Proteomic and phenotypic profiling of the amphibian pathogen Batrachochytrium dendrobatidis shows that genotype is linked to virulence

Molecular Ecology (2009) 18, 415–429 doi: 10.1111/j.1365-294X.2008.04041.x

© 2009 The AuthorsJournal compilation © 2009 Blackwell Publishing Ltd

Blackwell Publishing LtdProteomic and phenotypic profiling of the amphibian pathogen Batrachochytrium dendrobatidis shows that genotype is linked to virulence

MATTHEW C. F ISHER,* JAIME BOSCH,† ZHIKANG YIN,‡ DAVID A. STEAD,‡ JANET WALKER,‡ LAURA SELWAY,‡ ALISTAIR J . P. BROWN,‡ LOUISE A. WALKER,‡ NEIL A. R . GOW,‡ JASON E. STAJICH§ and TRENTON W. J . GARNER¶*Department of Infectious Disease Epidemiology, Imperial College London, St. Mary’s Hospital, Norfolk Place, London W2 1PG, UK, †Museo Nacional de Ciencias Naturales (CSIC), José Gutierrez Abascal 2, 28006 Madrid, Spain, ‡School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK, §Department of Plant and Microbial Biology, University of California, 111 Koshland Hall, Berkeley, CA 94720, USA, ¶Institute of Zoology, Zoological Society of London, Regent’s Park, London NW1 4RY, UK

Abstract

Population genetics of the amphibian pathogen Batrachochytrium dendrobatidis (Bd) showthat isolates are highly related and globally homogenous, data that are consistent with therecent epidemic spread of a previously endemic organism. Highly related isolates are pre-dicted to be functionally similar due to low levels of heritable genetic diversity. To test thishypothesis, we took a global panel of Bd isolates and measured (i) the genetic relatednessamong isolates, (ii) proteomic profiles of isolates, (iii) the susceptibility of isolates to theantifungal drug caspofungin, (iv) the variation among isolates in growth and phenotypiccharacteristics, and (v) the virulence of isolates against the European common toad Bufobufo. Our results show (i) genotypic differentiation among isolates, (ii) proteomic differen-tiation among isolates, (iii) no significant differences in susceptibility to caspofungin, (iv)differentiation in growth and phenotypic/morphological characters, and (v) differentialvirulence in B. bufo. Specifically, our data show that Bd isolates can be profiled by theirgenotypic and proteomic characteristics, as well as by the size of their sporangia. Bd geno-typic and phenotypic distance matrices are significantly correlated, showing that less-relatedisolates are more biologically unique. Mass spectrometry has identified a set of candidategenes associated with inter-isolate variation. Our data show that, despite its rapid globalemergence, Bd isolates are not identical and differ in several important characters that arelinked to virulence. We argue that future studies need to clarify the mechanism(s) and rateat which Bd is evolving, and the impact that such variation has on the host–pathogendynamic.

Keywords: Batrachochytrium dendrobatidis, chytrid, proteome, infectious, virulence, panzootic, amphibian

Received 3 September 2008; revision revised 28 October 2008; accepted 5 November 2008

Introduction

Emergence of the chytrid Batrachochytrium dendrobatidis(Bd) is now widely recognised as a proximate driver of theglobal decline in amphibian species (Berger et al. 1998;Stuart et al. 2004). Until now, research into chytridio-mycosis has focused on analysis of surveillance data (Ron2005), ecological synthesis (Mendelson et al. 2006; Pounds

et al. 2006; Lips et al. 2008) and mathematical modelling(Briggs et al. 2005; Mitchell et al. 2008) in order to identifythe main factors that are driving the emergence of thisdisease. Except for two studies where putative differencesin virulence were identified between Australian and USAisolates of Bd (Berger et al. 2005; Retallick & Miera 2007),there has been no attempt to identify whether differentgenotypes of Bd are biologically similar to one another. Nostudy exists examining how biological variation may belinked to ecological background, host specificity or differentialhost–pathogen outcomes. Perhaps, the hypothesis that Bd

Correspondence: Matthew C. Fisher, Fax: +44 207 5943693; E-mail:[email protected]

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is a rapidly spatially emerging pathogen has led to a beliefthat an organism with low global genetic diversity(Morehouse et al. 2003; Morgan et al. 2007) will have lowphenotypic diversity. This is not a logical standpoint fortwo reasons: first, there are several known instances wheredifferent fungal genotypes lead to differential host–pathogenoutcomes following infection, even in pathosystems wheregenetic diversity is low (McDonald & Linde 2002; Keiperet al. 2006). Second, there is evidence to suggest that differentlineages of Bd might be causing differential mortality innature. Support for this second point comes from observa-tions that infection by Bd can follow varied trajectorieswithin different spatial and temporal settings (Alford &Richards 1997; Retallick et al. 2004; Pounds et al. 2006;Walker et al. 2008). While differences in disease outcome maybe due to changes in ‘classical’ epidemiological parameters,such as host density and environmental constraints, studiesto date have been confounded by not addressing the issueof what Bd genotype is infecting animals, and whether thisvariable is a proxy for quantitative differences between thephysical characteristics (phenotypes) of lineages that mayeffect host/pathogen dynamics.

The infectious life cycle of Bd progresses from a motileaquatic zoospore that infects an amphibian, then sub-sequently develops into a cutaneous zoospore-releasingsporangium (Longcore et al. 1999). By using minimal media,it is possible to propagate the entire life cycle of the organismin the absence of a host. This has the advantage of allowingthe identification of traits that differ among isolates andlineages, enabling evolutionary processes to be dissected.There are two broad classes of evolutionary models for thedivergence of quantitative morphological traits betweenorganismal lineages. The first process is neutral diver-gence, where the balance between the twin stochasticforces of mutation and random genetic drift govern popu-lation differentiation (Wright 1951; Nei 1986). The secondprocess is natural selection, where differential selection onquantitative genetic traits between populations drivesdivergence (Wright 1951; Merila & Crnokrak 2001).

