Research Article Sinefungin, a Natural Nucleoside …downloads.hindawi.com/journals/bmri/2014/156987.pdfResearch Article Sinefungin, a Natural Nucleoside Analogue of S-Adenosylmethionine,

Research ArticleSinefungin a Natural Nucleoside Analogue ofS-Adenosylmethionine Inhibits Streptococcus pneumoniaeBiofilm Growth

Mukesh Kumar Yadav1 Seok-Won Park2 Sung-Won Chae1 and Jae-Jun Song1

1 Department of Otorhinolaryngology-Head and Neck Surgery Korea University College of Medicine Seoul 135-705 Republic of Korea2Department of Otorhinolaryngology-Head and Neck Surgery Dongguk University Ilsan Hospital GyeonggiGoyang 410-773 Republic of Korea

Correspondence should be addressed to Jae-Jun Song jjsong23gmailcom

Received 5 February 2014 Revised 5 June 2014 Accepted 8 June 2014 Published 23 June 2014

Academic Editor Meirong Zhao

Copyright copy 2014 Mukesh Kumar Yadav et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Pneumococcal colonization and disease is often associated with biofilm formation in which the bacteria exhibit elevated resistanceboth to antibiotics and to host defense systems often resulting in infections that are persistent and difficult to treat We evaluatedthe effect of sinefungin a nucleoside analogue of S-adenosylmethionine on pneumococcal in vitro biofilm formation and in vivocolonization Sinefungin is bacteriostatic to pneumococci and significantly decreased biofilm growth and inhibited proliferationand structure of actively growing biofilms but did not alter growth or the matrix structure of established biofilms Sinefunginsignificantly reduced pneumococcal colonization in rat middle earThe quorum sensing molecule (autoinducer-2) production wassignificantly reduced by 92 in sinefungin treated samples The luxS pfs and speE genes were downregulated in biofilms grownin the presence of sinefungin This study shows that sinefungin inhibits pneumococcal biofilm growth in vitro and colonizationin vivo decreases AI-2 production and downregulates luxS pfs and speE gene expressions Therefore the S-adenosylmethionine(SAM) inhibitors could be used as lead compounds for the development of novel antibiofilm agents against pneumococci

1 Introduction

Streptococcus pneumoniae is a major cause of pneumoniameningitis bacteremia and otitis media (OM) in youngchildren and the elderly [1 2] Pneumococcal colonizationand disease is often associated with biofilm formation [3]in which the adherent bacteria are attached to the hostsurface and to each other to form multilayer structurescovered by an extracellular matrix (exopolysaccharide (EPS))[4 5] Bacteria in biofilms exhibit elevated resistance both toantibiotics and to host defense systems which often resultsin infections that are persistent and difficult to treat [6 7]Various studies have previously reported that pneumococ-cal biofilm exhibits significantly higher resistance to peni-cillin tetracycline rifampicin amoxicillin erythromycinclindamycin and levofloxacin [8 9] Marks et al reportedthat pneumococcal biofilms formedonnasopharyngeal tissueof mouse were more resistant to gentamicin and penicillin

G than did planktonic cell [10] Therefore there is an urgentneed to search for alternative antimicrobial target to developantimicrobial compound against pneumococcal biofilms

The effective antibiofilm strategies should include inhi-bition of microbial adhesion to the surface and of coloniza-tion and interference with the signal molecules modulatingbiofilm development and the disaggregation of the biofilmmatrix [11 12] In S pneumoniae quorum sensing (QS)signaling regulates biofilm communities and plays a keyrole in coordinating the spatial disposition aggregation ofcells and exopolysaccharide formation [13 14] Autoinducer-2 (AI-2) is the only QS molecule in pneumococci which issynthesized by S-ribosylhomocysteine lyase (LuxS) throughactivated methyl cycle (AMC) [15] In AMC the S-adenosyl-L-methionine (SAM) is a central molecule which donatesmethyl group for methionine recycling methylation ofbiomolecules and biosynthesis of AI-2 [16 17] Furthermore

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 156987 10 pageshttpdxdoiorg1011552014156987

2 BioMed Research International

the byproducts of AMC such as 51015840-methylthioadenosineS-adenosylhomocysteine (MTASAH) are toxic to the bacteria[15] In S pneumoniae detoxification of SAH is carried outby MTASAH nucleosidase (Pfs) and S-ribosylhomocysteinelyase (LuxS) and this pathway is absent in human [17ndash19]

The SAM is a central molecule of AMC involvedin recycling of methionine methylation of biomoleculesbiosynthesis of AI-2 and polyamine biosynthesis [20 21]Therefore we hypothesized that altering the SAM activitycould have adverse effect on pneumococcal biofilms Here weevaluated the effect of sinefungin a nucleoside analogue of S-adenosylmethionine on pneumococcal in vitro biofilm

Sinefungin is a natural nucleoside and a structural analogof SAM It inhibits transmethylation reactions related toDNA RNA proteins and other molecules [22ndash25] Anti-fungal antiviral and antiprotozoal activities of sinefunginhave been reported [26 27] Previously Parsek et al stud-ied effect of sinefungin on Pseudomonas acyl homoserine-lactone quorum-sensing signal generation [28] However theeffect of sinefungin on S pneumoniae has not been studiedIn this study we examined the effect of sinefungin on Spneumoniae particularly on biofilm growth

2 Materials and Methods

21 Bacteria Strains and Culture Conditions S pneumoniaeD-39 strain (NCTC 7466) is an encapsulated serotype 2pathogenic strain It was obtained from theHealth ProtectionAgency Culture Collections (HPA Salisbury UK) Bacteriawere routinely grown in tryptic soy broth (TSB BD DifcoDetroit MI USA) or on blood agar (BA Hye In SeoulKorea) supplemented with 5 vv sheep blood at 37∘C in anatmosphere of 5 CO

2 Vibrio harveyi (V harveyi) strains

MM32 (ATCC BAA-1121) [29] were grown on autoinducerbioassay (AB) medium at 30∘C [30] Sinefungin (sc-203263)was purchased from Santa Cruz Biotechnology (Santa CruzCA USA)

22 Effect of Sinefungin on Planktonic Cell Growth S pneu-moniae were grown with different concentrations of sine-fungin (10 20 30 40 and 50 120583gmLminus1) at 37∘C in 5 CO

2

for different time points (0 hndash10 h) And optical densityat 600 nm (OD

600) was measured after 2 h time interval

using a SpectraMax plus 384 automated microplate reader(Molecular Devices Sunnyvale CA USA) After 6 h ofincubation 100120583L aliquots was serially diluted and platedon BA for enumeration of colony forming units (CFU) Thepercentage decrease in planktonic cell growth was calculatedby subtracting the values of sinefungin treated samples fromcontrol (untreated) samples The experiment was replicatedthree times with triplicate samples at each time point

23 Effect of Sinefungin on In Vitro Biofilm Growth In vitropneumococcal biofilm growth was carried out in 96-wellflat-bottom polystyrene microtiter plate (BD falcon SparksMD USA) using a static model [31] Briefly a fresh colonyof S pneumoniae grown overnight on BAP was scraped

and grown in TSB+1 glucose medium [32] The mid-logarithmic phase cell suspension (1 times 108 CFUmLminus1) wasdiluted 1 100 with fresh sterile TSB+1 medium and 200120583Laliquots were inoculated in wells of 96-well microtiter platesand incubated at 37∘C in 5 CO

2 After incubation the

medium was discarded and plates were gently washed threetimes with 200120583L sterile phosphate buffered saline (PBS)Thereafter plates were air-dried and stained with 50120583Lcrystal violet (CV 01) for 15min Excess stain was decantedoff and plates were washed three times with sterile distilledwater The biofilm was dissolved in 200120583L of 95 ethanoland the OD

570nm was measured using the aforementioned

automated spectrophotometerThedata represent the averagevalue of three replicates

