Biochem.J.(2002)368,611–620(Printed in Great Britain)
611
Anti-microbial properties of histone H2A from skin cretions of rainbow trout,Oncorhynchus mykiss
Jorge M.O.FERNANDES*,Graham D.KEMP †,M.Gerard MOLLE ‡and Valerie J.SMITH*1
*Gatty Marine Laboratory,School of Biology,University of St Andrews,Fife KY168LB,Scotland,U.K.,†Centre for Biomolecular Sciences,School of Biology,University of St Andrews,Fife KY169QT,Scotland,U.K.,and ‡Centre de Biochimie Structurale,UMR 5048CNRS,554INSERM,29rue de Navacelles,34090Montpellier,France
Skin exudates of rainbow trout contain a potent 13.6kDa anti-microbial protein which,from partial internal amino acid quencing,peptide mass fingerprinting,matrix-associated lar desorption \ionization MS and amino acid analysis,ems to be histone H2A,acetylated at the N-terminus.The prot广州华章
ein,purified to homogeneity by ion-exchange and reverd-pha chromato-graphy,exhibits powerful anti-bacterial activity against Gram-positive bacteria,with minimal inhibitory concentrations in the submicromolar range.Kinetic analysis revealed that at a con-centration of 0.3µM all test bacteria lo viability after 30min incubation.Weaker activity is
also
displayed against the yeast Saccharomyces cere isiae .The protein is salt-nsitive and has no
INTRODUCTION
The last few years have en a great burgeoning of reports of the occurrence and characterization of low-molecular-mass anti-microbial peptides from a wide variety of organisms [1].The molecules have attracted much rearch interest becau of their biochemical diversity,broad specificity against bacteria and \or fungi [1]and also becau some have anti-viral [2],anti-tumoural [3]or wound-healing effects [4].Undoubtedly,they are important components of the innate host defences and reprent a source of potentially uful natural antibiotics for pharmaceutical ap-plication.To date,over 750such eukaryotic peptides,mostly isolated from mammals,amphibians,incts or other inverte-brates,have been described and listed on the anti-microbial quences databa (http:\\www.
ieste.it \"tossi \).Certainly,the teleostei is an important and worthy taxon for further study in this respect.Fish are of great economic im-portance in aquaculture throughout the world.They have a long evolutionary history,display huge species diversity and have wide distribution in marine and other aquatic habitats.Moreover,they live in a microbe-rich environment and are vulnerable to invasion by pathogenic or opportunistic micro-organisms.Non-specific microbicidal compounds are also likely to be especially important for fish,as their adaptive immune system is structurally simpler than that of mammals or amphibians,and is not fully effective in young fry or at low environmental temperature [5].The skin epithelium and other mucosal surfaces of fish are the sites in which anti-microbial peptides are particularly likely to be abundant,as the mucosae are constantly expod to water-borne micro-organisms,rendering the fish at risk of infection through grazes or cuts.In many other animal groups the surface mucosa
Abbreviations ud:MALDI-TOF MS,matrix-assisted lar desorption/ionization time-of-flight MS;MBC,minimal bactericidal concentration;MIC,minimal inhibitory concentration;MHB,Mueller–Hinton broth;TFA,trifluoroacetic acid;c.f.u.,colony-forming units.1
To whom correspondence should be addresd (e-mail vjs1!st-and.ac.uk).
bdp
The partial internal amino acid quence of histone H2A from trout skin has been deposited in the Swiss-Prot databa under accession number P83327.
haemolytic activity towards trout erythrocytes at concentrations below 0.3µM.Reconstitution of the protein in a planar lipid bilayer strongly disturbs the membrane but does not form stable ion channels,indicating that its anti-bacterial activity is probably not due to pore-forming properties.This is the first report to show that,in addition to its classical function in the cell,histone H2A has extremely strong anti-microbial properties and could therefore help contribute to protection against bacterial invasion.Key words:anti-bacterial protein,epithelium,mucosal immun-ity,nucleosome.
produces a battery of non-specific microbicidal proteins to prevent harmful systemic infections that would result if microbes breached this barrier [6,7].