In order to determine whether lineages are undergoingadaptive divergence via natural selection, neutral expecta-tions (measured by the between-population variation inneutral marker loci; Wright 1951) are compared againstestimates of between-population variation in quantitativetraits (Spitze 1993; McKay & Latta 2002). There are threeobservable outcomes from comparisons between quantita-tive traits and marker loci: divergence in traits equalsdivergence between neutral loci; this is evidence that traitsare behaving in a neutral manner. Functional traits havediverged to a greater extent than marker loci; this suggeststhat directional or disruptive selection is occurring. Finally,marker divergence exceeds that of adaptive traits; thissuggests that stabilising selection is occurring and thatselection is favouring the same phenotype in different

environments. Discriminating between neutral and adap-tive processes in infectious diseases is key to identifyingthe underlying biological mechanisms that are importantin the process of infection. This statement is supported byobservations that differential immune-selection on influ-enza genotypes governs which lineages cause infection insubsequent years (Grenfell et al. 2004). Conversely, stabilisingselection on trichothecene virulence-associated genes inFusarium graminearum has maintained these chemotype-alleles as trans-specific polymorphisms (Ward et al. 2002).

To address whether there is a link between Bd genotypeand a pathologically relevant phenotype, we determinedthe degree of marker divergence among isolates and devel-oped proteomic approaches to identify candidate phenotypicmarkers that distinguish between varied Bd genotypesfrom around the world. In addition to proteomic studies,we developed methodologies that identified differentmorphological characteristics between Bd genotypescollected from our European study-sites. Finally, we usedan amphibian model system to show that there is anassociation between phenotypic/morphological variationand the virulence of Bd. Our results show that the ‘pathogenprofile’ of Bd, comprising proteomic, genotypic and pheno-typic/morphological data, is important in determining theoutcome of infection, and that future studies need to focuson the biological determinants that underlie this keyepidemiological variable.

Methods

Bd Isolates and genetic relatedness

Six isolates of Bd were cultured in 2007 from infected Alytesmuletensis tadpoles from the Spanish island of Mallorca (fivefrom Torrent des Ferrerets, TF, and one from Coco de sa Bova,CCB). One isolate was cultured from a common newt,Trituris vulgaris in the UK (Kent). We included isolates fromearlier time periods that we acquired from infected Alytesobstetricans in mainland Spain (C2A, Penalara natural Park,Madrid; and IA042, Ibon Acherito, Spanish Pyrennees). Allisolates were named according to the scheme suggested byBerger (Berger et al. 2005). As outgroups, we included anisolate from Australia, Au-Lcaerulae98, isolated from Litoriacaerulea and from North America, JEL270, isolated fromRana catesbeiana, Point Reyes, California (Table 1). All isolateswere maintained at 21 °C in mTGhL broth (Longcore et al.1999) in 75 mL tissue culture flasks (NUNC). DNA wasextracted from subcultured Bd isolates following the protocolof Boyle et al. (Boyle et al. 2004). Subsequently, the followingfive SNP-containing loci were amplified by polymerasechain reaction (PCR) and sequenced using standardconditions: BdC5, BdC18.1, BdC18.2, BdC24, 8702X2, 8392X2(Morgan et al. 2007). Concatenated multilocus genotypeswere subsequently compared using the neighbour-joining

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algorithm in paup*4.0 using the pairwise genetic distanceDAS. Here, DAS = 1 – (the total number of shared alleles at allloci/n) where n is the total number of loci compared(Stephens et al. 1992) and is calculated using the ‘meancharacter distance’ in paup*4.0.

Proteomics

Three biological replicates of each isolate were grown at21 °C in 180 mL of mTGhL broth for 9 days. Cultures werescraped with a sterile loop then pelleted at 657 g for 5 minand resuspended in 10 mL of a protease inhibitor solu-tion (PMSF 1 mm; Pepstatin 1 mg/mL in DMSO; 200 μLLeupeptin, 200 μL Calcyulin; EDTA 0.5 m), frozen at –80 °Cthen lyophilized under vacuum. Protein extracts weremade essentially using previously described procedures(Yin et al. 2004). Briefly, cells were washed twice withice-cold water, resuspended in 160 μL of 7.5 m urea, 5 mthiourea, 1.25 mm EDTA, 1.75 mg/mL pepstatin A, proteaseinhibitor cocktail (Roche Diagnostics), and disrupted byshearing with 0.25 g glass beads (0.4 mm diameter; BDH).Then 40 μL of 20% w/v CHAPS, 50% v/v glycerol, 10% v/vcarrier ampholytes was added. Samples were vortexedthoroughly for 20 s, and then shaken six times for 30 s with1-min intervals on ice. Cell debris and glass beads wereremoved by centrifugation at 10 000 g for 10 min at 4 °C,and the resulting supernatant stored at –80 °C. Proteinconcentrations were measured using a modified CBB proteinassay kit (Pierce Biotechnology).

Two-dimensional gel electrophoresis was performed asdescribed previously (Yin et al. 2004). Briefly, about 500 mgof protein was loaded on Immobiline DryStrip gels (24 cm,pH 4–7 linear; Amersham Biosciences) and run on an EttanIPGphor Isoelectric Focusing Unit (Amersham Biosciences).

Precast SDS 12.5% polyacrylamide gels (26 × 20 cm, Amer-sham Biosciences) were used for the second dimension,using an Ettan Dalt System (Amersham Biosciences).Proteins were visualised by colloidal CBB staining.

Gels were scanned using a UMAX Powerlook 1120, andthe images analysed using Progenesis SameSpots software(Nonlinear Dynamics) to identify features that werereproducibly up- or down-regulated. Spot volumes werenormalised against total spot volume and total spot areaafter background subtraction (mode of non-spot). Featuresthat displayed statistically significant changes in meannormalised spot volume on the three replicate gels (P ≤ 0.05;Student’s t-test) were selected for protein identification.

