Immunologic recognition of a 25 amino acid repeat arrayed in tandem on a major antigen of Blastomyces dermatitidis

Immunologic recognition of a 25-amino acid

repeat arrayed in tandem on a major antigen of

Blastomyces dermatitidis.

B S Klein, … , L H Hogan, J M Jones

J Clin Invest.

1993;

92(1)

:330-337.

https://doi.org/10.1172/JCI116571

.

A 120-kD glycoprotein antigen abundantly expressed on Blastomyces dermatitidis yeasts is

a target of cellular and humoral immune responses in human infection. To investigate the

antigen and immune response more carefully at the molecular level, we screened an

expression library from B. dermatitidis to identify clones that encode this antigen,

designated WI-1. A 942-bp cDNA was isolated by immunologic screening with polyclonal,

rabbit anti-WI-1 antiserum. Northern hybridization analysis showed that the cDNA

hybridized to yeast message approximately equal to 3.9 kb. DNA and deduced protein

sequence analysis of the clone demonstrated a 25-amino acid repeat arrayed in tandem,

present in 4.5 copies near the 5' end, and rich in predicted antigenic epitopes. Further

analysis showed strong homology in these tandem repeats with invasin, an adhesin of

Yersiniae. Cloned cDNA was used to express a 30-kD fusion protein strongly recognized in

western blots by rabbit anti-WI-1 antiserum, and by sera from all 35 blastomycosis patients

studied. The fusion protein product of subcloned cDNA encoding only the tandem repeat

also was strongly recognized in western blots by sera from the 35 blastomycosis patients,

but not by sera from 10 histoplasmosis and 5 coccidioidomycosis patients. An

antigen-inhibition radioimmunoassay showed that the tandem repeat alone completely eliminated

rabbit and human anti-WI-1 antibody binding to radiolabeled native WI-1. From these

results, we conclude that the […]

Research Article

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Immunologic

Recognition of

a

25-Amino

Acid

Repeat

Arrayed

in Tandem

on a

Major Antigen of Blastomyces dermatitidis

BruceS.Klein,*"Laura H. Hogan,* and Jeffrey M.Jonesil

Departmentsof*Pediatrics, $Internal Medicine, and

WMedical

Microbiology and Immunology, UniversityofWisconsinMedical School

and University ofWisconsinHospital and Clinics; and

IlResearch

Service, WilliamS.Middleton Memorial VeteransHospital, Madison, Wisconsin53705

Abstract

A120-kDglycoprotein antigen abundantly expressed on

BWas-tomyces

dermatitidis

yeasts is a target of cellular and humoral immuneresponses in human infection. To investigate the anti-gen and immune response more carefully at the molecular level, we screened an expression library from B. dermatitidis to iden-tify clones that encode this antigen, designated WI-1.A942-bp cDNA wasisolated by

immunologic

screening with polyclonal, rabbit

anti-WI-i

antiserum. Northern hybridization analysis showed that the cDNA

hybridized

toyeast message_3.9 kb.

DNA anddeduced protein sequence

analysis

of the clone dem-onstrateda25-amino acid repeatarrayed in tandem, present in 4.5

copies

nearthe

5'

end, and rich in

predicted antigenic

epi-topes. Further analysis showed strong homologyinthese

tan-dem repeats with invasin, an adhesin of

Yersiniae.

Cloned cDNA was used to express a 30-kD fusion

protein

strongly recognized in western blots by rabbit

anti-WI-i

antiserum, and by sera from all 35

blastomycosis

patients

studied. The fusion protein product of subcloned cDNA

encoding only

the tandem repeat also was strongly

recognized

inwestern blots bysera

from the

35 blastomycosis patients,

butnot byserafrom 10

histoplasmosis

and5

coccidioidomycosis

patients.

An

antigen-inhibition radioimmunoassay showed that the tandem repeat alonecompletely

eliminated

rabbit and human

anti-WI-i

anti-body

binding

toradiolabeled native WI-1. From these

results,

we conclude that the

25-amino

acid repeat of WI-1

displays

an

immunodominant

B cell epitope, and that the

carboxyl-ter-minus of the molecule

exhibits

anarchitecture that may

pro-moteadhesion of

Blastomyces

yeaststohost cellsor extracellu-larmatrix

proteins

and

ultimately

provide

aclearer

picture

of the molecular

pathogenesis

of

blastomycosis. (J.

Clin. Invest. 1993.

92:330-337.)

Keywords: blastomycosis * tandem repeata

serodiagnosis

*dimorphic fungus * cDNA

Introduction

Blastomycosis,

an

endemic

systemic

mycosis accompanied by

protean

manifestations

involving

the

lung, skin, bone,

genito-urinary

tract, and

brain,

results from infection with the thermal

dimorphic fungus Blastomyces

dermatitidis. Humans and

AddresscorrespondencetoDr. Bruce S.Klein, Universityof Wiscon-sinHospitalandClinics, K4/434,600HighlandAvenue,Madison,WI

53792.

Receivedfor publication28August1992and inrevisedform3 Feb-ruary1993.

loweranimals such as dogs are primarily infected by inhaling aerosolized conidia from soil, where the organism dwells as a saprophyte. At

body

temperature, the conidia convert to the parasitic or invasive yeast forms that cause disease.

Alack of understanding of the antigens of B. dermatitidis yeasts has hampered the development of reliable assays for diagnosis and study of immunologic features of blastomycosis. We recently described a novel 120-kD surface protein abun-dantly expressed onB.dermatitidis yeasts (1) and have demon-strated thattheantigen isa targetof antibody(1) and

cell-me-diated immune responses in infected humans (2). Based on

theseobservations, we developed a radioimmunoassayto reli-ably diagnose infection by detecting antibody against the pro-tein and havecharacterizedTcellsdirected againstprocessed

forms of the proteininthe course of human disease. Results from these preliminary studies indicatethat the antigen holds

considerablepromise for improved diagnosis and study of the immunologic features of blastomycosis.

