Protease Inhibitors from Marine Actinobacteria as a Potential Source for Antimalarial Compound

13 Pages·2014·1.3 MB·English

Protease Inhibitors from Marine Actinobacteria as a
Potential Source for Antimalarial Compound
L.Karthik1,GauravKumar1,TarunKeswani2,ArindamBhattacharyya2,S.SarathChandar3,K.V.Bhaskara
Rao1*
1EnvironmentalBiotechnologyDivision,SchoolofBioSciencesandTechnology,VITUniversity,Vellore,Tamilnadu,India,2ImmunologyLab,DepartmentofZoology,
UniversityofCalcutta,Kolkata,WestBengal,India,3GeneticsDivision,SchoolofBioSciencesandTechnology,VITUniversity,Vellore,Tamilnadu,India
Abstract
Thestudywasplannedtoscreenthemarineactinobacterialextractfortheproteaseinhibitoractivityanditsanti-Pfactivity
under in vitro and in vivo conditions. Out of 100 isolates, only 3 isolates exhibited moderate to high protease inhibitor
activitiesontrypsin,chymotrypsinandproteinaseK.Basedonproteaseinhibitoractivity3isolateswerechosenforfurther
studies.ThepotentialisolatewascharacterizedbypolyphasicapproachandidentifiedasStreptomycesspLK3(JF710608).
The lead compound was identified as peptide from Streptomyces sp LK3. The double-reciprocal plot displayed inhibition
modeisnon-competitiveanditconfirmstheirreversiblenatureofproteaseinhibitor.ThepeptidefromStreptomycesspLK3
extract showed significant anti plasmodial activity (IC : 25.78 mg/ml). In in vivo model, the highest level of parasitemia
50
suppression (<45%) was observed in 600 mg/kg of the peptide. These analyses revealed no significant changes were
observedinthespleenandlivertissueduring8dpi.Theresultsconfirmedup-regulationofTGF-banddownregulationof
TNF-aintissueandserumlevelinPbAinfectedpeptidetreatedmicecomparedtoPbAinfection.Theresultsobtainedinfer
that the peptide possesses anti- Pf activity activity. It suggests that the extracts have novel metabolites and could be
considered asa potentialsource fordrug development.
Citation:KarthikL,KumarG,KeswaniT,BhattacharyyaA,ChandarSS,etal.(2014)ProteaseInhibitorsfromMarineActinobacteriaasaPotentialSourcefor
AntimalarialCompound.PLoSONE9(3):e90972.doi:10.1371/journal.pone.0090972
Editor:OlivierNeyrolles,InstitutdePharmacologieetdeBiologieStructurale,France
ReceivedSeptember20,2013;AcceptedFebruary6,2014;PublishedMarch11,2014
Copyright: (cid:2)2014 Karthik et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:Theseauthorshavenosupportorfundingtoreport.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected]
Introduction DuetotheevolutionofdrugresistanceinPlasmodiumsp,which
necessitates the need for new drugs, ideally directed against new
Malaria is a highly infectious disease caused by a protozoan targets such as heme and malarial proteases. The life cycle of
parasiteofthegenusPlasmodium.Theseparasitesaretransmitted malarial parasite exhibits two stages: exoerythrocytic cycle and
bythebiteofinfectiousfemaleAnophelesspmosquitoes.Thereare erythrocytes lifecycle.Theerythrocytes lifecyclewasresponsible
totally five species of Plasmodium associated with malarial fever for all clinical manifestations and it begins when free merozoites
viz., P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi. invadeerythrocytes.ThefreemerozoiteswillenterintotheRBC
Among them, P. falciparum is highly virulent and it is the cellsanddevelopfromsmallring-stageorganismstolarger,more
predominantagentinAfrica.While,P.vivaxiscomparativelyless metabolically active trophozoites followed by multinucleated
virulent and is more prevalent throughout the world and schizonts [5]. The schizonts will ruptures the erythrocytes and
remaining three species are associated with the minor outbreaks releases 30,000 invasive merozoites in P. falciparum, 10,000 for P.
inseveralpartsoftheworld.Malariaisamajorcauseofmorbidity vivax and P. ovale and 2,000 for P. malariae. This step is called as
and mortality and it is projected that around 3.3 billion people egress. At this stage, proteases are required for the rupture and
were at risk of malaria in 2010. Likewise, among 91% of deaths subsequent invasion of erythrocytes by merozoite stage parasites
are estimated in the WHO African Region, with children under and for the degradation of hemoglobin by intraerythrocytic
fiveyearsofageandpregnantwomenbeingseverelyaffected[1]. trophozoites.
