Central European Journal of Biology

New effects and applications of thioflavins

* E-mail: ludovit.varecka@stuba.sk

Received 17 July 2009; Accepted 06 January 2010

Abstract: The thiazol dye Thioflavin T (ThT), which is used to stain amyloid fibrils, was found to have strong inhibitory effects on both growth and conidiation of the deuteromycete Trichoderma viride at concentrations between 10-100 μg/ml (ca. 30-300 μmol/l). Thioflavin S (ThS), also known to stain amyloid fibrils, had no significant effect at these concentrations. Both stains yielded a fluorescence response, but their distributions were different. ThT was non-homogenously distributed throughout the cytoplasm, whereas ThS fluorescence was strongly bound to septal regions. The effect of ThT was studied on several model microorganisms. It exerted a strong inhibitory effect on Staphylococcus aureus (Gram-positive bacterium) (MIC=10 μmol/l), but the effect on Escherichia coli (Gram-negative bacterium) was one order of magnitude less pronounced. The effect on Candida albicans was also very strong (MIC=50 μmol/l). The dermatophytic fungus Microsporum gypseum and deuteromycete Alternaria alternata were less affected by ThT (MIC=250 μmol/l and >500 μmol/l, respectively). These results show that ThT could be a useful inhibitor of selected microorganisms, whereas ThS could be a useful agent for monitoring formation and maintenance of intrahyphal septa without inhibiting the growth of the microorganism.

© Versita Sp. z o.o.

Keywords: Thioflavin T • Thioflavin S • Trichoderma viride • Growth • Conidiation

Department of Biochemistry and Microbiology,Faculty of Chemical and Food Technology,Slovak University of Technology, 812 37 Bratislava, Slovak Republic

Boris Lakatoš, Barbora Kaliňaková, Daniela Hudecová, Ľudovít Varečka*

Research Article

1. IntroductionNatural and synthetic dyes often display antimicrobial effects. Examples include triphenylmethane dyes [1] which were used for surface treatment against bacterial and fungal infection, and trypan blue used for the treatment of parasitic infections [2]. Acridine dyes also have antimicrobial properties [3]. Recently, some textile dyes with antimicrobial properties were developed in order to provide textile materials with antifouling properties [4,5]. Antimicrobial aspects of dyes were recently thoroughly reviewed by Wainwright [6]. ThioflavinsSandT(ThTandThS)aredyesusedforstaining histological samples [7-9]. The benzothiazole dyeThT(Figure1)isusedtovisualizeplaquescomposedof β-amyloid found in brain tissue of Alzheimer’sdisease patients [10-12] and amyloid fibers in vitro [13,14].WhenThTbindstoβ-sheets,suchasthoseinamyloid oligomers, the dye undergoes a characteristic

120 nm red shift of its excitation spectrum that may be selectivelyexcitedat450nm,resultinginafluorescencesignal at 482 nm [13,15]. ThT also binds rapidly and specifically to the anti-parallel β-sheet fibrils formedfrom synthetic β-amyloid (1-40), but does not bind tomonomer or oligomeric intermediates [16].ThioflavinThas been used as a dye for silk, cotton, wool, nylon and acetate(thelattertwowithfluorescence),andisusedtocolourballpoint-penink.ThTisalsousedinfluorescentstaining of viruses [17]. ThS is a thiazole dye with adifferentstructurethanThT,containingtwoquarternarynitrogen atoms [9] (Figure 1), which is occasionallyconfused with ThT. However, our observations using thin layerchromatography (notshown)have indicatedthatThioflavinSisamixtureofseveralmolecules,onecomponent of which appears to stain amyloids. We used thioflavin dyes in the screening ofphotochemical inhibitors of blue-light photoreceptors of thefilamentousfungusTrichoderma viride(unpublished).

Cent. Eur. J. Biol. • 5(2) • 2010 • 143–150DOI: 10.2478/s11535-010-0008-2

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New effects and applications of thioflavins

Inthecourseofthisscreeningwefoundthatthioflavinshave an antimicrobial effect on this fungus. Here we describe characterization of this antimicrobial effect on T. viride and other fungi, as well as several representative bacteria.