Inourinitial work with the 120-kD antigen, which we have designated WI-1, we employed crude techniques to isolate small amounts ofpartially purified protein and provided lim-ited

characterization of

the molecule. In the present paper, we reportthe

cloning of

cDNA encoding a portion ofWI-1. In

addition

to

providing

anabundant source ofrecombinant anti-gen and detailed structural information about the molecule,

ourwork demonstrates that thecarboxyl-terminus of WI-1 en-compasses antigen-rich, tandem repeats and an immunodomi-nant epitope against which human B cell responses are di-rected.

Methods

Fungi. AmericanType CultureCollection(ATCC)strains60636 and 26199wereusedforthesestudies (ATCC,Rockville,MD). These

repre-sent virulent isolates that have beenassociatedwith human disease. Stock cultures were maintained in the yeast form on 7H10 agar

enrichedwith oleic acid-albumincomplex(Sigma Chemical Co.,St.

Louis,MO)at37°C. Yeasts were grown in Erlenmeyer flasks

contain-ing brainheart infusion(BHI)broth(Difco Laboratories, Detroit,MI)

at370Cinagyrator shakerat120 rpmfor72h.Cellswereharvestedby

filtrationthroughasintered glass filter and washed with saline.

Extraction ofRNA andsynthesisofcDNA. Total RNAwas ex-tractedfromfreshlygrownB.dermatitidisyeastsusinga

phenol-chloro-form technique (3).Diethylpyrocarbonate (DEPC)-treatedwaterand RNAse-free glassware andplasticwarewereusedforextracting, purify-ing,andanalyzingRNA. A 10-gwet-wtpelletof yeastswassuspended

in10mllysisbuffer(0.1MTris, 0.1Mlithiumchloride, 0.01M

dithio-threitol, pH 7.0). The cellsuspensionwastransferredtoaprechilled

metal cannistercontaining 10 ml 0.45-0.50-mm glass beads, 15 ml

phenol-chloroform-isoamylalcohol(24:24:1), and2ml10%SDS. The cannisterwasplacedintoanMSKhomogenizer (BraunBiotechIntl.,

Allentown, PA) fitted withacarbondioxidecoolingdevice andagitated

at2,000 strokes/minfor 30sintervalsbeforecooling. Nearlycomplete

disruptionof cellswasaccomplished afterfive intervals. The chilled

330 B.S.Klein,L.H.Hogan,andJ.M. Jones

J.Clin. Invest.

© The AmericanSocietyforClinical

Investigation,

Inc.

0021-9738/93/07/0330/08

$2.00

mixture wascentrifugedat5,000gfor 15 minat4VC.The aqueous layerofeach tubewasextractedtwice withphenoland thenrepeatedly

withphenol-chloroform-isoamylalcoholtoclear the interface

ofprecip-itate. Nucleic acidwasprecipitatedwith100%ethanol,rinsed with 70% ethanol, dried, and diluted and stored in DEPC-treated water at

-70'C.Poly(A)'mRNA wasisolated from whole cell RNA witholigo

(dT) celluloseaffinitycolumns(BoehringerMannheimBiochemicals, Indianapolis,IN)using previouslydescribed methods(4). Composition ofwhole cell RNA andpoly

(A)'

mRNApreparationswasfollowedby

agarosegelelectrophoresis and

_OD26&280

wasusedtoestimate RNA concentration andpurity.A20-gwet-wtpelletof B. dermatitidis yeasts strain ATCC 60636yielded50 mg total RNA with

OD2W.2go

ratio of 2.17.Affinitypurification of10 mg total RNAyielded - 100Agpoly

(A)'mRNA.Gelelectrophoresisof total RNA showed abundant and distinct 28S and 18S ribosomal bands.Poly(A)'mRNA showed little

remainingribosomalRNAandasizerange upto - 14kb.

Startingwith 5 gg poly(A)'RNAfrom strain 60636, first- and second-strand cDNAweresynthesizedusingmurinemaloney leuke-mia virusreverse-transcriptase, oligo(dT)primer,and other reagents and methodsspecifiedinacommercialsynthesiskit(PharmaciaLKB

Biotechnology, Piscataway, NJ). The ends of double-stranded cDNA werefilled in with KlenowfragmentandligatedtoEcoRI/NotI

adap-torswith the sequenced(5'-AATTCGCGGCCGC-3'). SephacrylS-400 spun columns (PharmaciaLKB Biotechnology)were usedtopurify

cDNAfragments>400 bp. Eachaliquotof cDNAwasligatedinto 100 ngof a dephosphorylatedphagemidvector,pBluescriptII

(KS')

(Stra-tagene, LaJolla,CA),which containstranscriptionalandtranslational

startsequences from thelacZ gene. Afterligation,cDNAwas

electro-porated into competent Escherichia coliXL1-Blue(Stratagene),which wereprepared asdescribed (5).

Screening the cDNA expression library withantibody and colony hybridization. TransformedE.coliwereplatedon LBagarin 150-mm-diameterpetri plates (Falcon Plastics, Cockeysville, MD). After

over-night growthat37°C, bacteria werereplica-plated onto afresh agar plate, andonto a 150-mmnitrocellulose sheet(Schleicher&Schuell,

Inc., Keene,NH).After coloniesgrewto1-2mmonnitrocellulose,the filterwastransferredto afresh LB agar plate supplemented with 5 mM

isopropylthiogalactoside

(IPTG)'

andincubated foranadditional 2-4 hours. Bacteriaonthefilterswerelysed and the filters processed for

immunoscreeningasdescribed (6). Rabbit antiserumspecificforWI-I (anti-WI-I titer,1:10,240,asmeasuredbyradioimmunoassay [1])was

usedto screen aportionoftheexpression library.Theserum was preab-sorbed with E.coli lysate(PromegaBiotec,Madison, WI) overnightat

4°C.Preliminary experiments determinedthatpreabsorbed antiserum diluted 1:2,000coulddiscriminate50 ng WI-I spottedonto

nitrocellu-losefrombackgroundstaining oflysedE.coli proteinsonthefilter. Immunoscreeningwasperformedwith thisantiserumaccordingto pre-viously described methods (6). Rabbitanti-WI-I bound to nitrocellu-lose was detectedwith goatanti-rabbit immunoglobulin G conjugated with alkaline phosphatase (Promega Biotec). Positive colonies were isolated from original replica plates, replated, and rescreened with

anti-bodyuntil theywerepurified.