World Malaria Report (2012) summarizes that 106 countries are The merozoites form of P. falciparum express a number of
malaria-endemic in 2011[2]. merozoite surface proteins (MSPs). These may be considered as
Three different approaches were considered for the control of target antigens for vaccine preparation [6]. The merozoites
more virulent malarial parasite, Plasmodium falciparum. They are synthesize a B195-kDa glycosyl phosphatidy- linositol-anchored
widelyexploiteddevelopmentofeffectivevaccines,vectorcontrol precursor that assembles as a complex with two peripheral
and development of new drugs. It is very difficult to develop a membrane proteins such as MSP6 and MSP7 [7–10]. This
vaccineduetotheirexhibitionoftheirmultipleantigenicity.Based complex(MSP1/6/7)isuniformlypresentinthemerozoitesurface
onseveralfactors,thevectorcontrolshowslimitedsuccess.Onthe and it initiates the erythrocyte invasion [11]. This complex was
other hand, there is an increasing resistance of malarial parasites involving ‘primary’ proteolytic cleavage events earlier to egress
totheexistingdrughence,thereisanurgentneeddemandfornew stage [12] and the cleavage products remain associated with the
antimalarial agents [3,4]. surfaceofthereleasedmerozoite,tothecomplexisfinallyshedat
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ProteaseInhibitorforAntimalarialTreatment
the point of erythrocyte invasion in an essential secondary actin, AP-linked anti-rabbit, AP-linked anti-mouse secondary
processing step by the action of a membrane-bound parasite antibodies were purchased from the Cell signaling Technology
protease called PfSUB2 [13]. The primary proteolysis and the Cell Signaling Technology, Inc (Danvers, MA, USA). Pre stain
positionalconservationofthecleavagesitesinMSP1orthologues molecularweightproteinmarkerswerepurchasedfromBangalore
acrossthePlasmodiumgenus[14]proposedthatprimeprocessingis Genei (India) andtheremaining chemicals were purchased in an
essential for the function of the MSP1/6/7 complex and for analytical grade of the highest purity (India). All solutions were
merozoite viability. preparedwithcommercialreagentsofatleastpro-analysisquality
The exonemes, specialized merozoite organelles releases the and with sterilized 18 MV milliQ water. When necessary, the
subtilisin-like serine protease called PfSUB1 [15] and it mediates specific originsof reagents arelisted inthetext.
the proteolytic maturation of members of a family of abundant,
papain-likeputativeproteasescalledSERA,previouslyimplicated In silico molecular docking analysis
in egress [16]. The inhibition of PfSUB1 prevents SERA Topredicttheinhibitingcharacteristicsofrespectiveinhibitors/
maturation and blockegress. This indicates a role for PfSUB1 in ligands (S. albogriseolus, S. longisporus, S. fradiae, S. lividans and S.
triggering egress, probably through activation of the SERA griseus)onPLASMEPSIN-IIandFALCIPAIN-2receptors,insilico
enzymes. molecular docking approach was opted. Initially before docking
Enzyme inhibitors are the third important product of marine calculations, both receptors and ligands were subjected to energy
actinobacteria.Sofar,itisusedforthestudyofenzymestructures minimization by GROMOS97 available in a Swiss PDB viewer.
and reaction mechanisms, but recently it has been used in For the above in silico approach, Hex Server (http://hexserver.
pharmacology [17]. These selective inhibitors can be used as a loria.fr), an interactive molecular graphics program was used for
powerful tool for inactivating target proteases in the pathogenic calculating and visualizing feasible docking modes of pairs of
processesofhumandiseasessuchasmalaria,emphysema,arthritis, macromolecules. It adopts Spherical Polar Fourier (SPF) correla-
pancreatitis,thrombosis,highbloodpressure,musculardystrophy, tionstoincreasespeedofthedockingcalculations.Inthefirststep
cancer, and AIDS [18]. Enzyme inhibitors from marine micro- as an input, respective PDB files of ligands and receptors were
organisms were sparsely studied. However, terrestrial isolated specified inthe Hex server [21].Further, the docking correlation
Streptomycesisaoneofthepotentialproducersofenzymeinhibitors typecalculationparametersweresetto‘‘Shapeandelectrostatics’’
[19]. The isolation of novel enzyme inhibitor from terrestrial mode. Later, the origin and interface residues for the docking
sources is rare hence marine actinobacteria will provide new calculationsweresettotheirrespectivedefaultvalues.Further,the
potential inhibitors. range angles for receptor and ligands were set to 180u. The step
Proteasesareessentialconstituentsfoundinprokaryotes,fungi, sizes for the docking calculations were set to 10. Finally, the
plants and animals. Serine, cysteine, metalloproteases is widely docking evaluation solutionswere set toevaluate 10best docking
spread in many pathogenic parasites, where they play critical solutions.AllthedockingcalculationswereperformedusingGPU
functions related to evasion of host immune defenses, acquisition mode. The best docking orientations were selected based on the
of nutrient for growth and proliferation, facilitation of dissemina- bindingenergy(kJ/mol)forthefurtherintermolecularinteraction
tionortissuedamageduringinfection[20].Thus,proteasesplaya studies. The results were analyzed with Hex 6.0, a stand-alone
foremost role in pathogenesis. Moreover, protease enzymes are graphicalapplicationtovisualizetheintermolecularinteractionsof
used for a long time in various forms of clinical therapies. Their receptors and ligands.
