Research Article Characterization of the algC Gene Expression Pattern in the Multidrug Resistant ...

15 Pages·2015·6.32 MB·English

Hindawi Publishing Corporation
e Scientific World Journal
Volume 2014, Article ID 593546, 14 pages
http://dx.doi.org/10.1155/2014/593546
Research Article
Characterization of the algC Gene Expression Pattern in
the Multidrug Resistant Acinetobacter baumannii AIIMS 7 and
Correlation with Biofilm Development on Abiotic Surface
PraveenK.Sahu,1,2PavithraS.Iyer,1SagarH.Barage,3
KailasD.Sonawane,4andBaluA.Chopade1,5
1InstituteofBioinformaticsandBiotechnology,UniversityofPune,Pune411007,India
2IspatGeneralHospital,SAIL,Rourkela769005,India
3DepartmentofBiotechnology,ShivajiUniversity,Kolhapur416004,India
4StructuralBioinformaticsUnit,DepartmentofBiochemistry,ShivajiUniversity,Kolhapur416004,India
5Dr.BabasahebAmbedkarMarathwadaUniversity,Aurangabad431001,India
CorrespondenceshouldbeaddressedtoPraveenK.Sahu;[email protected]
Received30July2014;Revised10November2014;Accepted10November2014;Published3December2014
AcademicEditor:JoseCorreaBasurto
Copyright©2014PraveenK.Sahuetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,
whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
RelativequantificationofalgCgeneexpressionwasevaluatedinthemultidrugresistantstrainAcinetobacterbaumanniiAIIMS
7 biofilm (3 to 96h, on polystyrene surface) compared to the planktonic counterparts. Comparison revealed differential algC
expressionpatternwithmaximum81.59-foldincreaseinbiofilmcellsversus3.24-foldinplanktoniccells(𝑃 < 0.05).Expression
levelsstronglycorrelatedwithspecificbiofilmstages(scaleof3to96h),coincidingmaximumatinitialsurfaceattachmentstage(9h)
andbiofilmmaturationstage(48h).Cloning,heterologousexpression,andbioinformaticsanalysesindicatedalgCgeneproductas
thebifunctionalenzymephosphomannomutase/phosphoglucomutase(PMM/PGM)of∼53kDasize,whichaugmentedbiofilms
significantlyinalgCclonescomparedtocontrols(lackingalgCgene),furtherlocalizedbyscanningelectronmicroscopy.Moreover,
moleculardynamicsanalysisonthethree-dimensionalstructureofPMM/PGM(simulatedupto10ns)revealedenzymestructureas
stableandsimilartothatinP.aeruginosa(synthesisofalginateandlipopolysaccharidecore)andinvolvedinconstitutionofbiofilm
EPS(extracellular polymericsubstances).Our observationondifferential expressionpatternofalgC havingstrongcorrelation
withimportantbiofilmstages,scanningelectron-microscopicevidenceofbiofilmaugmentationtakentogetherwithpredictive
enzymefunctionsviamoleculardynamic(MD)simulation,proposesanewbasisofA.baumanniiAIIMS7biofilmdevelopment
oninanimatesurfaces.
1.Introduction biofilmformation[7]oneitherabiotic[8,9]orbioticsurfaces
[10,11].BiofilmformationisavirulencetraitinA.baumannii
In recent years, Acinetobacter baumannii has been listed as whichisofmultifactorialnature[4,12].Theprocessofbiofilm
oneofthemostimportantnosocomialpathogens[1–3].The development in A. baumannii is a highly regulated process
pathogen has become a universal challenge to treatment, and could be the interplay of several genetic determinants
owingtoitsmultidrugresistant(MDR)natureandaplethora [13].Theextracellularmatricesofbacterialbiofilmcomprise
of virulence attributes [2, 4, 5]. Associated mortality up ofproteins,nucleicacids,andpolysaccharides[14]whichare
to 30% is seen with A. baumannii infections [6], such as often considered as ideal start-points to further investiga-
ventilator-associatedpneumonia,bacteraemia,urinarytract tion towards effective treatment measures against biofilm-
infections, burn wound infections, endocarditis, secondary associatedpathogens,suchasMDRA.baumannii.
meningitis,andsepticemiaespeciallyinintensivecareunits Acinetobacterspp.andPseudomonasaeruginosatogether
[1]. A. baumannii infection and colonization often involve are known to be responsible for a significant proportion of
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nosocomialinfections[15]withcrudemortalityratesof30% biofilmdevelopment.Moleculardynamics(MD)simulation
to75%incaseofnosocomialpneumoniaonly[16].Inpatients wasperformedonthethreedimensional(3D)structureofthe
with cystic fibrosis, alginate production by P. aeruginosa is geneproduct(enzymePMM/PGM)toconfirmthestability
foundtobeassociatedwithhighmorbidityandmortality[17, and to compare with that of P. aeruginosa, besides phylo-
18].Productionofalginate,anexopolysaccharide,isrespon- geneticanalysis.Subsequently,biofilmaugmentationdueto
siblefordevelopmentofmucoidbacterialphenotype[17,19] algC gene was evaluated using cloning and heterologous
which is associated with biofilm formation in P. aeruginosa expressionstudiesfollowedbyscanningelectronmicroscopy.
underironlimitingconditions[20];althoughproportionsof
alginate in the extracellular polysaccharide/polymeric sub-
2.MaterialsandMethods
stances(EPS)ofbiofilmcouldvarysignificantlyinstrainslike
PA14andPA01ofP.aeruginosa[21].Itactsasanintercellular
2.1. Bacterial Strains and Culture Conditions. Multidrug
material in complex biofilm structures and facilitates non-
resistant clinical isolate of Acinetobacter baumannii (strain
specific attachment of bacteria to surfaces, thus increasing
AIIMS 7) was used in this study, as described previously
cohesion [22]. Biosynthesis of alginate is well characterized
[9]. The bacterium was grown on CLED (cysteine-lactose
in P. aeruginosa [23, 24] and Escherichia coli [25]. The gene
electrolyte-deficient) agar and Luria Bertani (LB) broth
algCinP.aeruginosacodesforacrucialbifunctionalenzyme (HiMedia,India).E.coliDH5𝛼wasusedforthecloningand
phosphomannomutase/phosphoglucomutase (PMM/PGM),
heterologous expression experiments. Clones were main-
belongingtothe𝛼-D-hexomutasesuperfamily,synthesizing
tainedonLuriaagarplatescontaining100mg/Lampicillin.
alginateandlipopolysaccharide(LPS)core,respectively[24,
26,27].ThegeneticregulationofalgCgeneinP.aeruginosa
2.2.NucleicAcidsPurificationandPCR. GenomicDNAwas
[23, 24, 28] could be dependent on surface attachment and
purified using a DNA isolation kit (Sigma Aldrich, USA).
otherimportantfactors.