A 942 bp WI-l-cDNAidentified through immunoscreening was usedsubsequently to screen the library by colony hybridization to at-tempttoidentify additional,longer cDNAs. The entire 942-bp

frag-ment wasremovedfrom the vector by digestion withNotI(Bethesda ResearchLaboratory, Gaithersburg,MD), andpurified from low

melt-ing point agarose (Bethesda Research Laboratory) using Geneclean (BIO 101, Inc.,LaJolla, CA). A 100-ngaliquot of the fragment was radiolabeled with

_[a-32P]dCTP

toaspecificactivityof

109

cpm/ug usingrandomoligonucleotidesasprimers (IBI-Kodak Co. New Haven,

CT). Colony hybridization was performed as described (6). Positive colonies were isolated from original replica plates, replated, and re-screenedwith probe until they were purified.

Northernhybridizations. Total RNA samples (20

_Ag)

were

electro-1.Abbreviation usedinthis

_{paper: IPTG,}

_{isopropylthiogalactoside.}

phoresed through 1% agarosegelsinMOPS/2.2Mformaldehydefor

16-18 h at 30 V and transferred by capillary action to Nytran

(Schleicher&Schuell, Inc.)membranesusing lOX SSC. DNA

frag-mentsforhybridizationwereradioactivelylabeled with[a-32P]dCTP using random oligonucleotides as primers (IBI-Kodak Co.). WI-i cDNAprobewasobtainedasdescribed forcolony hybridization.An actin DNAprobefromHistoplasma capsulatumwasthe generousgift

of E.Keithandwasusedto assessloadingvariation. RNA molecular

weightmarkerswerepurchased from BRL-Gibco.Probeswere

hybrid-izedtofilters in 40% formamide forhighstringencyor20% formamide for lowstringency,0.25 MNa' (P04),1 mMEDTA,7% SDSat420C for 16-24 h. Hybridizedfilterswerewashed first in2x SSCatroom

temperaturefor2-5min,then in 0.25 MNa'(P04),1 mMEDTA,2% SDSat640Cforhigh stringencyor550Cfor lowstringencytwice for 10 min,nextin 0.04 MNa' (P04),1mMEDTA,1% SDSat640C forhigh stringencyor550C forlowstringencyoncefor 40-60min,andfinally

rinsed in 4x SSCat roomtemperature(7).Washed blotswereusedto

expose XAR-5film,EastmanKodakCo.,Rochester,NYwith

intensi-fyingscreens at-80'C.

Sequenceanalysis. Single-stranded anddouble-strandedplasmid sequencingwasperformedaccordingtotheprotocolssupplied usinga

Sequence2.0 polymerasekit(UnitedStates Biochem.Corp.,

Cleve-land, OH).Single-strandedDNAwaspreparedfrompKS+phagemid

clonesbystandard methods(8). Sequenceprimers homologoustothe

pKS+ vectorpolylinkerweretheT3 and SKprimers (UnitedStates Biochem.Corp.). Three setsofprimers homologousto internal

se-quences in the cDNA insertwerepurchased from Operon

Technolo-gies,Inc.(Alameda,CA).Thecompletesequenceofboth strandswas

determined. Both nucleotide sequences and the derived amino acid sequenceswere usedtosearch the GeneBank and EMBL databases

usingprogramssupplied bytheGenetics Computer Group(8).

Expressionandimmunologicassessmentoffusionprotein.

Trans-formedE.coliwasgrown in LBmediacontaining

50Mgg/ml

ampicillin

and 12.5 ,g/mltetracycline inagyrator shakerat 37°C. Whenthe

density ofthe culture reached

_OD6w

_0.6(after - 4hofgrowth), 5

mMIPTGwasadded.Optimal lacZ geneexpressionandproduction

of the fusion proteinwasexaminedat i-hintervalsafterinduction. Bacteriawerecentrifugedat5,000g for 15 minat4°C.Alysatewas

prepared byresuspendingthepelletin IXgelloading buffer (50mM

Tris, pH 6.8, 100 mMdithiothreitol,2%SDS,0.1%bromphenol blue,

10%glycerol)equivalentto0.1 voloftheoriginalculture. Thelysate

washeated to100°Cfor 3 min andcentrifugedat12,000 gfor1 min at

roomtemperature.LysatesofE.coli containingvectoralonetobe used

as antigen inhibitors of antibody binding in radioimmunoassays

(RIAs)were prepared without reducingagents. Bacteriawere

resus-pended 1:4(wt/vol) in alysis buffer (50mM Tris, pH 8.0, 1 mM

EDTA, 100MuMPMSF,and 10%sucrose)towhichlysozyme (1mg/ml)

andTriton x-100(0.1%)wereaddedbeforecentrifugationat12,000g for 30 min.LysateswereexaminedbySDS-polyacrylamide gelas de-scribedby Laemmli (9).Gelswerestained forproteinwith Coomassie

brilliant blue.