application in medicine is gaining more and more attention as
several clinical studies are indicating their benefits in oncology, Actinobacteria strains
inflammatory conditions, blood rheology control and immune Hundredactinobacteriastrainswereisolatedfromtheislandof
regulation. Hence,thisstudy wasfocused on thescreening of the Nicobar (9u 099 N, 92u 499 E). These isolation procedures were
proteaseinhibitorfrommarineactinobacteriaforanti-Pfactivity already published [22]. Further, these isolated strains were used
under invitroandinvivo conditions. for screening to detect the protease inhibitor production. These
strains were designated as LK1-LK100. All the strains were
Materials and Methods inoculated into 50ml SS medium in 250ml Erlenmeyer flasks
containing the sea water 50%, distilled water 50%, pH 7.5 and
Ethics statement incubated for 2 days in a rotary shaker incubator (200 rpm) at
Animalexperimentswerecarriedoutaspertheguidelinesofthe 28uC. The seed inoculums (10%) were transferred into 200ml
Committee for the Purpose of Control and Supervision of production medium in 1 L Erlenmeyer flasks. The inoculated
Experimental Animals (CPCSEA), Government of India (Regis- cultures inthe production medium were incubated for 72h on a
tration No: 885/ac/05/ CPCSEA) and as approved by the rotary shaker (2000rpm) at 28uC. After fermentation the broth
Institutional Animal Ethics Committee (IAEC) University of was centrifuged at 4000rpm for 10min and the filtrate was
Calcutta, and confirms with the Guide for the Care and Use of separated.
Laboratory Animals published by the US National Institutes of
Health (NIHPublication No.85–23, revised 1985). Assay for protease inhibitor activity
Toamixtureofmercuricchloride,phosphatebufferandtrypsin
Media, chemicals and reagents solution, marine actinobacterial extracts were added. After
All the media were purchased from Himedia, India. Giemsa’s adjusting the pH to 7.5 BAPNA (N-a-benzoyl-DL-arginine-p-
azur eosin methylene blue solution and Hematoxylin and eosin nitroanilide)wasaddedandincubatedat37uCfor20mins.After
stains for microscopy were obtained from Merck KGaA Frank- incubation, 5% of the TCA was added and incubated at room
furter Str., Germany. A phosphate buffer solution (PBS), sodium temperature for 20mins. Later, the solution was filtered with
bicarbonate,sodiumazide,RNaseA,NBT(nitro-bluetetrazolium Whatmann no.1 filter paper and the absorbance was taken at
chloride) and BCIP (5-bromo-4-chloro-39-indolyphosphate p- 280nm. The inhibiting activity of protease inhibitor was
toluidine salt) (Cat# RM 578, RM 2577) were procured from expressed usingfollowing formula.
Himedia chemicals, India. Antibodies against TGF-b, TNF-a, b- Inhibiting activity (%)= C-T/C X 100
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Table1. Insilicostudiesofprotease inhibitor from marine actinobacteria.
Receptors Ligands-BindingAffinity(kJ/mol)
A.S.albogriseolus B.S.longisporus C.S.fradiae D.S.lividans E.S.griseus
PLASMEPSIN-II 2692.4540 2458.0594 2440.7606 2574.2310 2673.7805
FALCIPAIN-2 2192.4911 2590.0973 2269.6229 2420.6248 2491.3755
ThedockingrevealedtheS.albogriseolushasatendencytoinhibitthePLASMEPSIN-IIreceptoreffectively.Inaddition,S.longisporoustendstoinhibitFALCIPAIN-2
receptors.
doi:10.1371/journal.pone.0090972.t001
Kinetics of protease inhibition by protease inhibitor primers. The sequence was analysed by ABI3730XL capillary
The mode of inhibition of protease inhibitor against trypsin DNAsequencer(ABIPrism310GeneticAnalyzer,Tokyo,Japan).
activity was measured with increasing concentrations of BAPNA Further, thegenus andspecies were successfully identified.
