Peptidases Compartmentalized to the Ascaris suum Intestinal Lumen and Apical Intestinal Membrane

14 Pages·2014·5.01 MB·English

Peptidases Compartmentalized to the Ascarissuum
Intestinal Lumen and Apical Intestinal Membrane
Douglas P. Jasmer1.*, Bruce A. Rosa2, Makedonka Mitreva2,3.
1Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington, United States of America, 2The Genome Institute,
WashingtonUniversitySchoolofMedicine,St.Louis,Missouri,UnitedStatesofAmerica,3DepartmentofMedicineandDepartmentofGenetics,WashingtonUniversity
SchoolofMedicine,St.Louis,Missouri,UnitedStatesofAmerica
Abstract
Thenematodeintestineisatissueofinterestfordevelopingnewmethodsoftherapyandcontrolofparasiticnematodes.
However,biologicaldetailsofintestinalcellfunctionsremainobscure,asdotheproteinsandmolecularfunctionslocated
on the apical intestinal membrane (AIM), and within the intestinal lumen (IL) of nematodes. Accordingly, methods were
developedtogainacomprehensiveidentificationofpeptidasesthatfunctionintheintestinaltractofadultfemaleAscaris
suum.PeptidaseactivitywasdetectedinmultiplefractionsoftheA.suumintestineunderpHconditionsrangingfrom5.0to
8.0. Peptidase class inhibitors were used to characterize these activities. The fractions included whole lysates, membrane
enriched fractions, and physiological- and 4 molar urea-perfusates of the intestinal lumen. Concanavalin A (ConA) was
confirmed to bind to the AIM, and intestinal proteins affinity isolated on ConA-beads were compared to proteins from
membrane and perfusate fractions by mass spectrometry. Twenty-nine predicted peptidases were identified including
aspartic, cysteine, and serine peptidases, and an unexpectedly high number (16) of metallopeptidases. Many of these
proteins co-localized to multiple fractions, providing independent support for localization to specific intestinal
compartments, including the IL and AIM. This unique perfusion model produced the most comprehensive view of likely
digestivepeptidasesthatfunctionintheseintestinalcompartmentsofA.suum,oranynematode.Thismodeloffersameans
to directly determine functions of these proteins in the A. suum intestine and, more generally, deduce the wide array
functionsthat existin thesecellularcompartments ofthe nematodeintestine.
Citation:JasmerDP,RosaBA,MitrevaM(2015)PeptidasesCompartmentalizedtotheAscarissuumIntestinalLumenandApicalIntestinalMembrane.PLoSNegl
TropDis9(1):e3375.doi:10.1371/journal.pntd.0003375
Editor:JohnPiusDalton,McGillUniversity,Canada
ReceivedAugust26,2014;AcceptedOctober27,2014;PublishedJanuary8,2015
Copyright: (cid:2)2015 Jasmer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
DataAvailability:Theauthorsconfirmthatalldataunderlyingthefindingsarefullyavailablewithoutrestriction.Allprocesseddatausedfortheanalysisis
availableinS1andS2Tables.Ascarissuumproteindesignationsfordatapresentedinthepaperandsupplementaltableswerederivedfrompublicdatabase
entriesforthegenomesequenceofthisparasite.
Funding:TheresearchwassupportedbyNHGMSgrantGM097435toMM,andasubcontractfromthatgranttoDPJ.Thefundershadnoroleinstudydesign,
datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*Email:[email protected]
.Theseauthorscontributedequallytothiswork.
Introduction Consequently, knowledge of functions sited at this interface,
and cellular processes needed to maintain it, could lead to
Parasitic nematodes cause major diseases of humans, directly approaches that disrupt intestinal cell functions critical for
affecting more than two billion people on a global scale [1]. parasite survival.
Diseases they cause in food animal species also pose significant Research on the AIM of the parasitic nematode Haemonchus
constraints to agricultural production and thus, indirectly impact contortus, a gastrointestinal nematode of small ruminants, has
humanhealthinregionsoftheworldwherenutritionalresources demonstrated theimportance of AIMglycoproteins astargets for
arelimited.Acquisitionofresistancetocontemporaryanthelmin- vaccines [4–6], advances on which have been extended to
tics by many species of parasitic nematodes is on the rise [2,3]. hookworms [7], and in stimulating host mucosal immune
Hence,theneedforresearchtoidentifynewtargetsfortherapies responses during an infection [8]. These advances have under-
totreatandcontrolinfectionsbythesepathogenshasneverbeen scored the unique value of the nematode intestine as a target for
greater.Thenematodeintestineisonetissueofimportanceinthis vaccines.ImportanceoftheH.contortusintestineasadrugtarget
context. was demonstrated relative to a benzimidazole anthelmintic [9],
The intestine of parasitic nematodes is formed by a single which when used to inhibit AIM biogenesis caused catastrophic
cell layer. The apical intestinal membrane (AIM) forms an damagetotheH.contortusintestine.ThenematodeAIMisalsoa
intestinal lumen (IL), and both the AIM and IL are accessible primarytargetforcrystalproteintoxinsproducedbyBacillusspp.,
from the outside host environment. In combination, these two which have efficacy against multiple parasitic nematodes [10].
intestinal cell compartments form a major parasite interface Propertiesuniquetothenematodeintestineappeartoaccountfor
with the host that performs a wide range of biochemical and anthelminticeffectsofboththebenzimidazoleandcrystalprotein
cellular functions essential for survival of these pathogens. treatments. Consequently, the nematode intestine presents multi-
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AscarissuumIntestinalPeptidases
Author Summary Methods
Past research has demonstrated that the nematode Additional details are provided in Supporting Information (S1
intestinehasvaluefordevelopingnewmethodsoftherapy Text).