2. Experimental Procedures2.1 Strains and mediaTrichoderma viride Persoon: Fries, strain CCM F-534fromtheCzechCollectionofMicroorganisms(MasarykUniversity,Brno,CzechRepublic)wasused.ModifiedCzapek-Dox (Cz-D) medium contained the followingcomponents(ing/l):NaNO3 (2),KCl(0.5),MgSO4.7H2O(0.5), FeSO4.7H2O (0.01), K2HPO4 (1), sucrose (30),andyeastextract(5),withdistilledwaterto1litre.ThepHwas adjusted to 6.5-6.7 prior to sterilization.Solidmedium contained 2% (w/v) agar.Conidia ofT. viride were prepared from a 5-day culture (malt agar, roomtemperature) by scraping into 5 ml of a 2% Tweensolution and removing mycelia by filtration througha nylon mesh. All steps were performed under sterile conditions. The number of conidia was determined using a haemocytometer. Staphylococcus aureusCCM3953andEscherichia coli CCM 3988 were purchased from the CzechCollectionofMicroorganisms(MasarykUniversity,Brno,Czech Republic) and were cultured in Mueller-Hintonmedium. The yeast Candida albicans was purchased from the mycological laboratory of Slovak MedicalUniversity, Bratislava, Slovakia, and was cultivated inSabouraud-dextrosemedium.Bothbacteriaandyeastwere incubated under vigorous shaking. Filamentousfungi Rhizopus oryzaeCCM8284,Alternaria alternata CCMF-128andMicrosporum gypseum were obtained from the mycological laboratory of Slovak MedicalUniversity,Bratislava,Slovakia,andwerecultivatedonSabouraud-dextrose agar (dermatophytes) and maltagar(otherfungi).

2.2 Surface cultivationFor dosage experiments,T. viride was grown in Petri dishes(ø120mm)onCz-Dagarat25°C in thedark.Theplates(induplicatesortriplicates)wereinoculatedby soaking paper discs (ø 5 mm) in the conidiasolution, placing the discs at the centre of the plate, and incubatingplates in thedarkat25°C.For thestudyofthioflavineffectsonphoto-inducedandageing-inducedconidiation, paper discs covered with 24-hour old dark-grown mycelia were placed on larger paper discs (ø5cm)onto freshPetriplatescontainingsolidCz-Dmedia with different concentrations of thioflavins, andcultivated in the dark for 24, 48 and 72 h when ageing-induced conidiation was observed. For the study ofphoto-induced conidiation, inoculated Petri dishes were illuminated with white light for 10 min and incubated for 30-36 h in the dark. Growth of the culture was assessed as a diameter of colony.

2.3 Fluorescence microscopy of myceliaMycelia resuspended in sterile physiological solutionwere placed in Eppendorf tubes in a total volume of 500μlandwereincubatedwithThioflavinSorTalone(final concentration 3 μg/ml) or in combination withHoechst 33342 and propidium iodide (both at finalconcentration10μg/ml)for15minatroomtemperature(25°C).Followingstaining,myceliawereplacedonglassmicroscopeslidesandobservedonAxioImager1(CarlZeiss, Jena,Germany)usingobjectiveswith40or63opticalmagnification(totalmagnification400×or630×).Photographs were processed using Paint Shop Proversion8.1(JASCsoftwareInc.,Prairie,MN,USA).

2.4 Morphogenesis quantification and growth kinetics

For quantitative evaluation ofmyceliamorphologyweused the method described by Trinci [18]. We measured lengthoftheprincipal(longest)hyphae(PL)withinthestudied mycelia and the total length of mycelia, i.e., the

Figure 1. Structures of Thioflavin T and Thioflavin S according to Wu et al. [9]

S

N+

NCH3

CH3CH3

S

N+CH3

CH3

S

N+

NCH3

CH3CH3

-O3S

Thioflavin T Thioflavin S

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sum of the lengths of principal hyphae and all branches of givenmycelia (TL). The hyphal growth unit (HGU)was calculated as the ratio ofTL to number of apicaltips, i.e., the average length of hyphae belonging to one apicaltip.Forgrowthkineticsevaluationwecalculatedparameter E

H0 – total length of mycelia at time 0,Ht - total length of mycelia at 1 hour,B0 – number of apical tips at time 0,Bt – number of apical tips at 1 hour.These characteristics were measured and calculated for three independent cultivations of Trichoderma viride in the presence or absence of different levels of thioflavins.