(0.5,1,2and4mM)asasubstrateintheabsenceandpresenceof
proteaseinhibitorsat0.5 mg/mland1 mg/ml.Optimalamounts Extraction, purification and characterization of protease
of protease inhibitor were determined based on the enzyme inhibitor
inhibitory activity assay. Mode of inhibition of protease inhibitor TheStreptomycesspLK3wassubjectedtofermentationfor7days
was determined by Lineweaver–Burk plot analysis of the data in production medium. After fermentation, the supernatant was
calculated following Michaelis-Menten kinetics. collectedandpurifiedbyfoursub-sequentialstepsviz.,ammonium
sulphateprecipitation,dialysis,Ionexchangechromatographyand
Dialysis for reversibility of protease inhibitor action preparative HPLC chromatography. Thefunctional groupof the
Trypsin (100 U/ml) was incubated with protease inhibitor compound was measured using Fourier transform infrared (FT-
(23.5 mg/ml) in 0.5ml of tris HCL buffer (10mM, pH 7.5) for IR)spectroscopy(ThermoNicolet-330,USA)usingaKBrpellet.
2 hat37uCanddialyzedagainsttrisHCLbuffer(1 mM,pH 7.5) The structure of the compound was established by using the
at 4uC for 24h, changing the buffer every 12h. Another spectral data obtained from gas chromatography (Perkin Elmer,
premixed-enzyme solution (0.5ml) was kept at 4uC for 24h Claeus 680) -mass spectrometry (Claeus 600) at a flow rate of
without dialysis for the control experiment. Reversibility of 1 ml/min with a carrier gas of helium and Liquid Chromatog-
proteaseinhibitorhasbeendeterminedbycomparingtheresidual raphy-Mass Spectrometer (LC-MS) (Thermo LCQ Deca XP
enzyme activity afterdialysis with that of non-dialyzedone. MAX).TheprotonNMR(1HNMR),carbonNMR,H,H-COSY
andHSQC(13CNMR,VBrukerAvanceIII500MHz(AV500))
Identification of the actinobacterial Isolate spectra of the compounds were obtained by using a dimethyl
The strain was screened based on the protease inhibitor sulfoxide d6(DMSO-d6) assolvent.
production and the efficient isolate was subjected to molecular
characterization based on 16s rRNA sequencing chromous In vitro anti- Pf activity
biotech,Bangalore,India.The16srRNAfragmentwasamplified P. falciparum isolates NZR-2 was obtained from National
using PCR polymerase. The PCR product was sequenced bi- InstituteofMalariaResearch,NewDelhi,India.Theexperiment
directionallyusingtheforward(59-AGAGTRTGATCMTYGCT- was carried out in a 96-well microtitre plate by adding 150ml of
WAC-39) and reverse (59-CGYTAMCTTWTTACGRCT-39) infected human red blood cell suspension to each well. The
Figure1.IntermolecularinteractionsbetweenPLASMEPSIN-IIandProteaseinhibitorofmarineactinobacteriaA.S.albogriseolusPI;
B.S.longisporusPI;C.S.fradiaePI;D.S.lividans;E.S.griseus.EnzymePLASMEPSIN-II(indicatedinredcolor)andtheircorrespondinginhibitors
(indicatedingreencolor)arerepresentedinribbonconfiguration.
doi:10.1371/journal.pone.0090972.g001
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(IAEC) University of Calcutta, and confirms with the Guide for
Table2. Proteaseinhibitor activity ofmarine actinobacteria.
the Care and Use of Laboratory Animals published by the US
NationalInstitutesofHealth(NIHPublicationNo.85–23,revised
1985).
LK1 90 89 30
ParasitestrainPlasmodimbergheiANKAobtainedfromNational
LK2 91 91 42
InstituteMalariaResearchCenter,NewDelhi.Parasitizedmouse
LK3 98 95 40
red blood cells (pRBC) from a liquid N preserved stabilize were
2
injected (16106 pRBC, in 100ml phosphate buffer solution) into
doi:10.1371/journal.pone.0090972.t002
miceofthesamebackground.MicewereinfectedwithPbA(16106
pRBC), in 100ml PBS by intraperitoneal injection, after ampli-
peptide was dissolved separately in RPMI-1640 to obtain a
ficationanequalnumberofuninfectederythrocyteswereinjected
concentrationrangeof20,40,60,80and100mg/mland50mlof
into control mice. Parasitemia was monitored daily in all
each dilution was poured into individual wells separately.
experimental groups by Giemsa-stained thin blood smears made
Artemisininwasusedasapositivecontrolandwaterasanegative
fromtailsnips.Survivabilityandparasitemiaofmice(n=60)were
control. The microtitre plate was incubated for 48hours. The
alsoobserveddaily.Thepercentageofparasitemiawascalculated
percentage of parasitemia was determined microscopically after
asfollows:Parasitemia(%) = [(numberofinfectederythrocytes)/
Giemsa staining.
(total number of erythrocytes counted)]x 100.