and control of parasitic nematodes, as related to both
vaccines andotheranthelmintics.Yet,informationrelated Parasite material and intestinal fractionation
to basic intestinal cell biology is very limited. Research AdultfemaleAscarissuumwereobtainedfromswineinfectedas
progressreportedheremovestowardsthecomprehensive weanling pigs (mixed breed, Swine Center, Washington State
identification of proteins (peptidases and others), and University) 60 to 70 days post-infection. Infections were initiated
hence functions, that are sited on the apical intestinal
with A. suum eggs collected from uteri of three or more patent
membraneandwithintheintestinallumenofadultfemale
female worms. Eggs were cultured in distilled water at room
Ascaris suum. These advances provide an unprecedented
temperature for 30 days to allow eggs to embryonate, and then
research model to determine critical functions sited at
treatedwith0.25%sodiumhypochloriteforuptosevenminutesto
theselocationsandtodevelopapproachestoinhibitthose
decoat the eggs, followed by three rounds of washes in 50ml
functions. Comparative analysis among diverse parasitic
distilledwater,andthenstoredat5Cintherefrigeratoruntiluse.
species raises expectations that the results from A. suum
Parasiteintestinalsamplesweredissectedfromtwoormorefreshly
can be applied to many parasitic nematodes for which
similar researchis technicallyimpossible to perform. isolated worms maintained in ice cold phosphate buffered saline
(PBS, pH 7.4). Intestinal samples were stored 280C until used.
Intestinal tissue samples used for centrifugal fractionation and
plebiologicalcharacteristicsthatcanbetargetedbynewtherapies ConcanavalinA(ConA)isolationandanalysiswerenotnecessarily
for treatment andcontrol ofparasitic nematodes. fromthesame wormpreparations.
Nevertheless, details of intestinal cell functions remain
obscure, in part due to research challenges imposed by the Protein fractions
smallsizeofmanyparasiticnematodes,includingH.contortus. Whole intestine and pseudocoelomic fluid. To generate
In this context, thesizeof adultAscarissuum,the large round protein samples for peptidase experiments, frozen intestinal
wormofswine(20–35 cmcomparedtoca.2.5 cmforadultH. samplesweregroundinliquidnitrogenusingamortarandpestle.
controtus), offers research capabilities absent with many other Approximately50mlofgroundfrozensampleweretransferredto
nematodespecies.ThecloserelationshiptoA.lumbricoides,the amicrofugetubeand1 mlofPBSwasaddedtothesamplewhich
most globally prevalent parasitic helminth of humans [11], was briefly vortexed and then treated in three rounds of a freeze
makes A. suum research particularly relevant to this human (220C)thaw(4C)cycle.Thehomogenatewasthencentrifugedat
pathogenandglobalhealth. 5,0006g for 10minutes, which produced a pellet (P1) and
Previous comparisons of transcripts expressed in the intestines supernatant (S1). The S1 supernatant was centrifuged at
of H. contortus, Caenorhabditis elegans and A. suum identified 50,0006g for 30minutes producing a 5,000 to 50,0006g pellet
likely, orthologous intestinal proteins in common among these (P2)andsupernatant(S2).Pseudocoelomicfluid(PF)wasobtained
species, which represent a phylogenetic distance spanning an bymakinganincisionwithirisscissorsalongthebodywalllocated
estimated350millionyears[12].Thesesimilaritiesweredetected between thelateral attachments of theintestine to thebody wall.
despite the utilization of markedly different nutrient sources by This methodavoided damage totheintestine andallowed access
these species, e.g. host blood, H. contortus; bacteria, C. elegans; to pseudocoelomic fluid, which was collected with a needle and
andhostintestinalcontent,A.suum.Thiscomparativeapproach, syringe fromthebody cavity.
coupled with larger genome projects [13], has begun to clarify Cannulationandcollectionofintestinalperfusates. Adult
orthologous protein groups with potential for broad biological A.suumwormswerecollectedfrominfectedpigsandimmediately
application to many species of nematodes, whether parasitic or placed in 37C PBS. They were processed within three hours for
not. cannulationandcollectionofperfusates.Theanteriorendsofadult
Inresearchreportedhere,wecoupledadvantagesconferredby female A. suum worms were removed by scalpel just below the
the large size of A. suum, and past progress made with H. esophagus.Abluntneedlecannula(25gauge),bluntedbygrinding
contortus, to develop an improved model for investigating withaDremelgrindingwheel,wasinsertedintotheanteriorendof
functionslocatedontheAIMandintheILofA.suum.Previous theintestine.Supergluegelwasappliedaroundthecannula,about
researchclarifiedpropertiesofsomeglycoproteinsandpeptidas- three quarters of the way up the cannula prior to insertion. The
es located on the H. contortus AIM surface [4,6,14,15], and cannulawasinserteduntilwormtissuecameintocontactwiththe
intestinal transcript analysis identified candidate, orthologous superglue.Liquidsupergluewasthenappliedaroundthejunction
intestinal proteins from A. suum [12], which we hypothesize wheretheanteriorendofthewormencounteredthesupergluegel.