24 Effect of Different Concentrations of Sinefungin onBiofilmGrowth To study the concentration-dependent effectof sinefungin on pneumococcal biofilms S pneumoniaebiofilms were grown with 10ndash50120583gmLminus1 sinefungin for18 h as described above Biofilm biomass was detected byCV-microtiter plate assay as described above The percent-age decrease in biomass was calculated by subtracting thebiomass of control biofilms grown without sinefungin Enu-meration of biofilmbacteriawas done as described above (ieCFU)

25 Effect of Sinefungin on Established Biofilms Pneumococ-cal biofilms were established in microtiter plate wells for 12 hThe formed biofilmswere exposed to different concentrationsof sinefungin for 6 h and analyzed by CV-microtiter plateassay and the cell viability was determined by CFU counts

26 Effect of Different Exposure Times of Sinefungin on BiofilmFormation Pneumococcal biofilms were generated in thepresence of 35 120583gmLminus1 sinefungin for 6 12 18 and 24 hAt each time point biofilm biomass was determined asdescribed above

27 Scanning Electron Microscopy (SEM) Biofilms In vitrobiofilms grown with 35 120583gmLminus1 sinefungin and withoutsinefungin in 24-well tissue culture plates for 18 h wereanalyzed by SEM The medium was removed and the plateswere gently washed two times with sterile PBS to removeplanktonic cells The samples were prefixed by immersion in2 glutaraldehyde in 01M phosphate buffer and postfixedfor 2 h in 1 osmic acid dissolved in PBS Samples weretreated in a graded series of ethanol and t-butyl alcohol driedin a model ES-2030 freeze dryer (Hitachi Tokyo Japan)platinum coated using an IB-5 ion coater (Eiko KanagawaJapan) and observed using a S-4700 field emission scanningelectron microscopic (Hitachi)

28 Autoinducer Assay V harveyi MM32 was used as thequalitative reporter strain to detect the changes in the produc-tion of AI-2 V harveyi MM32 (BB120 luxNTn5 luxSTn5)is an ideal reporter strain as it can sense AI-2 but cannotsynthesize AI-2 of its own [29]The reporter strain was grown

BioMed Research International 3

in AB medium for 16 h and then diluted 1 5000 A total of90 of the diluted reporter strain was then added to a 96-wellplate S pneumoniae biofilms were grown with 35 120583gmLminus1sinefungin and without sinefungin as described earlier for18 h and sonicated for 3 seconds to disperse the adherent cellsThen 1mL of the cell suspension was centrifuged and filtersterilized And 10 of the filtered supernatant was added tothe 90 reporter stain Media without bacteria were negativecontrol The plates were incubated at 30∘C for 15 h and theluminescence of the reporter strain was monitored usinga luminometer (GloMax Multi-Detection System PromegaMadison WI USA)

29 In Vivo Pneumococcal Colonization For in vivo coloniza-tion study we used rat OM model The animal experimentprotocol was reviewed and approved by the animal researchand care committee at Dongguk University Ilsan Hospital(Gyeonggi South Korea) Twenty pathogen-free SpragueDawley rats weighing 150ndash200 g were obtained from OrientBio (Gyeonggi South Korea) All rats were examined priorto use by otomicroscopy to document abnormal middle earand were kept isolated in an infection-free zone for 2 weeksRats were assigned randomly to groups that received bacteria(119899 = 8) bacteria with 35 120583gmLminus1 sinefungin (119899 = 8) orno procedure (control group 119899 = 4) Fifty microliters ofcell suspension containing 3 times 107 CFU S pneumoniae wasinjected into the middle ear cavity through the tympanicmembrane of the right ear using a tuberculin syringe anda 27-gauge needle [33] Animals were sacrificed 1 weekafter inoculation and the middle ear bulla was asepticallyacquired The tympanic membrane was removed and earswere irrigated to remove planktonic bacteriaThe bullae werehomogenized and serially diluted and plated on BAP forenumeration of CFU

210 Quantification of Gene Expression of In Vitro FormedBiofilms Using Quantitative Real-Time RT-PCR Expressionsof luxS pfs and speE genes in biofilms grown for 18 h withoutand with (35 120583gmLminus1) sinefungin were quantified by real-time RT-PCR The biofilms were washed and the adherentcells were scraped and immediately processed for RNAextractionThe cells were lysed by incubation of the cell pelletin 100 120583L (3mgmLminus1) lysozyme (Sigma-Aldrich St LouisMO USA) for 4min The total RNA was extracted using aRNeasy Total RNA Isolation System Kit (Qiagen ValenciaCA USA) according to the manufacturerrsquos instructions withfew modifications On-column DNAse (Qiagen) treatmentwas performed for 10min at 20ndash25∘C RNA quality wasassessed spectrophotometrically

cDNA was synthesized using the ImProm-II ReverseTranscriptase Kit (Promega Madison WI USA) accordingto the manufacturerrsquos instructions Primers for speE genewere designed by standard procedures from the nucleotidesequence of S pneumoniae D39 strain (Table 1) The primersused for luxS pfs and gyrB (house-keeping) geneswere previ-ously reported [31 34] Real-time RT-PCR was carried out intotal volume of 20120583L consisting of 10 120583L of 2X SYBR GreenPCR Master Mix (Roche Applied Science Indianapolis IN

Table 1 Primers used for gene expression study

Genes Primer sequences Amplicon size(base pair)

speE F-51015840-GACTTTGCTGCAGGGCTAGA-31015840R-51015840-AAATGGATCTGTCGCATCGT-31015840 120

luxS F-51015840-TATGTTCGCTTGATTGGG-31015840R-51015840-GCCGGCAGTAGGGATAGAGT-31015840 105

pfs F-51015840-TTGCTGCTATGCCAGAAGAA-31015840R-51015840-TTCCCCAAAACAACTTGCTC-31015840 76

gyrB 51015840-CAGATCAAGAAATCAAACTCCAA-3101584051015840-CAGCATCATCTACAGAAACTC-31015840 172

USA) 3 pmol of each forward and reverse primers 4 120583LcDNA and nuclease-free water PCR conditions includedinitial denaturation at 95∘C for 10min followed by 45 cyclesof denaturation at 95∘C for 15 sec annealing at 56∘C for 10 secextension at 72∘C for 15 sec and final extension at 72∘C for5min followed by melting curve analysis from 60 to 95∘CNegative controls containing nuclease-free water and noreverse transcriptase control were included in each RT-PCRexperiment The relative gene expression was analyzed usingthe 2minusΔΔCT method [35]The reference gene was gyrB and thestandard condition was biofilms grown without sinefungin

211 Statistical Analysis The values were calculated as themean of individual experiments performed in triplicate andcompared with those of the control groups Differencesbetween two mean values were calculated by Studentrsquos 119905-testThe statistically significant tests were set at a 119875 value lt005

3 Results

31 Effect of Sinefungin on Planktonic Cell Growth Thegrowth of pneumococci with different concentrations ofsinefungin in time course experiment showed bacteriostaticeffect of sinefungin The growth of bacteria in sinefungintreated samples was slow in comparison to control samples(untreated) And a significantmaximum growth difference insinefungin treated and control samples was detected at mid-log phase (4 h after inoculation) However at the end of thelog phase (6 h of inoculation) there was no significant plank-tonic bacteria growth inhibition (Figure 1(a))TheCFUcountof 6 h grown bacteria also detected no significant differencein sinefungin treated and untreated samples (Figure 1(b))

32 Effect of Sinefungin on In Vitro Biofilm Growth

321 Sinefungin Inhibits In Vitro Formation of Pneumococ-cal Biofilms Significantly (119875 lt 005) decreased biofilmbiomass and CFU counts were detected in the samplesgrown with sinefungin The inhibitory effect of sinefun-gin was concentration-dependent (Figures 2(a) and 2(b))Biofilms grown with 10 120583gmLminus1 (lowest concentration) and50 120583gmLminus1 (highest concentration) sinefungin demonstrateda significant (119875 lt 005) biomass decrease of 15 and 53respectively