In vivo anti- Pf activity
Experimental groups parasite clearance and curative
Male Swiss albino mice (,25g each; aged 6 to 8 weeks) were
efficacy
maintained in sterilized cages and absorbent media; food and
Foroptimizationofdrugconcentration,astocksolutionofLK1,
waterwereprovidedadlibitum.Animalexperimentswerecarried
LK2andLK3(5000mg/ml)weredissolvedin(PBS),and50mice
out as per the guidelines of the Committee for the Purpose of
received 100ml i.p. injections each of LK1, LK2 and LK3 (600,
Control and Supervision of Experimental Animals (CPCSEA),
800, 1000and 1200mg/kg/ BW respectively) from2 dpi tillthe
Government of India (Registration No: 885/ac/05/ CPCSEA)
last day of survival. The control group received injections of
and as approved by the Institutional Animal Ethics Committee
vehicleonthesametreatmentschedule.Parasitologicalevaluation,
Figure2.StreptomycesspLK3strainshowedhighproteaseinhibitoractivityagainsttrypsinandchymotrypsinwithIC valuesof
50
LK3strainA.trypsin(96.77mg/ml)andB.chymotrypsin(161.8mg/ml)B.Modeofproteaseinhibitionbypeptide.Thedouble-reciprocalplot
displayed non-competitive inhibition mode C. Reversibility of peptide action was confirmed by the continuous steep decline in Vmax with an
increaseininhibitorconcentrationwithKmvalueremainingconfined.
doi:10.1371/journal.pone.0090972.g002
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methanolandstainedwithGemsaatpH 7.2,forparasitemia.The
Table3. Kineticanalysis ofprotease inhibitionbypeptide.
microscope had an Ehrlich’s eyepiece and a nose diaphragm
showing about 100 red blood cells per field. The number of
parasitized erythrocytes in each of the 10–50 such fields were
Peptide(mg/ml) Vmax(mM/min) Km(mM)
counted three times and the average was calculated to give the
0 0.24 1.64 Parasitemia of each individual animal. Percentage of suppression
0.5 0.19 1.54 was calculated byusing thefollowing formula[23,24,25].
1 0.14 1.65
2 0.10 1.7 %Suppression~Parasitemiainnegativecontrol(cid:2)
doi:10.1371/journal.pone.0090972.t003 Parasitemiainstudygroup=Parasitemiainnegative
control
survivability indicated that LK1 (800mg/kg/BW), LK2
(1000mg/kg/BW) and LK3(600 mg/kg/BW) was thebest drug
concentration. Parasitamia and survivability in mice were mon-
itoreddaily (n=60).Theexperimental animalsweredividedinto
Histopathology of spleen
groups such as (1) uninfected control, (2) PbA (16106 pRBC)
The spleen was aseptically removed from each host and
infected,PbAinfectedandtreatedwithLK1(800mg/kg/BW)(3)
immediately washed in phosphate buffered saline (PBS, pH 7.4).
PbAinfectedandtreatedwithLK2(1000mg/kg/BW)and(4)PbA
The tissues were then fixed for 24h in buffered formaldehyde
infected andtreated withLK3(600 mg/kg/BW). Foran analysis
solution(10%inPBS)atroomtemperature,dehydratedbygraded
on respective dpi, 5 mice from each experimental group were
ethanol, andembeddedinparaffin (MERCK,solidification point
used.
60–62uC). Tissue sections (5-mm thickness) were then deparaffi-
The‘8-daysuppressivetest’wasadoptedforthedetermination
nized with xylene, re-hydrated with graded alcohols (100–50%
of the parasite clearance and curative efficacy, respectively. The
ethanol), stained with eosin and hematoxylin, and then mounted
dosesforLK1(800 mg/kg/BW)(3)PbAinfectedandtreatedwith
in DPX resin (Merck, Mumbai, India). Digital images were
LK2 (1000mg/kg/BW) and (4) PbA infected and treated with
captured with Olympus CAMEDIA digital camera, Model C-
LK3(600mg/kg/BW).Groupsof5miceeachwereadministered
7070wide zoom.
vehicle only. Tests were performed in a 8 day Suppressive
standard test. The Plasmodium berghei is most widely employed in
Western blot analysis and cytokine assays
therodent malaria parasite.
ThemechanismswereelucidatedbyWesternblotanalysisand
Thin smears of blood films were obtained from the peripheral
cytokineassays asdescribed earlier [26].
blood on the tail from each mouse on day four after infection
[23,24].Thesmearswereplacedonmicroscopeslides,fixedwith
Statistical analysis
Valuesbetweengroupsonsameordifferentdpiwereanalyzed
usingoneortwowayANOVA.Allvaluesareshownasmean6
SD, except where otherwise indicated. Data were analyzed and
when appropriate, the significance of the differences between
meanvalueswasdeterminedusingStudent’stest.*Pvalues,0.05
were considered significant for all statistical analysis, otherwise
stated.