It is well established that skin cretions of fish contain a number of innate humoral defence factors,including complement factors [8],lectins [9],proteas [10]and lysozymes [11,12].More recent investigations have started to reveal that the surface mucosa of some fish also contains a range of anti-microbial peptides [12–17].So far the number of species studied and the range of proteins recorded remains small and relatively few have been directed at salmonids,one of the chief groups obec商务英语听力
f fish intensively farmed for commercial purpos.To address this issue we initiated an investigation into the prence,character and diversity of low-molecular-mass anti-microbial proteins in skin cretions of fish,focusing on the rainbow trout Onco -rhynchus mykiss as the model species.In a previous paper [12]we reported the isolation of two constitutively expresd anti-bacterial proteinaceous factors from detergent extracts of skin mucus from this species:a novel muramida with an unusually low isoelectric point and a cationic peptide of approx.3kDa.As our studies also indicated that other anti-microbial factors might be prent,the prent investigation was aimed at examining the prence and character of acid-soluble anti-microbial proteins in O .mykiss skin cretions.
EXPERIMENTAL Animals
Female rainbow trout,weighing 400–500g,were purchad from College Mill Trout Farm,Perthshire,U.K.They were maintained in flow-through freshwater tanks (14p 1m C)and fed daily with
612J.M.O.Fernandes and others
Table1List of the micro-organisms ud for anti-bacterial assays
Original sources,identification codes and culture conditions are also indicated.Yeastrel medium:0.5%(w/v)Lab-Lemco(Oxoid),0.7%(w/v)yeastrel(Natex,Gloucs.,U.K.),0.95%(w/v)peptone (Difco,West Moley,Surrey,U.K.)and0.5%(w/v)NaCl.KDM-2medium:1%(w/v)peptone,0.05%(w/v)yeast extract(Sigma),0.1%(w/v)cysteine hydrochloride(Sigma)and20%(v/v) foetal calf rum(Invitrogen,Paisley,U.K.),pH6.5.Yeast extract medium(ATCC1067):0.05%(w/v)yeast extract and1%(w/v)D-(j)-gluco(Sigma).Blood ba was from Difco.BKD,bacterial kidney dia;ERM,enteric red mouth dia.
Species Identification code Original source Culture conditions(medium,temperature)
Aerococcus viridans NCIMB1120Moribund lobsters Nutrient(Oxoid),30m C
华中师范大学自考
Aeromonas hydrophila NCIMB1134Mouth lesion of rainbow trout Nutrient,30m C
Aeromonas salmonicida004MT004*–Yeastrel,20m C
A.salmonicida849MT849*Diad salmon Yeastrel,20m C
Bacillus subtilis ATCC6051–Nutrient,30m C
Escherichia coli NCIMB12210–Nutrient,37m C
Listonella anguillarum NCIMB2129Farmed rainbow trout Blood ba,20m C
L.anguillarum01MT1637*–Blood ba,20m C
Micrococcus luteus NCIMB376–Blood ba,20m C
Planococcus citreus NCIMB1493–Nutrient/1.5%NaCl,20m C
Renibacterium salmoninarum NCIMB1114Salmonid with BKD KDM-2,15m C
Staphylococcus aureus NCIMB6571–Nutrient,37m C
Saccharomyces cerevisiae––Yeast extract,37m C
Yersinia ruckeri MT252*Salmonid with ERM Nutrient,25m C
*Strains kindly provided by Dr Tony Ellis(Marine Laboratory,Aberdeen,U.K.).
the commercial diet Dynamic Red M(Ewos,Bathgate,West Lothian,U.K.).
Preparation of skin mucus extracts
A total of10fish were humanely killed by immersion in an anaesthetic bath containing0.6g of3-aminobenzoic acid ethyl ester\l of solution,4h after gentle surface stimulation with ultrafine sandpaper to enhance mucus cretion.Samples of mucus and associated epidermal cells were collected by scraping the skin dorso-lateral surfaces(total volume of approx.150ml) and homogenized1:4(v\v)in a solution of50%(v\v)ethanol (Merck,Poole,Dort,U.K.),3.3%(v\v)trifluoroacetic acid (TFA;Sigma,Poole,Dort,U.K.)and2%(v\v)general-u protea inhibitor cocktail(Sigma).Following extraction by stirring for60min at4m C,the preparation was centrifuged at 29000g for60min at4m C and the supernatant lyophilized.The resulting extract was resuspended in100ml of20mM Hepes (Acros,Loughborough,Leics.,U.K.)and the pH adjusted to7.0 with5M NaOH(BDH,Poole,Dort,U.K.)before centrifuging at29000g for30min at4m C.