2.5 Antimicrobial assayThe antimicrobial activity of Thioflavin Twas evaluated by a macrodilution method in L-shapedtubesadaptedforthedirectmeasurementofabsorbance using G+ bacteria Staphylococcus aureus CCM3953,G- bacteria Escherichia coliCCM3988andyeast Candida albicans [19]. Bacterial and yeast cultures, in Mueller-Hinton and Sabouraud-glucose media,respectively, were incubated with vigorous shaking. TheeffectofThioflavinTonthegrowthoffilamentousfungi Rhizopus oryzaeCCM8284,Alternaria alternata CCMF-128andMicrosporum gypseum was observed usingamacrodilutiontechniqueonSabouraud-glucoseagar(dermatophytes)andmaltagar(otherfungi)duringthe static cultivation. The diameters of growing fungal colonies were measured at intervals as previously described [20].

The antimicrobial activity of Thioflavin T wascharacterized by the IC50 values (concentration of acompound inhibiting the growth of microorganisms to 50%incomparisontothecontrol),MICvalues(minimalinhibitory concentration of a compound inhibiting a microbial growthby100%)andMMCvalues (minimalmicrobicidalconcentrationofcompound).TheIC50 values weredeterminedfromtoxicitycurves.SubcultureswithnogrowthinthepresenceofThioflavinTwerepreparedseparately in Petri dishes containing agar medium and incubatedat30°Cfor48h(forbacteriaandyeast)orat25°Cfor96h(forfilamentousfungi).TheMICvaluewastaken as the lowest concentration which showed visible growth of microbial colonies in the subculture dishes, and theMMCvaluewastakenasthelowestconcentrationwhich showed no visible growth of microbial colonies. ThioflavinTwasdissolvedindimethylsulfoxide(DMSO).Final concentration of DMSO never exceeded 1.0%(v/v)ineithercontrolortreatedsamples.ConcentrationofThioflavinTwas in the range0.01-500μmol/l inallexperiments.

2.6 ChemicalsThioflavin T from Fluka, Thioflavin S from Merck,Darmstadt, Germany, agar from Difco, SabouraudDextrose Broth and Sabouraud Dextrose Agar fromHiMediaLaboratoriesPvt.LtD,Mumbai,India,allotherchemicalswerepurchasedfromLachema,Brno,CzechRepublic.

t 0

0 t

2 (H +H )E = ,(B +B )∗ where

Figure 2. Effect of Thioflavin S (panel A) and Thioflavin T (panel B) on T. viride growth. Concentrations of Thioflavins : Control (-□-); 0.1 mmol/l (-■-); 0.3 mmol/l (-●-); 1.0 mmol/l (-▲-), 10.0 mmol/l (-▼-).

0 10 20 30 40 50 60 700

20

40

60

80

100

Colonydiameter,m

m

Time, h

Colonydiameter,m

m

Time, h

A

0 10 20 30 40 50 60 700

20

40

60

80

100B

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New effects and applications of thioflavins

Figure 3. Effect of different concentrations of Thioflavin T on photo-induced conidiation (A-D) and ageing-induced conidiation (E-H) of T. viride. ThT was dissolved in sterile water and added to the sterile culture media before congelation. Final concentrations of ThT in media were same for PIC and AIC: (A, E) – control (H2O); (B, F) – 0.03 mmol/l; (C, G) – 0.1 mmol/l; (D, H) – 0.3 mmol/l.

A

C

B

D

F

E

G

H

A

B

C

D

E F

Figure 4. Effect of different concentrations of Thioflavin S on photo-induced conidiation (A-D) and ageing-induced conidiation (E, F) of T. viride. ThS was dissolved in sterile DMSO and added to the sterile culture media before congelation. Final concentrations of ThS in media were: (A, E) – control (DMSO); (B) – 1.0 mmol/l; (C) – 3.0 mmol/l; (D, F) – 10.0 mmol/l. Final concentration of vehicle (DMSO) in all samples did not exceed 1%.