perform related functions in these two species. In addition to Theposterioronesixthofthewormwasthenremovedtoeliminate
peptidases, many other proteins are expected to reside at the a fragilesection oftheintestine. Approximately onecentimeterof
nematode AIM, or in the IL, fulfilling roles in digestion and theremainingposteriorendofthebodywasresectedtoexposethe
nutrient transport, as examples. The approach reported here intestine. Perfusates injected into the anterior end of the intestine
facilitatedidentificationofanextensivesetofapparentA.suum werecollectedfromtheposteriorendthatwasplacedontoParafilm
peptidasesthataresitedattheAIMandIL,inadditiontomany M (Pechiney Plastic Packaging Co., Chicago, IL), which allowed
other putative AIM and IL proteins. The results have greatly manipulation of the posterior end of the intestine in positions to
expanded concepts on apparent functions that reside at the A. reducecontaminationofpseudocoelomicfluid(PF).Perfusionwith
suumAIMandintheIL.TheapproachalsoestablishedA.suum dyeindicatedalumenalcontentofca.50ml.Cannulatedintestines
as a unique model to directly investigate i) functions that are were perfused with approximately 300 to 500ml of PBS (PBS
located in these nematode intestinal cell compartments and ii) perfusate),followedbyasimilarvolumeof4MureainPBS(4MU
methods to inhibit those functions, in context of improved or perfusate),eachdeliveredwithatuberculinsyringeattachedtothe
noveltherapiesandmethodsofparasitecontrol. cannula. Maintenance of worms at 37C prior to cannulation
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AscarissuumIntestinalPeptidases
appeared to enhance the flow of perfusates through the intestine. 100mMphosphate(pH8.0).SampleswereincubatedwithBodipy
Perfusate samples were stored at 220C until used in various FL casein (E6638, Life Technologies, Grand Island, NY) for two
experiments. Protein concentrations for all samples were deter- hours in a C-1000 Touch thermal cycler with a CFX96 Optical
minedusingabicinchoninicacidassay(MicroBCAProteinAssay Reaction Module (Bio-Rad, Hercules, CA) at 37uC, in a total
Kit, Thermo Scientific, Rockford, IL). The PBS and 4MU volumeof50ml.Assayswereconductedintriplicateusing96well
perfusates were collected as a paired set from individual worms. PCR plates (Bio-Rad, Hercules, CA). Fluorescence signal was
ResultsfrompeptidaseassaysandConAblotswerepresentedfora measured(excitation490nm;emission530nm).Netfluorescence
setofperfusates.Apairedsetofperfusateswasalsousedforanalysis signal was determined by subtraction of starting values from end
by mass spectrometry. Perfusate samples were derived from values. Mean fluorescence was calculated for no protein controls
differentwormsusedtogenerateintestinaltissuesamplesforother afterincubationwithBodipyFLcaseinatagivenpHfortwohrs.
experimentsinvolvingwholetissueorfractionsfromwholeintestinal Thisbackgroundvaluewassubtractedfromeachfluorescencevalue
tissue. obtainedforsampleswithprotein.Activitywasexpressedasmean
ConcanavalinA(ConA)bindingproteins. Wholeintestinal relativefluorescenceunitspermgofprotein.
lysate was prepared with peptidase inhibitors (1mM Pepstatin A, ForanalysisofConAbindingproteins,intestinalsupernatantS1
1mMPMSF,10mM1,10Phenanthroline,5mMIodoacetamide, solubilized in 1% TX-100 was incubated with beads (1mg per
Sigma, St. Louis, MO) and solubilized with 1% sodium dodecyl 100mlpackedbeads)for2hourswithinversion,thenwashedwith
sulfate(SDS).Thelysatewasclarifiedbycentrifugationat5,0006g Binding Buffer containing divalent cations (50mM TRIS,
and then the supernatant was diluted to 0.25% SDS with Binding [pH7.4],500mMNaCl,1mMeachMgCl ,MnCl ,andCaCl ).
2 2 2
Buffercontainingdivalentcations(50mMTRIS[pH7.4],500mM ConA-agarosebeadswithboundproteins(10ml)weretransferredto
NaCl,1mMeachMgCl2,MnCl2,andCaCl2).ConA-agarosebeads wellsof96wellflat-bottomedplates(CorningCostar,Corning,NY)
(Sigma,St.Louis,MO)wereincubatedwithsolubilizedlysate(2mg
in 50ml total volume for assays as described for peptidase assays.
per200mlpackedbeads).Themixturewasincubatedwithinversion Beadswithnoproteinswereusedfornoproteincontrols.The96
fortwohours,andthenwashedthreetimeswithninebeadvolumesof
well plates were rotated during incubation (50rpm) to ensure
BindingBuffer(50mMTRIS,pH7.4,500mMNaCl),then300ml
mixing. Reaction supernatants from which beads were eliminated
washesofpotassiumthiocyanate(0.25M,0.5M,1Mand2M)in
weretransferredtoa96-wellPCRplate(Bio-Rad,Hercules,CA)for
20mM TRIS [pH7.4], 0.2% Triton X-100, two washes per
fluorimetric measurements, as described for peptidase assays.
concentration),andthentwoBindingBufferwashes(9beadvolumes
Alternatively, SDS (1.0%) solubilized intestinal lysates were used
each).Proteinsremainingonthebeadswereelutedbyboilingin0.1%
for ConA bead isolation and analysis by mass spectrometry and
SDS for separation by SDS-polyacrylamide gel electrophoresis
testinginpeptidaseassays,butproducednodetectableactivity.
(PAGE)andmassspectrometricanalysis.
Statistical analysis
Histological analysis
Meanfluorescenceunitsgeneratedfromtreatmentsinpeptidase
10% formalin fixed adult female worms embedded in paraffin
assays were compared first by analysis of variance (ANOVA),
(Histology Laboratory, Washington State University) were sec-
followedbyTukey’smultiplecomparisonofmeans.ANOVAwas
tioned, attached to glass slides and deparaffinized and steam
conducted among treatment groups for individual pHs. For
treated. To assess specificity of ConA binding, sections were
inhibitionexperiments,ANOVAandmultiplemeanscomparisons
treated with sodium periodate (5 mM in 50mM sodium acetate
wereconductedtoidentifymeansofinhibitortreatedgroupsthat
buffer,pH 4.5),followedbysodiumborohydride(50 mMinPBS,
differed from the untreated control group at a given pH. For
pH 7.4) to disrupt carbohydrates containing vicinyl hydroxyl
significancewithConA-beadisolatedproteins,meanfluorescence
groups. Slides were ten treated with 0.3% hydrogen peroxide in
at each pH tested was assessed by 95% confidence intervals to
methanolfor30minat25uCtoeliminateendogenousperoxidases
determine iftheintervalswere above zero.
andthenincubated withConA-horseradishperoxidase(HRPO).