Total RNA and plasmid purification was done using Trizol
Earlierstudyshowsbiofilmformationbyclinicalstrains
reagent(Invitrogen,USA)andGenEluteplasmidextraction
A.baumanniionabioticsurface(urinarycatheters)asamajor
kit (Sigma Aldrich, USA) respectively, according to manu-
reasonfordevice-relatedinfections[29].Toexplorethebasis
facturer’s instructions. Concentration and purity criteria of
ofpersistenceonsuchclinicallyimportant(abiotic)surfaces,
the nucleic acids were quantified in a BioPhotometer Plus
wecharacterizedtheroleofextracellularmacromoleculeslike
(Eppendorf, Germany). Primers used for amplification of
extracellularDNA(eDNA),amajorcomponentofthebiofilm
directPCRforalgCandRT-PCRofinternalregionsofalgC
EPS matrix and evaluated its role in biofilm formation [9].
are listed in Table1. Primers were designed using Primer
Besides eDNA, other extracellular macromolecules such as
Quest (IDT, USA) and synthesized (Sigma Aldrich, USA).
exopolysaccharides also play significant role in the consti-
Foramplificationofthe1781bpfragmentalgCfromgenomic
tutionofthebiofilmEPS.RecentfindingsontheEPSof A.
DNA,PCRconditionsusedwereinitialdenaturationof5min
baumanniiindicateitasauniversalprotectorfromantibiotics ∘ ∘
at94 C,followedby30cycles of30secat94 C,20seconds
liketobramycin[30]andoneofitscapsularpolysaccharides ∘ ∘
at 54.5 C, and 45 seconds at 72 C with final extension of 5
(K1)beinghighlyimmunogenicandapotentialtherapeutic ∘
minutesat72 C.AmplificationofalgCwasalsoverifiedwith
target via passive immunization [31]. The synthesis of spe-
nativeplasmidsasPCRtemplate.PCRassayswereperformed
cific exopolysaccharides like alginate and LPS core is well
inanep-gradientPCR(Eppendorf,Germany);productswere
characterized in P. aeruginosa along with their involvement
separatedonagarosegelsstainedwithethidiumbromideand
inbiofilmformationasmentionedearlier;however,remains
documented in a gel documentation system (Alpha Imager
unclear in A. baumannii, which is a close relative and a
HP, Alpha Innotech, USA). DNase-, RNase-, Protease-free
globally important nosocomial pathogen. Presumably, the
water was used as negative control. All PCR chemicals and
geneticbasisoftheassociationofexopolysaccharidessuchas
reagentswerepurchasedfromSigmaunlessstatedotherwise.
alginateandLPScorewithbiofilmformationmayexacerbate
A. baumannii infections on clinically important surface,
which can multiply the intrinsic antibiotic resistance and 2.3. Confirmation of Internal Regions of cDNA and DNA
worsenthetreatmentscenario.However,therearenoreports Sequencing. ToconfirmthetranscriptionofalgC,twointer-
as yet, which describe the genetic association of algC with nalregionsofalgCcDNA(1360bpand463bp)wereampli-
biofilmformationinA.baumannii.Thereforeinthisstudy,we fied using upstream primer 363F and CD1701R and inter-
setouttocharacterizetheA.baumanniialgCgeneexpression nal nested primers 1260 nest1F and CD1701R, respectively
patternduringbiofilmformationanditsassociationwiththe (Table1).TotalRNAsamplesweretreatedwithDNaseIand
biofilmdevelopment,whichcouldfurtherpavewayforfuture thenusedinRT-PCR,whichwasperformedusingasingle-
research on potential drug target(s) for biofilm-associated stepReverseTranscriptionkit(Promega,USA).Totestpossi-
infectionscausedbyMDRA.baumannii. bleDNAcontaminationinRNAsamples,directPCRoftotal
In the current study, we identified the algC gene in RNA without reverse transcription was performed. DNase-
the MDR strain of A. baumannii AIIMS 7 genome and , RNase-, Protease-free water was used as negative control.
characterized its quantitative gene expression patterns in RT-PCRwasperformedwithone𝜇LofcDNAtemplateand
growingbiofilmcellscomparedtotheplanktoniccellsusing 100pm of primers. cDNA from E. coli DH5𝛼 with pGEM-
therelativequantification(ΔΔCtmethod)inreal-timePCR. 3Zf (+) plasmid as emptyvector(AppliedBiosystem, USA)
Geneexpressionpatternwascorrelatedwithspecificstagesof servedasnegativecontrol.DNAsequencingwasperformed
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Table1:Oligos/probesusedforPCR,RT-PCR,andreal-timePCRanalysis.
󸀠 󸀠
Gene Primer 5-3 Nucleotidesequence
118 F TTAGAACCGGGTGAGCGTTTAGCA
algC
1897 R AGATGCTGATCTTGTGGCATTGCG
ALGC1 F GCTGTGAAGTCATTGCTTTACGT
𝑎𝑙𝑔𝐶𝜓
ALGC1 R TGAAGGGTCTGGTGCATGATC
ALGC1 MFAM 6FAM-CAAATGGTGAGTTCCC-TAMRA
CD363F CCGTGCTTACGATATTAGAGGCAA
algCcDNAint region1
CD1701R AATTTGCGGATAACGCTCTTGC
1260nest1 CTTCCTCCGCGCGTATTTATC
algCcDNAint region2
CD1701R AATTTGCGGATAACGCTCTTGC
16B-F TGGCTCAGATTGAACGCTGGCGGC
16SrRNA
16B-R TACCTTGTTACGACTTCACCCCA
16SRR F TGTCGTGAGATGTTGGGTTAAGTC
16𝑆𝑟𝑅𝑁𝐴𝜓
16SRR R CCGAAATGCTGGCAAGTAAGGA
16SRR MFAM 6FAM-ACGAGCGCAACCCTTT-TAMRA
𝜓
Primersforreal-timePCRassays.
in the Applied Biosystems 3730 DNA Analyzer platform. 2.5.RelativeQuantificationofalgCExpressionPatternbyReal-
Sequenceswereanalyzedbysequenceanalysissoftwarev5.1.1 TimePCR. SpecificprimersandprobesforalgCgenealong
(Applied Biosystems, USA) and assembled using software with 16SrRNA gene (endogenous control) were designed
ContigExpress (Vector NTI Advance v11.5.0, Invitrogen). and synthesized as “assay-by-design” from Applied Biosys-
Promoter and the ribosome binding sites of algC were pre- tems as listed in Table1. Prior to proceeding with relative
dictedusingonlinetoolNeuralNetworkPromoterPrediction quantification, the cDNA template of 24 hour old biofilm
program(http://www.fruitfly.org/seq tools/promoter.html). sampleswith10-foldserialdilutionswasusedtoanalyzethe
standard curves of both algC and 16srRNA gene. Real-time
2.4. Total RNA Extraction and cDNA Synthesis from Biofilm PCRwasperformedin10𝜇Lreactionsin96-wellPCRplates
andPlanktonicA.baumanniiCells. Tocomparegeneexpres- using100ngcDNA,2XTaqMangeneexpressionmastermix
sionpatternofalgCinbiofilmversusplanktonicA.bauman- (Applied Biosystems, USA) and 20X algC assay mix (20X
niiAIIMS7,totalRNAwasextractedfromcellsgrowingin 16SrRNA assay mix in case of controls) in a 7500 fast real-
biofilmandplanktonicmodeandthenreal-timequantitative time PCR system (Applied Biosystems, USA). The relative
RT-PCRwasperformed.Biofilmswereallowedtoformin6- numberofalgCmRNAwasdeterminedusingΔΔCt-method
wellpolystyreneplates(Tarsons,India)beginningat3hours (comparative threshold cycle) by normalizing Ct values of
6
upto96hours.A.baumanniiAIIMS7cultures(10 CFU/mL, algCmRNAwiththatof16srRNAinbiofilmandplanktonic
overnightgrown)wereinoculatedontothewellsanddiluted samples at various time points from 3 to 96 hours. Relative
initially with sterile distilled water (without LB broth) at a quantification (RQ) represented in “fold over increase” was
∘
dilutionof1:40.Afterincubationfor3hoursat37 Cunder determined according to methods described elsewhere [32]
static conditions, the supernatant was discarded and fresh andtheformulausedforcalculationwasRQ=2−ΔΔCt.Cutoff
sterile nutrient medium (Luria broth) was added at a final
Ctwaskept35,withanautomaticthresholdof0.2andbase-
dilutionof1:40andthenincubatedundersimilarconditions
linefromcycle3–15with95%confidencelevel.The24-hour
toallowbiofilmformation.Afterappropriateincubation(s),
biofilmsamplewassetasthecalibratorforthiscomparative
platesweretakenout;nonadherentcellswereaspiratedand
geneexpressionanalysis.StatisticalcomparisonofRQvalues
discarded after brief sonication. Plates containing biofilms
(between biofilm and planktonic samples) was performed
were washed twice with phosphate buffer saline (PBS).