Toassessantibody recognition ofthefusionprotein,westernblots

of gelswerepreparedasdescribed(10).Humanantisera used in these

experimentswerefrom blastomycosis patientsin whomantibody

re-sponsestoWI-i hadpreviouslybeen demonstratedbyRIA(1),and from seronegativecontrol subjects with otherfungal diseasesor no

illness.Sera had been storedat-70°Cforup to8 yprior to testing. Thefusion proteinwaselectroelutedfrompolyacrylamidegels

us-inganelectrophoretic concentrator(Isco, Inc., Lincoln, NE) as de-scribed(1)for furtherimmunologicstudies. To determine the relative

importanceofimmunologic determinantsonthefusionprotein,an

antigen-inhibitionRIA wasused. Itexamined theabilityof samples of elutedproteinorcrudeE.colilysatetoblock anti-WI-Iantibody

bind-ingtoradiolabeled WI-1.Briefly,RIAs were constructedusing 0.1-5ng radiolabeled WI-I as atarget. Assays were done in 12X75mm acid-washed glass tubes. We used0.1%BSA in 0.04 M Na phosphate, 0.15

consisted of 0.1 ml ofdiluted serum and 0.1 ml of radiolabeled antigen. When anantigen-inhibition RIA was done,0.1 mlof the inhibitor or BSA-PBS wasadded todiluted antibody in appropriate tubes. All tubes wereincubated 4-18 h at40Cbefore radiolabeled antigen was added. Foreachantigen-inhibition RIAperformed,a preliminaryRIAwas

done todetermine the dilution of serum that bound - 50%of

radiola-beledantigentarget. This dilution of serumwasadded totubesofthe

antigen-inhibition RIA. For RIAs andantigen-inhibitionRIAs, 2 mg insoluble staphlococcalproteinA(Sigma ChemicalCo.) was added to each sample and control tubeafter overnightincubation at 4VC.

Precip-itates werecentrifuged at 2,000 g for 10-40 min and a volume of super-natant wasremovedfromeach tubeforcountinginagamma counter. Wecorrectedforcoprecipitationin RIAs by the methodofMindenand Farr(1 1).

Results

GGATCCATATCATGAGAAGTATGATTGGGATCTCTGGAATAAGTGGTGCAACAAGGACCC 60

D P Y H E K Y D W D ... 20

Invasin-CTACAACTGCGACTGGGACCCATCTCATGAGAAGTATGATTGGGATCTCTGGAATAAGTG 120

Y ii g ii " a. S H E K Y D W D

like Invasin

-GTGCAACAAGGACCCCTACAACTGTGACTGGGACCCATATCATGAGAAGTATGATTGGGA 180 like

TCTCTGGAATAAGTGGTGCAACAAGGACCCCTACAACTGTGACTGGGACCCATATCATGA

:., .... ....'.. ...K: Y H E

Invasin-like

240

80

GAAGTATGATTGGGATCTCTGGAATAAGTGGTGCAACAAGGACCCCTACAACTGTGACTG 300

K Y D W D . ... 100-W : :

Invasin- like

*

GGACCCATCTCATGAGAAGTACGATTGGGATCTCTGGAGTAAGTGGTGCAACAAGCACGA 360

.nl.w S H E K Y D W D L W S K W C N K H D 120

CGAGCACGACAAACATCCATTGTGCCCTGTCTGTGACCCCCTCTCAGGCAAAAATCACTG 420 E H D K H P L C P V C D P L S G K N H C 140

Identification

of

WI-IcDNAclones. Approximately106 recom-binants were obtained in theexpression library. Rabbit anti-WI-1 antibody was used to screen 2,000 recombinants and

identified twopositiveclones. The cDNA inserts inplasmids from these clones were estimated to be approximately 700 and 950 bp by NotI digestion of the plasmids; EcoRI digestion

yieldeda commonfragment of approximately 430 bp. Colony hybridizationwasusedto screen anadditional 10,000

recombi-nants and identified an additional 25 independent, positive

clones. NotI

digestion

demonstrated each cDNAtobe 950

bp and contain thecommonenzymerestriction site for EcoRI. Northernhybridizations usingWI- 1 cDNA toprobeB.

der-matitidis RNA illustrate that the full-length message forWI-1

is - 3.9 kb(Fig. 1). WI- 1 messageis abundantly expressedin

the yeast strain 60636, from which the partial cDNA was

cloned, as well as in the unrelated strain 26199 (data not

shown).

WI-IcDNA sequence. DNA sequenceanalysis ofthe largest

clone isolated by immunoscreening is shown in Fig. 2. The

942-bpsequencecontainstwoopenreadingframes. One open

reading

frame

predicts

an amino acid sequence

conforming

verycloselywith theamino acid

composition

ofWI- 1 in

con-taining

largeamountsof

cysteine, tyrosine,

and

proline (Klein,

B.,

manuscript

in

preparation).

The

predicted

molecularmass

of the recombinant antigen is 25.5 kD; 3.5 kD encoded by

Figure 1. WI- 1 gene

ex-pression

in

_Blastomyces

dermatitidis yeasts.

4111 Northern_analysisof total RNAhybridization

(20,ug)from ATCC strain 60636probed

witha942-bp WI-l

cDNAfragment (lane

2).Ethidium bromide

stainingof gelpriorto

transfer shows28Sand

18Sribosomal bands

(lane3). Molecular size 3, standardsareshown in 2

z o lanei.

TCATCCAACCACTTCCTGTGTCAGCACAGGCCACCACTACCACTGCGCCTGCCGCGCTGG 480 H P T T S C V S T G H H Y H C A C R A G 160

CTACAAGGCTAGCCATTATAGCCATGACCACAAGCATTTCCGCATGCCAGTCAAAGGCTA 540 Y K A S H Y S H D H K H F R M P V K G Y 180

TGAATTCCTTGTTTTCACCGGCCCGCATACCAAGTGCAATGTCCTTTGCGACGGTTACCC 600 E F L V F T G P H T K C N V L C D G Y P 200

GCACAAGCCAGCCCATGAGCTTTGCGGCGAGGTTAAGGTTCATAATTATTGCGGGCCATG 660 H K P A H E L C G E V K V H N Y C G P 219 AGCTTCCTTTGAGGGAAAGCGCAGAAAGAGGGAAATTTGGGGTGATTTTGGAGTACTTGA 720 ACAGATTTATTTTCCTCGTTTTGAGGGAGGGAGAAAGCGAACATGTAATTTTGTAGATGG 780