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3. Results

3.1 Effect of thioflavins on growth and conidiation

Initial experiments with different concentrations of the thioflavins showed thatThioflavinS (ThS) did notsignificantly affect the growth ofT. viride, even at the highestconcentrationused(10mmol/l)(Figure2A).ThSalso did not affect photo-induced nor ageing-induced conidiationofthefungus(Figure4). On the other hand, ability of ThioflavinT (ThT) toinhibit growth and conidiation of T. viride was markedly stronger;aconcentrationof0.3mmol/lresultedintotalgrowtharrest (Figure2B).The IC50 value for ThT was calculatedtobe43μmol/l. To evaluate the effect of ThT on photo-induced and ageing-induced conidiation, we used concentrations up to 0.3 mmol/l. Results show that even the lowestconcentrationofThTusedinthisexperimentsignificantlydecreased both photo-induced and ageing-induced conidiation, but the growth of fungus was only slightly inhibited (Figure 3). Higher concentrations markedlyinfluencedthegrowthandconidiationofthefungus.

3.2 Effect of thioflavins on morphology and hyphal growth parameters

For the study of morphological changes in growth of T. virideinducedbythioflavins,weusedconcentrations in the range 1 – 10 μmol/l due to the preservation of fungal growth ability at these concentrations. Calculations of the parameters concerning themorphology (HGUandE)werebasedonmicroscopicobservations of fungal colonies cultivated in the presence of varying thioflavin concentrations at the24th and 25thhourofcultivation(forE)and48th hour of cultivation(forHGU).ThSatanyconcentrationusedinthe experiments did not induce morphological changes of fungal growth. The most distinguished property of ThT was the ability to delay conidia germination (notshown)andtheconsiderablyhigherfrequencyofhyphalbranching (ramification) observed at the lower ThTconcentrationscompared to thecontrol (Table1).Thehighest concentration of ThT used in this experiment (10μmol/l)evokedinhibitionofbranching,whichcouldbetheconsequenceoftheinhibitoryeffectofThTonthegrowth ability of the fungus.

Concentration, μmol/l

Main Hyphae Lenght,μm

Whole Mycelium Length, μm

Number of Apical Tips

E*,μm tip-1h-1

HGU*, μm

0 36000 ± 2080 62855 ± 3632 5 1063 ± 83 12571 ± 7261 40000 ± 3175 80000 ± 6350 13 763 ± 73 6153 ± 4883 48000 ± 5600 68000 ± 8001 17 531 ± 76 4000 ± 47110 20222 ± 3363 28000 ± 4657 6 360 ± 59 4666 ± 776

Table 1. Effect of Thioflavin T on T. viride morphology.

HGU - hyphal growth unit. * - for the way of calculations see Experimental Procedures. Values represent mean ± SD calculated from three independent experiments.

C D A

B

Figure 5. Fluorescent staining of T. viride mycelia using Thioflavins. Staining of mycelia was performed as mentioned in Experimental Procedures. Photographs A, B represent mycelia stained with ThT. Picture A was made in visible light. Photographs C, D represent mycelia stained with ThS. Picture C was made in visible light.

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3.3 Thioflavin staining and fluorescent microscopy of T. viride

ThioflavinTisutilizedasaprobeforfluorescentstainingofamyloidfibrilsandproteinaggregatesinanimalcellsand yeasts [21,22]. In addition, ThT has been used for hydrophobin staining in filamentous fungi [23-26]. We were interested to determine if T. viride forms protein aggregates similar to amyloid fibrils. After stainingofmycelia byThT orThS,we observed fluorescencelabelling of hyphae but not of conidia. However, the distributionof fluorescenceof both dyeswasdifferentfrom each other. ThT was distributed throughout the hyphaeanddisplayedafluorescenceaggregationwithinthe lumen of hyphae, suggesting the dye accumulates in nuclei or vacuoles (Figure 5A,B). ThS, however,strongly labelled intercellular septa along the hyphae. Stronger fluorescence was observed in the vicinity ofchlamydosporesandnearsitesofhyphaeramification(Figure 5C,D). Thus, it seems that each dye has auniqueeffectonT. viride.

