wetweight), 2.10. Oxygen Consumption. It was measured with a Clark
and different amounts of chitosan. The electric potential electrode connected to a polarization and measurement
differenceappliedwas100V. device(YellowSprings),collectingthedatawithacomputer.
The incubation medium was 10mM MES-TEA buffer in a
86 + 45 2+ 86 + 45 2+
2.7. Rb and Ca Uptake. Uptakeof Rb or Ca was final volume of 5.0mL, with 20mM glucose as substrate.
measured by adding 12.5mg of starved cells (25mg⋅mL−1) Tracings were started with 25mg (wet weight) (5mg⋅mL−1)
to 10mM MES-TEA, pH 6.0, and 20mM glucose, 0.5mL ofstarved cells to followthe oxygen consumptionrate. The
final volume, plus the indicated amounts of chitosan in a experimentswereconductedinaclosedchambermaintained
∘ 86 ∘
waterbathat30 C.After2min,0.1to0.6mMof RbClor at30 Cundercontinuousstirring.Inonesetofexperiments,
50𝜇M45CaCl2wasadded.Aftertwomoreminutes,aliquots a mixture of 20mM potassium phosphate, 1.4mM NAD+,
of 0.1mL were taken. They were filtered through a 0.25𝜇m 1.8mM ATP, and 5mM MgCl2 was also added. Variable
nitrocellulosefilterandwashedtwicewith0.5mLof10mM amountsofchitosanwereaddedasindicatedinthefigures.
CaCl2. The filters were then dried and placed in vials with
scintillation liquid and counted in a Beckman scintillation 2.11. Fermentation. Ethanol production was measured by
counter. incubating 12.5mg of starved cells, wet weight, in a final
volume of 1.0mL containing 10mM MES-TEA, pH 6.0,
2.8.EffectsonCellViability. Toexplorethisparameter,in96- 20mMglucose,andvariableamountsofchitosanasindicated
well plastic plates and using a multichannel pipette, 5𝜇L of in Figure11. Incubation was performed in test tubes in a
∘
thenonstarvedworkingsuspension(2.5mgofcells)andten- water bath at 30 C. After 10min, the tubes were cooled in
foldsuccessivedilutionswasaddedto200𝜇L(12.5mg⋅mL−1) anicewatermixtureandcentrifuged.Ethanolmeasurements
of 10mM MES-TEA, pH 6.0 plus the following additions: were performed in aliquots of the supernatants with an
(1) 20mM glucose; (2) 20mM glucose and 10mM KCl; (3) EthanolKit(BoehringerMannheim/R-Biopharm.Enzymatic
110mMglucose;(4)20mMglucoseand2%bactopeptone;(5) BioAnalysis)asrecommendedbythemanufacturers.
20mMglucosewith1%yeastextract;or(6)200𝜇LofYPD.In
eachcase,theindicatedamountofchitosanwasadded,and 2.12. Binding of Chitosan to the Cells. Cells (12.5mg, wet
the cells were incubated for 10min at 30∘C, under shaking. weight) were incubated for 10min at 30∘C in 10mM MES-
After incubation, the corresponding yeast suspensions at TEA buffer, pH 6.0, or 2% bactopeptone; or in 1% yeast
the same time were spotted in solid YPD medium, using a extract; or in YPD, plus the indicated additions of chitosan
solidpinmultiplatereplicator.Growthwasobservedafter48 (final volume 1mL), and then centrifuged in a bench cen-
hours. trifuge (1625g) to measure the chitosan remaining in the
Also,colonyformingunits(CFUs)weredeterminedafter supernatant. The method consisted in measuring the fluo-
incubation of C. albicans, with chitosan at variable conc- rescence increase (350–440nm, excitation-emission wave-
entrations under different conditions. Nonstarved cells lengths, resp.) of a 10𝜇M solution of calcofluor white 28
(12.5mg⋅mL−1)wereincubatedfor10minat30∘Cin10mM (Sigma Cat. number F-3397) in 2.0mL of a 10mM bicine-
MES-TEAbuffer,pH6.0plustheadditionsindicatedinthe TEAbuffer,pH8.0.Tothatpurpose,a1mMstocksolutionof
previous experiment, or in YPD, in a final volume of 1mL. thedyewaspreparedindimethylsulfoxide.Itistobenoted
Then,5𝜇Lofa10,000-folddilution(approximately200cells) thataftertheadditionofthedyetothemeasuringmixture,
wasspreadinsolidYPDmedium,andcolonieswerecounted averylargefluorescencewasobserved,whichdecayedwith
after48h. time;so,itwasletunderthelightuntilitattainedalowand
GrowthcurveswereperformedinaBioscreenCinstru- steadyvalueofthefluorescence.Then,insuccessive,alternate
ment. For that purpose, after growing the cells in YPD for additions,concentrationsof 1𝜇g⋅mL−1ofchitosanor20𝜇Lof
24hasusual,theyweresuspendedatafinalopticaldensity thesupernatantobtainedafterincubationandcentrifugation
of 0.03 units at 600nm in 300𝜇L of YPD medium, with of the cells were added to the spectrofluorometer cell to
variableamountsofchitosan(5,10,25,50,100,200,300,and recordthefluorescenceincreases.Fromtherelativevaluesof
400𝜇g, equivalent to 3.33 times those values, as 𝜇g⋅mL−1). thefluorescenceincreasesresultingfromstandardadditions
Microplateswerecontinuouslyshaken,andtheopticalden- and those of the supernatant, the remaining concentration
sityofeachwellwasmeasuredat600nmevery20minfor48 of chitosan in the supernatant after incubation could be
∘
hoursat30 C. calculated.