GTGGAAAGCTTCATCTGACCTCTGGACGAGGTCATTTTTTCCCTTTTATTATGGATAAAT 840 ATTGATGAAGTTGGATAATAGGGAGACATCTTTTTGCAAAATTAATAGTTATATATATGA 900

GTTAGATTACCTTCTTAATTAGATGCTTTAATGATTTTTGTG 942 Figure2.Nucleotideandpredicted proteinsequence ofpartialcDNA

encodingWI- 1.Thepredicted proteinsequenceisindicated below thenucleotidesequence.Numberingtotherightindicates the

nu-cleotideandproteinpositions.The25-aminoacid repeatsarrayedin

tandem,present in 4.5copiesnearthe 5'end,areoverlined witharrow

bars. WI- 1 sequenceshowingsimilarity with invasinappears shaded. Theasteriskoverlyingnucleotideposition359 denotes theFokI re-striction site usedto cutand subclone cDNAencoding onlythe

tan-dem repeats.Apolyadenylation site,located in the 3' nontranslated

region, is comprised ofthetripartatestructure TAGT(nucleotides 886-889),anupstream sequence TAG(859-861)andadownstream sequenceTTT(927-929).This issimilartothepolyadenylationsite

describedfor the CYC 1 gene locus inSaccharomycescerevisiae(29).

plasmid j3-galactosidase

fusionsequences and the remainder

by

theWI- 1 insert. TheDNA sequence

predicts

anamino acid sequence

comprising

a 25-aminoacidrepeat

arrayed

in

tan-dem, present in 4.5 copiesnearthe 5'end, withamino acid

variations in onlytwo

positions (serine

vs.

tyrosine,

residues

3,28,53,78,103;andserine for

asparagine,

residue

113).

DNAanddeduced amino acidsequencesofWI-1 showed

similarity

toothersequences in the GeneBank database

(Fig.

2

and

3).

17amino acid residuesof

WI-1

spanning

the endofone

tandem repeat and the

beginning

of another showed35%

iden-tity and 88%

homology

tothe

protein

adhesion ofYersiniae, invasin(12). 36 amino acid residues outside the tandem repeat ofWI- 1

(spanning

nucleotides 391 to

498) comprise

a

cysteine-richregion

showing

27%to41%

identity

anda

high degree

of

similarity

to anEGF-like domain

expressed

on numerous

pro-332 B.S.Klein,L. H.Hogan,and J.M.Jones

kB

7.5-4.4-*

2.4

-

4Al

A WI-1 LWNKWCNKDPYNCDWDP _{Figure 3.Homology of}

Invasin LWGEWGS LTAYSSDWCS WI-1 sequencewith other sequences in the

B WI-1 CDPLSGKN-HCHPTTSCVST GenBankandEMBL

EGF Consensus C--L--N N G GCSO -X C X N T data bases.InA andB,

* _{sequenceidentity is}

in-GHHYHCACRAGYKASHY.... dicated by_avertical

XGGYXCXCXEGYXLXDGKxcx line.A conservative

F _{aminoacid substitution}

is indicated by a colon. Asteriskscorresponding to EGF consensus sequence denote the pres-enceandspacing of cysteine residues, a prominent feature of the motif.

teinsthatconfer adhesion to cells or extracellularmatrix(13). This similarity with EGF-like domains expressed in at least sevenotherproteins isnotlikelydue tochancealone (P values

ranging from< 1.7 X 10-8 to < 0.01),accordingto a recently

describedalgorithm (14).

Thepredicted structure and antigenicity of the protein en-coded by the WI- 1 insert was analyzed using algorithms of Chou andFasman(15) and KyteandDoolittle (16).The

pro-tein, which corresponds to the carboxyl terminus, is hydro-philic alongmostof its lengthexceptin theportion showingan

EGF-likedomain,inwhichthe moleculeexhibitsa

hydropho-A

B

Mr(kD)'

116-

84-

58-

48.5-

36.5-bF, .

-VW W

...

4o__ l_

66-

45-

36-29-

4

24-

20-bic region. Residues in this area are predicted to form a beta-pleated sheet, a common structuralmotif of EGF-like domains

based on conserved sequence and dictated by the position of the disulfide bonds (13). In contrast, residues in each region

showing similarity with invasin arepredictedtoform several turns and anahelixat theinitiation. The 25-amino acid repeat

arrayedintandem folds back on itself severaltimes,and these repeats, which also encompass the invasin-like domains, are packedwithpredicted antigenic sites.

Expression of the fusion protein. Lysates of E. coli XLl-blue transformed withthe pBluescriptII vector alone orthe

vector plus 942-bp WI- 1 insert, studied by SDS-PAGE, show that a fusionprotein of - 30 kD is expressed by 2 h and

pro-ducedmaximally by4 hafterinductionwith IPTG(Fig.4 A, lane2, see arrow). This protein may represent WI-1 cleaved from the 3.5 kD (3-galactosidase fusion partner, since the same

lysatealsocontainssmaller amountsof33.5 kDproteinthatis

enriched in comparisonto control lysates. Rabbit anti-WI- 1 specifically recognizes both of these proteins in a western blot

(Fig.

4

_B).

Human immune responses to WI-Ifusion proteins. 46 sera

from35patientswithculture-confirmedcasesofblastomycosis

were testedfor recognition of proteins inthe E.colilysates. 19

(54%) ofthepatientshad isolated pulmonarydisease, the

re-mainderhaddisseminated disease involving skinalone(8

pa-tients); lungandskin (5); lung,skin,andbrain (1); brainalone (1); and eye alone (1).Titersof antibodyto WI- 1 inthese pa-tients ranged from 1:100 to 1:34,000 (geometric mean titer,

1:850).Allsera testedreactedspecifically withWI- 1 fusion pro-teininWestern blots(Fig. 5).Somebackground reactivitywith E. coli proteinsin the lysatesisseen in alllanesoftheblots.