3.4 Effect of ThT on growth of other model organisms

In order to determine whether the inhibitory effect of ThT wasspecifictoT. viride or if it is a general property of the compound, we used a variety of microorganisms including the Gram-negative bacterium Escherichia coli, Gram-positive bacterium Staphylococcus aureus and various fungi: the pathogenic yeast Candida albicans, dermatophyte Microsporum gypseum, phytopathogenic fungus Alternaria alternata and the saprophytic, non-septate fungus Rhizopus oryzae. We found that ThT exerted the strongest antimicrobial effect on Staphylococcus aureus and Candida albicans, withMICvalues ranging inmicromolar concentrations (Table 2). TheMIC value forE. coli was significantlyhigher than that observed in S. aureus. The sensitivity of M. gypseum to ThT was comparable to that of T. viride. Alternaria alternata and Rhizopus oryzae were resistant

to ThT, with MIC values in the submilimolar range (Table2).

4. DiscussionResults demonstrate that ThT has a strong inhibitory effect on both growth and conidiation of T. viride. Figure2BandFigure3 show that these processes are inhibited in parallel. Therefore, ThT is not a selective inhibitor of either growth or conidiation. Restricted growth is likely the cause of decreased conidiation. Similar results were observed in T. viride nourished by various carbon and nitrogen sources [27]. It is also clearthatThSdoesnot inhibitanyoftheseprocessesat concentrations up to 10 mmol/l (Figure 2A and Figure4). Results also demonstrated that ThT possesses strong antimicrobial activity against several model microorganisms. The bactericidal effect of ThT on S. aureus is as high as that of many commercially used antimicrobial agents. Similarly, ThT has fungistaticeffects against C. albicans, and is comparable to many agents used for suppressing growth of this pathogenic fungus. ThT was not as effective against filamentousfungi, especially Rhizopus and Alternaria. As a result, ThT may only be effective as an antimicrobial agent against a limited number of species. In the future, the antimicrobial properties of ThT should be tested using a wider panel of microorganisms. Future studies can also further elucidate themechanism of antimicrobial action of ThT. We obtained some pertinent information only with T. viride. It is not certainthattheinteractionofthedyewithamyloidfibrilscauses inhibitionbecauseThS,whichalso isused forthestainingofamyloidfibrils,doesnothaveaninhibitoryeffect. Results indicate it is not the binding to some hypothetical amyloid structure essential for the growth of microorganisms that is important for the inhibitory action of ThT.

Studied Microorganism Staphylococcusaureus

Escherichiacoli

Candidaalbicans

Parameter IC50 MIC MMC IC50 MIC MMC IC50 MIC MMC

Values,μmol/l 6 10 25 44 117 250 5 50 >500

Studied Microorganism Rhizopusoryzae

Alternaria alternata

Microsporum gypseum

Parameter IC50 MIC MMC IC50 MIC MMC IC50 MIC MMC

Values,μmol/l 200 >500 >500 142 >500 >500 40 250 500

Table 2. Effect of Thioflavin T on growth of selected microorganisms.

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An alternative mechanism which could explain the effect of ThT could be its interactionwith DNA. SuchaninteractionhasbeenobservedbyCañeteet al. [28] and recently by Ilanchelian and Ramaraj [29], where the authors showed an ability of ThT to bind DNA,and proposed that this is due to groove binding and electrostatic interactions between ThT and DNA [29]. ThefluorescencedistributionofThTthatweobserved(Figure5)corroboratesthesedata. The observation that ThS enables visualizationof septa without inhibiting growth and conidiation is interesting and indicates that this compound could be

used for monitoring the development of septa without exerting a harmful effect on the organism.

AcknowledgementsThisworkwas supported byScience andTechnologyAssistance Agency under the contract No. APVT-20-003904, APVV 0642-07 and VVCE-0064-07, andSlovakGrantAgency for Science (VEGA, project No.1/0434/08).Dr.WalterVollekisacknowledgedforcolourphotographs of surface cultivations.

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