usingStudent’s𝑡-testand𝑃value<0.05wasconsideredtobe
Surface-attachedbacteria(biofilmforming)werescrapedoff
statisticallysignificant.
andtotalRNAwaspurifiedusingTrizolreagent(Invitrogen,
USA)aspermanufacturer’sinstructions.Similarly,totalRNA
2.6.BiofilmDevelopmentAssay. Biofilmdevelopmentassays
waspurifiedfromplanktoniccellcultureatthecorrespond-
wereperformedtoassessthepatternofbiofilmformationof
ing time points (3–96h). RNA samples were treated with
A.baumanniiAIIMS7onpolystyrenesurfaceandforfurther
DNaseI(Sigma,USA).TotestpossibleDNAcontamination
in RNA samples, direct PCR of total RNA without reverse correlationwithgeneexpressionplotoverthesametimeline.
transcriptionwasdone.cDNAwassynthesizedusingVerso Beginning at 3h up to 96h, quantitative biofilm assay was
cDNA synthesis kit (Thermo, USA) as per manufacturer’s performedon96-wellmicrotitreplates,accordingtoearlier
instructions. cDNA concentrations were determined using methods [10] with appropriate modifications. Briefly, A.
6
BioPhotometerPlus(Eppendorf,Germany). baumanniiAIIMS7cultures(10 CFU/mL,overnightgrown)
4 TheScientificWorldJournal
wereinoculatedontopolystyrenemicrotitrewellsofa96-well USA).AminoacidsequencesweretranslatedusingTranslate
plate(Tarsons,India)anddilutedinitiallywithsteriledistilled tool of the Expert Protein Analysis System (ExPASy, Swiss
water(nonutrientbroth)atadilutionof1:40.Afterastatic InstituteofBioinformatics).Proteinsequencesretrievedfrom
∘
incubationof3hoursat37 C,thesupernatantswereaspirated NCBIwerealignedusingCLUSTALW[33]andphylogenetic
andthensterileLBbrothwasaddedtothewellswithafinal tree was constructed with the help of analysis software
dilutionof1:40withsterileLBbroth.Foralltimepoints(3– MEGA v5.0 [34]. Bootstrapping was performed with 1,000
96h),thiswasrepeatedinordertoallowA.baumanniicells replicates for checking relative support for the branches
toattachontopolystyreneinitiallybutnottogrowandthen in the phylogenetic tree. Comparison was restricted to
tosubstituteitwithadditionoffreshLuriabrothtofacilitate sequences of close organisms, for example, Pseudomonas
adherentandmicro-aggregatedcellstodevelopbiofilms.All aeruginosa,Pseudomonasputida,Pseudomonassyringae,and
∘
plates were incubated at 37 C under static conditions for previously predicted PMM sequence from whole genome
biofilm development, including negative controls (no cells, sequence of Acinetobacter baumannii strain AYE, Acineto-
onlynutrientmedium)foreachtestsamplesintheplate.After bacterhaemolyticus,andAcinetobactercalcoaceticus.Higher
appropriateincubationperiod(s),platesweretakenoutand eukaryote sequences, for example, Oryctolagus cuniculus
nonadherentanddeadcellswereremovedbybriefsonication PGM isoform 1 and PMM from Homo sapiens, were also
and aspiration carefully. The wells were washed thrice with includedforcomparison.
sterile PBS, dried, and stained with 0.1% Gentian Violet
(HiMedia, India). Excessive stain was removed by sub- 2.10.CloningandHeterologousExpression. Upstreamprimer
merging the plate in a water trough and then dried in (alg 118F)anddownstreamprimer(alg 1897R)wereusedin
laminarairflowfollowedbyaddition200𝜇Lof100%ethanol aPCRprogramwithanadditional15minfinalextensionfor
for solubilizing the stained biofilm matrices. To measure producingpoly-AtailsinthePCRproductstoaidwhileTA
the absorbance, plates were read in a Multi-Plate Reader cloning. Amplified algC was purified using gel purification
(MolecularDevices,USA)at570nmbeforebeingshakenat kit (Bangalore GeNei, India) and ligated into pGEMT-Easy
1020rpmfor10seconds.Allbiofilmdevelopmentassayswere vector (Promega, USA) to produce resultant recombinant
performedinreplicateof12andrepeated.Absorbancevalues plasmid pGEalgCA7. The plasmid was transformed into
weretermedasbiofilmgrowthindexafternormalizationwith chemically competent E. coli DH5𝛼. Colonies were picked
LB control, and values were plotted in Excel spreadsheets anddirectPCRwasperformedtoconfirmpresenceofalgC
(Microsoft,USA)forfurtheranalysis. gene.
2.7. Statistical Correlation of Biofilm Formation with algC 2.11. Assessment of AlgC (PMM/PGM) Protein Expression.
Gene Expression. To find the concurrence of algC gene Mid-log phase grown (with 100mg/L ampicillin in Luria
expression pattern with biofilm formation, RQ values (fold broth,undershakingconditions)E.coliDH5𝛼-pGEalgCA7
overincreaseinalgCgeneexpression)inbiofilmmodewere andE.coliDH5𝛼-pGEM-3Zf(+)atOD600=0.8wereused
correlatedlinearlywiththebiofilmindicesatcorresponding topreparewholecellextracts.Thecellextractswereanalyzed
timepointsandcorrelationcoefficientsweredeterminedusing bySDS-PAGE(12.5%)andproteinbandsweredocumentedin
Excel spreadsheet (Microsoft, USA). Correlation statistics ageldocumentationsystem(AlphaInnotech)afterstaining
wasrestrictedtothreemajorstages,thatis,initialattachment with0.2%Coomassiebrilliantblue(Himedia).