Chitosan (low molecular weight) was obtained from
2.9. Chitinase Assay. Cells were grown as for the growth Sigma-Aldrich (Cat. number 448869) and dissolved at
curvesmentionedbefore,withvariableamountsofchitosan 10mg⋅mL−1 of water to which lactic acid was added to
(25, 50, 100, 200, 400, 600, and 800𝜇g, equivalent to 3.33 reach a pH around 4.5. From this stock solution, other
times those values, as 𝜇g⋅mL−1). Samples were taken after dilutionswerepreparedfortheexperiments.Molecularmass
4 BioMedResearchInternational
2.5 50mg
2 10𝜇g mL−1 1.9
Chitosan
1.5 5𝜇g mL−1 1.4 Chitosan
1 5𝜇g mL−1 125mg
Chitosan
0.9
mV) 0.50 2.5𝜇g mL−1 20𝜇g mL−1 (mV) 0.4 250mg
(
−0.5
0 −0.1
−1 Cells 125 mg Control 1𝜇Eq H+ 2.5𝜇g mL−1 −0.6 Cells
−1.5 10𝜇M K+ 10𝜇g mL−1 5𝜇g mL−1 10𝜇M K+
−2 −1.1
0 200 400 0 200 400 600 0 200 400
Time (s) Time (s) Time (s)
(a) (b) (c)
+
Figure1:EffluxofK andeffectsonacidificationofC.albicansstrainATCC10231producedbyvariableconcentrationsofchitosan.In(a)
and(b),theincubationmediumwas2mMMES-TEAbuffer,pH6.0,and20mMglucose.Atthestartofthetracings,10𝜇MKClwasaddedas
+
aconcentrationreference,followedbystarvedcells(125mg,wetweight).AftertheuptakeofK wascomplete,theindicatedconcentrations
ofchitosanwereadded.In(b),thetracingsoftheacidificationofthemediumareshown.In(c),differentamountsofcellswereaddedas
indicatedwithaconstantchitosanconcentration.
was determined by mass spectrometry and found to be In view of the limited sensitivity range of the elec-
96.5kDa. trode, experiments were performed incubating the cells for
All experiments were performed at least three times, 10min in test tubes with a wider concentration range of
although in some cases, only representative data are pre- chitosan,andaftercentrifugation,analyzingthesupernatant
+
sented. for nucleotides (material absorbing at 260nm), K , and
phosphate.ResultsareshowninFigure2;theconcentrations
ofthethreeparametersinthesupernatantshowedanincrease
3.Results thatdependedonthechitosanconcentration.
Sincetheseeffectsindicatedthepermeabilizationofthe
3.1. Permeability Changes. Chitosan mostly being a glu- plasmamembrane,itwasalsoconsideredinterestingadding
cosamine polymer, a strongly cationic compound, our first 0.8Msorbitolwiththeideaofexploringtheintegrityofthe
considerationwasthatitmighthaveeffectssimilartothatof internalcompartments.Theexperimentsshowedthatwhen
othercationicpolymers,likethosereportedbyYphantisetal. thecellswereincubatedwithsorbitoltheeffluxofnucleotides
[3],ordiethylaminoethyl-dextran,increasingthepermeabil- andpotassiumwassimilarinitsabsenceorpresence(Figures
ityoftheplasmamembrane[4,9,11].Inpreliminaryexperi- 2(a)and2(b)),butitsignificantlypreventedphosphateefflux
ments,wefoundthatlowconcentrationsofchitosan,around (Figure2(c)).