BindingwaslocalizedbydevelopmentwithMetalEnhancedDAB
Mass spectrometry – sample preparation
Substrate (Thermo Scientific, Rockford, IL.). Sections were then
counterstained with Mayer’s haematoxylin (Thermo Scientific, ConA bead-isolated proteins were separated on SDS-PAGE
Fremont, CA). gelsintwolanes,oneofwhichwasCoomassieBluestainedand
thesecondwastransferredtonitrocelluloseandprobedwithCon
SDS-PAGE analysis A-HRPO.Bandsdetectedinblotswereusedtoalignwithstained
bandsinthegel,whichwereexcisedandthenpreparedforinsitu
Protein samples were separated by SDS-PAGE under non-
trypsin digestion and analysis by LC-MS/MS as described [8].
reducing conditions, using 17% to 7% polyacrylamide gradient
Proteins in PBS and 4MU perfusates, PF, and P2 pellets were
gelsusingpreviouslydescribedmethods[15].Proteinswereeither
precipitated with the ‘‘2-D Clean-up kit’’ (GE Healthcare Life
stained with Coomassie Brilliant Blue R-250 or transferred to
Sciences), and then resolubilized in 8M urea, 100 mM Tris,
nitrocellulose filters. Filters were then incubated with ConA-
HRPO to localize glycoproteins by chemiluminescence (Pierce pH 8.5 (20 ml) for 30 min at 37uC. Samples were then reduced
ECLWesternBlottingSubstrate,ThermoScientific,RockfordIL) with1 mMtris(2-carboxyethyl)phosphineTCEP(2 mlof10 mM
recorded on x-ray film (Kodak O-MAT). To assess specificity of TCEP stock) at room temperature for 30 minutes, followed by
ConA binding, replicate nitrocellulose filters were treated with alkylation with 20 mM Iodoacetamide for 30 minutes in the
sodiumperiodateasdescribedfortissuesections,andcontrolfilters dark. Then, samples were quenched with 10 mM DTT for
were treated thesame,butexcluding sodiumperiodate. 15 minutesanddiluted1:4dilutioninTris(pH 8.5).
Peptidase assays Liquid chromatography, tandem mass spectrometry (LC-
Foranalysisofsolubleprotein,sampleswereadjustedto1%TX- MS/MS)
100andusedinassaysat0.5to4mgs(dependingonassay)ineach Proteins were digested sequentially with endoprotease Lys-C
well.Buffersusedwere100mMcitratephosphate(pH5.0–7.0)and (cleaving lysine at the C-terminus) and trypsin as previously
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Table1. IntFam-241proteins relatedto Haemonchus contortus, Ascaris suumand Caenorhabditis elegansintestinal peptidases.
Family1 Protein2 Cluster3
A1 GS_12574 AS00118.cl;
AS00709.cl
GS_15316 AS05229.cl
GS_19445 AS02056.cl
GS_21229 AS00866.cl
C01 GS_06461 AS00443.cl;
AS05277.cl
M1 GS_04166 AS01463.cl
M20 GS_16898 AS01671.cl
S28 GS_16223 AS00974.cl
1PeptidasefamilyasdefinedintheMEROPSdatabase[27].2ProteindesignationbasedonBlastXoftranslatedproteinsequences[18]usingcDNAclustersequencedata
[12].3AscarissuumintestinalcDNAclustersdescribedin[12].Proteinshighlightedinbold-italicswerenotdetectedinfractionsanalyzedinthispaper.
doi:10.1371/journal.pntd.0003375.t001
described [16] and then processed for liquid chromatograpy- A. suum intestinal peptidase activity
tandem massspectrometry(Supplemental Materials). In previous research, intestinal homogenates and lysates from
adultfemaleH.contortusprovedvaluablefordissectingbiological
Mass spectrometry data processing and analysis propertiesofAIMproteins,ofwhichalargefamilyofcathepsinB-
LC-MS data files (MS2 centroided) were used for database like (CBL) cysteine peptidases comprise a prominent component
searching with MASCOT (Matrix Science, version 2.3.0.0) [4,13,15,22,23]. This general approach was used to assess
using previously described software settings [17], against the peptidase activity in the adult female A. suum intestine. Activity
deduced A. suum proteome [18] and the Sus scrofa proteome was detected in A. suum intestinal lysates using a Bodipy casein
(Uniprot, downloaded Sept. 2012) to identify potential host substrate predominately atpH 5.0and6.0,withloweractivityat
contamination.Thresholdsfordetectionweresetinaccordance pH 7.0and 8.0(Fig.1A).
to the suggestions by the Scaffold documentation, and as The potential for A. suum peptidases to exist as membrane
described in other recent proteomics studies [19–21]. Proteins associated proteins was determined by enrichment for peptidase
that contained similar peptides and could not be differentiated activity in pellet fractions P1 (large debris) and P2, (5,000 to
based on MS/MS analysis alone were grouped to satisfy the 500006g) (Fig.1A). Although not exclusive to this fraction, the
principles of parsimony. resultsshowthatintestinalpeptidaseactivitywasenrichedinboth
P1 and P2 pellets relative to lysate of whole intestine or
Ethics statement supernatantfractionsintested,whichislikelytoreflectmembrane
Theresearchinvolvinguseofswinewasreviewedandapproved associated proteins.