Preabsorption of rabbit and human anti-WI-1 antisera with either the elutedfusion proteinorwithE.coli lysatecontaining

the fusion protein nearly eliminated recognition of radiola-beled WI- 1 in the RIA, whereas preabsorption with E. coli

lysate without the fusion protein had no influence on WI- 1

recognition (Fig.6, A and B). Thefusion protein further inhib-ited anti-WI-1 bindingto '251-WI-1 inadose-dependent man-ner(Fig.6,

C),

with2 ugprotein inhibiting100% of bindingby

eitherrabbitorhumanantiserum.

30

kD-

14.2-1

2

3

4

Figure4. SDS-PAGE analysisof recombinant

WI-l

protein. (A) 10% acrylamidegel of E. coli lysatecontainingpBluescript II plasmid alone(lane 1) or plasmid plusWI-I cDNAinsert (lane 2) 4 hafter induction with IPTG. Molecular weight standards are indicated to theleft of lane1.Coomassie bluewasusedtostain gel. (B) Immuno-blots of E. coli lysatescontainingplasmidalone(lane 3) or

recombi-nantWI-I (lane 4) probed with rabbitanti-WI-i antiserum (RIA

titer, 1:10,240)diluted 1:2,000. Molecularweightstandardsare indi-catedtotheleftof lane 3.

12 3 4 5 6 7 8 9 10 11 12131415 16

Figure 5.AntibodyrecognitionofrecombinantWI-I inblastomycosis

patients.ImmunoblotsofE.colilysatescontainingrecombinantWI-I (lanes1-8)or vectorcontrol(lanes9-16)4hafter induction with IPTG, probed with antisera fromblastomycosis patients. Serawere

diluted 1:1,000.Resultsareshownfor 8patients(similarresultswere

Mr(kD)

66-

45-

36-

29-

24-

30-

20- $J,_~.f':-.i,<.:.-N

14-2

116- w

-_. .s,. !

S... w.

t .: :: w ..:M

'%.;: '.

...

:

...

.:: .A,

*S,

*: .:

.:iiS

.x... .:.::.,s

S

Ev

,X,:

_E _|:

3

B

1

0

c

a 3

E.CaII E.ocll 30 kD Neilve

Yeat yseatY Fusion Wil1

*30kD Protein

Fuslon

Protein

C

100

80~~~~~~~~~

80o

-I*

...

...

Z0

a

c 40 0 20

0.125 0.250 0.500 1.000

30 kD Fusion Protein(g)

2.000

Figure6. Human and rabbitantibody recognitionof recombinant

andnative WI-Iby radioimmunoassay.An RIAinhibitiontest

mea-sured inhibition ofantibody bindingto 23I-WI-Ibyvariousantigen preparations. (A) SDS-PAGE gelsofantigensusedasinhibitorsinthe

assay:representative E.coli lysatecontaining plasmidvectoralone

(lane 1), electroeluted30-kD recombinantWI-I (lane 2),and

HPLC-purifiednative WI-I (lane 3).(B)Inhibitionofantibody

bind-ingto 1251-WI-l by E.colilysate (50ggprotein),E. colilysate "spiked"withelectroeluted recombinantWI-1 (2ugof fusionprotein

addedtolysate),electroeluted recombinantWI-Ialone(2_ggprotein), and nativeWI-1(500 ng). (C) Dose-dependentinhibition ofantibody bindingto1251-WI-l byelectroeluted 30-kD recombinant WI-1.

Dilu-tions of human andrabbit antiserayielding50%radiolabeledantigen bindingwereusedin theassays.

Subcloning

and

immunologic study of the

tandem repeat.

We examined whether the tandem repeat of cloned WI-1 cDNAwasanimportantsiteforanti-WI-I antibody

recogni-tion. Restriction sitesbounding therepeatswereusedto sub-clone a 372-bp fragment encoding 4.5 copies ofthe tandem

repeatinto pBluescript II (Fig. 2).AFokIdigest ofWI-I cDNA

yieldeda 1,678-bpfragment including the tandemrepeatsplus

five additional basesatthe3'end andaportion ofvector atthe

5'end. Afterthe endsweremade blunt with Klenowtreatment,

a HindIII digest, which cuts in the polylinker site of pBlue-script,wasusedto separatethe cDNA andvector.The372-bp

fragment was purified and cloned directionally into

pBlue-scriptIIcutwithHind III and SmaI.Ligated cDNAwas

elec-troporated into E. coli XL-I Blue and the transformantswere

assessed for recombinants by blue/white color selection. Fu-sion protein encoded by subcloned cDNAwasexpressedinE.

coliasabove. When studied by SDS-PAGE, the fusion protein

comprised by tandemrepeatsis produced maximally by 4 h

afterinductionwithIPTG,migratesat 21kD,andis

recog-nizedspecifically by rabbit anti-WI-I antiserum inaWestern

blot (Fig. 7).

Serafrom the 35 blastomycosis patients studied abovewere

examined for recognition of the tandem repeat by Western

blot.All ofthem reacted strongly with the recombinant protein

(Fig.8).Noneof theserafromtenpatientswithhistoplasmosis

(complement fixation titers, 1:32to1:512; median titer, 1:128)

orfivepatients with coccidioidomycosis (complement fixation titers, 1:32to1:64; median titer, 1:64) reacted with the

recombi-B

A

Mr

(kD)

45-

36-29-

6.0

24-

20-....

if

14O-

66-

45-

36-

29-

24-i_

_.0-4

",

_0

2_

~~Oiz

_ _

,*....