0

005

01

015

02

025

03

035

0 2 4 6 8 10

Control

Opt

ical

den

sity

of p

lank

toni

c cel

l

Time (h)

grow

th at

600

nm

10120583g20120583g

30120583g40120583g50120583g

(a)

Control

(CFU

mL)

10120583g 20120583g 30120583g 40120583g 50120583g100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

100E + 09

Sinefungin concentration

(b)

Figure 1 (a)Growth of Streptococcus pneumoniaewith different concentrations of sinefungin (10120583g to 50120583gmLminus1) in time course experiment1 100 diluted cell suspensions were grown with different concentrations of sinefungin at different time points (0 2 4 6 8 and 10 h) at 37∘Cin 5 CO

2and optical density was measured at 600 nm (b) CFU counts of pneumococci grown with different concentrations of sinefungin

at 6 hours After 6 h of incubation 100 120583L aliquots were serially diluted and plated on blood agar plate to enumeration bacteria Error barsrepresent the standard deviation of the mean values

0005

01015

02025

03035

04

Control

Opt

ical

den

sity

of cr

ysta

l vi

olet

at570

nm

lowastlowast lowast

lowastlowastlowastlowast

10120583g 20120583g 30120583g 40120583g 50120583gSinefungin concentration (120583gmL)

(a)

(CFU

mL)

100E + 06

100E + 05

100E + 04

100E + 03

100E + 02

100E + 01

100E + 00

lowast lowast lowast lowast lowast

Control 10120583g 20120583g 30120583g 40120583g 50120583gSinefungin concentration (120583gmL)

(b)

0

005

01

015

02

025

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035

Opt

ical

den

sity

of cr

ysta

l vi

olet

at570

nm

lowast


(c)

(CFU

mL)

100E + 08

100E + 07

100E + 06

100E + 05

100E + 04

100E + 03

100E + 02

100E + 01

100E + 00

lowast


(d)

Figure 2 Effect of sinefungin on pneumococcal in vitro biofilm growth (a) (b) Pneumococcal biofilm growth with different concentrationsof sinefungin for 18 h and detection of biofilm biomass by crystal-violet microtiter plate assay and CFU counts of pneumococcal biofilms(c) and (d) show the effect of different concentrations of sinefungin on already established biofilms (c) Detection of biofilm biomasses withcrystal-violet microtiter assay (d) CFU counts of pneumococcal biofilmsThe error bars represent the standard deviation of the mean valuesSignificance was determined by Studentrsquos 119905-test (lowast119875 lt 005 lowastlowast119875 lt 001)


0

005

01

015

02

025

03

035

Control Sinefungin

Opt

ical

den

sity

of cr

ysta

l

lowastlowast

viol

et at

570

nm

(a)

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005

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015

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sity

of cr

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at570

nm

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(b)

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of cr

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et at

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(c)

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018

Control Sinefungin

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sity

of cr

ysta

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lowastvi

olet

at570

nm

(d)

Figure 3 Inhibitory effect of sinefungin (35120583gmLminus1) on Streptococcus pneumoniae in vitro biofilms grown at different time intervals (6 12 18and 24 h) Pneumococcal biofilms were grown with 35 120583gmLminus1 sinefungin at different time points and the biofilm biomasses were quantifiedby crystal-violet microplate assay (a) Biofilms grown at 6 h (b) biofilms grown at 12 h (c) biofilms grown at 18 h and (d) biofilms grown at24 h The experiments were repeated three times in triplicate The error bars represent the standard deviation of the mean values The resultswere significant by Studentrsquos 119905-test (lowast119875 lt 005 lowastlowast119875 lt 0005)

322 Effect of Sinefungin on Established Biofilms Pneumo-coccal biofilms were generated for 12 h and then incubatedwith different concentrations of sinefungin for 6 hNo signifi-cant decrease of biofilm biomass and viable count was evidentat 10 20 30 and 40 120583gmLminus1 of sinefungin while 50120583gmLminus1sinefungin produced significant decrease in biofilm biomassand viable counts although the percentage decrease was low(only 5 of biofilm decreases) (Figures 2(c) and 2(d))

323 Biofilm Inhibition with Time To determine theinhibitory effect of sinefungin on biofilms grown at differenttimes biofilms were generated for 6 12 18 and 24 h withand without sinefungin A significant (119875 lt 005) decrease ofbiofilm biomass was evident at all time points (Figure 3)

33 SEM of Pneumococcal Biofilms Biofilms grown in theabsence and presence (35120583gmLminus1) of sinefungin were exam-ined by SEM Highly organized biofilm was produced in theabsence of sinefungin These biofilms were heterogeneous in

nature with cells embedded in thematrix and connectedwitheach other and to the base of the plate The adherent cellsformed a three-dimensional structure of significant depth(Figures 4(a) 4(b) and 4(c)) In contrast biofilms grownwith 35 120583gmLminus1 sinefungin were thin and the bacteria werescattered in clumps Cell-to-cell connections were not visibleand the biofilms were disorganized (Figures 4(d) 4(e) and4(f))

34 Autoinducer Assay Thecell-free supernatants of biofilmsgrown with and without sinefungin were examined forAI-2 production via the V harveyi MM32 reporter strain(Figure 5) After 15 h the reporter strain detected significantlyhigh AI-2-induced luminescence response in the supernatantfrom the untreated samples than samples grown with sine-fungin The luminescence responses were significantly 92less in sinefungin treated samples in comparison to untreatedsamples These results indicate that sinefungin interferes anddecreases the production of AI-2 in pneumococcal biofilms


(a) (b) (c)

(d) (e) (f)

Figure 4 Scanning electron microscope (SEM) images of biofilms grown with and without sinefungin Panels (a) (b) and (c) arerepresentative of SEM images of biofilms without sinefungin the biofilm is thick and well organized with a three-dimensional structurePanels (d) (e) and (f) are representative of SEM images of biofilm grown with 35120583gmLminus1 sinefungin biofilms are thin and scattered andclumps of cells are seen with no organized structure The scale bars of the figure are 10 5 and 3 120583m

0500000

100000015000002000000250000030000003500000400000045000005000000

Without sinefungin With sinefungin

Lum

ines

cenc

e (RL

Us

)

lowast

Figure 5Measurement of autoinducer-2 production changes in cellfree supernatant of biofilms grown with 35 120583gmLminus1 sinefungin andwithout sinefungin using vibrio harveyi reported strain V harveyireporter strain was grown with cell free supernatant for 15 h andluminescence response was measured The error bars represent thestandard deviation of the mean values Significance was determinedby Studentrsquos 119905-test (lowast119875 lt 005)

35 Sinefungin Inhibits In Vivo Pneumococcal ColonizationThe in vivo experiment showed less bacterial recovery inbulla lysate isolated from rat inoculated with sinefungin after1 week of bacterial inoculation The mean CFU counts ofbacteria only treated group were 598 times 103 (SD = 4649) andof sinefungin treated group were 170 times 103 (SD = 2533) Asignificant (119875 lt 005) 70 less bacteria were recovered insinefungin treated rat bulla (Figure 6)

Bacteria Sinefungin

CFU

coun

tsbu

lla

100E + 05

100E + 04

100E + 03

100E + 02

100E + 01

100E + 00

lowast

Figure 6 Quantification of pneumococcal colonization in themiddle ear of rats inoculated with 35 120583gmLminus1 sinefungin CFUcount was used to enumerate bacteria in whole bulla lysate ofsamples treated with bacteria suspension supplied with sinefunginwith respect to control groupsThe error bars represent the standarddeviation of the mean values The results were significant byStudentrsquos 119905-test (lowast119875 lt 005)

36 Quantification of Gene Expression of In Vitro BiofilmsQuantitative RT-PCR revealed a significant (gt2-fold)decreased gene expressions of luxS pfs and speE inbiofilms grown with sinefungin compared to biofilm growthwithout sinefungin The expression of luxS was significantlydecreased by 853-fold (119875 = 0001) in the biofilms grownwith sinefungin Expression of the pfs gene was decreasedby 288-fold (119875 = 0006) Similarly the speE gene was