(3–9 hours), consolidation of the surface-attached micro-
aggregation (12–24 hours), and maturation of biofilm (36– 2.12.AssessmentofBiofilmAugmentation. Biofilmaugmenta-
48 hours). 24-hour biofilm gene expression data (calibrator tionwasvisualizedbyscanningelectronmicroscopy.Briefly,
sample)wasexcludedfromthecorrelation.𝑃valueof<0.05 overnightgrowncells(3×109 cells)wereinoculatedon(1×
wasconsideredtobestatisticallysignificant. 1cm)sterileglassslidesinside12-wellcultureplates(Tarsons,
∘
India)andincubatedat37 Cstatic.After24hofincubation,
2.8.MicroscopicExaminationofA.baumanniiBiofilmDevel- culture supernatant was removed; slides were immediately
opment. To localize the stages of biofilm growth over a flooded with 2.5% glutaraldehyde in PBS and incubated at
temporal scale (3–96h), biofilms were formed on sterile room temperature for 2 hours, followed by rinsing with
polystyrene culture dish, 55mm × 15mm (Tarsons, India), steriledistilledwater,andseriallydehydratedwithanethanol
∘
and incubated at 37 C under similar culture conditions as gradient (25–100%), CO2-critical point dried and coated
describedabove.Afterappropriateincubationperiods,plates with platinum in an Auto Fine Coater (JFC-1600, JEOL,
were taken out; supernatants were aspirated followed by Japan).Theslideswerethenobservedinascanningelectron
washingthreetimeswithphosphatebuffersalineforremoval microscope (Vega, Tescan, USA) with 30KV input voltage.
of nonadherent cells and fixed with 1mL of methanol, air- For quantitative biofilm augmentation in the algC clones,
dried,andobservedwithoutstainingunderamodularbright biofilms were formed in microtitre plates and quantified as
fieldmicroscope(AxioscopeA1,Zeiss,Germany)withbright permethodsdescribedearlier[10].
fieldsettingsatmagnificationof100x.
2.13.SequenceAlignmentandBuildingModelforPMM/PGM.
2.9. Bioinformatics and Phylogenetic Analysis. Nucleotide PMM/PGMsequenceofA.baumanniiAIIMS7(AEC46864)
and protein sequence comparisons were performed using wasusedasatargetsequenceinBLASTpprogramtoidentify
BLASTn (National Center for Biotechnology Information, possibletemplatestructuresavailableinProteinDataBank.
TheScientificWorldJournal 5
Theselectedtemplateshavinggoodalignmentscorewiththe 90
target sequence were aligned using CLUSTALW. Template
80
proteinstructure1K2Y.pdb(P.aeruginosaPMM/PGMS108A
mutant),havingmaximumsequencealignmentscore(32%) ase) 70
e
withtargetsequence,wasusedasthefinaltemplatetobuild cr 60
n
homologymodel of PMM/PGM. Three-dimensionalmodel er i 50
v
ofPMM/PGMwasconstructedusingMODELLER9v7[35] d o 40
boyf tchoensmidoedreinlgw1aKs2tYe.spteddbbasyautseimngpl1a0tensstrmuoctleucrue.laThr deysntaabmiliictys Q (fol 30
R 20
(MD)simulation.
10
2.14.MolecularDynamics(MD)Simulation. MDsimulations 0
3 6 9 12 18 24 36 48 72 96
were performed on the homology model of PMM/PGM
Time (hours)
from A. baumannii AIIMS 7 in a HP workstation with the
help of GROMACS 4.0.4 program using the GROMOS96 Planktonic
45a3 force field [36]. The structure was fully solvated Biofilm
+
with water (SPC) and system was neutralized with 9Na
Figure1:Comparisonof algC geneexpressionpatterninbiofilm
ions. The solvated structure was minimized by steepest
andplanktonicA.baumanniiAIIMS7.Real-timequantitativePCR
descent method for 1000 steps at 300K temperature and resultsshowingrelativequantificationofalgCgeneexpressionlevels
constant pressure. The LINCS algorithm with 8.0A˚ cutoffs (calculated according to ΔΔCt-method and represented as “fold
was used for energy minimization of PMM/PGM whereas overincrease”)atcorrespondingtimepoints.Expressionlevelswere
PME algorithm with 8.0A˚ nonbonded cutoffs was utilized normalized with indigenous control (16SrRNA gene expression)
similarly as used in an earlier MD simulation study [37]. in both biofilm and planktonic cells (calibrator: 24-hour biofilm
AfterequilibrationperiodproductionMDwasrunfor10ns sample,foldoverincrease=1).
at300Ktemperature,pressure,andconstantvolumeensem-
ble. Solvent accessible surface (SA, Richards’ surface) and
molecular surface (MS, Connolly’s surface) areas were cal- showedsignificantvariationinthethresholdcycle(Ct)values
culated using CASTp analysis [38]. Structural comparison inbiofilmandplanktonicmodeofgrowthofA.baumannii,
aftermoleculardynamicssimulationofinitialstructurewith whichdescribeddifferentialexpressionalpatternsofthetwo
final structure was done using PDBeFOLD software [39]. modesofgrowth(Figure1).NumberofcopiesofalgCmRNA
The MD simulation trajectory was then visualized using were calculated using 16SrRNA gene as internal control at
theVMD(visualmoleculardynamics)package[40].Three- eachtimepoint(ΔCt).Toevaluatetherelativegeneexpres-
dimensionalimagesofenzymePMM/PGMafterMDsimu- sion in biofilm and planktonic modes of growth over the
lationrunweregeneratedusingtheUCSF-Chimeratool[41] temporalscaleof3to96hours,the24-hourbiofilmsample
andPymol(http://pymol.sourceforge.net/). was used as calibrator (ΔΔCt). Relative quantification (RQ)
plot for gene expression at various time points from 3 to
2.15.NucleotideSequenceAccession. TheGenBankaccession 96 hours showed low basal (almost linear) expression of
number for A. baumannii AIIMS 7 algC gene sequence algCinplanktonicmode(maximumof3.24-fold,48hours)
is JF701279 and AEC46864 for the protein (PMM/PGM) whereas highly variable and elevated algC expression was
sequence. seen in biofilm mode (maximum 81.59-fold, 48 hours) and
displayingadefinitepattern.Theinitialattachmentstage(9
3.Results hours) and maturation stage (48 hours) of biofilm showed
maximalexpressionofalgC,whichwasincontrastandcould
3.1.IdentificationofalgCGeneinA.baumanniiAIIMS7. PCR be seen as steadily low expression at almost all time points
with genomic DNA yielded a fragment of 1781bp (Supple- in planktonic mode of growth (Figure1). This observation
mentary Figure S1 in the Supplementary Material available affirmsthatthealgCgeneishighlyup-regulated(atthetran-
onlineathttp://dx.doi.org/10.1155/2014/593546).PCRampli- scriptionlevel)whiletheA.baumanniicellsgrowinabiofilm
fications with plasmids were unsuccessful, confirming that mode (i.e., attached to an abiotic surface) and follows a
the gene is not plasmid-borne. Reverse transcription PCR pattern as depicted (Figure1); however in planktonic (free
using total RNA showed amplification of internal coding form,inasuspension,unattachedtosurface)thealgC gene
regions1(1360bp)and2(463bp),whichconfirmedtheactive istranscribedatabasalrate.
transcription of the algC ORF (Supplementary Figure S2).