1

2

3

4

5

Figure7. Fusionproteinproductof subcloned372-bpWI-I cDNA

encodingtandem repeat.(A) 12.5%SDS-PAGEgelofE.colilysate containingpBluescriptIIplasmidalone(lane 1), plasmid plusWI-I cDNA insert(lane 2)4 h after induction withIPTG,andelectroeluted

fusionprotein (lane 3).Molecularweightstandardsareshown to the

left of lane 1. Coomassiebluewasused to staingels.(B)Immunoblots

ofE.colilysate containingrecombinant WI-I tandemrepeat(lane

4)orplasmidalone(lane 5) probedwith rabbit anti-WI-I antiserum

(RIAtiter1:10,240)diluted 1:2,000.

334 B.S.Klein,L. H.Hogan,and J.M.Jones

1 2 3 4 5 6 7 8 9 10 11 12 13 15 1718 192021 22 3242526

Figure8.Antibodyrecognitionof recombinant WI- I tandem repeat in blastomycosis patients and control subjects. (A) Immunoblots of E. coli

lysatescontaining WI-I tandem repeat (lanes 1-11) probed with antisera from blastomycosis patients. Sera were diluted 1:50. Results are shown forI I representativepatients (similar results were obtained for an additional 24 patients). (B) Immunoblots of E. coli lysates containing WI-I

tandem repeatprobed with antiserafrom 10 histoplasmosis patients (lanes 12-21) and five coccidioidomycosis patients (lanes 22 to 26). Sera were diluted 1:50.

nantprotein,althoughsomebackground reactivitywith E.co/i

proteinsinthelysatesis evident(Fig. 8).

To assess the relativeimportanceof the tandem repeatin

antibody recognitionof WI-1,we testedthe21-kD fusion

pro-tein in the antigen-inhibition RIA using anti-WI-I antisera

from tenblastomycosispatientsand an immunized rabbit.The tandem repeat inhibiteda mean of 96%(range85-100%)ofthe

antibody binding to radiolabeled WI-I in theblastomycosis patients, and87% of thebindingofantibody inthe immune

rabbit

(Fig.

9).The resultswereuninfluenced bywhether the

fusion protein was tested after purification by elution,orinthe presence of E. colilysate, whereas E. colilysate alone without the fusionprotein hadnoinfluence whatsoeveronanti-WI-I antibody binding of radiolabeled WI-1.

Discussion

The diagnosis and basic investigation ofblastomycosis has beenseriously hamperedbythe lack ofwell-defined antigens. Werecently described a novel antigen,WI-1,a 120-kD glyco-protein that is abundantly displayedonthe surfaceof B.

der-I I I

EL-n E-.oi 21kD

lysate lysate Fusion

+21kD Protein

Fusion Protein

-*Rabbt Ab tHumanAb

0 %

Pt.1 Pt.2 Pt.3 Pt.4 Pt.5 Pt.6 Pt.7 Pt.8 1t9 Pt.10

21 kDFusion Protein(Tandem repeat)

Pt.10

Naive

W)91

Figure 9. Human and rabbit

antibodyrecognition of combinant WI-i tandem re-peatbyradioimmunoassay.

AnRIAinhibition test mea-suredinhibition of antibody

bindingto '25I-WI-1 by

variousantigen preparations.

Antigensused asinhibitors were: crude E. colilysate containing plasmid vector alone(see Fig. 7, lane 1), electroeluted recombinant

WI-l tandem repeat (seeFig.

8,lane 3), thelysateand eluted proteinpreparations

combined,and HPLC-puri-fied native WI-I (seeFig.7, A, lane3).Protein content of

inhibitorswere50ug crude E.coli lysate,2gg

recombi-nantWI-I tandem repeat, and 500 ng native WI-i.

Di-lutionsofrabbitand human antiserayielding50% radio-labeledantigen binding were used in the assay.

100

80f

- 60

' 40

20

matitidisyeasts(1), shedintothemedium of activelygrowing

cells (Klein, B., manuscript submitted for publication) and

highly conserved in

wild-type

strains(1). Theinteraction ofB.

dermatitidis yeasts with the human immune system elicits a vigorous humoral and cell-mediatedresponsedirected against

WI- 1andits

epitopes

during acquisition ofresistanceto infec-tion (1, 2). Because of the possible importance of

WI-l

with

respect toimprovedserodiagnosis,studyofhost-pathogen

in-teractions, and

understanding

of the biology of thefungus,we

have soughttolearnmoreaboutthis antigenatthemolecular level.

Inthis report, wedescribe the cloning of cDNA that

en-codesaportion

of

WI-1.Several lines of evidencesupportthis

conclusion.

First, rabbit

anti-WI- 1

antiserum

was used to screen anexpressionlibrary for clones that produce immunore-active

protein,

and the cDNAisolated and characterized

pro-ducesa

fusion

protein

thatreacts

specifically

andstronglywith

rabbit antiserum

in bothaWesternblot and

radioimmunoas-say. Second,

antisera from blastomycosis patients

with

high

antibody

titersto

native

WI-1 also reactstrongly and

specifi-cally with the fusion

protein

in both of these assays,whereas

sera

from

15

histoplasmosis

or

coccidioidomycosis

control subjects donot.Although the number of controls

studied

here is limited, the specificity is consistent with previous work, in whichonly two (3%)of 73 control subjects withotherfungal infections had detectable

antibody

to

radiolabeled,

nativeWI-1

(1). Third, the resultsof analysis of the DNA sequence and deduced amino

acid

sequence

of

the cDNA are remarkably

consistent with

previous analysis

of the amino acid

composi-tion

of

nativeWI-1, suchasthe

high

content

of

aromatic resi-dues and

especially

the

unusually

large amount of

cysteine

(Klein, B.,

manuscript

in

preparation).

Finally,

northern

hy-bridization

using

the cloned cDNA

probe identifies

amessage

- 3.9 kb,

approximately

the

size

predicted

for

native

WI- 1.

Despite

theselines of

evidence,

formal

proof

thatwehave

iso-latedWI- 1 cDNAawaitsa

comparison

of the DNA sequence

with amino acidsequence from native WI-1. In view of this

fact,

itmaybe

appropriate

torefertothecDNAand

recombi-nant

protein

wedescribeas a WI-

1-like

antigen

sequence.