S-Adenosylmethionine SAM

S-Adenosylhomocysteine(SAH)

Substrate

Methylatedproduct

Adenine

S-Ribosylhomocysteine(SRH)

45-Dihydroxy-23 pentanedione(autoinducer 2)

Homocysteine

Methionine

Decarboxylated SAM

Putrescine

Spermidineother polyamines

5-Methylthioribose (MTR)

Adenine

ATPSAM synthetase

(MetK)

Spermidine synthase(speE)

Methylthioadenosine

MTASAH nucleosidase (pfs)


SAM-dependent transmethylase

Methionine synthases MetE or MetH

S-S-ribosylhomocysteinelyase (LuxS)

PPi + Pi

NH2

NH2S+

O

O

HO OH

NN

NN

NH2

S

O

O

HO

HO OH

OH

NH2

NH2

S

O

OHO

OH OH

N

NN

NH

O

OHOH

N N

NN

NH2

H3CS

NH2

NH2

S

O

O

OH

OHH3C

HS

CO2

minusO

H2O

H2O

Figure 7 Systematic diagram of cyclic production of homocysteine and activated methyl cycle in Streptococcus pneumoniae Methionine isconverted into SAM by SAM synthetase (MetK) Donation of the methyl group of SAM to a variety of methyl acceptors results in SAH theMTASAH nucleosidase (pfs) first converts SAH into S-ribosylhomocysteine (SRH) which is then recycled back to homocysteine by LuxS Asa byproduct of this reaction 45-dihydroxy 23-pentanedione is produced which spontaneously forms autoinducer-2The decarboxylation ofSAM produces MTA (methylthioadenosine) as catalyzed by spermidine synthase (speE) Spermidine (a polyamine) is the byproduct of thisreaction from putrescine

downregulated by 322-fold (119875 = 001) Gene annotationrevealed the identity of the luxS and pfs genes as methioninepathways genes that recycle cysteine through AMC and theQS molecule AI-2 is the byproduct of AMC (Figure 7) Inthis pathway SAM acts as a methyl donor The luxS and pfsgenes encode enzymes that catalyze removal of SAH andSRH which are toxic to bacteriaThe speE gene is involved inbiosynthesis of spermidine catalysis by decarboxylated SAM

4 Discussion

SAM is an important nucleoside that serves as an activatedmethyl group donor in the methylation of macromoleculesto yield SAHMTA which leads to biosynthesis of AI-2and polyamine Therefore inhibition of SAM activity bycertain substrate analogs could decrease luxS and pfs genesexpression and limits synthesis of autoinducer-2 and hence

causes reduction in biofilm formation and may attenuatevirulence

In this study we examined the effect of sinefungina nucleoside analogue of SAM and a methytransferaseinhibitor on pneumococcal biofilm growth in vitro and invivo colonization

Planktonic growth of pneumococci with different con-centrations of sinefungin demonstrated that sinefungin isbacteriostatic The significant inhibitory effect of sinefunginwas on actively growing cells (log phase) No significantplanktonic cell growth inhibition was detected at concen-trations that inhibited biofilm growth which indicates thatsinefungin may be a biofilm growth inhibitor The inhibitoryeffect of sinefungin on in vitro biofilms was concentration-dependent We tested the inhibitory effect of sinefungin withtime on in vitro biofilms grown [36] At all the time pointstested there were low biofilm biomasses and CFU counts


in the biofilm samples grown with sinefungin Probablysinefungin inhibits quorum sensing systems (luxS-AI-2) thathas been reported to control pneumococcal biofilm forma-tion [14 37] No significant decrease in the biofilm biomassand the CFU counts in established biofilms treated withsinefungin indicates that sinefungin is unable to kill bacteriaThe inhibitory effect of sinefungin has been reported in virusfungi and protozoa [26 38]

The difference in the architecture of the biofilms grownwith and without sinefungin was verified with SEM SEMconfirmed that the biofilms grown with sinefungin were sig-nificantly different in terms of their connection to substrateand thickness and were unorganisedThey also lost their cell-to-cell connections andwere devoid ofmicrocolonies similarto a prior report [14]

Using the rat OM model we detected significantly lowrecovery of pneumococci in rat bulla treated with sinefunginThis indicates that sinefungin inhibits in vivo colonization ofpneumococci It is previously reported that mutant bacterialstrains defective in QS create less potent infections [39] QS-deficient intranasal S pneumoniae infections in mouse wereless effective at spreading to the lungs or the bloodstream[40]

The QS regulates pneumococcal biofilms and AI-2 inthe only QS molecule in S pneumoniae Various studiesreported the prevention and treatment of infectious diseasesby inhibiting bacterial QS [12] Therefore our next aim wasto detect the changes in AI-2 biosynthesis Here we detectedsignificantly low AI-2 induced luminescence response inthe sinefungin treated samples that indicates that sinefungininterferes in AI-2 biosynthesis Similarly Stroeher et al 2003[40] reported the significantly reduced ability of luxSmutantstrain to elicit bioluminescence in a V harveyi AI-2 reporterstrain

Next we detected changes in gene expression of luxSand pfs genes The significantly decreased expressions ofluxS and pfs genes could be due to inhibition of SAM-dependent methyltransferase activity The encoded Pfs andLuxS enzymes are necessary for biofilm formation and pro-duction of AI-2 [14 41] In S pneumoniae luxS is a virulentgene and a central regulator of competence fratricide andbiofilm formation and a luxSmutant strain displays a signifi-cant decrease in pneumococcal biofilm formation [13 14 42]The downregulation of luxS and pfs genes may decrease thebiosynthesis of AI-2 in sinefungin treated samples [40] In Spneumoniae AI-1 is absent and AI-2 is the only QS moleculeinvolved in expression of the enzymes for biofilm formationexotoxin synthesis and antibiotic resistance factors [19]Thus the downexpression of luxS and pfs genes leads todecreased biosynthesis of AI-2 which in turn disrupts spatialarrangement andbuild-up of the organized biofilms Previousstudies reported that cultures of Vibrio cholerae and entero-hemorrhagic E coli (EHEC) strain O157H7 treated withMTASAH nucleosidase inhibitors did not synthesize AI-2and showed reduced biofilm formation [39] In a previousstudy 5-azacytidine a hypomethylating drug demonstratedsimilar downregulation of genes of AMC [34]

The other reason for inhibitory effect of sinefungin maybe accumulation of toxic byproducts of AMC Recent reports

have suggested that substrates of AMC such as MTA andSAH are toxic to cells and that bacteria possess MTASAHnucleosidase andor a combination of SAH hydrolase andMTA phosphorylase to remove these inhibitory nucleosides[21] In S pneumoniae methionine is converted to SAM bySAM synthetase (encoded bymetK) and the SAM-dependentmethyltransferase produces SAH and methylated productsThe detoxification of SAH is carried out by MTASAHnucleosidase (Pfs) to produce S-ribosylhomocysteine (SRH)[17 19]Then the enzyme S-ribosylhomocysteine lyase (LuxS)further cleaves SRH to homocysteine and 45-dihydroxy-23-pentanedione the precursor of the AI-2 QS molecule(Figure 7) Sinefungin is the analog of SAM in which theSndashCH

3(sulfonium moiety) of SAM is replaced by a Cndash

NH3(amine)Therefore sinefungin inhibits SAM-dependent

nucleic acid methyltransferases by competing with SAM foroccupancy of the methyl donor site on the enzyme [27 4344] Thus the presence of sinefungin may interfere with themethylation process and downregulate luxS and pfs genesresulting in an imbalance of SAH SRH and MTA Similarlythe growth defectswere observed in theNeisseriameningitidispfs and luxS mutants either due to the accumulation of toxicSAH and MTA or due to metabolic imbalances within thebacteria [45]

The decarboxylated SAM is involved in biosynthesis ofspermidine a polyamine involved in biofilms Spermidinesynthase encoded by speE catalyzes the production of spermi-dine fromputrescine and decarboxylated SAM [46] Previousstudies reported speE a virulence gene involved in pneu-mococcal colonization pneumonia and invasive infections[47] The low expression of speE gene in biofilms grownwith sinefungin indicates that sinefungin also interfere inspermidine biosynthesis