bytheWashingtonStateUniversityInstitutionalAnimalCareand General inhibitors effective against distinct peptidase classes
Use Committee, protocol #04097-004., approved on 12/19/ were used to identify peptidases that might contribute to the
2013.GuidelinesareprovidedbytheFederalAnimalWelfareAct, activities detected in whole lysates (Fig. 1B) and the membrane
USA. enrichedfractionP2(Fig.1C).Inhibitorsofaspartic,metalloand
serine peptidases each caused inhibition of activity in intestinal
Results lysates, and often in a pH dependent manner (Fig.1B). For
instance, the aspartic and metallo peptidase inhibitors, pepstatin
A. suum homologues of previously reported, broadly and 1,10 phenanthroline, respectively, each inhibited activity at
conserved, intestinal peptidases themoreacidicrange.Incontrast,theserinepeptidaseinhibitor,
Peptidases fromfour different major classes (Aspartic, cysteine, phenylmethylsulphonylfluoride (PMSF) inhibited activity at neu-
metalloandserine)havebeendirectlyorindirectlylocalizedtothe tral and basic pH conditions, while some inhibition by 1,10
AIMofH.contortus[14,15,22].Predictedhomologsororthologs phenanthrolinewasalsoobservedunderthesepHconditions.No
of these H. contortus AIM peptidases are members of intestinal significant inhibition (p.0.05) was observed with an inhibitor of
proteinfamilies(IntFam-241,[12])foundtobeconservedamong cathepsinB-likecysteinepeptidases(E-64),andsimilarresultswere
this parasite, A. suum and C. elegans. Nucleotide sequences for obtainedwith thegeneralcysteine peptidaseinhibitor, iodoaceta-
these H. contortus AIM proteins encode signal peptides, but not mide(S1Fig.).ResultswiththeP2pelletweresimilartothelysate,
always hydrophobic sequences that are expected for integral except that PMSF showed greater inhibition under acidic
membrane proteins, suggesting a peripheral association with the conditions bycomparison tothewhole lysate.
AIM.ESTclustersequencesthatencodetheA.suumIntFam-241 The samples that were assayed are very complex. Although
homologuesofH.contortusAIMpeptidaseswerenextmappedby distinct effects were observed relative to pH and inhibitors, the
BLASTN to A. suum gene models from the published A. suum resultsshouldbeviewedasprovidinggrossindicationsofpeptidase
genome sequences [18]. The A. suum gene and corresponding activity in the intestinal homogenate and other fractions of
proteindesignations fortheseIntFam-241peptidases arelistedin interest,describedbelow.Theinhibitorresultsprovidedasenseof
Table 1.Accordingly,wehypothesizedthatthededucedA.suum diversity relative to peptidase class and pH dependency for the
proteins listedare AIMor ILpeptidases. peptidase activities detected, which will be evaluated in more
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introduced constraints on interpretations of those results. Never-
theless, inhibitors were routinely tested at pH 5.0 to assess
peptidase involvementinproducing signal intheassays.
Concanavalin A binding proteins from A. suum intestine
Severalknownproteins,includingpeptidases,locatedontheH.
contortusAIMareglycosylated[4,14].Hence,weinvestigatedthe
use of a lectin, concanavalin A (ConA), to identify glycosylated
proteins on the A. suum AIM. Glycans located on the A. suum
AIMwerepreviouslydetectedbyConA[24],andthisobservation
was confirmed here (Fig.2A, B). Pretreatment of tissue with
periodate prevented binding of ConA to the AIM surface,
indicating specificity of ConA binding to the A. suum glycans.
ConAbindingwasmostobviousonandatthebaseoftheA.suum
AIM. There was also evidence of ConA binding to material that
appeared loosely attached to the AIM and extended into the
lumen, which may reflect insoluble cellular material on the AIM
surface that isreleased fromtheAIM intothelumen.
ConA was used to probe blots of SDS-PAGE gels in which
proteins of intestinal lysates were separated (Fig.2C). A large
number of protein bands were identified that ranged inMr from
14toover225kDa.BindingofConAtothesebandswasinhibited
by pretreatment of the blot with periodate, which supported the
dependence of binding on the presence of periodate-sensitive
glycans.
Next, ConA-agarose beads were used to isolate ConA binding
proteins from Triton X-100 lysates of the intestine. Proteins that
remainedonthebeadsfollowingwasheshydrolyzedBodipycasein
at pHs that ranged from 5.0 to 8.0 (Fig.2 D). In this case,
inhibitors of aspartic, cysteine, metallo and serine peptidases
significantly reduced peptidase activity associated with beads.
(Fig.2E).AlthoughtheextendedtimerequiredforConAbinding
mayhavedifferentiallyaffectedthevariouspeptidaseactivities,the
results support that peptidases were isolated on ConA beads. On
the contrary, methods used to isolate ConA binding proteins for
analysis by mass spectrometry included SDS treatment, and no
peptidase activity was detected with beads from these prepara-
tions.