2.0𝜇g⋅mL−1 in 10mL of incubation medium produced the
+
effluxofsignificantamountsofK fromacommercialstrain 3.2. Glucose-6-phosphate Dehydrogenase. After incubating
of S. cerevisiae (not shown). So, we decided to investigate cells with low or moderate concentrations of chitosan, no
+
whetherinC.albicansitalsoproducedtheeffluxofK and absorbancepeakat280nminthesupernatantwasdetected;
changes of pH of the medium with selective electrodes. In so,wedecidedtomeasuretheactivityofglucose-6-phosphate
theseexperiments,theconcentrationof12.5mg⋅mL−1ofcells dehydrogenase directly in whole cells in the presence of
wasmaintained.Atthestartingofthetracings,asareference, variable amounts of chitosan. This was performed with the
we added 10𝜇M KCl; the addition of the cells produced ideathateventhoughproteinsatintermediateconcentrations
+
another small increase of the potassium ion concentration ofchitosandidnotleakout,smallmoleculessuchasNADP
ofthemedium,followedbyitsuptaketotheinitiallevelsor and glucose-6-phosphate might enter the cells and reach
lower.Then,theadditionofchitosan(2.5,5.0,or10𝜇g⋅mL−1) the enzyme inside, allowing to find out about its integrity.
producedacleareffluxofthecation(Figure1(a)),aswellasan Incubating whole cells with chitosan, activity was detected
increaseoftheacidificationrateofthemedium(Figure1(b)). whentheamountofchitosanreached50𝜇g⋅mL−1intheassay
Large chitosan concentrations produced an inhibition and mixture.Inthesupernatantsafterincubationwithchitosan,
reversaloftheacidificationofthemedium. the activity of the enzyme could only be detected in those
It was found that the effect of chitosan depended on from cells previously incubated with 250𝜇g⋅mL−1 chitosan
the amount of cells used. Using a lower amount of cells, or higher, indicating that in fact (a) the disruption caused
a proportionally larger efflux was observed (Figure1(c)), by chitosan on the cell membrane did not result in leakage
indicatingastrongbindingofthepolymer. ofenzymesoutofthecellbutallowedtheentranceofsmall
BioMedResearchInternational 5
100
100 120
G+Sorb G
100 80
80
G
G G + Sorb
80
%) 60 %) %) 60
Total ( 40 Total ( 60 Total ( 40
G + Sorb
40
20 20
20
0 0 0
0 25 50 75 100 125 0 25 50 75 100 125 0 25 50 75 100 125
Chitosan (𝜇g mL−1) Chitosan (𝜇g mL−1) Chitosan (𝜇g mL−1)
(a) (b) (c)
+
Figure2:Effluxofnucleotides(materialabsorbingat260nm),K ,andphosphateproducedbychitosanatvariableconcentrationsonthe
∘
strainC.albicansATCC10231.Cells(50mg,wetweight)wereincubatedfor10minat30Cin10mMMES-TEA,pH6.0,and20mMglucose,
with(G+Sorb)orwithout(G)0.8Msorbitol,inafinalvolumeof4.0mLandtheindicatedconcentrationsofchitosan.Afterincubation,
thecellswerecentrifuged,andthedifferentmaterialsweremeasuredinthesupernatantasdescribedunderexperimentalprocedures.(a)
Materialabsorbingat260nm;(b)potassium,and(c)phosphate.
molecules(substrates),and(b)muchhigherconcentrations K+20 mM
ofchitosanwererequiredtoallowtheeffluxofproteins(not
shown). This experiment also showed that even though the 25𝜇g mL−1
plasma membrane had been disrupted at intermediate or 1
moderatechitosanconcentrations,enzymesremaininginside 50𝜇g mL−1
couldbestudiedinsitu. AU)0.8 10𝜇g mL−1
m
n 0
+ 0
3.3. The Plasma Membrane Potential. K efflux appearedto 9
be accompanied by the measured anions (nucleotides and 05–0.6 Chitosan or K+20 mM
4
5
inorganicphosphate);however,thepossibilitythatchitosan e (
produced an electrogenic efflux of the cation was explored. nc0.4
e
c
For this purpose, the effects of the polymer were studied es
by estimating the membrane potential by the fluorescence uor0.2 Cyanine
Fl
changes of DiSC3(3). This method can be used in this Cells
yeast,asshownbythefluorescenceincreaseobservedwhen
0
adding glucose to starved cells, as well as by the decrease
+ 0 100 200 300 400
produced by the addition of K on this parameter [17].