Results and Discussion
Estimation of protease inhibitors efficacies using in silico
molecular docking
To validate the efficacy of protease inhibitors from marine
actinobacteria as a suitable targets for the malarial protease, five
inhibitors (S. albogriseolus, S. longisporus, S. fradiae, S. lividans and S.
griseus)wereassayedagainstPLASMEPSIN-IIandFALCIPAIN-2
using Hex server (Table.1). Docking calculations revealed that S.
albogriseolus have a probable tendency to inhibit the PLASMEP-
SIN- II receptor effectively and may outperform the other
inhibitors. On the other hand, S. longisporous tends to inhibit
FALCIPAIN-2receptors,whencomparedtotheotherinhibitors.
The preliminary study revealed that the protease inhibitors from
fiveactinobacteriainhibittherespectivereceptorsmoreeffectively
(Fig.1).Hence,wehadhypothesizedtheinhibitingcharacteristics
of protease inhibitors obtained from marine actinobacteria
comparatively and their roles in malaria chemotherapeutic
Figure3.The16sRNAsequencingconfirmedthestrainLK3isa development were elucidated usinginsilico studies.
novel species with G+C content of the isolated DNA at 57.29
mol%.
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Figure4.Purificationofproteaseinhibitor.A.ElutionpatternofproteaseinhibitoronDEAE-Sepharoseanionexchangecolumn;B.Elution
patternofproteaseinhibitoronC18-RPHPLCcolumn;C.LCMSanalysisofpurifiedcompound.
doi:10.1371/journal.pone.0090972.g004
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Figure5.Characterizationofproteaseinhibitor.A.MassspectrumofpeptidecompoundobtainedfromtheGCMSclearlyrevealedthatitmay
beasmallpeptidewith5or6aminoacids(MolecularWeight-568da)Theaminoacidsmaybepresentinthepeptideare:AILKRVMGNC;B.FTIR
spectrumofpeptidecompound.Absorbanceat1632correspondedtothecarbonylfunctionswhereastheabsorbancefrom3300–3600cm-1maybe
duetotheNHfunctionfromtheaminoacidfunctionalgroups.
doi:10.1371/journal.pone.0090972.g005
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Figure6.CharacteristicsofPbAinfectioninSwissAlbinoMiceandeffectofLK1(800mg/kg/BW),LK2(1000mg/kg/BW)andLK3
(600mg/kg/BW) respectively. (A) Resulting parasitemias were expressed as a percent parasitemia (mean 6 SD), measured daily on Giemsa
stained blood smears. Parasitemia was evaluated statistically by Two-way ANOVA with Bonferroni post-test showed that the parasitemias were
significantlydifferent(*p,0.05)betweenPbAinfectedgroupsandLK3treatedgroups,whereasparasitemiaswerenotsignificantlydifferentinthe
LK1,LK-2groupw.r.t.PbAinfectedcontrolsandthematchedcontrols(P.0.05).(B)EffectofLK1(800mg/kg/BW),LK2(1000mg/kg/BW)andLK3
(600mg/kg/BW)respectivelyonsurvivalofmiceinfectedwithP.bergheiANKA.EachvalueinYaxisrepresentsthepercentage(%)ofsurvivalofthe
treatedgroupscomparedtoPbAtreatedcontrolmice.Thetreatmentgroupsrepresentmiceinjectedintraperitoneallyfrom2dpitillthelastdayof
survival.Resultsarepresentedasarithmeticmean(6SE)offivemicepergroup.(C)PercentageparasitemiasuppressionafteradministrationofLK1
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(800mg/Kg),LK2(1000mg/Kg)andLK3(600mg/Kg)respectively.Datafromfivereplicatesarepresentedasarithmeticmean6standarddeviation
andindicatep,0.05withrespecttothecontrols.Datashownareonerepresentativeexperimentoffive(n).
doi:10.1371/journal.pone.0090972.g006
Screening of protease inhibitor producing marine isolated from the marine Salinispora tropica possessed antimalarial
actinobacteria activity [31]. It is also acts as a potent proteasome inhibitor in
development for treatingcancer [32].