Test micro-organisms
The various strains of micro-organisms,their original source and culture conditions ud in the prent study are listed in Table1. Each micro-organism was grown to exponential pha before washing in sterile saline[approx.3.2%(w\v)NaCl(Sigma) for marine strains;0.8%(w\v)NaCl for non-marine strains] and resuspension in Mueller–Hinton broth(MHB)(Oxoid, Basingstoke,Hants.,U.K.)to a concentration of10&colony-forming units(c.f.u.)\ml.
Anti-bacterial assays
Anti-bacterial activity was assd using a modification of the two-layer radial diffusion assay of Lehrer et al.[18],as described previously by Smith et al.[12].The Gram-positive bacterium Planococcus citreus was ud as the test organism throughout the protein purification procedure.
Minimal inhibitory concentration(MIC)assays of the purified
protein against each of the bacteria listed in Table1were
performed by microtitre broth dilution,bad on that devid by
Friedrich et al.[19].Briefly this entailed diluting the test protein
in0.2%(w\v)BSA(Sigma)\0.01%(v\v)acetic acid(BDH)to
give a ries of eight2-fold dilutions.Volumes of11µl of each
dilution were added to individual wells of a polypropylene96-
well microtitre plate(Corning Costar,Cambridge,U.K.).Each
suspension of washed bacteria(100µl),grown under the
conditions in Table1and diluted in MHB to a concentration of
10&c.f.u.:ml−"(as above),was then added to each well in duplicate,and the trays were incubated at the appropriate
temperature for each bacterium(Table1)until the attenuance at
570nm(read with an MRX II plate reader;Dynex,Billinghurst,
West Susx,U.K.)reached0.2in the positive control well,
containing bacteria and diluent only.The MIC was considered to
be the lowest concentration of protein that reduced growth by
50%compared with the control well.The minimal bactericidal
concentration(MBC)was obtained by plating out the contents
of each well showing no visible growth.The MBC was taken as
the lowest concentration of protein that prevents any residual
colony formation after incubation for24h at the appropriate
temperature.Cecropin P1(Sigma)was ud as reference. Protein purification
The reconstituted protein extract was fractioned by cation-
exchange chromatography using a CM Macro-Prep1cm i10cm
Econo-column(Bio-Rad,Hemel Hempstead,Herts.,U.K.),
previously equilibrated with20mM Hepes\0.1M NaCl,pH7.0bilingual
(buffer A).Elution was performed with a linear gradient of buffer
A to buffer B(where buffer
B is20mM Hepes\1M NaCl,
pH7.0)over90min,followed by35min of buffer B,at aflow
rate of1ml:min−".Fractions eluting between80%and100% buffer B were collected and applied to Sep-Pak Vac5g t C") cartridges(Waters,Watford,Herts.,U.K.)equilibrated in0.15% (v\v)TFA.Two successive stepwi elutions were performed with20ml of20%(v\v)and70%(v\v)acetonitrile(BDH)in acidified water.The latter fraction was lyophilized,resuspended in acidified deionized water(Elga,High Wycombe,Bucks.,最好的英国留学中介
613 Anti-microbial properties of trout histone H2A
U.K.)and loaded on to an ODS2-Inertpak C")reverd-pha HPLC column(particle size5µm, 4.6mm i250mm; Capital HPLC,Broxburn,U.K.).The HPLC system comprid a ries410LC pump(Perkin Elmer,Beaconsfield,Bucks.,U.K.) coupled with a996photodiode array detector(Waters)and a 2110fraction collector(Bio-Rad).Elution was executed at25m C with a biphasic gradient of0.1%(v\v)TFA in water and0.09% (v\v)TFA in acetonitrile(e Figure1,top left-hand panel, below)at aflow rate of1ml:min−".Active fractions of interest were further chromatographed by reverd-pha HPLC on the same column but under a shallower gradient(30–55% acetonitrile over50min at aflow rate of1ml:min−").
At each step,protein profiles were determined by SDS\PAGE using the Tris\Tricine system with a16
%parating gel,14% spacer gel and5%stacking gel,as described by Scha gger and von Jagow[20].
Protein quantification
Total protein was estimated by the method of Bradford[21] using BSA(Pierce,Rockford,IL,U.S.A.)as standard.Amino acid analysis of the purified protein was performed at the Protein and Nucleic Acid Chemistry Facility,University of Cambridge, Cambridge,U.K.,using the post-column ninhydrin method or the pre-column AccuTag system when picomolar nsitivity was required.