Proteins were identified by peptide fragment finger-printing essentially as described elsewhere (Wilm et al.1996). Spots (1.2 mm diameter) were cut from gels andtransferred to 96-well format microtitre plates using anInvestigator ProPic robotic workstation (Genomic Solutions).Proteins in gel plugs were digested with trypsin (PromegaUK) using an Investigator ProGest robotic workstation(Genomic Solutions). Peptides were extracted, dried in aSpeedVac SC110A (Savant Instruments) and dissolved in0.1% formic acid for LC-MS/MS analysis. The LC-MSsystem comprised an UltiMate 3000 LC system (Dionex)coupled to an HCTultra ion trap mass spectrometer withESI source fitted with a low-flow nebuliser (Bruker DaltonicsLtd). Peptides were separated on a PepSwift monolithiccapillary column (Dionex) at 2 μL/min using a lineargradient of acetonitrile. Solvent A was 3% acetonitrile in0.05% formic acid and solvent B was 80% acetonitrile in0.04% formic acid. The gradient was 3–45% solvent Bover 12 min, followed by 90% solvent B for 1 min and re-equilibration of the column in 3% solvent B for 5 min beforethe next injection. Tandem mass spectra were acquired in

Table 1 Geographical Bd Isolates used, dateisolated and their associated echinocandin-resistance profiles

Isolate Region Date isolated Caspofungin IC50 (μg/mL)

Ma-Amuletensis-TF1.1-p2 Mallorca 2007 16Ma-Amuletensis-TF1.2-p2 Mallorca 2007 4Ma-Amuletensis-TF5a1-p2 Mallorca 2007 4Ma-Amuletensis-TF5a2-p2 Mallorca 2007 8Ma-Amuletensis-TF5a3-p2 Mallorca 2007 NGMa-Amuletensis-CCB1-p2 Mallorca 2007 NGIb-Aobstetricans-C2A-p12 Spain 2002 8Ib-Aobstetricans-IA042-p12 Spain 2004 8UK-Lvulgaris-TvB-p2 UK 2007 16Au-Lcaerulae98-1385 12 16/10/00 Australia 2000 16USA-Rcatesbeiana-JEL270 USA 1999 16

IC50, concentration at which there is 50% reduction in growth relative to controls. NG, no growth. Differences in growth between isolates were not statistically significant. European isolates are named according to the nomenclatural scheme suggested by Berger et al. (Berger et al. 2005) denoting region-species-isolate-passage no. Ma, Mallorca; Ib, Iberian Peninsula; Au, Australia; USA, North America.

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autoMS(2) mode. The three most abundant precursors,excluding singly charged ions, were selected for fragmen-tation in each autoMS(2) cycle, with active exclusion of pre-cursors already selected within a 1-min window. Mascotgeneric files were generated automatically by the DataA-nalysis software (Bruker Daltonics) and limited to 300MS/MS spectra per file. These were submitted to MascotMS/MS Ions searches on a local Mascot server using adatabase constructed from a FASTA file provided withAssembly 1 of the Batrachochytrium dendrobatidis SequencingProject [Broad Institute of Harvard and MIT (http://www.broad.mit.edu/)]. The search parameters used were:enzyme = trypsin; fixed modifications = carbamidomethyl(C); variable modifications = oxidation (M); Mass values =monoisotopic; peptide mass tolerance = 1.5 Da; fragmentmass tolerance = 0.5 Da; max missed cleavages = 1; instru-ment type = ESI-TRAP.

Caspofungin susceptibility testing

Minimum inhibitory concentrations (concentrations at whichgrowth is reduced by 50% relative to controls; IC50) weredetermined by broth microdilution testing using theClinical and Laboratory Standards Institute [CLSI, formerlyNational Committee for Clinical Laboratory Standards(NCCLS)] guidelines M27-A2 (National Committee forClinical Laboratory Standards002). Caspofungin (MerkResearch Laboratories) concentrations ranged from 0.032 μg/mL to 16 μg/mL. Each Bd culture (Table 1) was diluted to5 × 105 zoospores/mL with fresh mTGhL and 200 μL ofdiluted culture was added to each well in triplicate. Plateswere incubated for 21 days at 21 °C. After incubation, opticaldensities were read in a VERSAmax tunable microplatereader (Molecular Devices) at 492 nmol.

Samples were fixed in 10% (v/v) neutral buffered formalin(Sigma) and examined by phase differential interferencecontrast (DIC) microscopy. Cell morphology was assessedto determine whether there was a visible effect of drugtreatment on Bd. All samples were examined by DIC micro-scopy using a Zeiss Axioplan 2 microscope. Images wererecorded digitally using the Openlab system (Openlabversion 4.04, Improvision) using a Hamamatsu C4742-95digital camera (Hamamatsu Photonics).

Sporangia/zoospore size and fecundity

European Bd isolates were grown in mTGhL broth for7 days at 21 °C and active zoospores counted using ahaemocytometer and an inverted microscope (OlympusCK40). They were then centrifuged at 657 g for 5 minand resuspended to 10 000 zoospores/mL in fresh broth.Subsequently, the isolates were aliquoted in quadruplicateinto wells of a 12-well tissue culture plate (NUNC)containing 4 mL of broth at the following dilutions; 1000

zoospores/well, 10 000 zoospores/well. On day 21 post-inoculation, the bottom of each well where the sporangiahad settled was photographed using a Canon EOS 350D(3456 × 2304 pixel field). Images were analysed using ImageJsofware (http://rsb.info.nih.gov/ij/) and the followingparameters recorded: (i) the number of zoospores in thecentral 1000 × 1000 pixel square, (ii) the area of the 10largest sporangia contained in the field of view, and (iii) thearea of 10 arbitrarily chosen zoospores per view. Resultswere analysed by analysis of variance (anova) using the Rstatistical package (version 2.6.2).