DNAsequenceofthegenewasanalyzedtopredictregulatory 3.3.BiofilmGrowthPatternCorrelateswithQuantitativealgC
regions such as promoter, ribosomal binding site, transla- Gene Expression. Biofilm growth pattern A. baumannii on
tion start, and end, which were identified and annotated polystyrene surface can be seen in Figure2, which can be
(SupplementaryFigureS3). explained in three distinct stages. First, an exponential rate
of increase was observed at 3 to 9 hours, which defined
3.2.ElevatedLevelsofalgCGeneTranscriptionduringBiofilm the initial attachment and micro-colony aggregation stage
Mode of Growth. Quantitative real-time PCR experiment (Figure2).After9hourtill36hoursitshowedsteadylevelof
6 TheScientificWorldJournal
3 followedbyappearanceofrobustthree-dimensionalbiofilm
structures, which were found persistent till 72 and 96h
2.5
(Figures 3(i) and 3(j)). Overall, visualization of biofilm
x 2 growthatdevelopingstageswasintandemwiththepatterns
e
d ofbiofilmgrowthaswellasalgCgeneexpression.
n
m i 1.5
ofil 3.5. In Silico Analysis of Protein Reveals Highly Conserved
Bi 1
Identity. TocompareandevaluatetherelatednessofA.bau-
0.5 manniiAIIMS7algCencodedPMM/PGM,insilicoanalyses
were performed. BLASTn results showed 100% similarity
0
with the predicted PMM/PGM sequence in whole genome
3 6 9 12 18 24 36 48 72 96
sequences of A. baumannii available in NCBI database.
Time (hours)
Alignment results also displayed significant similarity with
Figure 2: Pattern of biofilm growth of A. baumannii AIIMS 7. PMM/PGMnucleotidesequencesofothergenera.472amino
Graphshowingcorrespondingbiofilmindicesofbiofilmsformedon acid residues could be theoretically translated in one frame
󸀠 󸀠
polystyrenemicrotitresurface,calculatedafternormalizationwith (5 –3 )oftheDNAsequence.BLASTpshowedsimilarresults
controlandplottedversusrepresentativetimepoints(threeto96h). asinBLASTnalignment.Themolecularweightoftheprotein
was predicted to be 52.94kDa with isoelectric point (pI)
of 5.67. CLUSTALW alignment with selected bacterial and
increaseinbiofilmindices,indicatingtheconsolidationstage eukaryoticPMM/PGMsequencesshowedconservedregions
oftheattachedmicro-coloniesandapproachingmaturation in the protein as shown in Figure4(a) (active site, Mg2+
of biofilm. Third, maximum production of biofilm matrix binding site, and sugar binding site marked as colored
could be seen at 36 to 48 hours, marking the maturation rectangles) in the protein as shown. Figure4(b) shows the
stage. Lastly, as like the plateau phase in the planktonic phylogenetictreeindicatingrelatednessbetweenPMM/PGM
growth curve of A. baumannii AIIMS 7 (data not shown), enzymesfrompseudomonadsandhighereukaryotes(rabbit
the biofilm growth pattern also exhibited a steady state
andhuman).PMM/PGMsequencefromA.baumanniihad
and persistent nature after maturation (after 48 hour till
less than 25% identity with rabbit muscle PGM isoform
96 hours). To find the association of algC gene expression
1, whereas it had a mere 10% identity with human PMM
patternattranscriptionlevel(asrevealedfromreal-timePCR
(Figure4(b)).
analysis)withthatofbiofilmgrowth,correlationcoefficients
weredeterminedusingtheRQvaluesinbiofilmsamplesand
3.6. Assessment of Protein. To demonstrate the synthesis of
biofilm indices at corresponding time points. High corre-
PMM/PGM protein, whole cell extracts of cloned E. coli
lation was observed at all three major stages of biofilm. DH5𝛼withresultantrecombinantplasmidpGEalgCA7cells
Correlation coefficients were 0.902, 0.925, and 0.983 (𝑃 <
were used in SDS-PAGE analysis, which showed a clearly
0.05) corresponding to initial attachment, consolidation of overexpressedproteinof∼53kDasuggestingthePMM/PGM
the surface-attached micro-aggregation, and maturation of protein in question (Figure5). Control E. coli DH5𝛼 (with
biofilmstages.Correlationwasfoundtobemaximum(0.983,
emptyvectorpGEM-3Zf+lackinganalgCgene)didnotshow
𝑃<0.05)atstageswherebiofilmsweremature.Thisindicated
anyPMM/PGMproteinband.
astrongpossibilitythattranscriptionofalgCcouldbeinclose
associationwithbiofilmformation,especiallythematuration
3.7.BiofilmAugmentationbyalgCafter24h. Electronmicro-
stage(36–48hours)andalsotheinitialattachmentstage(3–9
graphs of biofilm formed by the algC clones (Figure6)
hours).
showed significant increase in biofilm formation compared
tocontrols,probablyindicativeoftheoverexpressionofthe
3.4. Microscopic Visualization of Biofilm Development.
PMM/PGM protein and the resultant exopolysaccharides.
To visualize the growth pattern of biofilm, bright-
Thicker biofilms could be seen in the algC clones having
field microscopy was performed on biofilms growing
clear dense matrices (Figures 6(a), 6(c), and 6(e)) with a
on polystyrene surface, which showed varied biofilm
intracellularcementingmaterialclearlyvisible(Figures6(a)
morphologyatgradualtimepoints(Figure3)withastrong
and6(e))indicativeofthebiofilmEPS.OverallSEManalysis
concurrence with quantitative evaluation of biofilm growth
pattern(Figure2,asdescribedabove).Atinitialattachment atagradientofmagnificationsandconcurrentcomparisons
stages, planktonic cells were seen scantily aggregated with control clones (lacking functional copy of algC) sug-
(Figures3(a)and3(b))butgraduallythemicro-aggregation gestedsubstantiallythatthereissignificantfacilitationofthe
on the surface increased, subsequently making completely overallbiofilmformation,emphasizingtheenrichmentofthe
attached micro-colonies at 9h which could be seen clearly biofilmmatrices,whichcouldbeduetothecohesiveactivity
(Figure3(c)). Further stages of growth (12, 18, and 24h) of EPS including alginate and LPS cores. The quantitative
showed consolidation of micro-colonies leading to stable biofilmassayconcurredwiththemicroscopicanalysis,with
structures of biofilm (Figures 3(d)–3(f)). At 36–48h, fully augmentation of biofilm up to 3.87-fold in the algC clones
mature biofilm communities of A. baumannii were seen, compared to the control cells lacking algC gene (𝑃 < 0.02,
enriched with thick EPS matrices (Figures 3(g) and 3(h)), datanotshown).
TheScientificWorldJournal 7
(a) (b) (c)
(d) (e) (f)
(g) (h) (i)
(j)
Figure 3: (a)–(j) Microscopic visualization of A. baumannii AIIMS 7 biofilms. Representative bright field micrographs depicting static
biofilmsformedonpolystyrenemicrotitresurfaceatrespectivetimepoints(3,6,9,12,18,24,36,48,72,96h)(magnification,100x).