Time (s)
It was found (Figure3) that the addition of chitosan in
factproducedanincreaseofthefluorescence,inagreement Figure 3: Effects of chitosan on the estimated electric plasma
+
with what was suspected, that is, that the efflux of K is membrane potential difference on the strain C. albicans ATCC
electrogenicandhyperpolarizestheplasmamembrane.The 10231.Thecells(25mg)wereaddedtothespectrofluorometercell
resultsoftheseexperimentsshowedafluorescenceincrease containing 10mM MES-TEA buffer, 20𝜇M CCCP, 10𝜇M BaCl2,
atallconcentrationsused,althoughatthehighestones,the and20mMglucose,inafinalvolumeof2.0mL.After30s,200nM
tracingsbecamenoisybecauseoftheflocculationofcells. of the cyanine DiSC3(3) was added. Where indicated, 20mM
KClorvariableamountsofchitosanwasadded.Thefluorescence
changes,540–590nm,forexcitationandemission,respectively,were
3.4. Z Potential. Considering that chitosan might bind to followed.AU:arbitraryunits.
the cell surface due to its positive charge and modify it, we
measured the zeta potential as an indicator of the electric
86 + 45 2+
surfacecharge.Theeffectsofchitosanonthezetapotential, 3.5. Uptake of Rb and Ca . The possible mechanisms
+
as calculated from their mobility in the electrophotemeter, consideredfortheeffluxofK producedbychitosanwerethat
revealedthatincreasingconcentrationsofchitosanwithinthe (a)theneutralizationofthesurfacechargemaydecreasethe
+
rangeusedinotherexperimentsproducedacleardecreaseof actualconcentrationofK inthevicinityofitstransporter(s)
thisparameter(Figure4). resultinginitseffluxthroughacarriersimilarordifferentto
6 BioMedResearchInternational
0 3.5
3
−20
2.5
−40
1) 2
−
g
volts) −60 ·moles 1.5
Milli 𝜇(
( 1
−80
0.5
−100 0
0 40 80 120 160 200 240 280 320
Chitosan (𝜇g mL−1)
−120
45 2+
0 2 4 6 8 10 Figure5:Effectsofchitosanon Ca uptakebyCandidaalbicans
Chitosan (𝜇g mL−1) strainATCC10231.Starvedcells(12.5mg,wetweight)wereaddedto
10mMMES-TEA,pH6.0,and20mMglucoseandconcentrationsof
Figure 4: Effects of chitosan on the 𝜁 potential of C. albicans chitosanasindicated,in0.5mLfinalvolume,inawaterbathat30∘C.
ATCC 10231 cells. The mobility of the cells was measured against After2min,50𝜇M45CaCl2wasadded,andaftertwomoreminutes
time in a Zeta Meter ZM-75 cell electrophotemeter, to calculate of incubation, aliquots of 0.1mL were filtered through a 0.25m
the electrophoretic mobility. From this last parameter, the zeta nitrocellulosefilterandwashedtwicewith0.5mLof10mMCaCl2.
potentialwasobtainedbytheHelmholtz-Smoluchowskiequation. The filters were then dried and placed in vials with scintillation
The medium (13mL final volume) contained 10mM MES-TEA liquidandcountedinaBeckmanscintillationcounter.
buffer,2mMglucose,12.5mgofcells(wetweight),andtheindicated
amountsofchitosan.Thepotentialdifferenceappliedwas100V.
Table 1: Kinetic constants of 86Rb+ uptake; effect of chitosan on
thestrainC.albicansATCC10231.Uptakeof86Rb+ wasmeasured
incubating12.5mgofstarvedcellsin10mMMES-TEA,pH6.0,and
20mMglucose,0.5mLfinalvolume,intheabsenceorpresenceof
thatusedfor+theuptake,and(b)itmaybeduetothestimula- chitosan, 20𝜇g⋅mL−1 plus the indicated amounts of chitosan in a
tionoftheH /cationantiporteralreadydescribedbyKrauke waterbathat30∘C.After2min,0.1to0.6mMof86RbClwasadded.