Assessment of virulence of European Bd isolates using larval challenge

Common toad (Bufo bufo) egg strings were subsampled inApril 2007. Once tadpoles had fully developed operculaand no visible gills (Gosner stage 25), 40 were randomlyassigned to each treatment. Tadpoles were transferredto individual Nunc 175 cm2 EasY Flasks (Nalge, NuncInternational) without lids containing 140 mL of aged tapwater. We added 300 mg of ground Tetra Tabi Min (TetraGmbH) to each flask every second day as food. Flasks weredosed according to one of seven treatments: (i) high dose ofUK Bd UKTvB, (ii) low dose of Bd UKTvB, (iii) high dose ofMallorca Bd TF5a1, (iv) low dose of Bd TF5a1, (v) high doseof Pyrenneen Bd IA042, (vi) low dose of Bd IA042, (vii)control sham infection using filtrate from live Bd cultures.Zoospore concentration of Bd cultures was assayed beforeinfection using a haemocytometer and by counting onlyactive zoospores. We diluted an aliquot of stock culture(high dose, 2500–7500 zoospores total) by 1/100 usingfilter-purified media to obtain the 1/100 (low dose, 25–75 zoospores total). Cumulative exposure for high-dosegroups = 19 000 zoospores; low dose = 190 zoospores.Negative controls (sham infections) were obtained byfiltering active Bd culture using a 25-mL sterile syringe anda 0.2-μm sterile filter disk (Nalgene, Nunc).

Tadpoles were exposed to repeated infections every 4days to coincide with water changes and mortality wasrecorded every day. Each experimental group receivedfour infections on days 0, 6, 9 and 13. Once the forelimbs ofa tadpole had emerged, the water in the flask was reducedby 70%, the flask tilted to provide both aquatic and terres-trial habitat and infections ceased. When an individual’stail had receded to a stub (Gosner stage 45), we classifiedthe individual as a metamorph, removed it from the flaskand transferred it to a 1-L box lined with moist paper towel.Small numbers of pinhead crickets were added as food toeach box every 4 days following the replacement of thepaper towelling and mortality recorded on a daily basis.The entire experiment was completed in a climate-controlledroom kept at 21 °C and with a 12:12 h day/night lightschedule. The experiment lasted 80 days after administering

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the initial infectious dose and was subjected to a survivalanalysis using R.

All experiments involving animals were performedfollowing full ethical review by Imperial College Londonand the British Home Office.

Results

Comparison between Bd genotypes and their associated proteomic fingerprints

Phylogenetic analysis of the ten Bd isolates showed thatthey were all unique except for five isolates recoveredfrom the Spanish island of Mallorca in 2007, which wereall identical at the five sequenced loci (Fig. 1). We sub-sequently focused our study of the Bd proteome on fiveglobal isolates, and a single representative isolate fromMallorca, TF5a1.

To compute the fold changes in protein expressionamong our panel of six isolates, we ran 18 2D gels comprisingthree biological replicates for each isolate. Analysis usedthe Australian isolate, Au-Lcaerulaea98, as an arbitrarilychosen standard against which all other comparisons weremade. Pattern matching between the gels identified 2885

spots in total, of which a core set of 90 spots were up- ordown-regulated among isolates. Here, 70 (2.42%) of theseinter-isolate changes were statistically significant (P < 0.05;Table 2). Global relationships among and between sampleswere visualised using a 3D plotting of the three mainprincipal components (Fig. 2). The principal componentanalysis (PCA) showed that biological replicates for eachisolate were resolved into six distinct groups, demonstratinghigh reproducibility between the biological replicants inour system (Fig. 2). Isolates JEL270 and IA042 were groupedvery close to each other, and the PCA showed that themaximum amount of variation in the experiment is foundbetween the isolates from Mallorca (TF5a1) and Australia(Fig. 2).

We then asked the question ‘does genetic distance corre-late with phenotypic distance?’ Here, ‘genetic distance’ wasmeasured using DAS. ‘Phenotypic distance’ was defined asthe pairwise matrix of the averaged PCA values betweeneach group of biological replicates and was given the valueDPROT. In order to compare values of DAS and DPROT using asimilar scale, values were scaled to between 0 and 1 bydividing each observed value by the maximum recordedvalue (DAS/DAS max and DPROT/DPROT max). A significantpositive correlation was found when these two matriceswere compared (correlation coefficient = 0.665, mantel testP = 0.021; Fig. 3a). The slope of the trendline was ~1 showingthat, unit for unit, genetic distance (DAS) and phenotypicdistance (DPROT) increased in a broadly similar manner.

Identification of differentially expressed proteins

Patterns of up- and down-regulation of the 90 core dif-ferentially expressed Bd proteins are shown in Fig. 4. Here,9% of proteins were up-regulated relative to the standardisolate, Au-Lcaerulae98, and 24% were down-regulated.The remaining 66% of proteins were variable in theirexpression relative to the standard (Fig. 4, Table 2). Relativeto the standard, the Mallorcan isolate TF5a1 was the mostdifferentiated, with 23% and 33% of the 90 core gene

Fig. 1 Neighbour-joining (NJ) tree of the distance coefficient, DAS,between Bd isolates at five loci: BdC5, BdC18.1, BdC18.2, BdC24,8702X2, 8392X2. Isolates with identical genotypes are collapsedonto a single branch.

Table 2 Numbers of proteins out of the core-set of 90 (Fig. 4) thatare differentially expressed relative to the standard isolate, Au-Lcaerulae98 for each isolate. Figures in brackets are the numbersof significantly differentially expressed proteins for each isolate atP < 0.05

SpainC2A

USAJEL270

SpainIA042

UKTvB

MallorcaTF5a1

Up-regulated (P < 0.05)

38 (5) 37 (11) 39 (7) 31 (8) 35 (21)

Down-regulated (P < 0.05)

43 (2) 46 (6) 44 (11) 54 (16) 51 (30)

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products significantly up- and down-regulated, respectively(Table 2).