3.8. Molecular Dynamics Simulation Study of PMM/PGM. eachdomainsequentially(Figure7(a))formingheartshaped
To predict the functional association between PMM/PGM geometry. Sequence alignments (Supplementary Figure S4)
enzyme (encoded by algC gene) resulting in alginate/LPS and model building study showed that the active site was
core mediated biofilm formation in A. baumannii, molec- located at the centre of the two domains (domains 1 and
ular dynamics (MD) simulations up to 10ns were carried 2) in a deep cleft formed by Ser104, Asp244, Asp246, and
out on homology model of PMM/PGM of A. baumannii Asp248. PMM/PGM sequence of A. baumannii AIIMS 7
AIIMS 7 using GROMACS v4.0.4 program. The generated showedconservedsequencemotifindomain3fromresidues
PMM/PGMmodelofA.baumanniiAIIMS7containedfour 327-331(GEYAGH)whichwouldactasasugarbindingsite.
domains of equal size arranged in “heart shaped” manner A cluster of positively charged conserved residues found in
(Figure7(a)) which form a compact structure, similar to P. domain 3 (Lys287) and domain 4 (Arg427, Arg438) could
aeruginosa [42]. The polypeptide chain proceeds through be involved in phosphate binding. The cleft showed solvent
8 TheScientificWorldJournal
H.sapiens ----------mavtaqAarrrerVlclfDvdGtL---------------------tpaRQ
O.cuniculus ----mEegPlplltirtapYhdqkpgtsglRkKt-yyfedkpcylenFiqsiffsidlKd
A.haemolyticus -------------mnIkhkFPlNIFRAYDIRGKL-TnLTptiiRsIavA----LAAQYtE
A.calcoaceticus -------------mnVrhsFPkSIFRAYDIRGKL-SyLTtDvvRsIaYg----LAqQYKQ
AB AIIMS 7 -------------mnVrhsFPkNIFRAYDIRGKL-SyLTtDvvRsIaYg----LAqQYKQ
AB AYE -------------mnVrhsFPkNIFRAYDIRGKL-SyLTtDvvRsIaYg----LAqQYKQ
P.aeruginosa -----------mstakAptlPaSIFRAYDIRGvvGdtLTAETAywIGrA----igSEsla
P.syringae ---------mnspasVApnlPeTIFRAYDIRGvvGdtLnAETAywIGrA----igSEsla
P.putida --------mndmahlVpaalPdSIFRAYDIRGvvGktLhAETAywIGrA----igAQsla
H.sapiens kidPEva--------------aflQkLr----SrvqI---GvVggsdyckIAeqLGdGDe
O.cuniculus rqgssLVvGgDGRyfnkSaietIlQmaaanGigrlvIgqnGilsTPavscIiRkIkaigG
A.haemolyticus agQkQiVIGYDaRLtSPtyAniIqQifehqGleVinI---GCcsTPMMYYIARdya-GNG
A.calcoaceticus vkQtQLVIGYDaRLtSPAyAylIeeiLiEqGlNVTnI---GCcssPMMYYIARdfG-GNG
AB AIIMS 7 aeQtQLIIGYDaRLtSPAyAhlIeeiLvEqGlNVTnI---GCcsTPMMYYIARefG-GNG
AB AYE aeQtQLIIGYDaRLtSPAyAhlIeeiLvEqGlNVTnI---GCcsTPMMYYIARefG-GNG
P.aeruginosa rgEPcvavGrDGRLSgPeLvkqliQgLvDcGCqVSDv---GmVPTPVLYYaAnvLegkSG
P.syringae qnEPnvsvGrDGRLSgPeLvqqliQgLhDsGCHVSDv---GlVPTPaLYYaAnvLagktG
P.putida qgEPQvsvGrDGRLSgPmLveqlikgLvDsGCHVSDv---GlVPTPaLYYaAnvLagkSG
H.sapiens Viekfdyvfaen----GtvqykhGrlLSkqtIQnh-------LgEellq--d--------
O.cuniculus iilTASHNPggpNgdfGiKFnIsnggpaPEAItdkifqisktieEyAic--PDlkvdlgv
A.haemolyticus iMVTASHNPKSdN---GiKWILkGEPpSPEAIQQ--------vglyAegftdqiieiqnl
A.calcoaceticus iMVTASHNPKSdN---GiKWILkGEPpSPEmIQQ--------vgEeAqtYVPNhsisvle
AB AIIMS 7 iMVTASHNPKSdN---GiKWILrGEPpSPEmIQQ--------vgEfAqtYVPthtislle
AB AYE iMVTASHNPKSdN---GiKWILrGEPpSPEmIQQ--------vgEfAqtYVPthtislle
P.aeruginosa VMlTgSHNPpdYN---GFKiVvaGEtLanEqIQa--------LrEriek--nDl--asgv
P.syringae VMlTgSHNPKdYN---GFKiVIaGDtLanEqIQa--------LhErikt--nNl--tsqk
P.putida VMlTgSHNPsdYN---GFKiVIaGDtLanEqIQa--------Lltrlkt--nDl--tlaq
H.sapiens ----li--nFClsY---------------------------mallRLpkkrgtfIefrNG
O.cuniculus lgkqQF--DlenKfkpftveivdsveayatmlrnifdfnAlkellsgpnrLKIrIDamhG
A.haemolyticus dqlhkiipqYClQY-----------------------qQAllSDIhLskPLKIVLDglhG
A.calcoaceticus lTlpQFkAeFCqQY-----------------------qQAIfkDIQLkRPLKVVLDglhG
AB AIIMS 7 lStpQFnSeFCkKY-----------------------qQAIfNDIQLkRPLKVVLDglhG
AB AYE lStpQFnSeFCkKY-----------------------qQAIfNDIQLkRPLKVVLDglhG
P.aeruginosa gSveQv--DilpRY-----------------------fkqIrDDIamakPmKVVvDCgNG
P.syringae gSitQv--DileRY-----------------------fQqIkNDIvmaRkLKVVvDCgNG
P.putida grvekv--DilgRY-----------------------fQqIvgDvKLakkLKVVvDCgNG
H.sapiens mlni-------spiGrs------CtLEeri-----efSeldkkEkireKfvE---Dlkte
O.cuniculus vvGpyvKKiLcEeLGapanSavnCvpledFggHHPDP-nltyaaDLvetmKsgehDfGaA
A.haemolyticus SAGrCAKsvL-EkLGCdVIALr-CEanGhFPdHaPDPSHAehLKqLqqaIisenADlGIA
A.calcoaceticus SAGhCsKliL-EkmGCEVIALr-tnpnGeFPdHaPDPSHAAhLisLrkaVvEqqADIGIA
AB AIIMS 7 SAGhCsKlvL-EkmGCEVIALr-ttpnGeFPdHaPDPSHAAhLKeLrktIiEqGADIGIA
AB AYE SAGhCsKlvL-EkmGCEVIALr-ttpnGeFPdHaPDPSHAAhLKeLrktIiEqGADIGIA