and Sychrova [18]. The second possibility was tentatively
Aftertwomoreminutes,aliquotsof0.1mLweretakenwhichwere
discardedbecauselowtomoderatechitosanconcentrations filteredthrougha0.25𝜇mnitrocellulosefilterandwashedtwicewith
ofchitosanproducedanincreaseoftheacidificationrateof 0.5mLof10mMCaCl .Thefilterswerethendriedandplacedin
2
the medium and only high concentrations of the polymer; vialswithscintillationliquidandcountedinaBeckmanscintillation
alreadyproducingthecelldisruptionresultedinadecrease counter(𝑛=7).
of the acidification rate of the medium. The first possibility
could be tested by analyzing the effects of the polymer on Km,mM 𝑉max,𝜇Eq⋅g−1in2min
86 + Control 0.41±0.08 8.7±1.24
the uptake of Rb . If chitosan produces the reversible
displacementofthemonovalentcationfromthesurface,its Chitosan 2.30±0.05 8.9±3.8
effect should be reflected in an increase of the Km for its
transport,butnochangeinits𝑉maxResultsoftheexperiments
showed that low amounts of chitosan (20𝜇g⋅mL−1/12.5mg increaseoftheuptakeofthedivalentcation;however,higher
+ concentrations, presumably by producing the disruption of
ofcells)producedaclearinhibitionofRb uptake(Table1).
Variabilityofdatafor𝑉max waslarge,notsoforKm,which the plasma membrane resulted in a large decrease of the
uptake (Figure5). However, the decrease of the divalent
wasfoundtoincreasebytheadditionofthisconcentrationof
chitosanwithoutanysignificantchangeof 𝑉max.Asexpected, cationuptakedidnotreachzero.
this polymer concentrationproduced an inhibitionclose to
thecompetitivetype,thatis,anincreaseofapparentKmand 3.6. Effects of Chitosan on Cell Viability and Growth. The
nochangeof 𝑉max. experimentstoanalyzetheeffectsofchitosanoncellviability
In the study of amiodarone, another agent that also showed that spotting dilutions (with a solid pin multiplate
+
produces K efflux from yeast cells [19–21], it was found replicator) of the cell suspension after incubationwith very
that parallel to the increased plasma membrane potential small amounts of chitosan in simple buffer (between 2.5
producedbythedrug,aseveralfoldincreaseof 45Ca2+uptake and5.0𝜇g⋅mL−1 addedtotheincubationmixture)causeda
takes place. In a similar experiment with C. albicans, we clear decrease of growth (Figure6(a)). However, the effect
found that, as expected from the hyperpolarization of the clearly depended on the amount of cells. At the higher
cells,lowconcentrationsofchitosanproducedaremarkable cell concentration, the cells grew at all concentrations of
BioMedResearchInternational 7
Buffer-G Buffer G+K YPD
100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
0 0
0
2.5
2.5
250
5
5
−1𝜇·(gmL) 1200 −1𝜇·(gmL) 1200 −1𝜇·(gmL) 357050
25
25
1000
50
50
100 1250
100
(a) (b) (c)
Buffer-G
Buffer-G 110 mM Buffer-G-peptone Yeast extract
100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
0 0 0
25 25 25
50 50 50
−1𝜇·(gmL) 110205 −1𝜇·(gmL) 110205 −1𝜇·gmL) 110205
(
250 250 250
500 500 500
1000
1000 1000
(d) (e) (f)
Figure6:ViabilityofC.albicans,strainATCC10231afterincubationwithvariableconcentrationsofchitosanunderdifferentconditions.
Nonstarved cells (2.5mg) and successive tenfold dilutions in sterile water were incubated in 96-well plastic plates as described under
∘
experimentalproceduresfor10minat30Cin10mMMES-TEAbuffer,pH6.0plus(a)20mMglucose;(b)20mMglucoseand10mMKCl;
(c)YPDmediumonly;(d)110mMglucose;(e)20mMglucoseand2%bactopeptone;(f)20mMglucoseand1%yeastextract,andeachone
withvariableamountsofchitosanin𝜇g⋅mL−1asindicated,inafinalvolumeof0.2mL.Then,theywerespottedusingasolidpinmultiplate
replicatorinsolidYPDmedium.Imageswerecapturedafter48hofgrowth.
chitosan, up to 100𝜇g⋅mL−1. This was also observed at the MESTEA10mM,pH6.0with110mMglucose,orwith2%
firsttenfolddilutionofthecells.However,atthehighestcell bactopeptone and 20mM glucose, or with 1% yeast extract
dilutions, growth was inhibited even after incubation with and 20mM glucose. Increasing the glucose concentration
2.5𝜇g⋅mL−1.Experimentswithotherpolycationicmolecules to 110mM (the concentration in YPD) did not result in
[3]havedemonstratedthattheireffectscanbeinhibitedby any significant reversal of the chitosan effect, but on the
KCl;inthepresenceof10mMKClintheincubationmedium, contrary,growthinhibitionwasmoreevident(Figure6(d)).