Among the 100 isolates tested for protease inhibitor activity,
LK-1,LK-2andLK-3strainshowedsubstantialproteaseinhibitor
activity. The optimized 3 strains were subjected to fermentation Mode of protease inhibition
and the protease inhibitor extracts were lyophilized. These 3 The mode of inhibition of peptide on trypsin activity was
strains were initially assayed against trypsin, chymotrypsin and analyzedusingLBplot.Thedouble-reciprocalplotdisplayednon-
proteinaseK(table2).Inthese,StreptomycesspLK3strainshowed competitiveinhibitionmode(Fig.2B)asthereisacontinoussteep
high protease inhibitor activity against trypsin and chymotrypsin. declinein Vmaxwith anincrease ininhibitorconcentration with
Thepotentialstrainwith50%inhibitonwerefurthersubjectedto Kmvalueremainingconfined(Table3).Enzymeactivityoftrypsin
determine an IC value. The LK3 extract inhibited trypsin and was completely recovered after the dialysis, as showed by the
50
chymotrypsin with IC values of 96.77mg/ml and 161.8mg/ml enzyme mixed inhibitor curve (EID) which was similar to the
50
respectively (Fig.2 A). curves of enzyme control without dialysis (EC) and dialysis (ED)
Out of 100, 3 isolates exhibited moderate to high protease (Fig. 2C). A dialyzed mixture of enzyme and extract confirmed
inhibitor activity of trypsin and chymotrypsin. Many reports are that processes are not affected the enzyme activity dialysis.
available on the production of inhibitors from Streptomyces sp viz., However, the non-dialyzed mixture of enzyme and extract (EIC)
S.mauvecolor, S.michiganensis, S.violaceus and S. yokosukaensis were showed less inhibited activity and it confirms the irreversible
produced antipain against papain enzyme. It is also capable to nature of protease inhibitor.
inhibit trypsin and cathepsin B. S.hygroscopicus and S.lavendulae The main role of irreversible inhibitor is once target enzyme
producedchymostatinagainstchymotrypsin.S.griseoruberproduced inhibited, it cannot reactivate and the organism must reproduce
elastatinal against elastase [27,28]. So far, only few reports only the enzyme. Several reversible protease inhibitors reached the
available for anti- Pf activity of marine actinobacteria. Maskey et market as a drug but only few drugs of irreversible protease
al.(2004)reportedthatthetrioxacarcinsAandDisolatedfromthe inhibitors are available such as aspirin and b-lactam antibiotics.
marine Streptomyces sp. isolate B8652 BCC 5149 possessed Sajid and McKerrow (2002) reported irreversible inhibitor could
‘‘extremely high antiplasmodial activity’’ against the parasite P. beusedinbacterial,viralandparasiticdiseasesandinthefuture,
falciparum K1 and NF54 strains which was much higher than the irreversible inhibitors are promising source for disease treatment
clinically used compound chloroquine [29]. Peraud (2006) [33].
reported that the manzamines isolated from the marine Micro-
monospora sp. strain M42 possessed antimalarial activity [30]. Identification of potential strain
Prudhomme et al (2008) reported that the salinosporamide A, The 16s rRNA sequencing is a method of choice for tracing
bacterial phylogeny and definite the taxonomy [34]. Hence, the
potentialstrainwasidentifiedasStreptomycesspLK-3through16S
rRNA Sequencing. The BLAST search showed only 93%
similarity with Streptomyces mutabilis. The strain was deposited in
the Bankit (GenBank, NCBI, USA) under the accession number
JF710608. The 16s rRNA sequencing of strain LK3 was
confirmed that it occupies a distinctive phylogenetic position
within the radiation including representatives of the family
Streptomycetes using neighbor-joining tree (Fig. 3) and it forms
aseparatesubcladefromothermemberofStreptomyces.Itconfirms
thestrainLK3isanovelspecies.TheG+Ccontentoftheisolated
DNAis57.29mol%(http://tubic.tju.edu.cn/GC-Profile/).Based
on the chemotaxonomy, morphological and 16S rRNA gene
sequence data suggest that strain LK3 represents a novel species
whencomparedwithtypestrainsofspeciesinnonomurakey.The
polyphasic taxonomic study of strain LK3 merits classification as
Figure 7. Histopathological changes in hepatic and splenic
thetypestrainofanovelspecieswithinthegenusStreptomyces,and
tissues in response to LK3 treatment during PbAinfection.
Hematoxylinandeosinstainswereusedtopreparesectionsoftheliver hence thestrain werenamed asStreptomyces spLK3.
andspleenfrommicetreatedwithvehicle,PbAinfected+600mg/Kg
LK3andPbAinfectedrespectively.Sectionsofcontrol(A)andPbA+LK3 Purification and characterization of protease inhibitor
infected(B)dpilivershowing,surroundinghepatocyteswithnucleiand
The RSM optimized Streptomyces sp LK3 [35] was subjected to
blood sinusoids lined with Kupffer cells. Liver from infected mice (C)
shows,moriformvacuolizationofhepatocytesandhemozoinpigment- fermentation(7days)andextractedproteaseinhibitorwaspurified
edKupffercellson8dpi.Incontrol(D)andPbA+LK3infectedspleen by a four sub-sequential steps viz., Ammonium sulphate precip-
tissue section (E), distinct and defined separate cluster of white pulp itation, dialysis, Ion exchange chromatography and preparative
markedbyyellowarrows.Intreatmenttissuesectionswhitepulpand HPLCchromatography.Theproteaseinhibitorwasachieved80%
redpulparenotdistinct(F).Appearancesofhollowspaceswithoutcells
saturation. Following this, dialysis and anion exchange chroma-
observed in spleen tissue sections compared to that of control (red
tography wasperformed.