Matrix-assisted lar desorption/ionization time-of-flight MS (MALDI-TOF MS)
Information concerning the molecular mass and purity of the anti-bacterial factors was determined by MALDI-TOF MS.The sample of interest(0.5µl,estimated concentration10pmol:µl−" in deionized water)was applied to the target plate along with 0.5µl of a saturated solution ofα-cyano-4-hydroxycinnamic acid (Fluka,Poole,Dort,U.K.)in acetonitrile(Ultrafine, Manchester,U.K.)\0.1%TFA(35:65,v\v).Cytochrome c and haemoglobin(both from Sigma)were similarly applied to an adjacent spot for calibration.Mass spectra were obtained on a TofSpec2E instrument(Micromass,Manchester,U.K.)using a 337nm lar and operated in linear mode.Peptide mapping of tryptic digests was performed by MALDI-TOF in reflectron mode.
Partial primary structure determination
In order to determine partial N-terminal amino acid quence, the purified protein was subjected to standard automated Edman degradation on a Proci Sequencer(Applied BioSystems, Warrington,Cheshire,U.K.).Internal quence information was obtained following digestion of the purified protein with chymo-trypsin for18h at37m C using an enzyme\substrate ratio of approx.1:20.The digests were fractionated on a100mm i1mm C")microbore column(Brownlee,Cheshire,U.K.)using a linear biphasic gradient of0.1%(v\v)TFA in water(buffer C)and 0.1%(v\v)TFA in acetonitrile(buffer D),in the range5% buffer D to35%buffer D over30min.The purified peptide was also quenced by standard automated Edman degradation on a Proci Sequencer.
Sequence analysis
Homology arches were performed against the SwissProt,NR and Month databas with the basic local alignment arch tool (BLAST)[22],provided by the NCBI rver(http:\\ bi.v\BLAST).The Mascot algorithm[23]was employed for protein identification by peptide massfinger-printing.Amino acid composition,protein mass and isoelectric point were predicte
d by the Expert Protein Analysis System (ExPASy)proteomics rver of the Swiss Institute of Bio-informatics(http:\\pasy.ch\).Protein identification by amino acid composition and molecular mass was performed with the cond constellation of the AACompIndent tool(ExPASy). Sequence alignments were executed with the Omiga2.0quence analysis software(Oxford Molecular\Accelrys,Cambridge, U.K.),using the Clustal W1.6algorithm[24].The Omiga2.0 software was also ud to arch for proteolytic cleavage sites within the protein quence.
Proteolytic digestion
Anti-bacterial activity of the purified protein was assd by radial diffusion assay as above,following digestion with afinal concentration of60µg:ml−"proteina K(Sigma)for60min at 37m C.
Kinetic assay
Washed P.citreus cell suspensions(90µl)containing 10&c.f.u.:ml−"(as above)were incubated at20m C for different time periods with10µl of anti-bacterial protein at two differ-ent concentrations(mean value of MIC interval or MBC).As a control,the anti-bacterial protein solution was replaced with 10µl of MHB.The reactions were terminated by diluting the samples1:100in MHB and plating on MHB aga
r plates in triplicate.Plates were incubated for18h at20m C.
Haemolysis assay
载体英文Haemolytic activity of the purified protein was tested against trout erythrocytes.The erythrocytes were obtained from whole freshly kiss blood withdrawn into syringes coated with heparin(Sigma;500units\ml of blood)and extensively washed with10mM PBS containing0.9%NaCl,pH7.4,to remove the leucocytes and plasma.Erythrocytes were then packed by centrifugation at800g for10min at4m C.For the assay,the test sample was rially diluted to give a range of protein concentrations from0.2to12µM.Aliquots of11µl of each dilution were added to100µl of a2%(v\v)packed cell volume of trout erythrocyte suspension in PBS and incubated for 30min at37m C.For negative controls,11µl of PBS replaced the protein sample under test,while for positive controls it was substituted with0.2%(v\v)Triton X-100(Sigma).Following incubation,all samples were centrifuged at1000g for5min at room temperature and100µl of the supernatant from each well was diluted with800µl of PBS before absorbance measure-ment at545nm using a Ultrospec3300pro spectrophotometer (Amersham Biosciences,Little Chalfont,Bucks.,U.K.).The supernatant from the negative control rved as a reference. Percentage haemolysis was defined as the ratio of absorbances between each sample and the positive control.
Muramida activity
Muramida activity was tested by radial diffusion assay as described in Smith et al.[12]using hen egg-white lysozyme (Sigma;50µg:ml−")as standard.