Only a few molecules have proven effective againstpneumococcal biofilms Domenech et al reported 80reduction of pneumococcal biofilms growth with theamidase LytA and recently Sumitomo et al reported thatS-carboxymethylcysteine inhibits pneumococcal adhesion tohuman alveolar epithelial cells [48 49] Similarly Trappettiet al showed that neuraminidase inhibitors DANA (ie 23-didehydro-2-deoxy-N-acetylneuraminic acid) zanamivirand oseltamivir inhibit the capacity of pneumococci toform sialic acid-dependent biofilms [50] Here we first timeshowed that sinefungin significantly inhibits in vitro biofilmgrowth and in vivomiddle ear colonization of pneumococci

In conclusion sinefungin decreases in vitro pneumococcibiofilm growth decreases formation of organised biofilmsdecreases AI-2 production and downregulates expressions ofluxS pfs and speE genes Sinefungin also decreases in vivocolonization of pneumococci in themiddle earTherefore theS-adenosylmethionine (SAM) inhibitors can be used as leadcompound for the development of novel antibiofilm agentsagainst pneumococci

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper


Acknowledgment

This research was supported by Basic Science ResearchProgram through the National Research Foundationof Korea (NRF) funded by the Ministry of Education(2013R1A1A2A10004451)

References

[1] P Ispahani R C Slack F E Donald V C Weston and NRutter ldquoTwenty year surveillance of invasive pneumococcal dis-ease in Nottingham serogroups responsible and implicationsfor immunisationrdquo Archives of Disease in Childhood vol 89 no8 pp 757ndash762 2004

[2] A Kadioglu J N Weiser J C Paton and P W AndrewldquoThe role of Streptococcus pneumoniae virulence factors inhost respiratory colonization and diseaserdquo Nature ReviewsMicrobiology vol 6 no 4 pp 288ndash301 2008

[3] L Hall-Stoodley F Z Hu A Gieseke et al ldquoDirect detectionof bacterial biofilms on the middle-ear mucosa of childrenwith chronic otitis mediardquo Journal of the American MedicalAssociation vol 296 no 2 pp 202ndash211 2006

[4] J W Costerton P S Stewart and E P Greenberg ldquoBacterialbiofilms a common cause of persistent infectionsrdquo Science vol284 no 5418 pp 1318ndash1322 1999

[5] R D Wolcott and G D Ehrlich ldquoBiofilms and chronic infec-tionsrdquo Journal of the AmericanMedical Association vol 299 no22 pp 2682ndash2684 2008

[6] R E Kania G E Lamers M J Vonk et al ldquoCharacterization ofmucosal biofilms on human adenoid tissuesrdquoTheLaryngoscopevol 118 no 1 pp 128ndash134 2008

[7] P V Vlastarakos T P Nikolopoulos P Maragoudakis ATzagaroulakis and E Ferekidis ldquoBiofilms in ear nose andthroat infections how important are theyrdquo The Laryngoscopevol 117 no 4 pp 668ndash673 2007

[8] G del Prado V Ruiz PNaves V Rodrıguez-Cerrato F Sorianoand M del Carmen Ponte ldquoBiofilm formation by Streptococcuspneumoniae strains and effects of human serum albuminibuprofen N-acetyl-l-cysteine amoxicillin erythromycin andlevofloxacinrdquo Diagnostic Microbiology and Infectious Diseasevol 67 no 4 pp 311ndash318 2010

[9] M Garcıa-Castillo M I Morosini A Valverde et al ldquoDif-ferences in biofilm development and antibiotic susceptibilityamong Streptococcus pneumoniae isolates from cystic fibro-sis samples and blood culturesrdquo Journal of AntimicrobialChemotherapy vol 59 no 2 pp 301ndash304 2007

[10] L R Marks G I Parameswaran and A P HakanssonldquoPneumococcal interactions with epithelial cells are crucial foroptimal biofilm formation and colonization in vitro and invivordquo Infection and Immunity vol 80 no 8 pp 2744ndash27602012

[11] A Bala R Kumar and K Harjai ldquoInhibition of quorumsensing in Pseudomonas aeruginosa by azithromycin and itseffectiveness in urinary tract infectionsrdquo Journal of MedicalMicrobiology vol 60 no 3 pp 300ndash306 2011

[12] C A Martin A D Hoven and A M Cook ldquoTherapeuticfrontiers preventing and treating infectious diseases by inhibit-ing bacterial quorum sensingrdquo European Journal of ClinicalMicrobiology and Infectious Diseases vol 27 no 8 pp 635ndash6422008

[13] C Trappetti L Gualdi L di Meola et al ldquoThe impact of thecompetence quorum sensing system on Streptococcus pneumo-niae biofilms varies depending on the experimental modelrdquoBMCMicrobiology vol 11 article 75 2011

[14] J E Vidal H P Ludewick R M Kunkel D Zahner andK P Klugman ldquoThe luxs-dependent quorum-sensing systemregulates early biofilm formation by Streptococcus pneumoniaestrain D39rdquo Infection and Immunity vol 79 no 10 pp 4050ndash4060 2011

[15] A L Beeston and M G Surette ldquopfs-dependent regulationof autoinducer 2 production in Salmonella enterica serovarTyphimuriumrdquo Journal of Bacteriology vol 184 no 13 pp3450ndash3456 2002

[16] B L Bassler E P Greenberg and A M Stevens ldquoCross-speciesinduction of luminescence in the quorum-sensing bacteriumVibrio harveyirdquo Journal of Bacteriology vol 179 no 12 pp4043ndash4045 1997

[17] G D Markham and M A Pajares ldquoStructure-function rela-tionships in methionine adenosyltransferasesrdquo Cellular andMolecular Life Sciences vol 66 no 4 pp 636ndash648 2009

[18] J Sun R Daniel I Wagner-Dobler and A P Zeng ldquoIsautoinducer-2 a universal signal for interspecies communica-tion a comparative genomic and phylogenetic analysis of thesynthesis and signal transduction pathwaysrdquo BMCEvolutionaryBiology vol 4 article 36 2004

[19] K Winzer K R Hardie N Burgess et al ldquoLuxS its role incentral metabolism and the in vitro synthesis of 4-hydroxy-5-methyl-3(2H)-furanonerdquoMicrobiology vol 148 part 4 pp 909ndash922 2002

[20] R T Borchardt ldquoS-adenosyl-L-methionine-dependent macro-molecule methyltransferases potential targets for the design ofchemotherapeutic agentsrdquo Journal of Medicinal Chemistry vol23 no 4 pp 347ndash357 1980

[21] N Parveen and K A Cornell ldquoMethylthioadenosineS-adenosylhomocysteine nucleosidase a critical enzyme for bac-terial metabolismrdquoMolecular Microbiology vol 79 no 1 pp 7ndash20 2011

[22] C Barbes J Sanchez M J Yebra M Robert-Gero and CHardisson ldquoEffects of sinefungin and S-adenosylhomocysteineon DNA and protein methyltransferases from Streptomyces andother bacteriardquo FEMS Microbiology Letters vol 69 no 3 pp239ndash243 1990

[23] RW Fuller and RNagarajan ldquoInhibition ofmethyltransferasesby some new analogs of S-adenosylhomocysteinerdquo BiochemicalPharmacology vol 27 no 15 pp 1981ndash1983 1978

[24] M T McCammon and L W Parks ldquoInhibition of steroltransmethylation by S-adenosylhomocysteine analogsrdquo Journalof Bacteriology vol 145 no 1 pp 106ndash112 1981

[25] P Paolantonacci F Lawrence and M Robert-Gero ldquoDifferen-tial effect of sinefungin and its analogs on the multiplication ofthreeLeishmania speciesrdquoAntimicrobial Agents andChemother-apy vol 28 no 4 pp 528ndash531 1985