arrows). Black arrows represent hemozoin accumulation in liver and
spleentissuesections.Magnificationindicated406. The eighth peak contains the highest protein concentration
doi:10.1371/journal.pone.0090972.g007 (0.15mg/ml). Similarly, Angelova et al (2005) was purified and
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ProteaseInhibitorforAntimalarialTreatment
Figure8.Celllysatefromrespectivecontrol,treatmentspleenandliverweresubjectedtowesternblotanalysisandELISA(A)liver
and(B)spleenduringPbAinfectionandtreatedwithpeptide(C)ELISAresults(p,0.05,ANOVAfollowedbyposthocLSDtest).
doi:10.1371/journal.pone.0090972.g008
characterizedanovelproteaseinhibitor(PISC-2002)isolatedfrom InhibitorsfromStreptomycesspwasclassifiedasSSIfamilyinhibitors
culture supernatants of Streptomyces chromofuscus using DEAE- and designated as SSI –like [39]. Hence, the protease inhibitor
Sepharose [36]. Figure 4A shows the elution pattern of the fromStreptomycesspLK3wasbelongstotheSSIfamilyinhibitors.
proteaseinhibitorandabroadactivepeakwasobtained,whenthe The microbial origin inhibitors are low molecular weight
dialyzed sample was passed through a DEAE Sepharose column peptides of unusual structures [40]. Similarly, protease inhibitor
with 0.4M NaCl Tris-HCl buffer with NaCl pH 7.5. Further from Streptomyces sp LK3 is a low molecular weight peptide of
purification was carried out by preparative HPLC on a C unusual structure. In 1969, Umezawa discovered first low
18
column (Fig. 4B) and a single peak was confirmed by LCMS molecular weight enzyme inhibitor from Streptomyces sp [41]. At
(Fig. 4C). About 10mg of pure protease inhibitor was obtained present,morethan100inhibitorswerereportedbutthisisthefirst
from3 g of lyophilisedsupernatant. report on marine Streptomyces sp.
MassspectrumobtainedfromtheGCMSclearlyrevealedthatit
may be a small peptide with 5 or 6 Aminoacids (Molecular In vitro anti- Pf activity
Weight-568da) (Fig. 5A). The 1H, 13C, H,H-COSY, HSQC also A total of 3 different actinobacterial isolates were chosen for
showed the chemical shift values in the aliphatic region (2.5–4.0 anti- Pf activity based on the protease inhibitor activity. Extracts
and 5.0). This chemical shift may be due to the alpha hydrogens were tested at five initial concentrations: 20 mg/ml, 40mg/ml,
and NH hydrogens. The compound doesn’t have any chemical 60mg/ml,80mg/mland100mg/ml.Threeextractsshoweda50
shiftvaluesinthearomaticregion.ThecarefulexaminationofIR to 85% inhibition. The peptide from Streptomyces sp LK3 extract
spectra also revealed the presence of peptides. The IR spectra showed significant anti plasmodial activity (IC : 25.78 mg/ml).
50
showedabsorbanceintheregionof1632cm21,3300–3600cm21 Therestoftheotheractinobacterialextractsshowedweakactivity
(Fig. 5B). The absorbance around the 1632 corresponded to the (IC .200mg/ml).
50
carbonyl functions where as the absorbance between 3300–
3600cm21maybeduetotheNHfunction.Apart fromthistwo In vivo anti- Pf activity
characteristic peak, no other characteristic peaks have been Effect of LK1, LK2 and LK3 on survival and parasite
observed. The amino acids may be present in the peptide are: clearance efficacy during Plasmodium berghei ANKA
AILKRVMGNC. infection. The clinical course of infection in the experimental
Manyreportsareavailableontheproductionofinhibitorsfrom mice is summarized in Figure 5. Mice from both experimental
Streptomyces sp viz., S.mauvecolor, S.michiganensis, S.violaceus and groupswereagematchedandhad,PbAinfected+LK2treatedset
S.yokosukaensis were produced antipain against papain enzyme andinPbAinfected+LK3treatedset(Fig.6A)andcontinuedto
anditcanalsocapabletoinhibittrypsinandcathepsinB[37,38]. risetillmicedies.MostofthemicesuccumbedtoCMduring4–9
PLOSONE | www.plosone.org 10 March2014 | Volume 9 | Issue 3 | e90972