Fig. 1. Intestinal peptidase activity. A. Whole intestinal cell
Collectively,theresultsshowedthati)ConAboundbothtothe
fractionation of peptidase activity. Peptidase activity was determined
using a Bodipy casein substrate with 4mg of sample,as described in AIM and IL content of A. suum, ii) multiple ConA binding
methods, for whole intestinal homogenates (WL), 5,0006g pellet (P1) proteinsexistintheA.suumintestine,andiii)intestinalpeptidase
and supernatant (S1), and a further 50,0006g pellet (P2) and activity wasisolated on ConA-agarose beads.
supernatant (S2) derived from S1. All samples were solubilized in 1%
TX-100 prior to running assays. pH at which reactions were run is
Peptidase activity in perfusates of the A. suum intestinal
indicatedonthexaxis.Peptidaseactivity(yaxis)isreportedasRelative
Fluorescent Units (RFU) mg-protein21. Means that differ from one lumen
another(p,0.05)areindicatedbydifferentletterdesignations(A,B,C, The foregoing experiments provided general information on
D)belowthexaxis,andwereanalyzedaccordingtopHconditions.B, characteristicsofA.suumintestinalproteinsthatmayincludeAIM
C. Inhibition of peptidase activity in the whole lysate (B) and P2 (C)
orILglycoproteinsandpeptidases.Cannulationandperfusionof
fractions by pepstatin (aspartic proteases, Pep), phenylmethylsulfonyl
chloride(serinepeptidases,PMSF),1,10phenanthroline(metallopepti- the intestinal lumen provided a more direct approach uniquely
dases,1,10Phen)andtrans-epoxysuccinyl-L-leucylamido(4-guanidino)- supported by the large size of A. suum, as compared to other
butane, L-trans-3-carboxyoxiran-2-carbonyl-L-leucylagmatine, N-(trans- nematodes such as H. contortus and C. elegans, Fig.3 A-C
epoxysuccinyl)-L-leucine4-guanidinobutylamide(C1cysteinepeptidas- indicates the steps utilized in cannulation to perfuse the A. suum
es (including cathepsin B) cysteine peptidases, E-64). Percentage
intestine.Injectionofdyeintotheintestinallumenofwormswith
inhibition(yaxis)ofthemeanactivityuninhibitedcomparedtomean
an intact posterior end demonstrated confinement of dye to the
activityoftheinhibitedisshownforeachpH(xaxis)tested.Anasterisk
denotestreatmentsthatweresignificantlydifferent(p,0.05)fromthe lumen (Fig.3D).
untreated control group for each pH indicated. All assays were IL content was first collected by perfusion with phosphate
conductedwiththreereplicates. bufferedsaline(PBSperfusate).Thisperfusionwasfollowedinthe
doi:10.1371/journal.pntd.0003375.g001
same worms by perfusion with PBS containing 4 M urea (4MU
perfusate). The urea chaotrope was perfused because ConA
detail by mass spectrometry in subsequent sections. Other binding was most evident on the AIM, by comparison to the IL,
intestinal fractions that will be described below were assayed for suggesting that at least some predicted AIM glycoproteins
peptidase activity when incubated with Bodipy casein. However, (peptidases) are peripheral AIM proteins that may be solubilized
use of denaturants with several samples described below by 4MUandcollected foranalysisinthisperfusate.
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AscarissuumIntestinalPeptidases
perfusate, significant inhibition was observed with PMSF and
1,10 phenanthroline at pH 5.0 (Fig. 3F). Although these results
suggest enrichment for different classes of peptidases, between
the PBS and 4MU perfusates, inhibitory effects of urea on
peptidases in the4MU fraction also seems likely, rendering this
point inconclusive.
Theseresultsshowedthat1)PBSand4MUintestinalperfusates
containedConAbindingproteins,2)ConAbindingproteinswere
differentially obtained from the intestine according to specific
perfusion conditions, and 3) both perfusates contained peptidase
activity that wasdistinct frompotential contamination by PF.
Mass spectrometric analysis of A. suum intestinal
fractions
Proteins from each of the fractions described above (ConA
agarose beads, PBS and 4MU perfusates, and P2 pellet) were
analyzedbyLC-MS/MSmassspectrometry.Acompletelistingof
results is provided in S1 Table. The smallest subset of proteins
identified wasgeneratedwithproteinselutedfromConAagarose
beads (full set in S2 Table, peptidases in Table 2). These bead-
isolated proteins were separated by PAGE, and ConA binding
bands located on nitrocellulose filters were then excised from
corresponding gels for analysis (as described in the methods).
Fig.2.ConcanavalinA(ConA)bindingproteinsfromAdultA. Twentysevenproteinswereidentified,mostindividualproteinsof
suumintestine.A,B.Histologicalsectionsweretreatedwithout(A)or which were confined to one or two unique PAGE gel slices (S2
with sodium periodate (B), as described in Methods, and then Table). Eighteen of the 27 proteins fell into the two major
incubated with the ConA-horse radish peroxidase conjugate (ConA- functional categories ofpeptidases (10)andO-glycosyl hydrolases
HRPO).Bindingwaslocalizedbyhistochemicaldetection.Blackarrows
(eight), of which the glycosyl hydrolases may be related to
point to intestinal microvilli, the white arrow points to material
saccharidase activity previously detected in A. suum intestinal
extending from microvilli into the lumen. C. Intestinal proteins
separatedbynon-reducingSDS-PAGEweretransferredtonitrocellulose brush border preparations [25,26]. Only three proteins
filters, and then untreated (NP) or treated (P) with sodium periodate. (GS_16354, GS_12574, GS_21785) from the entire ConA set
Filters were incubated with Con A-HRPO and ConA binding bands werenotdetectedineitheroftheperfusatefractions,andonlyone
localized by chemiluminescence. Molecular weight markers are (GS_21785) was absent from both the perfusate and P2 pellet
indicatedontheleftofthepanel.D.Peptidaseactivitywasevaluated
fractions (Table 2). For proteins detected in both the ConA
for intestinal proteins bound to ConA-agarose beads, as described in
binding proteins and the perfusates, representation was often the
Methods,andconductedatthepHconditionsindicatedonthexaxis.