averysmallprotectionwasseen(Figure6(b)).However,in Bactopeptoneproducedsomereversalofthechitosaneffect
thosecellsincubatedinYPD,chitosanshowedaverysmall (Figure6(e)).Theincubationinthepresenceofyeastextract
effect;somegrowthwasobservedevenatthehighestdilution resultedinalmostthesamegrowthaswithYPD(Figure6(f)).
ofthecells(104),1.25mg⋅mL−1 ofchitosan(Figure6(c)).To TheresultsoftheCFUexperimentsareshowninFigure7:
elucidate which component of the YPD medium conferred (a) when the cells were incubated with chitosan in buffer;
“protection”againstchitosan,thecellswerealsoincubatedin lowconcentrationsalreadydecreasedthenumberofcolonies;
8 BioMedResearchInternational
180 10
160
140 666𝜇g·mL−1
120 333𝜇g·mL−1
ol )
ntr100 m 1333𝜇g·mL−1
o n
CFUs (% c 6800 log OD600 1 167𝜇g·mL−1 1000𝜇g·mL−1
40
83𝜇g·mL−1
20
0 Control w/o cells
0
0 25 50 75 100 125 150 175 200 225 250 0.1
Chitosan (𝜇g·mL−1) 0 2 4 6 8 1012141618202224262830323436
Time (h)
B+G20 B+G20+K
B+G110 B+G110+K Figure 8: Growth curves of C. albicans strain ATCC 10231 with
B+G20+bact B+G20+YE
different concentrations of chitosan. The cells (0.03 final optical
YPD YD density units at 600nm) were placed into 300𝜇L of YPD in the
presence of indicated chitosan concentrations and growth was
followedfor40h.
Figure7:ColonyformingunitsofC.albicansstrainATCC10231
after incubation with variable concentrations of chitosan under
different conditions. Nonstarved cells (10mg wet weight) were
∘
incubatedfor10minat30CinYPD,or10mMMES-TEAbuffer, issecretedintothegrowthmediumwhencellsaregrownina
pH6.0plustheindicatedadditionsinafinalvolumeof1mLwiththe nutrient-richmedium,YPD[23].Withthetwoexochitinase
indicatedchitosanamounts.Then,5𝜇Lofa10,000-folddilutionwas
substrates of the assay kit, we observed first an increase of
spreadinplatesofsolidYPDmedium,andcolonieswerecounted
the activity with time in the absence of chitosan; however,
after 48h. Tracings of incubations as indicated in the chart with
anotherimportantincreaseoftheactivityathigherchitosan
buffer (B) plus: I: 20mM glucose (G 20); ◊: 20mM glucose and
10mMKCl(K);󳵳:110mMglucose(G110);◼:110mMglucoseand concentrations was observed (Figure9). Results with the
10mM KCl; ⧫: 20mM glucose and 2% bactopeptone (Bact); ×: exochitinasesubstratespresentedprovideinformationabout
20mMglucoseand1%yeastextract(YE);and◻:YPD;∙:YD. exosplitting activity on the polymer chains, as it happens
on higher analogs of chitin [24, 25]. Even though chitosan
is deacetylated, chitinases can degrade it [22], and chitosan
degradation by chitinases could explain why cells resumed
(b) in the presence of 10mM KCl, only a tendency was growthafteralongperiodoftimeathighconcentrations.
observed,whichwasinsignificantbecauseofthelargestan-
dard deviations, in the sense that for a similar decrease of
3.8.EffectsonOxygenConsumptionandFermentation. Since
the number of colonies, slightly higher concentrations of +
theK effluxproducedbychitosanappearstodepletethecells
chitosan were required; (c) when the cells were incubated +
ofK andothersmallmolecules,itwasdecidedtofindout
in buffer with glucose and yeast extract or with YPD, they
whateffectthismighthaveonthemainmetabolicpathways
resistedmuchhigherconcentrationsofthepolymer;(d)with
bactopeptone,thecellsresistedupto50𝜇g⋅mL−1;thatis,10- othfatthevecreyllslo,twhactoins,creenstpriartaiotinosnoafncdhfietromsaenn(ta2t𝜇iogn⋅m.ILtw−1a)ssftoimunud-
fold higher concentrations of chitosan than those effective latedrespiration,butalready10𝜇g⋅mL−1completelyinhibited
onlywithbuffer.
it(Figure10(a)).Itmustbenotedthatintheseexperiments,
Growth curves in YPD (Figure8) showed that chitosan
only inhibited growth starting at 666𝜇g⋅mL−1 or more. inordertobeabletofollowtheoxygenconsumptionrate,a
lower concentration of cells than that in other experiments
Besides, even at higher concentrations, after several hours, hadtobeused(5mg⋅mL−1).
growthinhibitionrecovered.