Planar lipid bilayer assay
To test the ion-channel behaviour of the anti-microbial protein purified from trout mucus,macroscopic and single-channel
614J.M.O.Fernandes and others
Figure 1Purification of an anti-microbial protein from skin cretions of rainbow trout
Top left-hand panel:acidic protein extracts of skin mucus were fractionated by cation-exchange chromatography on a CM Macro-Prep column and the active fractions were concentrated by solid-pha extraction using t C 18Sep-Pak cartridges;the 70%acetonitrile (CH 3CN)eluate was subjected to C 18reverd-pha HPLC.The chromatogram was obtained at 214nm (solid line).Acetonitrile con
centration gradient is reprented by the dashed line.Anti-bacterial activity profile against P.citreus is denoted by the histogram,which shows the area of the clear zones (A)on a radial diffusion assay.Top right-hand panel:active fractions labelled OM2were pooled and fractionated by C 18reverd-pha HPLC using a shallower water/acetonitrile gradient.Absorbance was monitored at
golden time
615
Anti-microbial properties of trout histone
H2A Figure 2Partial internal quence of the anti-microbial protein purified from skin mucus of rainbow trout and its homology with other histones or histone-derived peptides
elisabeth harnoisIdentical residues between the purified anti-microbial protein,histone H2A from rainbow trout [26],human histone H2A.5[44],buforin I [33]and parasin I [15]are reprented by shaded boxes.
experiments were carried out.For the conductance experiments,virtually solvent-free planar lipid bilayers were formed using the Montal and Mueller technique [25].The membrane was formed over a 100–150µm hole in a Teflon film (10µm thick),pre-treated with a mixture of 1:40(v \v)hexadecane \hexane,parating two half glass cells.Lipid monolayers were spread on top of the electrolyte solution (1M KCl \10mM Tris,pH 7.4)in both compartments.Bilayer formation was achieved by lowering and raising the electrolyte level in one or both sides and monitoring capacity respons.Asolectine IV-S from soya bean (Sigma)was ud as the lipid.Voltage was applied through an Ag \AgCl electrode in the cis side.
With regard to the macroscopic conductance experiments,the 13.6kDa protein was added in a concentration range between 5i 10−)and 5i 10−(M from a 10−&M stock solution in 0.3%(v \v)Triton X-100.The doped membranes were subjected to slow voltage ramps (6.6mV \s)and transmembrane
静雅思听下载currents were fed to a Keitley amplifier (model 427;Cleveland,OH,U.S.A.).Current–voltage curves were recorded on an x –y plotter.In single-channel recordings,the protein concentrations ranged from 2i 10−*to 10−)M.Currents were amplified and potentials were applied simultaneously by a patch-clamp amplifier (RK 300;Biologic Science Instruments SA,Claix,France).Single-channel currents were monitored on an oscilloscope (R5103N;
214nm (solid line).The peak fraction eluting at 34.6min was found to be anti-bacterial to P.citreus (e histogram).Bottom panel:Tris/Tricine SDS/PAGE analysis of the active fractions.Lane 1,markers;lane 2,crude extract;lane 3,pooled active ion-exchange fractions;lane 4,70%acetonitrile eluate from solid-pha extraction;lane 5,OM2C 18reverd-pha HPLC fractions;lane 6,purified anti-microbial protein after cond HPLC.Each lane contains 7.5µl of sample.The numbers on the left correspond to the molecular-mass markers (kDa).Protein of interest is indicated by an arrow.
Tektronix,Beaverton,OR,U.S.A.)and stored on a DAT recorder (DTR 1202;Biologic Science Instruments SA)for off-line analysis.Satori (V3.1;Intracel Software,Royston,Herts.,U.K.)was ud for downstream analysis.All experiments were performed at room temperature.
RESULTS Protein purification
Cation-exchange chromatography of the skin cretion extracts yielded active fractions eluting between 0.8and 1.0M NaCl (results not shown).The active fractions were pooled and
concentrated by solid-pha extraction on t C ")
Sep-Pak cartridges.Subquent chromatography by C ")
reverd-pha
HPLC resulted in two groups of active fractions,designated OM1(25–41min)and OM2(47–54min;Figure 1,top left-hand panel).Refractionation of OM2by HPLC with the same column,but using a shallower gradient,yielded a single peak with a retention time of 34.6min (corresponding to 42.3%acetonitrile)that exhibited anti-bacterial activity (Figure 1,top right-hand panel).SDS \PAGE revealed a single band with an apparent molecular mass of approx.15kDa (Figure 1,bottom panel).This activity was thermostable,remaining prent even after