[26] O Perez-Leal C Moncada A B Clarkson and S MeralildquoPneumocystis S-adenosylmethionine transport a potentialdrug targetrdquoAmerican Journal of Respiratory Cell andMolecularBiology vol 45 no 6 pp 1142ndash1146 2011

[27] S Zheng S Hausmann Q Liu et al ldquoMutational analysisof Encephalitozoon cuniculi mRNA cap (guanine-N7) methyl-transferase structure of the enzyme bound to sinefungin andevidence that cap methyltransferase is the target of sinefunginsantifungal activityrdquoThe Journal of Biological Chemistry vol 281no 47 pp 35904ndash35913 2006


[28] M R Parsek D L Val B L Hanzelka J E Cronan Jr and EP Greenberg ldquoAcyl homoserine-lactone quorum-sensing signalgenerationrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 96 no 8 pp 4360ndash4365 1999

[29] S T Miller K B Xavier S R Campagna et al ldquoSalmonellatyphimurium recognizes a chemically distinct form of thebacterial quorum-sensing signal AI-2rdquo Molecular Cell vol 15no 5 pp 677ndash687 2004

[30] T F Holzman P L Riley and T O Baldwin ldquoInactivationof luciferase from the luminous marine bacterium Beneckeaharveyi by proteases evidence for a protease labile region andproperties of the protein following inactivationrdquo Archives ofBiochemistry and Biophysics vol 205 no 2 pp 554ndash563 1980

[31] M R Oggioni F Iannelli S Ricci et al ldquoAntibacterial activityof a competence-stimulating peptide in experimental sepsiscaused by Streptococcus pneumoniaerdquo Antimicrobial Agents andChemotherapy vol 48 no 12 pp 4725ndash4732 2004

[32] M Moscoso E Garcıa and R Lopez ldquoBiofilm formation byStreptococcus pneumoniae role of choline extracellular DNAand capsular polysaccharide in microbial accretionrdquo Journal ofBacteriology vol 188 no 22 pp 7785ndash7795 2006

[33] M K Yadav S Chae and J Song ldquoIn vitro Streptococcuspneumoniae biofilm formation and in vivo middle ear mucosalbiofilm in a ratmodel of acute otitis induced by S pneumoniaerdquoClinical and Experimental Otorhinolaryngology vol 5 no 3 pp139ndash144 2012

[34] M K Yadav S W Chae and J J Song ldquoEffect of 5-azacytidineon in vitro biofilm formation of Streptococcus pneumoniaerdquoMicrobial Pathogenesis vol 53 no 5-6 pp 219ndash226 2012

[35] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔ119862119879 methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[36] A Lizcano T Chin K Sauer E I Tuomanen and C JOrihuela ldquoEarly biofilm formation on microtiter plates is notcorrelated with the invasive disease potential of Streptococcuspneumoniaerdquo Microbial Pathogenesis vol 48 no 3-4 pp 124ndash130 2010

[37] J E Vidal K E Howery H P Ludewick P Nava and K PKlugman ldquoQuorum-sensing systems LuxSAutoinducer 2 andcom regulate Streptococcus pneumoniae biofilms in a bioreactorwith living cultures of human respiratory cellsrdquo Infection andImmunity vol 81 no 4 pp 1341ndash1353 2013

[38] J M Zingg J C Shen A S Yang H Rapoport and P AJones ldquoMethylation inhibitors can increase the rate of cytosinedeamination by (cytosine-5)-DNA methyltransferaserdquo NucleicAcids Research vol 24 no 16 pp 3267ndash3275 1996

[39] J Gutierrez T Crowder A Rinaldo-Matthis M C Ho S CAlmo and V L Schramm ldquoTransition state analogs of 51015840-methylthioadenosine nucleosidase disrupt quorum sensingrdquoNature Chemical Biology vol 5 no 4 pp 251ndash257 2009

[40] U H Stroeher A W Paton A D Ogunniyi and J C PatonldquoMutation of luxS of Streptococcus pneumoniae affects virulencein a mouse modelrdquo Infection and Immunity vol 71 no 6 pp3206ndash3212 2003

[41] S Schauder K Shokat M G Surette and B L Bassler ldquoTheLuxS family of bacterial autoinducers biosynthesis of a novelquorum-sensing signal moleculerdquoMolecular Microbiology vol41 no 2 pp 463ndash476 2001

[42] S Romao G Memmi M R Oggioni andM C Trombe ldquoLuxSimpacts on LytA-dependent autolysis and on competence inStreptococcus pneumoniaerdquoMicrobiology vol 152 no 2 pp 333ndash341 2006

[43] J R Horton K Liebert S Hattman A Jeltsch and X ChengldquoTransition fromnonspecific to specificDNA interactions alongthe substrate-recognition pathway of dam methyltransferaserdquoCell vol 121 no 3 pp 349ndash361 2005

[44] G Schluckebier M Kozak N Bleimling E Weinhold andW Saenger ldquoDifferential binding of S-adenosylmethionine S-adenosylhomocysteine and sinefungin to the adenine-specificDNA methyltransferase M TaqIrdquo Journal of Molecular Biologyvol 265 no 1 pp 56ndash67 1997

[45] K Heurlier A Vendeville N Halliday et al ldquoGrowth deficien-cies of Neisseria meningitidis pfs and luxS mutants are not dueto inactivation of quorum sensingrdquo Journal of Bacteriology vol191 no 4 pp 1293ndash1302 2009

[46] P Shah and E Swiatlo ldquoA multifaceted role for polyamines inbacterial pathogensrdquoMolecular Microbiology vol 68 no 1 pp4ndash16 2008

[47] P Shah B Nanduri E Swiatlo Y Ma and K PendarvisldquoPolyamine biosynthesis and transport mechanisms are crucialfor fitness and pathogenesis of Streptococcus pneumoniaerdquoMicrobiology vol 157 no 2 pp 504ndash515 2011

[48] M Domenech E Garcia and M Moscoso ldquoIn vitro destruc-tion of Streptococcus pneumoniae biofilms with bacterial andphage peptidoglycan hydrolasesrdquo Antimicrobial Agents andChemotherapy vol 55 no 9 pp 4144ndash4148 2011

[49] T Sumitomo M Nakata M Yamaguchi Y Terao and S Kawa-bata ldquoS-carboxymethylcysteine inhibits adherence of Strepto-coccus pneumoniae to human alveolar epithelial cellsrdquo Journalof Medical Microbiology vol 61 no 1 pp 101ndash108 2012

[50] C Trappetti A Kadioglu M Carter et al ldquoSialic acid apreventable signal for pneumococcal biofilm formation colo-nization and invasion of the hostrdquo Journal of InfectiousDiseasesvol 199 no 10 pp 1497ndash1505 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


the byproducts of AMC such as 51015840-methylthioadenosineS-adenosylhomocysteine (MTASAH) are toxic to the bacteria[15] In S pneumoniae detoxification of SAH is carried outby MTASAH nucleosidase (Pfs) and S-ribosylhomocysteinelyase (LuxS) and this pathway is absent in human [17ndash19]

The SAM is a central molecule of AMC involvedin recycling of methionine methylation of biomoleculesbiosynthesis of AI-2 and polyamine biosynthesis [20 21]Therefore we hypothesized that altering the SAM activitycould have adverse effect on pneumococcal biofilms Here weevaluated the effect of sinefungin a nucleoside analogue of S-adenosylmethionine on pneumococcal in vitro biofilm

Sinefungin is a natural nucleoside and a structural analogof SAM It inhibits transmethylation reactions related toDNA RNA proteins and other molecules [22ndash25] Anti-fungal antiviral and antiprotozoal activities of sinefunginhave been reported [26 27] Previously Parsek et al stud-ied effect of sinefungin on Pseudomonas acyl homoserine-lactone quorum-sensing signal generation [28] However theeffect of sinefungin on S pneumoniae has not been studiedIn this study we examined the effect of sinefungin on Spneumoniae particularly on biofilm growth