Relative fluorescence units (RFU) mg-protein21 from hydrolysis of highest in the 4MU versus PBS perfusate, which might be
Bodipy casein is indicated on the y axis. A 95% confidence interval expectedforperipheralmembraneproteins.Therefore,theConA
wasconstructedforgroupmeansateachpHtested,whichineachcase isolatedproteinsidentified appeartoaccount,at leastinpart,for
was greater than zero. E. Inhibition of peptidase activity isolated on ConA binding proteins located in situ on the AIM, and for
ConA agarose beads. Assays were conducted at pH5.0. Percentage
peptidase activity associated with ConA agarose beads and
inhibition indicated on the y axis was determined with inhibitors, as
perfusates.
described in Figure1, and means for each inhibitor treatment tested
were significantly lower(p,0.05)thanthe uninhibitedcontrolgroup. Amorecomplexgroupofpeptidaseswasdetectedinperfusate
All peptidase assays were conducted with three replicates. F. samples(Table2).ThesesampleswerealsocomparedtoLC-MS/
Coomassie blue stained SDS PAGE gel, as in panel C, of intestinal MS results obtained for the PF, a most likely contaminant of the
proteinsisolatedonConAagarosebeads.ConA,indicatesheavyConA perfusate samples. Predictedpeptidases identified inthe PBSand
proteins released from the beads. No bands were excised for mass
4MU perfusates by LC-MS/MS showed both similarities and
spectrometryatorbelowthispositioninthegel.
differences. Perfusate proteins annotated as peptidases corre-
doi:10.1371/journal.pntd.0003375.g002
sponded to aspartic, cysteine, metallo, serine carboxy and
threonine peptidases, or unassigned peptidases, according to
Blots of isolated proteins in both perfusates were probed with MEROPS [27]. These proteins were represented in one or both
ConA(Fig. 4A),whichshowedsomewhatsimilarbandingpatterns perfusates, although representation of mass spectra was better in
overall between the PBS and 4MU perfusates, but with different the 4MU perfusate for many proteins shared with the PBS
relative abundances of ConA binding proteins in each. Periodate perfusate. In total, 29 peptidases were detected in ConA and
pre-treatment of the nitrocellulose filter (blot) eliminated detect- perfusate fractions (Table2). All but four of these were also
able ConAbinding toproteinbandsfrom bothperfusates. detectedintheP2pellet.Thelargestgroupofpeptidasesidentified
In peptidase assays, mean activity of the PBS perfusate was in the Con A and perfusate fractions were categorized as
generally higher than the 4MU perfusate and the pseudocoe- metallopeptidases (M01, nine; M13, four; M17, one; M20, two),
lomic fluid (PF), in which negligible peptidase activity was followed by aspartic peptidases (A01, five), serine carboxy
detected, except for thelow level at pH 6.0.Theactivity in the peptidases (S10, three; S28, one), cathepsin B-like cysteine
PBS perfusate at pH 5.0 was largely inhibited by pepstatin peptidases (C01, one), threonine peptidase (T01A, one) and
(Fig. 3B, C). Despite treatment with denaturant, peptidase unassigned aminopeptidase N(-, two).
activity was detected in the 4MU perfusate under all pH Peptidase classes represented in the perfusates and ConA
conditions tested. Mean fluorescence was significantly higher binding fraction were largely consistent with our previously
than PF at both pH 5.0 and 8.0. In contrast to the PBS published predictions (Table 1), although the results significantly
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AscarissuumIntestinalPeptidases
Fig.3.CannulationoftheAscarissuumintestine.A.ThreecannulatedfemaleA.suumwithintactposteriorends.Numbersandarrowsreferto
stepsinthecannulationprocess:1,removaloftheanteriorendbelowtheesophaguswithascalpel;2,insertionofthebluntneedlecannula(25g),
withsupergluegelappliedtothecannula,intotheintestinallumen;3,removaloftheposterior1/6thoftheworm;4,resectionofthebodywallto
exposetheintestine,asinpanelsBandC.B.CannulatedwormprocessedasinA,butwithresectedposteriorbodyandexposedposteriorregionof
theintestine(arrow,5),laidontoparafilmforcollectionofperfusate.Asyringe(6)isattachedtothecannulahubtodeliverperfusate.C.Enlargement
oftheexposedposteriorregionoftheintestineisshowninpanelB.D.Cannulatedworm,withintactposteriorend,injectedwithmethyleneblue
dye.Whitearrowpointstoreproductiveorganswithinthepseudocoelomicbodycavity,blackarrowpointstotheintestinefilledwithdye.Notethat
thedyeisconfinedtotheintestine.
doi:10.1371/journal.pntd.0003375.g003
expanded the subclasses, increased the number and provided for the apparent serine peptidase activity detected at pH8.0.
direct evidence for A. suum AIM and IL peptidases identified Althoughhosttrypsinandchymotrypsinapparentlylocalizetothe
relative to these predictions (Table 3). The relatively high A. suum intestine [28], use of porcine trypsin for the mass
representation of some peptidase sequences in the 4MU fraction spectrometry analysis obviated clarification based on detection of
andoccurrenceintheConAfractionisconsistentwithlocalization thisprotein.Nevertheless,peptides ofporcine chymotrypsinwere
totheAIM asperipheral membrane proteins. also detected in perfusates, raising the possibility of host serine
Although information from the P2 fraction was not specific peptidase activityin perfusates analyzedat higherpHs.