Considering that inhibition of respiration might be due
to the efflux of small molecules (Figure10(a)); in another
3.7.ChitinaseAssay. GrowthcurvesinYPDshowedthatafter experiment,theeffectofthepolymeratdifferentconcentra-
16h, at high concentrations of chitosan, the cells started to tions was tested but adding also to the incubation medium
+
grow; so, we considered that the cells might be degrading amixtureofNAD ,potassiumphosphate,ATP,andMgCl2,
chitosananddecidedtomeasuretheactivityofchitinasein attempting to compensate for their efflux. It was found
thesupernatantsofexperimentsperformedasforthegrowth that in the presence of these metabolites, respiration was
curvesatdifferenttimes.Chitinasesshowactivityonchitosan preserved(Figure10(b)).Moreover,itssensitivenesstoCCCP
[22]andmostofitsactivityproducedinyeastcultures(90%) wasrecovered,indicatingtheintegrityofthemitochondrial
BioMedResearchInternational 9
0.12 0.12
4-Nitrophenyl- 4-Nitrophenyl𝛽-D
N-N󳰀-diacetyl-𝛽-D-chitobioside -N-N󳰀-N󳰀󳰀-triacetylchitotriose
0.10 0.10
0.08 0.08
−1L) −1L)
m m
·oles 0.06 ·oles 0.06
m m
𝜇 𝜇
( (
0.04 0.04
0.02 0.02
0.00 0.00
0 83 167 333 666 1333 2000 2666 0 83 167 333 666 1333 2000 2666
Chitosan (𝜇g·mL−1) Chitosan (𝜇g·mL−1)
10h 10h
24h 24h
48h 48h
(a) (b)
Figure9:ChitinaseactivityafterlongincubationsinYPD.Chitinasewasmeasuredwith4-nitrophenyl-N-N󸀠-diacetyl-𝛽-D-chitobiosideand
4-nitrophenyl𝛽-D-N-N󸀠-N󸀠󸀠-triacetylchitotrioseassubstrates.Samplesweretakenfromexperimentssimilartothoseforthegrowthcurves
withdifferentconcentrationofchitosan,atthreedifferenttimesofgrowth,asindicated.Activitywasmeasuredinaliquotsofthesupernatants
asdescribedunderexperimentalprocedures.
1.2 1.2
1 1
0.8 20,40𝜇g·mL−1 0.8
O
g O
000nat 0.6 Chitosan 10𝜇g·mL−1 0natg 0.6
2 0
= 20
1 0 =
0.4 4𝜇g·mL−1 1 0.4
Chitosan
10,20,40,
2𝜇g·mL−1 100𝜇g·mL−1
0.2 0.2
0 0
0 100 200 300 400 500 0 100 200 300 400 500
Time (s) Time (s)
(a) (b)
Figure10:EffectsofchitosanonC.albicansATCC10231respirationunderdifferentconditions.Cells(25mg,wetweight)wereaddedto10mM
MES-TEAbuffer,pH6.0,and20mMglucoseinafinalvolumeof5.0mL.Chitosan(𝜇g)wasaddedattheindicatedtimesandconcentrations
+
(a).In(b),asimilarexperimentwasperformedinthepresenceof20mMpotassiumphosphate,1.4mMNAD ,1.8mMATP,and5mM
MgCl2.
10 BioMedResearchInternational
4.Discussion
0.12 Aboutthepossibleuseofchitosanasanantimycotic,Rabea
etal.[2]stressedthefollowing:“Chitosanisinexpensiveand
nontoxic and possesses reactive amino groups. It has been
0.1 showntobeusefulinmanydifferentareasasanantimicrobial
compound in agriculture, as a potential elicitor of plant
defenseresponses,asaflocculatingagentinwastewatertrea-
0.08 tment,asanadditiveinthefoodindustry,asahydratingagent
) in cosmetics, and more recently as a pharmaceutical agent
1
−L in biomedicine.” However, these authors also stress the fol-
·ol (g 0.06 lowing:“...itisacurrentmatterofdiscussionastowhether
an thesebiopolymersmayhavethepotentialtoinfluencephys-
h
Et iological functions or metabolism in the microorganisms.