We successfully sequenced the peptides from 49 spotsusing LC-MS/MS, resulting in 79 protein fingerprintsbeing identified. These mass-spectrometry fingerprintswere used to query the Broad Institute Bd genome and toidentify the putative Bd loci that are being differentiallyexpressed (Table S1, Supporting information). Figure S1,Supporting information shows the physical location of the49 spots that were picked from the four 2D protein gels,and the 37 gene products that were subsequently assigned.In 19 cases (38%), the spot yielded more than one proteinsequence showing that co-migration of proteins hadoccurred (Table S1). In these cases, further expression anal-yses will be required to establish which of the proteins inquestion was responsible for the change in spot intensity.blast and GO annotations showed that differentiallyexpressed proteins were associated with multiple functions,ranging from catalysis (48%), cellular metabolic processes(20%), structural components (2%) and cellular componentssuch as ribosomes (20%; Table S1). A significant number ofproteins (29/79; Table S1) were identified in this analysisthat did not have any significant homology to knownproteins in GenBank. These data will be a valuable addition

to improving the annotation of the two Bd genomes as ourdata shows predicted Bd open-reading frames are real, andthat they are expressed in culture.

Treatment with caspofungin inhibits the growth of Bd isolates

The growth of nine of the 11 global Bd isolates wasinhibited by the antifungal drug, caspofungin, whichinhibits β(1,3) glucan synthesis (Table 1). The remainingtwo isolates did not show enough growth after 21 days tobe included in the analysis. Untreated sporangia werefilled with zoospores (Fig. 5a). Histological analysis showedthat, at sub-IC50 concentrations of caspofungin some zoo-spores within the sporangia appeared to lyse (Fig. 5b). AtIC50 concentrations of caspofungin, all zoospores withinthe sporangia had lysed and the sporangia itself had burst(Fig. 5c). There were measurable differences among theisolates in their susceptibility to caspofungin (Table 1).However, this was not statistically significant and Mann–Whitney tests showed no significant difference betweenisolates in their susceptibility to caspofungin, and therewas no significant difference between groups of isolatesfrom different regions in their drug-susceptibility profiles.

Fig. 2 3D plot of the principle componentsanalysis of the replicate data set for allsix global isolates. Axes show the scorenumber of principal components; M1.t(1)stands for Model 1, first principal com-ponent score; M1.t(2) stands for Model 1,second principal component score; Num isnumber (plotted using SIMCA-P software,UNIMETRICS).



Sporangia/zoospore size and fecundity

Cultures that were inoculated with 1000 zoospores/welland 10 000 zoospores/well had increased at least fivefoldin cell density after 21 days. anova of the numbers ofzoospores found in a 1000 × 1000 pixel field showed thatthe variance in zoospore production among isolates wassignificant for both treatments (1000 zsps/mL, P < 0.001;10 000 zoospores/mL, P < 0.001; Del). However, there wasno observable spatial pattern; when isolates were groupedinto three regions (Region 1, Mallorca; Region 2, MainlandSpain; Region 3, UK) there was no significant geographicaldifference in zoospore production for either treatments(data not shown).

Analysis of a second character, the average area of asingle sporangium, showed a dramatic pattern at both theindividual and regional level. Here, sporangia were signi-ficantly different in size among isolates for either treatment(1000 zoospores/mL, P < 0.001; 10 000 zoospores/mL,P < 0.001). However, unlike zoospore production, thiseffect was highly significant among geographical regions(1000 zoospores/mL, P < 0.001; 10 000 zoospores/mL,P < 0.001). Figure 6(a) shows that this regional difference isdue to those Bd isolates from Mallorca producing, on

average, sporangia that are significantly smaller than thosefrom other regions (Fig. 6b). This is the first time that a mor-phological difference among Bd isolates has been observedand described.

We then asked the question ‘does genetic distance corre-late with morphological distance?’. Here, as with ourproteomic analysis, genetic distance was defined usingDAS. ‘Morphological distance’ was defined for two charac-ters as either (I) the pairwise matrix of differences amongisolates in their average zoospore production, DZSP, or (II)the matrix of differences among isolates in their Log10

sporangia area, DSPOR. No significant correlation was foundwhen DAS was correlated against (I) DZSP for either treat-ment (1000 zoospores/mL correlation coefficient = –0.275,mantel test P = 0.1, Fig. 3b; 10 000 zoospores/mL correla-tion coefficient = –0.338, mantel test P = 0.06). On the otherhand, (II) DSPOR was significantly positively correlatedwith the genetic distance, DAS, for both treatments (1000zoospores/mL correlation coefficient = 0.825, mantel testP = 0.003, Fig. 3b; and 10 000 zoospores/mL correlationcoefficient = 0.789, mantel test P = 0.01). This result showsthat as isolates become more distantly related, there is anincreasing tendency for their sporangia to vary in their size.

We then analysed the fecundity of each isolate in relationto sporangial morphology by calculating the number ofzoospores produced as a function of the per unit area ofsporangia according to the relationship:

zsp per unit sporangia = (Nzsp/Nspor)/Aspor

where Nzsp = the mean number of zoospores per isolate,Nspor = the mean number of sporangia per isolate, andAspor = the mean size of a sporangia per isolate. Figure 7billustrates that isolates from Mallorca produced significantlymore zoospores relative to the size of their sporangia whencompared to isolates from mainland Spain and the UK,and that this effect is significant between regions (anova:P < 0.001). However, there was no clear relationshipbetween region and zoospore size showing that theobserved increase in fecundity per sporangia is not simplydue to a decrease in the size of the zoospores that theyproduce (Fig. 7c).

Assessment of virulence of European Bd isolates using larval challenge

To lessen the number of amphibians used while retaininghigh statistical power, we constrained our study tocomparing three isolates from three geographical regions(UK, mainland Spain and Mallorca) and using two dosingregimes (low and high doses of Bd).