regardingcompartmentalization,thisfractioncontained21addition- Several of the peptidase protein sequences were predicted to
al putative peptidases, including seven S10 and S28 serine have signal peptides for secretion, or are predicted non-classical
carboxypeptidases,whichmayalsofunctionontheAIMorintheIL. secretory proteins, each of which is consistent with compartmen-
In contrast to H. contortus in which cathepsin B-like cysteine talizationintheILorontheAIM.However,neitherofthesetwo
peptidases make up a prominent fraction of AIM-IL peptidases characteristicswaspredictedforthreeoftheConAandperfusate
[15,23], cysteine protease activity was variably indicated in peptidasesbasedonexistingA.suumproteinmodels(Table2).A
inhibitor assays and a single peptide was detected in the PBS recentpublicationontheA.suumsecretome[29]identified10of
perfusate for an A. suum cathepsin B-like (CBL) cysteine the ConA and perfusate peptidases identified here as excretory-
peptidase. Thus, A. suum CBLs appear to represent a relatively secretoryproductsofadultfemaleA.suum(Table 2),sixofwhich
minor constituent ofA.suum AIMandILdigestive peptidases. lack apparent signal peptides, and five of those were classified as
Also,basedonacidicpreferenceofserinecarboxypeptidases,no non-classicalsecretoryproteins.Whileourresultsclarifythelikely
AIMorILpeptidaseswereidentifiedthatwouldobviouslyaccount origin of these ‘‘secretory’’ products, their localization in
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AscarissuumIntestinalPeptidases
Fig.4.Peptidaseactivityinintestinalperfusates.A.ConA-HRPOblotofproteinsobtainedfromwholeintestinallysate(W),andPBS(P)or4M
urea(4MU)perfusatesoftheintestinallumenafterseparationbynon-reducingSDS-PAGEandtransfertonitrocellulose.B.Samples(4mg)ofthePBS
and 4MU perfusates, and pseudocoelomic fluid (Pf) were incubated with bodipy casein in peptidase assays, as described in Figs. 1 and 2.
MeasurementsinRelativefluorescenceunits(RFU)mg-protein21areshownontheyaxis.Meansthatdifferfromoneanother(p,0.05)areindicated
by different letter designations (A, B, C) below the x axis, C, D. Inhibition of peptidase activity in PBS (B) and 4MU (C) perfusates. Assays were
conductedatpH5.0.Percentageinhibitionindicatedontheyaxiswasdeterminedforwithinhibitors,asdescribedinFig.1.Meansforeachinhibitor
treatmentthatweresignificantlylower(p,0.05)thantheuninhibitedcontrolgroupareindicatedbyanasterisk.Allpeptidaseassayswereconducted
withthreereplicates.
doi:10.1371/journal.pntd.0003375.g004
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AscarissuumIntestinalPeptidases
e
al
M 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 -1 1 1 1 1 1 1 0 1 0 1 0 1
6
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Ex Fe 1 1 1 1 0 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 0 1 1 -1 1 1 1 0 1
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estin PBS 2 0 1 3 1 6 0 8 1 11 3 0 3 1 2 1 2 2 1 5 3 1 2 1 0 0 0 0 0 0 0 0 0 0
nt
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dAscaris 2Family - A01A A01A A01A A01A M01 M01 M01 M01 M13 S10 S10 - M01 A01A C01A M01 M01 M01 M13 M13 M13 M20A S28 M01 M17 M20A S10 T01A - S09 C13 C19 C95
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2. 1se 0 4 1 6 5 6 4 6 5 9 1 4 9 8 8 1 5 8 5 0 8 1 8 8 7 7 8 6 6 7 3 8 1 0
e da 92 57 90 31 44 16 58 74 28 21 84 70 45 51 42 46 55 51 51 14 34 70 89 17 30 49 00 67 01 47 60 79 48 94
Tabl Pepti GS_23 GS_12 GS_14 GS_15 GS_19 GS_04 GS_05 GS_05 GS_16 GS_08 GS_03 GS_22 GS_23 GS_00 GS_03 GS_06 GS_02 GS_21 GS_13 GS_19 GS_10 GS_02 GS_16 GS_11 GS_09 GS_14 GS_16 GS_09 GS_05 GS_11 Liv_01 GS_01 GS_03 GS_11
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AscarissuumIntestinalPeptidases
ale annotationinperfusates,ncethatis:1,
6Expression FemaleM 11 -10 11 11 01 00 11 -1-1 11 00 00 11 11 11 11 -1-1 unassignedfamily,seemorecompletePBSor4MU,PBSor4Mureaintestinal6thesecretome[29].Transcriptabunda
4ment 5NCTMSecreted Y-- Y-- Y-- --- -Y- Y-- --- --- --- -Y- YY- --- --- Y-- --- -Y- definedintheMEROPSdatabase[27];-,umberofspectraarelistedinTableS2);5mbrane.designatedasacomponentofedtoothertissuesinvestigated[30].
Compart P2PfSP 30- 10- 30- 20Y 20- 20- 10Y 11Y 20Y 10- 10- 30Y 30Y 30- 10Y 60- 2anL4genelibrary.Peptidasefamilyaspectra):ConA,ConcanavalinA(Y,yes,nNC,Non-classicalsecretion;TM,transme1lessabundantintheintestinecompar
3Fraction ConAPBS4MU 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 sequences[18],exceptLiv_01603identifiedindentified(numbersrefertoquantityofmasss4SP,signalpeptide;Pf,pseudocoelomicfluid.notdetectablydifferentintheintestine;and-
Cont.Table2. 12PeptidaseFamily GS_18334C95 GS_13060M67A GS_16309S09 GS_01230S09 GS_19153S09 GS_01127S10 GS_06945S10 GS_14352S10 GS_03565S12 GS_00889S16 GS_08933S26B GS_04641S28 GS_07735S28 GS_16223S28 GS_23795S28 GS_11889S33 1Proteindesignationfromtranslatedprotein3FractionsinwhichproteinswereiTableS1.respectively;P2,5K-50Kxgintestinalpellet;relativelymoreabundantintheintestine;0,doi:10.1371/journal.pntd.0003375.t002
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