0.04 Therefore, a significant increase in the number of scientific
studiestoobtainevidencetosupportthiscanbeexpected.”
Itiswellknownthatchitosanmaybeaneffectivefungi-
0.02 cidal, but ithasbeen foundthatratherhighconcentrations
are required to inhibit yeast growth [2, 6–8, 12, 13, 26–28].
However, the experiments reported here show a series of
0 interesting facts regarding its effects at low to intermediate
0 50 100 150 200 250 300 350 400
concentrations,eventhoughunderthoseconditionsandeven
Chitosan (𝜇g·mL−1) afterincubationinasimplebufferedmedium,thepolycation
didnotaffectcellviability.Besides,wefoundthattheeffects
depend on the incubation medium because of a series of
Figure 11: Effects of variable concentrations of chitosan on fer-
mentation of the strain C. albicans ATCC 10231. The incubation factors.
medium was 10mM MES-TEA, pH 6.0, and 20mM glucose in a When the cells were incubated in simple buffer, under
finalvolumeof1.0mLtowhich12.5mgoffastedcells(wetweight) conditionsnotverydifferenttothoseemployedbyCourch-
∘
wasadded.Afterincubationat30Cinawaterbathfor10min,the esne[19]andbyMaresovaetal.[20],theseauthorsproposed,
cellswerecooledinicewaterandcentrifuged.Ethanolwasmeasured analyzingtheeffectsofamiodarone,thattheincreaseofthe
in aliquots of the supernatants as described under experimental 2+
internalCa concentrationmaykillthecellsbyanapoptosis-
procedures.
like mechanism. Working with this same compound, we
proposedamechanism[21]thatmayalsobevalidforchitosan
asfollows.
function(notshown).Theadditionofphosphate-TEAalone
(1)The strong binding of chitosan, a polycationic mol-
(from5to50mM)didnotpreventtheinhibitionofrespira-
ecule, decreases the negative surface charge of the
toryactivityproducedbychitosan(notshown).
cells (to more positive values), as shown by the
Sincethisspecieshasasignificantfermentationcapacity decreasedmobilityofthecellsunderanelectricfield.
[15]; experiments were performed incubating the cells with
(2)This change decreases the actual concentration of
variable concentrations of chitosan. As Figure11 shows, +
chitosanatlowconcentrations(10to25𝜇g⋅mL−1)stimulated cationsatthesurface,mainlyK ,alteringthesteady
state balance between its internal/external concen-
fermentationandinhibiteditathigherones(50𝜇g⋅mL−1).
tration and the plasma membrane electric poten-
tial difference, resulting in its efflux. In fact, it is
knownthatthesurfacechargeofthecellsclearlycan
3.9.ChitosanBinding. Thefindingthattheeffectsofsimilar
influencethekineticpropertiesofmonovalentcation
amounts of chitosan were larger as the amount of cells was
86 +
uptake[29].Theexperimentsperformedwith Rb
decreased, suggested its strong binding to the cells. The
experiments performed to measure this parameter in fact showing an increased Km without a change of 𝑉max
indicatethatinfact,andpossiblyduetothedecreased
demonstratedthatthiswasthecase;freechitosanwasfound
effectiveconcentrationofthecationatthecellsurface,
onlywhenthecell/polymerratiowaslarge.Havingfoundthat
higher concentrations were required to achieve half
chitosaneffectsongrowthandviabilityweresmallerinYPD,
themaximalvelocityoftheuptake.
weexpectedthatbindinginthisincubationmediumwould
decrease;whatwefoundwasthatthefreepolycationinthe (3)This efflux then gives rise to a hyperpolarization of
medium did not increase but decreased. (Figure12(a)). So, the plasma membrane. This could be demonstrated
we did the same experiment but without cells, finding that by the estimation of electric plasma membrane
chitosan precipitates with YPD, and centrifuging it or not, potentialdifferencewithDiSC3(3).Weusetheterm
thefluorescenceproducedwithcalcofluorisquenched,indi- efflux (carrier mediated) and not leakage resulting
catinganinteractionofchitosanwiththeYPDcomponents fromthepermeabilizationofthemembrane,because
+
(Figure12(b)). although both mechanisms would increase K exit