Infection with a high dose of Bd (19 000 zoospores)showed a clear response: survival curves and logrank testsshowed that the Mallorcan isolate TF5a1 was significantly

Fig. 3 (a) Plot of scaled genotypic distance, DAS/DAS max, againstscaled proteomic distance, DPROT/DPROT max. (b) Plot of the scaledgenotypic distance, DAS/DAS max against scaled sporangial area,DSPOR/DSPOR max (circles) vs. scaled zoospore production DZSP/DZSP

max (crosses). Significance of the association between data points isassessed by the use of mantel tests with 1000 permutations of thedata and correlation lines are shown.

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Fig. 4 Up- and down-regulation of Bd protein spots relative to the reference isolate, Au-Lcaerulae98. Protein spots where there is nodifferential expression are shown in yellow. NS (legend) refers to not significant.



Fig. 5 Caspofungin inhibits the growth of Batrachochytrium dendrobatidis. USA isolate JEL270 after 21 days of growth in the absence (a) orpresence of 8 μg/mL caspofungin (b) and 16 μg/mL caspofungin (c). Scale bars = 5 μm.

Fig. 6 (a) Area covered by the sporangia(DSPOR) of nine European isolates of Bd for10 000 inoculated zoospores/mL. n = 40per isolate. (b) Grouped data for eachgeographical region.

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more virulent than a control sham infection of Bd, but wassignificantly less virulent than both the Spanish Pyreneanisolate IA042 and the UKTvB isolate (Table 3; Fig. 8). Therewas no significant difference between IA042 and UKTvB invirulence, with overall levels of mortality caused by eachisolate reaching 92.5% and 97.5% respectively by the end ofthe experiment (Day 80). anova demonstrated a significantinteraction between the weight of animals upon meta-morphosis and isolate (P < 0.012). Here, infection by IA042and TF5a1 both significantly lowered the weight of animalsrelative to the control group. However, infection by isolateUKTvB showed no effect on weight upon metamorphosis.

Infection with a low dose of Bd (190 zoospores) resultedin no significant difference in mortality among the fourgroups of animals. However, the ranked amount of mortalitythat was experienced by each group at the end of the experi-ment followed the same pattern for the high-dose infec-tions; namely the control group showed the least mortality(32%) followed by Mallorca TF5a1 (35%), Spain IA042(44.5%) then UKTvB (53.9%; Table 3, Fig. 8). For this low-dose set of infections, there was no evidence that Bd isolatehad an effect on the weight of animals upon metamorphosis.

However, there was a significant effect of weight onmortality, with lighter animals dying significantly earlierthan heavier animals (P < 0.0182).

Discussion

Linking genotype to phenotype in Bd

The most striking finding of our study is the demonstrationthat genetic differentiation among global Bd isolates at

Fig. 7 (a) Boxplot of mean zoospore countsfrom wells inoculated with 10 000zoospores/mL of each isolate. (b) Boxplotof mean zoospores produced per unitsporangial area from wells inoculated with10 000 zoospores/mL of each isolate. (c)Boxplot of mean zoospore diameter fromwells inoculated with 10 000 zoospores/mL of each isolate.

Table 3 Proportions of Bufo bufo toadlets surviving 80 daysfollowing infection with three isolates of Bd at high and low doses.Relative rankings are shown in brackets

Isolates

ControlMallorca TF5a1 Spain IA042 UK TvB

High dose (rank) 68% (1) 37.5% (2) 7.5% (3) 2.5% (4)Low dose (rank) 68% (1) 65% (2) 55.5% (3) 46.1% (4)



putatively neutral genetic markers is a proxy for diff-erentiation between isolates in measurable biological traitsthat have an effect on virulence. Our data show that, asBd isolates become more distantly related, their protein-expression profiles diverge in a broadly linear manner(Fig. 3a). To our knowledge, this is the first time that astudy has shown a link between the degree of geneticrelatedness among isolates of any infectious disease andtheir protein-expression profiles. Furthermore, we foundthat the size of the sporangia (DSPOR) is also linearlycorrelated with genetic differentiation between isolates,however changes according to a 2:1 ratio (Fig. 3b). This isevidence that this morphological trait is diverging fasteramong isolates than are our genetic markers, and suggeststhat this character may be under directional selection.While it is unclear what may be driving selection at thischaracter, the ecological zone within which the Bd infectionsare found in Mallorca are atypical: Bd growth is optimalbetween 17 °C and 23 °C (Piotrowski et al. 2004). The average

August maximum temperature for the Torrent des Ferreretsin Mallorca is 27.7 °C, 9.7 °C higher than that found inregions of mainland Spain where peak-August metamorphmortalities due to chytridiomycosis are observed, and4.7 °C higher than the known growth optima of Bd. Further-more, the infected species in Mallorca, Alytes muletensis, isthe only amphibian species found in these infected streams,and at a much lower altitude than is observed in mainlandSpain (185 m compared to an average of 1925 m). Therefore,this high-temperature/low-altitude/low-host species diver-sity landscape may have subjected Bd to strong selectionupon introduction to the island, the results of which aremanifested as a smaller-than-average sporangial size. Thishypothesis is supported by evidence of acceleratedsegregation of Bd genomes in a similar narrow host rangesystem in the Sierra Nevada, California (Morgan et al.2007).

When we compared the fecundity among isolates intheir zoospore production, relative to the number and sizeof their sporangia, we found that isolates from Mallorcaproduced significantly more zoospores (Fig. 7b). Researchby Woodhams et al. (2008) have shown that Bd uses life-history trade-offs to maintain high rates of zoosporeproduction across different temperatures. However, it is alsolikely that each genotype of Bd has a unique temperature-dependent reaction norm in fecundity; this possibility hasnever been examined. If such variation is found, then wewould expect to see not only temperature-dependentvariation in fecundity among lineages, but also temperature-dependent variation in fecundity-associated characters,such as the size of sporangia. Our data suggest that the sizeof Bd sporangia has been determined by natural selection.If this is the case, then it is likely that the plasticity inzoospore production observed by Woodhams et al. willvary widely among Bd lineages recovered from differentecological zones, and may have an impact on the host/pathogen dynamic due to the link between zoosporeproduction/longevity and pathogenicity (Mitchell et al.2008). However, these facets of Bd’s biology remain to beexplored.