P.aeruginosa vAGviApQLi-EALGCsVIpLy-CEVDGNFPNHHPDPgkpeNLKDLiAKVKaenADlGLA
P.syringae AAGviApQLi-EALGCEVISLf-aEVDGNFPNHHPDPgkleNLQDLiAKVKEtGADlGLA
P.putida AAGvvApQLi-EALGCEVIpLf-CEVDGNFPNHHPDPgkpeNLEDLiAKVKEtGADIGLA
H.sapiens FaGkG--------------------------LrfSrGgmIsFDV----------------
O.cuniculus FDGDGDRnmIlgkhGffVnPsdsvaviAaNIfsip-----yFqqtgvRgfarsmptsGal
A.haemolyticus lDGDGDRVvlldEqahIIsPDRLLsLFAQmcLQQHPhkEIVFDVKCSRmVadtVEQlnGQ
A.calcoaceticus lDGDGDRVvlVdEkaqIltaDRLLsLFAQmcLEQHPeqEIVFDVKCSRmVqetVEKlGGK
AB AIIMS 7 lDGDGDRVvlldEkanIltaDRLLsLFAQmcLEQqPdkEIVFDVKCSlmVqrtVERlGGK
AB AYE lDGDGDRVvlldEkanIltaDRLLsLFAQmcLEQqPdkEIVFDVKCSlmVqrtVERlGGK
P.aeruginosa FDGDGDRVGVVTntGtIIYPDRLLMLFAKDVvsRNPGADIIFDVKCtRrlialIsgyGGR
P.syringae FDGDGDRVGVVTnaGnVVYPDRLLMLFAlDVLKRNPGADIIFDVKCtRrltplIsehGGR
P.putida FDGDGDRVGVVTntGsIVYPDRLLMLFAQDVLsRNPGAEIIFDVKCtRrltplIEQhGGR
-------------fpeGwd-----------------------krYclds-----lDqdsf
H.sapiens
O.cuniculus drvanatkialyetpTGwkFfgnlmdaSk--lsLcGEeSfgtGsdhiRE------kDGLw
A.haemolyticus akM----------iRTGsSFLrSyLSQSnGNAvfgGEya---GHYvFndGRgFGyDDGLY
A.calcoaceticus akM----------iRTGsSFLrAyLSQSnGrAifgGEya---GHYvFndGRgFGyDDGLY
AB AIIMS 7 PkM----------iRTGsSFLrAyLSQSnGNAifgGEya---GHYvFndGRgFGyDDGLY
AB AYE PkM----------iRTGsSFLrAyLSQSnGNAifgGEya---GHYvFndGRgFGyDDGLY
P.aeruginosa PvM----------WKTGHSlIKkKmketG--AlLAGEMS---GHvFFKE-RWFGFDDGiY
P.syringae PvM----------WKTGHSlIKkEmkkSG--AlLAGEMS---GHiFFKE-RWFGFDDGiY
P.putida alM----------WKTGHSlIKkKmkQtG--slLAGEMS---GHiFiKE-RWyGFDDGiY
dtihffgneTspggnd-fEiFAd-----Prtv----------------------------
H.sapiens
O.cuniculus AvlawLsILatrKqsv-eDilkdhwhkFgrnffTrydYeeVeaegatkmmKdLeAlmfdr
A.haemolyticus AAlRvmEyLgQsEArclSELlAa----FPEryCTedIYIsthnasPqqVlnni-----Ei
A.calcoaceticus AAlRvmEyfTQssATtISELFAp----YPErCCTedtYIgtyQsDPkyVlQdi-----Ei
AB AIIMS 7 AAlRvmEyfTEssATtISDLFSn----YPErCCTedtYIgthQsDPkhVlQdi-----Ei
AB AYE AAlRvmEyfTEssATtISDLFSn----YPErCCTedtYIgthQsDPkhVlQdi-----Ei
P.aeruginosa sAARLLEILsQdQrds-ehvFSa----FPsdisTPEInItVtEdskfaIIEaL-----qr
P.syringae sAARLLEILsQesAna-eDLFet----FPndisTPEInIkVtDvtkfsIIKaL-----Et
P.putida sAARLLEILsktEqsa-enLFAa----FPndisTPEInIdVtDegkfsIIdaL-----qr
---------------------------------------------GhsVV----------
H.sapiens
O.cuniculus sfvgkqfsandkvytvekadnfeyhdpvdg-sVSKnqGlRLiFaDGsrIIfrlsgtgsAg
A.haemolyticus i-----------------------SHrlaA-RlSKIDGVRLDFDDGFGII-------RAS
A.calcoaceticus l-----------------------SHrlGA-RISKIDGVRLDFDDGFGII-------RAS
AB AIIMS 7 l-----------------------SHrlGA-RISKIDGVRLDFDDGFGII-------RAS
AB AYE l-----------------------SHrlGA-RISKIDGVRLDFDDGFGII-------RAS
P.aeruginosa d-----------------------Aqwgeg-nIttlDGVRvDypkGwGlV-------RAS
P.syringae d-----------------------AqwgdA-KlttIDGVRvDypkGwGlV-------RAS
P.putida d-----------------------AdwgeA-nlttIDGVRvDyanGwGlV-------RAS
------------spqdtvQRcrEiF----------------fPEtAhEa---
H.sapiens aTirlYIdsyEkDNakinQdpQvmlaplisialkvsqLQErtgrtAptvit-
O.cuniculus
NTgeyFtvRFdADhPdrLveIrQKF---------isMLQDqyPQIAqElaQS
A.haemolyticus
NTgeyFtvRFdADNPlrLkeIQQKF---------vDMLQEhyPQIAqElsEA
A.calcoaceticus
AB AIIMS 7 NTgeyFtvRFdADNPlrLkeIQQKF---------iDMLQEryPQIAqElmsl
AB AYE NTgeyFtvRFdADNPlrLkeIQQKF---------iDMLQEryPQIAqElsEA
NTTPVlVLRFEADteeeLERIktvF---------rNqLkavdssLpvpF---
P.aeruginosa
NTTPVlVLRFEAeteaeLQRIkdvF---------haeLkKvaPdLdlpF---
P.syringae
P.putida NTTPVlVLRFEADsdaeLQRIkdvF---------rtqLlrvePELqlpF---
(a)
Figure4:Continued.
TheScientificWorldJournal 9
92 A. baumanniiAIIMS7AlgC PMM
94 A. baumanniiAYE PMM
92 A. calcoaceticusPMM
76
A. haemolyticusPMM
36 O. cuniculusPGM isoform1
39
P. aeruginosaPAO1PMM
P. putidaPMM/PGM
P. syringaePMM
H. sapiensPMM
0.5
(b)
Figure4:(a)-(b)MultiplesequencealignmentandphylogeneticrelationshipofPMM/PGM.(a)CLUSTALWanalysesofPMM/PGMprotein
2+
sequencesdisplayingrepresentativecolorofboxwhichhighlightsconservedregions,thatis,activesite(red),Mg bindingsite(green),and
substratebindingsite(blue).(b)BootstrapconsensustreeforPMM/PGMenzymes,constructedbyMEGAv5.0usingtheneighbor-joining
(NJ)method(basedon1,000replicates).Numbersonthenodesarebootstrapvaluesandthebarrepresentsscaleofestimatedevolutionary
distance(20substitutionsatanyaminoacidpositionper100aminoacidpositions).