2 Materials and Methods

21 Bacteria Strains and Culture Conditions S pneumoniaeD-39 strain (NCTC 7466) is an encapsulated serotype 2pathogenic strain It was obtained from theHealth ProtectionAgency Culture Collections (HPA Salisbury UK) Bacteriawere routinely grown in tryptic soy broth (TSB BD DifcoDetroit MI USA) or on blood agar (BA Hye In SeoulKorea) supplemented with 5 vv sheep blood at 37∘C in anatmosphere of 5 CO

2 Vibrio harveyi (V harveyi) strains

MM32 (ATCC BAA-1121) [29] were grown on autoinducerbioassay (AB) medium at 30∘C [30] Sinefungin (sc-203263)was purchased from Santa Cruz Biotechnology (Santa CruzCA USA)

22 Effect of Sinefungin on Planktonic Cell Growth S pneu-moniae were grown with different concentrations of sine-fungin (10 20 30 40 and 50 120583gmLminus1) at 37∘C in 5 CO

2

for different time points (0 hndash10 h) And optical densityat 600 nm (OD

600) was measured after 2 h time interval

using a SpectraMax plus 384 automated microplate reader(Molecular Devices Sunnyvale CA USA) After 6 h ofincubation 100120583L aliquots was serially diluted and platedon BA for enumeration of colony forming units (CFU) Thepercentage decrease in planktonic cell growth was calculatedby subtracting the values of sinefungin treated samples fromcontrol (untreated) samples The experiment was replicatedthree times with triplicate samples at each time point

23 Effect of Sinefungin on In Vitro Biofilm Growth In vitropneumococcal biofilm growth was carried out in 96-wellflat-bottom polystyrene microtiter plate (BD falcon SparksMD USA) using a static model [31] Briefly a fresh colonyof S pneumoniae grown overnight on BAP was scraped

and grown in TSB+1 glucose medium [32] The mid-logarithmic phase cell suspension (1 times 108 CFUmLminus1) wasdiluted 1 100 with fresh sterile TSB+1 medium and 200120583Laliquots were inoculated in wells of 96-well microtiter platesand incubated at 37∘C in 5 CO

2 After incubation the

medium was discarded and plates were gently washed threetimes with 200120583L sterile phosphate buffered saline (PBS)Thereafter plates were air-dried and stained with 50120583Lcrystal violet (CV 01) for 15min Excess stain was decantedoff and plates were washed three times with sterile distilledwater The biofilm was dissolved in 200120583L of 95 ethanoland the OD

570nm was measured using the aforementioned

automated spectrophotometerThedata represent the averagevalue of three replicates

24 Effect of Different Concentrations of Sinefungin onBiofilmGrowth To study the concentration-dependent effectof sinefungin on pneumococcal biofilms S pneumoniaebiofilms were grown with 10ndash50120583gmLminus1 sinefungin for18 h as described above Biofilm biomass was detected byCV-microtiter plate assay as described above The percent-age decrease in biomass was calculated by subtracting thebiomass of control biofilms grown without sinefungin Enu-meration of biofilmbacteriawas done as described above (ieCFU)

25 Effect of Sinefungin on Established Biofilms Pneumococ-cal biofilms were established in microtiter plate wells for 12 hThe formed biofilmswere exposed to different concentrationsof sinefungin for 6 h and analyzed by CV-microtiter plateassay and the cell viability was determined by CFU counts

26 Effect of Different Exposure Times of Sinefungin on BiofilmFormation Pneumococcal biofilms were generated in thepresence of 35 120583gmLminus1 sinefungin for 6 12 18 and 24 hAt each time point biofilm biomass was determined asdescribed above

27 Scanning Electron Microscopy (SEM) Biofilms In vitrobiofilms grown with 35 120583gmLminus1 sinefungin and withoutsinefungin in 24-well tissue culture plates for 18 h wereanalyzed by SEM The medium was removed and the plateswere gently washed two times with sterile PBS to removeplanktonic cells The samples were prefixed by immersion in2 glutaraldehyde in 01M phosphate buffer and postfixedfor 2 h in 1 osmic acid dissolved in PBS Samples weretreated in a graded series of ethanol and t-butyl alcohol driedin a model ES-2030 freeze dryer (Hitachi Tokyo Japan)platinum coated using an IB-5 ion coater (Eiko KanagawaJapan) and observed using a S-4700 field emission scanningelectron microscopic (Hitachi)

28 Autoinducer Assay V harveyi MM32 was used as thequalitative reporter strain to detect the changes in the produc-tion of AI-2 V harveyi MM32 (BB120 luxNTn5 luxSTn5)is an ideal reporter strain as it can sense AI-2 but cannotsynthesize AI-2 of its own [29]The reporter strain was grown














3 Results





0

005

01

015

02

025

03

035

0 2 4 6 8 10

Control

Opt

ical

den

sity

of p

lank

toni

c cel

l

Time (h)

grow

th at

600

nm

10120583g20120583g

30120583g40120583g50120583g

(a)

Control

(CFU

mL)

10120583g 20120583g 30120583g 40120583g 50120583g100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

100E + 09


(b)




0005

01015

02025

03035

04

Control

Opt

ical

den

sity

of cr

ysta

l vi

olet

at570

nm

lowastlowast lowast



(a)

(CFU

mL)

100E + 06

100E + 05

100E + 04

100E + 03

100E + 02

100E + 01

100E + 00



(b)

0

005

01

015

02

025

03

035

Opt

ical

den

sity

of cr

ysta

l vi

olet

at570

nm

lowast


(c)

(CFU

mL)

100E + 08

100E + 07

100E + 06

100E + 05

100E + 04

100E + 03

100E + 02

100E + 01

100E + 00

lowast


(d)



0

005

01

015

02

025

03

035

Control Sinefungin

Opt

ical

den

sity

of cr

ysta

l

lowastlowast

viol

et at

570

nm

(a)

0

005

01

015

02

025

03

035

Control Sinefungin

Opt

ical

den

sity

of cr

ysta

l vi

olet

at570

nm

lowast

(b)

0

005

01

015

02

025

03

035

Control Sinefungin

Opt

ical

den

sity

of cr

ysta

l

lowastlowast

viol

et at

570

nm

(c)

0

002

004

006

008

01

012

014

016

018

Control Sinefungin

Opt

ical

den

sity

of cr

ysta

l

lowastvi

olet

at570

nm

(d)








(a) (b) (c)

(d) (e) (f)


0500000

100000015000002000000250000030000003500000400000045000005000000


Lum

ines

cenc

e (RL

Us

)

lowast



Bacteria Sinefungin

CFU

coun

tsbu

lla

100E + 05

100E + 04

100E + 03

100E + 02

100E + 01

100E + 00

lowast






Substrate

Methylatedproduct

Adenine



Homocysteine

Methionine

Decarboxylated SAM

Putrescine



Adenine

ATPSAM synthetase

(MetK)


Methylthioadenosine






PPi + Pi

NH2

NH2S+

O

O

HO OH

NN

NN

NH2

S

O

O

HO

HO OH

OH

NH2

NH2

S

O

OHO

OH OH

N

NN

NH

O

OHOH

N N

NN

NH2

H3CS

NH2

NH2

S

O

O

OH

OHH3C

HS

CO2

minusO

H2O

H2O



4 Discussion






















Acknowledgment


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














3 Results





0

005

01

015

02

025

03

035

0 2 4 6 8 10

Control

Opt

ical

den

sity

of p

lank

toni

c cel

l

Time (h)

grow

th at

600

nm

10120583g20120583g

30120583g40120583g50120583g

(a)

Control

(CFU

mL)

10120583g 20120583g 30120583g 40120583g 50120583g100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

100E + 09


(b)




0005

01015

02025

03035

04

Control

Opt

ical

den

sity

of cr

ysta

l vi

olet

at570

nm

lowastlowast lowast



(a)

(CFU

mL)

100E + 06

100E + 05

100E + 04

100E + 03

100E + 02

100E + 01

100E + 00



(b)