Bd, recombination and natural selection

Our findings are significant because they show thatBd is generating functional variation from a geneticallydepauperate background, and that at least one character,DSPOR may be under divergent selection in differentenvironments. Currently, the major hypothesis concerningthe emergence of Bd is that the organism is a single, diploid,sexless clone (Morehouse et al. 2003; James, unpublished olata)that is spreading globally via anthropogenic means (Fisher& Garner 2007; Walker et al. 2008). However, Morgan et al.(2007) found evidence for genetically diverse, recombiningpopulations infecting Rana muscosa in the Sierra Nevada,

Fig. 8 Changes in the survival probability for experimentalgroups of Bufo bufo tadpoles and metamorphs infected by threeisolates of Bd over 80 days. Low dose = 190 zoospores, highdose = 19 000 zoospores. n = 40 per experimental group.

426 M . C . F I S H E R E T A L .


suggesting that Bd may be able to segregate its genomeover time. If Bd is a genetically uniform clone, then it isunlikely that we would observe a positive correlationbetween genetic and phenotypic distance matrices. Thatwe observe such correlations suggests that the generation ofheritable genetic variation is occurring. Whether thisfunctional variation is being generated by a cryptic sexualcycle, or via mitotic recombination, is not possible to tell fromour analyses, and will entail comparisons of the patterns ofpolymorphism found throughout the Bd genome.

If, as we have shown, Bd is able to generate functionalgenetic diversity and thus increase its rate of adaptation tonew environments, then this pathogen is much more likelyto present a ‘shifting target’ by allowing the pathogen toadapt to new climates and/or host species. Interestingly,we found no evidence that genotypes differ significantly intheir resistance to caspofungin. The echinocandin class ofantifungal drugs inhibits the synthesis of a key componentof the fungal-cell wall, β(1,3) glucan via the inhibition of theenzyme β(1,3) glucan synthase, and this mode of actionlikely explains the widespread degeneration of the Bd spo-rangial and zoospore cell walls seen in Fig. 6. Comparisonof the growth inhibition for seven isolates of Bd by Ranapipiens antimicrobial skin peptides showed little variationamong isolates in their sensitivity (Woodhams et al. 2006);these observations suggest that not all Bd phenotypes showvariability among isolates. Arms races occurring betweenpathogens and their hosts rely on the generation of noveladaptive variation (Hamilton et al. 1990) and our demon-stration that isolates of Bd vary in proteomic and pheno-typic characters shows that the raw material upon whichnatural selection can work is available within this species.However, the degree to which the expression of geneticvariation in Bd is constrained will impact on the rate ofevolution of Bd in response to various selection pressures;this area will likely be a fruitful area for future investigation.

Linking Bd phenotype to virulence using infection models

Mathematical modelling has shown that increasedzoospore production by Bd increases the force of infectionin natural populations and thus increases the amount ofhost mortality (Mitchell et al. 2008). In this case, the rate ofproduction of zoospores is expected to be associated withthe virulence of an isolate. Our results showed that oneisolate, TF5a1 isolated from Alytes muletensis on the Spanishisland of Mallorca, was significantly less virulent than bothUKTvB (isolated from a UK newt) and IA042 (isolated fromAlytes obstetricans in the Pyrenees). These latter two isolateswere of equal virulence, although UKTvB caused slightlymore mortality in both experiments. The apparent hypoviru-lence of the Mallorcan isolate may be due to our choice ofan in-vivo model (UK Bufo bufo) that does not reflect theoriginal host species (A. muletensis); this choice was made

due to A. muletensis being an International Union for theConservation of Nature and Natural Resources (IUCN)Red List species. However, further experiments are plannedto attempt to recapitulate the observed hypovirulence byadditional experiments that use this host species.

Proteomic analyses showed that these three isolateswere all equally dissimilar from one another (Fig. 2). Incontrast, morphological analyses showed that while twocharacters, DSPOR and DZSP were not significantly differentbetween UKTvB and IA042, they were significantly differentwhen compared to the Mallorcan isolate TF5a1 showingthat there is a correlation between this morphological char-acter, and the virulence of the isolate. However, analysingthe fecundity of these isolates shows that Mallorcan isolatesproduce more zoospores as a proportion of their biomass(Fig. 7b) relative to other isolates, showing that the hypo-virulence of the Mallorcan isolate is not due to an attenu-ated ability to produce zoospores. We also showed thatzoospores of the Mallorcan isolates (and TF5a1) were notsmaller relative to those of other isolates, showing that theattenuation in virulence is not likely due to decreases in azoospores energetic ‘reserves’ (Fig. 7c). Instead, the aviru-lence of this isolate may be due to reduced pathogenicityupon infection as small sporangia are likely to cause lesshost tissue damage than large sporangia. Proof of thishypothesis requires a more detailed understanding ofthe pathology and pathophysiology of Bd infections insitu.

Continuous passage of pathogens results in attenuationas the organism becomes culture adapted (Nischik et al.2001). However, there has been no detailed analysis ofwhether this phenomenon occurs for Bd. In order tominimise this effect, we used isolates with low passagenumbers in our infection experiments, and our UK andMallorcan isolates were isolated in the year that theseexperiments took place. That Bd IA042 (passage 12) was ofsimilar virulence to UKTvB (passage 2) suggests that Bdhas not yet become culture adapted over the intervening10 passages. However, future Bd-virulence experimentsshould ensure that infection protocols are not overlyconfounded by differing passage histories.