1 2 M residues (Figure7(d); Supplementary Table S1). The metal
2+
ion Mg is required for enzymatic activity of PMM/PGM
2+
of A. baumannii AIIMS 7 (Figure7(d)), rather than Mn
2+ 2+
andZn .Specificinteractionsbetweenmetalion(Mg )and
94.3 protein, water-mediated H-bonds (so-called water bridges),
and hydrophobic (Lennard-Jones) interactions have been
identifiedandarelisted(SupplementaryTableS1).
66.0
3.9.StabilityofPhosphomannomutaseduringSimulation. To
verifythesimulationstabilityandtomeasurestructureand
dynamics of PMM/PGM model of A. baumannii AIIMS 7,
AlgC standardstructuralparameterslikeRMSD,rootmeansquare
44.0 fluctuation(RMSF),andradiusofgyration(RG)werecalcu-
latedandrepresentedinFigures8(a)–8(c).Duringmolecular
dynamicssimulationperiodof10ns,theaverageRMSDvalue
was 0.3nm as depicted (Figure8(a)). Steady RMSD result
showed for the atoms in the four domains of PMM/PGM
29.0 modelindicatedthatthecatalyticactivityisrelativelystable
duringthesimulation.Theresultforradiusofgyration(RG)
alsoindicatedthestabilityofmodeloverthewholesimulation
period (Figure8(b)). Minor fluctuations in C-alpha atoms
Figure5:SDS-PAGEanalysisofAlgC(PMM/PGM)protein.Lane1:
wholecellextractofcontrolE.coliDH5𝛼(lackingalgCgene);lane2: (RMSF) of helical and sheet structural elements could be
wholecellextractofE.coliDH5𝛼withalgCgeneshowingtheAlgC seenwhereasloopregionshowedvariabilitytosomeextent
proteinband∼53kDa;laneM:standardproteinmolecularweight (Figure8(c)). Overall, these results of RMSD, RMSF, and
marker(kDa). RGstronglyindicatedthestabilityofsystemovertheentire
simulationperiod.
accessible surface (SA, Richards’ surface) and molecular 4.Discussion
surface(MS,Connolly’ssurface)areasascalculatedbyalpha
shapemethod.Theenzymeshowedatotalcavityof3641.5A˚ Biofilm formation substantially aids to the spectrum of
andsphericalcentralcavityof1878.13A˚ wheresubstratecould multidrugresistancedisplayedbyA.baumanniiandisoften
bind as shown in Figure7(b). To compare the 3D struc- attributed as the major cause for antibiotic treatment fail-
tures obtained before and after MD simulations, structural ure [3, 5]. The process of bacterial biofilm development is
superpositionsweremadeusingPDBeFOLD,resultsofwhich believedtobeacomplexinterplayofthebiofilmEPSmatrix
showedrootmeansquaredeviation(RMSD)oftwoaligned components,thatis,aplethoraofproteinsandextracellular
structureswithintherangeof2.51A˚ (Figure7(c)).Metalion macromolecules,forexample,DNAandpolysaccharides.The
2+
Mg interacts with Asp244, Asp246, Asp248, and Ser104 synthesisofspecificexopolysaccharidessuchasalginateand
10 TheScientificWorldJournal
(a) (c) (e) (g)
(b) (d) (f) (h)
Figure6:(a)–(h)Visualizationofbiofilmaugmentationbyscanningelectronmicroscopy.Electronmicrographs(a,c,e,g)showingbiofilms
formedbyalgCclonesafter24h,atagradientofmagnifications(Bar=2𝜇m,5𝜇m,10𝜇m,and20𝜇m;absolutemagnificationsindicatedas
Kxintheverypicture)asobservedunderascanningelectronmicroscope.algCclonescanbeseenclearlyproducingdenseandrobustbiofilm
assuggestedbythethicknessandintegrityofthebiofilmmatrices,comparedtothecontrolbiofilmsbyE.coliDH5𝛼(lackingA.baumannii
algCgene)atcorrespondingcomparablemagnifications(b,d,f,h).
LPS core has been described in various bacterial systems reportergeneactivityincontinuousbiofilmculturecellstobe
[25,43,44],inparticularP.aeruginosa,withitscontribution 19-foldincreasedthanplanktoniccells.Earlier,Daviesetal.
in biofilm formation [20, 22]. However, algC gene function [46] have shown that expression of P. aeruginosa algC is
and its expression pattern have not been elucidated in A. upregulated after initial attachment of cells to teflon or
baumannii in order to understand the genetic basis of its glasssubstrata,wherecellsthatfailedtoupregulatealginate
associationwithbiofilmformation.Herein,aninitialgenetic biosynthesistypicallydetachedfromthesurface.Asreviewed
characterization of the algC gene and its association with earlierbyGacesa[47],itispresumedthatexopolysaccharide
biofilmformationisreportedintheMDRstrainAIIMS7of alginate plays a role in consolidation of the biofilm rather
A. baumannii, which depicts differential expression of algC than in the initial adhesion event and hence higher rate of
gene during the biofilm development. Besides, MD simula- algC transcription was not a prerequisite to surface attach-
tion was performed on the three-dimensional (3D) struc- ment by P. aeruginosa, indicating alginate biosynthesis not
ture of the gene product (enzyme PMM/PGM) to compare necessaryforitsattachmenttoglasssurface.Incontrast,our
with that of P. aeruginosa, followed by its cloning, heterol- data in support of the relative quantification of algC tran-
ogousexpression,andphylogeneticanalysis.Finally,biofilm scription by real-time PCR and biofilm augmentation by
augmentationbyalgCgenewasevaluatedquantitativelyand algC as visualized by SEM is indicative of the possible
confirmedbySEM. involvementofalgCexpressioninprobablyboththeevents,
Analysis of the algC gene expression pattern in the that is, initial attachment and consolidation of biofilms
temporalscale(upto96hours)revealsthatsurface-attached formed by A. baumannii. Furthermore, the upregulation of
bacteriahavesignificantlyhigherrateoftranscriptionlevels algC transcription at the maturation stages (36–48 hours)
than nonadherent or shaking cultures. The attachment or explains that it is strongly associated with maturation of
“surface sensing” could be the trigger for the activation of biofilms, as the correlation of algC expression with biofilm
algCpromoter,asaresultofwhichthealgCexpressioncould formationduringtheverystagewassignificantlyhigherthan
beseenconsistentlyhigher(maximum81.59-fold,𝑃 < 0.05) theinitialevents(0.983comparedto0.902,𝑃<0.05).During
inbiofilmformingA.baumanniiculturesthantheplanktonic planktonicmodeofgrowth,lowlevelsofalgCtranscription
counterparts (maximum 3.24-fold) as revealed by real time wereobservedascomparedtobiofilmformingA.baumannii,
PCR analysis (Figure1). This observation is in accordance whichshouldbeideallyduetolackofsurfaceattachment(less
withanearlierstudyinP.aeruginosa[45]whichshowsalgC promoter activity) and variations in the oxygen tension in