0

005

01

015

02

025

03

035

Opt

ical

den

sity

of cr

ysta

l vi

olet

at570

nm

lowast


(c)

(CFU

mL)

100E + 08

100E + 07

100E + 06

100E + 05

100E + 04

100E + 03

100E + 02

100E + 01

100E + 00

lowast


(d)



0

005

01

015

02

025

03

035

Control Sinefungin

Opt

ical

den

sity

of cr

ysta

l

lowastlowast

viol

et at

570

nm

(a)

0

005

01

015

02

025

03

035

Control Sinefungin

Opt

ical

den

sity

of cr

ysta

l vi

olet

at570

nm

lowast

(b)

0

005

01

015

02

025

03

035

Control Sinefungin

Opt

ical

den

sity

of cr

ysta

l

lowastlowast

viol

et at

570

nm

(c)

0

002

004

006

008

01

012

014

016

018

Control Sinefungin

Opt

ical

den

sity

of cr

ysta

l

lowastvi

olet

at570

nm

(d)








(a) (b) (c)

(d) (e) (f)


0500000

100000015000002000000250000030000003500000400000045000005000000


Lum

ines

cenc

e (RL

Us

)

lowast



Bacteria Sinefungin

CFU

coun

tsbu

lla

100E + 05

100E + 04

100E + 03

100E + 02

100E + 01

100E + 00

lowast






Substrate

Methylatedproduct

Adenine



Homocysteine

Methionine

Decarboxylated SAM

Putrescine



Adenine

ATPSAM synthetase

(MetK)


Methylthioadenosine






PPi + Pi

NH2

NH2S+

O

O

HO OH

NN

NN

NH2

S

O

O

HO

HO OH

OH

NH2

NH2

S

O

OHO

OH OH

N

NN

NH

O

OHOH

N N

NN

NH2

H3CS

NH2

NH2

S

O

O

OH

OHH3C

HS

CO2

minusO

H2O

H2O



4 Discussion






















Acknowledgment


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

005

01

015

02

025

03

035

0 2 4 6 8 10

Control

Opt

ical

den

sity

of p

lank

toni

c cel

l

Time (h)

grow

th at

600

nm

10120583g20120583g

30120583g40120583g50120583g

(a)

Control

(CFU

mL)

10120583g 20120583g 30120583g 40120583g 50120583g100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

100E + 09


(b)




0005

01015

02025

03035

04

Control

Opt

ical

den

sity

of cr

ysta

l vi

olet

at570

nm

lowastlowast lowast



(a)

(CFU

mL)

100E + 06

100E + 05

100E + 04

100E + 03

100E + 02

100E + 01

100E + 00



(b)

0

005

01

015

02

025

03

035

Opt

ical

den

sity

of cr

ysta

l vi

olet

at570

nm

lowast


(c)

(CFU

mL)

100E + 08

100E + 07

100E + 06

100E + 05

100E + 04

100E + 03

100E + 02

100E + 01

100E + 00

lowast


(d)



0

005

01

015

02

025

03

035

Control Sinefungin

Opt

ical

den

sity

of cr

ysta

l

lowastlowast

viol

et at

570

nm

(a)

0

005

01

015

02

025

03

035

Control Sinefungin

Opt

ical

den

sity

of cr

ysta

l vi

olet

at570

nm

lowast

(b)

0

005

01

015

02

025

03

035

Control Sinefungin

Opt

ical

den

sity

of cr

ysta

l

lowastlowast

viol

et at

570

nm

(c)

0

002

004

006

008

01

012

014

016

018

Control Sinefungin

Opt

ical

den

sity

of cr

ysta

l

lowastvi

olet

at570

nm

(d)








(a) (b) (c)

(d) (e) (f)


0500000

100000015000002000000250000030000003500000400000045000005000000


Lum

ines

cenc

e (RL

Us

)

lowast



Bacteria Sinefungin

CFU

coun

tsbu

lla

100E + 05

100E + 04

100E + 03

100E + 02

100E + 01

100E + 00

lowast






Substrate

Methylatedproduct

Adenine



Homocysteine

Methionine

Decarboxylated SAM

Putrescine



Adenine

ATPSAM synthetase

(MetK)


Methylthioadenosine






PPi + Pi

NH2

NH2S+

O

O

HO OH

NN

NN

NH2

S

O

O

HO

HO OH

OH

NH2

NH2

S

O

OHO

OH OH

N

NN

NH

O

OHOH

N N

NN

NH2

H3CS

NH2

NH2

S

O

O

OH

OHH3C

HS

CO2

minusO

H2O

H2O



4 Discussion






















Acknowledgment


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

005

01

015

02

025

03

035

Control Sinefungin

Opt

ical

den

sity

of cr

ysta

l

lowastlowast

viol

et at

570

nm

(a)

0

005

01

015

02

025

03

035

Control Sinefungin

Opt

ical

den

sity

of cr

ysta

l vi

olet

at570

nm

lowast

(b)

0

005

01

015

02

025

03

035

Control Sinefungin

Opt

ical

den

sity

of cr

ysta

l

lowastlowast

viol

et at

570

nm

(c)

0

002

004

006

008

01

012

014

016

018

Control Sinefungin

Opt

ical

den

sity

of cr

ysta

l

lowastvi

olet

at570

nm

(d)








(a) (b) (c)

(d) (e) (f)


0500000

100000015000002000000250000030000003500000400000045000005000000


Lum

ines

cenc

e (RL

Us

)

lowast



Bacteria Sinefungin

CFU

coun

tsbu

lla

100E + 05

100E + 04

100E + 03

100E + 02

100E + 01

100E + 00

lowast






Substrate

Methylatedproduct

Adenine



Homocysteine

Methionine

Decarboxylated SAM

Putrescine



Adenine

ATPSAM synthetase

(MetK)


Methylthioadenosine






PPi + Pi

NH2

NH2S+

O

O

HO OH

NN

NN

NH2

S

O

O

HO

HO OH

OH

NH2

NH2

S

O

OHO

OH OH

N

NN

NH

O

OHOH

N N

NN

NH2

H3CS

NH2

NH2

S

O

O

OH

OHH3C

HS

CO2

minusO

H2O

H2O



4 Discussion






















Acknowledgment


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b) (c)

(d) (e) (f)


0500000

100000015000002000000250000030000003500000400000045000005000000


Lum

ines

cenc

e (RL

Us

)

lowast



Bacteria Sinefungin

CFU

coun

tsbu

lla

100E + 05

100E + 04

100E + 03

100E + 02

100E + 01

100E + 00

lowast






Substrate

Methylatedproduct

Adenine



Homocysteine

Methionine

Decarboxylated SAM

Putrescine



Adenine

ATPSAM synthetase

(MetK)


Methylthioadenosine






PPi + Pi

NH2

NH2S+

O

O

HO OH

NN

NN

NH2

S

O

O

HO

HO OH

OH

NH2

NH2

S

O

OHO

OH OH

N

NN

NH

O

OHOH

N N

NN

NH2

H3CS

NH2

NH2

S

O

O

OH

OHH3C

HS

CO2

minusO

H2O

H2O



4 Discussion






















Acknowledgment


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Substrate

Methylatedproduct

Adenine



Homocysteine

Methionine

Decarboxylated SAM

Putrescine



Adenine

ATPSAM synthetase

(MetK)


Methylthioadenosine






PPi + Pi

NH2

NH2S+

O

O

HO OH

NN

NN

NH2

S

O

O

HO

HO OH

OH

NH2

NH2

S

O

OHO

OH OH

N

NN

NH

O

OHOH

N N

NN

NH2

H3CS

NH2

NH2

S

O

O

OH

OHH3C

HS

CO2

minusO

H2O

H2O



4 Discussion






















Acknowledgment


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


















Acknowledgment


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Acknowledgment


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Sinefungin, a Natural Nucleoside …downloads.hindawi.com/journals/bmri/2014/156987.pdfResearch Article Sinefungin, a Natural Nucleoside Analogue of S-Adenosylmethionine,