Bd proteomics

Further analysis of the Bd proteome is likely to provefruitful because such studies have the potential to uncoverdisease-associated phenotypic markers of chytridiomycosis.Currently, nothing is known about developmental regulationin Bd and its control of gene expression. Even less is knownabout the mechanisms by which the pathogen gainsentrance to the amphibian host and then causes disease.We have taken a single time point from mature cultures,and shown that more than 2885 proteins from the Bdproteome can be resolved using standard techniques, of



which 90 are identifiably differentially expressed betweenBd isolates. We used whole-culture extracts; therefore, theprotein profiles that we characterised represent mixed-developmental stages of Bd comprising both immature andmature sporangia and zoospores. In this case, we are notable to decipher whether the differences that we observedare due to variation in gene expression within zoospores orsporangia, and further development of our techniqueshould focus on separating different developmental stagesfrom one another before protein extraction, as well as focusingon the stages that are found pre- and post-infection.

According to GO annotations, identified Bd proteinscould be classified into eight categories that included struc-tural, catalytic and metabolic processes (Table S1). At leastone protein, HSP70, has been implicated as a potentialvirulence factor in other eukaryotic infectious diseases(Brochu et al. 2004; Zhang et al. 2006). HSP70 is significantlydown-regulated in isolates of Bd from Mallorca, suggestionthat moving these isolates from a high-temperature to alow-temperature culture environment may have effectedthe expression of this locus, albeit in an unexpectedmanner. We also identified two proteins with proteolyticfunction (26S protease regulatory subunit 6A-B and protea-some subunit beta type 3). Piotrowski et al. (2004) describethe production in culture of extracellular proteases withactivity against casein and gelatin (but not keratin) showingthat Bd has the ability to secrete proteolytic enzymes inculture. Given that cell-wall degradation via secretedproteases are likely to be a key component of the invasionprocess (Bremer 1995), that secreted proteinases contributeto the virulence of fungal pathogens (Hube et al. 1997; Nagliket al. 2003b), and that secreted proteinases are differentiallyregulated during disease development (Schaller et al. 1998;Naglik et al. 2003a), then proteolytic proteins deserve to beexplored further as potential virulence factors in Bd. Whilethese two proteases are not secreted, 36% of the identifiedproteins have no significant matches in GenBank showingthat the Bd proteome is extensively underdescribed, andlikely contains key virulence factors. Bd is a member of abasal fungal lineage, the euchytrids ( James et al. 2006) andthe species itself has no known close relatives, residing in itsown genus. Therefore, this novel emerging pathogen likelyholds many surprises in the type, and functions, of the pro-teins that it produces, and well deserves further scrutiny.

‘Pathogen profiling’ Bd

Identifying and classifying lineages is necessary to optimisedisease control strategies if genotypes vary in their epidemicpotential (Sintchenko et al. 2007); this strategy is exemplifiedby the strategy of tracking the evolution of methicillin-resistant Staphylococcus aureaus strains using multilocussequence typing (Robinson & Enright 2004). In addition togenomic techniques, other ‘omic’ technologies can be used

to profile pathogens, such as proteomics. Our analysis ofthe Bd proteome has shown that 2.42% of the 2885 resolvedprotein spots are significantly different in their expression,and that biological replicates of each isolate cluster moretightly than do the proteomic profiles of different isolates(Fig. 2). We also show that as the multilocus genotypes ofisolates become more distinct, then so do their proteomicprofiles. Therefore, it appears that lineages of Bd can beprofiled on the basis of their genotypes and phenotypes,enabling, at least for this set of global isolates andcharacters, easy discrimination.

In a perfect world, integration and sharing of Bd profilingdata using an informatics portal such as http://www.spatialepidemiology.net/bd/will allow optimum manage-ment strategies to be developed by conservation biologiststo manage the emergence of Bd based on the specific epide-miological profile of the introduced lineage. However, suchdata are unlikely to be available for some time; in theinterim however, Bd profiles are still able to provide infor-mation as to the likely virulence, or avirulence, of infectingisolates within specific disease settings. If, as we believe wehave shown, the lineage of Bd that has been introduced intoMallorca is relatively avirulent, then draconican responsesto the infection such as a widespread catch-and-treat strategymay not be necessary. As such, profiling Bd using the methodsthat we have detailed is already having an impact on disease-mitigation policy. On the other hand, our demonstrationthat Bd likely has the potential to adapt and evolve, raisesthe spectre that the pathogen may increase its fitness to newenvironments, or evolve drug resistance where treatmentis being widely used in captivity (Gewin 2008). Therefore,it is likely that Bd, like other pathogens, will present a movingtarget to management strategies and that our response toBd, like the pathogen itself, will need to be adaptive.

Acknowledgements

Alex Hyatt for providing the Australian Bd isolate, Susan Walkerprovided statistical advice and Ming-Shi Li provided the use oflaboratory facilities.

FundingNatural Environmental Research Council and the BritishBiotechnology and Biological Research Council for fundingthrough the Consortium for the Functional Genomics of MicrobialEukaryotes (COGEME) User Fund.

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This study is part of a wider research programme on the globalemergence of Bd (MCF, JB, JES and TWJG) and other emergingmycoses (MCF). ZY, DAS, JW, LS, AJPIB, LW and NARG studymycology and proteomics at the University of Aberdeen.

Supporting information

Additional supporting information may be found in the onlineversion of this article:

Fig. S1 Scans of the four 2D protein gels that were used forpeptide fragment fingerprinting Batrachochytrium dendrobatidis.Identified peptides are annotated onto the gels.

Table S1 Database search reports of tandem mass spectra for 49spots from Bd 2D gels SL0692 (Hyatt), SL0695, SL0712 (Spain C2A)and SL0709 (Mallorca)

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Proteomic and phenotypic profiling of the amphibian pathogen Batrachochytrium dendrobatidis shows that genotype is linked to virulence