ThecDNAclonestudied encodes onlyaportion ofWI- 1.

The

uniformity

in size of about Ikbfor nearly all the

indepen-dently

derived cDNA clones is

quite striking,

andsuggeststhat

RNA secondary structure may interfere with

transcription

beyond this

point.

This conclusion is

supported by

previous

difficulties

wehave had with in vitro translation of heat-dena-tured

Blastomyces

total or

poly

(A)'

mRNA:

Methyl mercury

hydroxide denaturation

ofRNA

secondary

structure was

re-quired

to

produce

large

(although

still

incomplete)

translation

products

(Klein,

B., and J. Jones,

manuscript

submitted for

publication).

Secondary structure commonly interferes with the

cloning

of

full-length

cDNAs

(6,

17).

Preliminary

evidence hasindicated thatwe canuse a custom

primer

derived from the

WI- 1cDNA sequence, rather than

oligo

(dT)121,8,

totranscribe beyond the presumed,

problematic

areaof secondarystructure

andisolate

larger

cDNAs.

The truncatedform of recombinantWI- 1produced bythe

clonedcDNA appears to be

highly antigenic

and tocontainan

immunodominantregion for antibody recognition by infected humans.The fusion protein contains219amino acidsat the

carboxy terminus ofWI- 1.Chou-Fasman

predictions

basedon

the deduced amino acid sequence ofthe fusion

protein

(15)

indicate that the 25-amino acid tandemrepeat is richin

anti-gens, probably B cell epitopes. These predictions are based

principallyonthehydrophilic character,andexposedand

ac-cessiblehighlycharged and polar residues,that appear to

com-prise theturnsandahelices within thisrepeat.Ourresults with

human anti-WI- 1 antisera are consistent with these

predic-tions. All patients tested strongly and specifically recognized the fusion

protein

in Western blots, and nearlyall antibody

reactivity against

WI-1 couldbe removedfromantiserum by

preabsorption with the fusion protein in theRIA.Our

experi-mental results

further

delineate the antibody recognitionsite to

within the tandemrepeatofthefusionprotein.Precise

identifi-cation of the residues that

comprise

theepitope shouldbe

rela-tively straightforward

with the

preparation

of synthetic

pep-tidesordeletionmutantsofWI-1 cDNA.

The themeof

immunodominant

Bcellepitopes contained withinmultiplerepeatsarrayedin tandemonmicrobial anti-genhas

previously

beendescribed inaconservedantigenic

epi-topeof

Trypanosoma

cruzi

(18)

and in the

circumsporozoite

antigen

of

Plasmodiumfalciparum

(19).

The

importance of

the

strong immuneresponse

directed against

thesedeterminants

remains

unclear. Nonetheless, identification ofthe

immuno-logic importance

of the tandem repeat on WI- 1 will permit

improved

serodiagnosis

of blastomycosis. This issue

notwith-standing,

it is well

accepted

that

antigen-specific

Tcellsconfer

acquired

resistance and protect

against

a lethal challenge of

Blastomyces

inanimal models

(20).

We have recently shown that

acquired

resistance in human infection is associated with

development

ofWI- 1

-responsive

Tcells. Further study

of

the tandemrepeatand the

products of larger

or

full-length

cDNAs ofWI-1 will

permit identification

ofTcellepitopes.

Anexamination of theDNAanddeducedaminoacid se-quence

of

WI-1

offers potentially

informative

insights into

an

abundantly expressed,

highly

conserved molecule of

Blasto-myceswhosefunction is unknown. The deduced amino

acid

sequence exhibitsan

interesting protein mosaic

architecture with tandem interaction domains containing regions ofgreat

similarity

with

invasin,

a 103-kD adhesin of Yersiniae,

adja-cent to an

EGF-like

domain,

amotif exhibited bynumerous

molecular

species

thatmediate

adhesive

interactions

with cells

or extracellular matrix

proteins.

Invasin- and EGF-like do-mains have been

extensively investigated. Invasin,

aproduct of theINV

locus,

isa

protein

virulence determinant ofYersiniae

thatpromotes

adhesion

of the bacterium and

penetration into

mammalian

phagocytic

and

nonphagocytic

cells

(21).

Several

receptorsthatbind invasin have been

identified

andeachisa

memberof theVLA

family

of

integrin

celladhesion molecules thatexpressesa common

_#I

chain

(22).

The

carboxy-terminal

192

amino acids

of invasin contain the

integrin

binding

do-main

(23),

even

though

this

region

doesnotcontain the

tripep-tide sequence

Arg-Gly-Asp (RGD),

a common motif forthe

binding

site of

ligands

with

fB integrins (24).

Invasinand fibro-nectin bindtothe same, or

closely

located siteson

_a541

inte-grins, although

binding by

fibronectin is mediated

by

the

tri-peptide

sequence

RGD,

whereas invasin binds with much

higher

affinity

than does fibronectin

(25).

The deduced

se-quenceof WI-l cDNA

predicts

four 17-amino acidrepeatsin

tandem thatexhibit 88%

homology

witha

portion

of invasinin the 192 amino acids ofthe

integrin-binding

domain.The

tri-peptide

sequence RGDis

similarly

notpresent in WI- 1 cDNA. It remains

possible

that WI-1 is

homologous

to invasin ina

region

thatis

completely

unrelatedto

_integrin

_binding.

How-ever, the

hydrophilic,

highly exposed

ahelicesthatturnback

and forth on themselvespredictedinthe invasin-like domains of WI- I cDNA would make thisregion of theproteinsuitable forligand-receptorinteractions.

TheEGF-like domain inWI-I cDNAconsists ofa

single-copyhomologueofthe EGF repeat unit, which preserves many

of theCysorGlyresiduescharacteristic of the structure,and

particularlythe presence of twoantiparallel,3-strandsbetween

Cys 3 andCys4believedtocomprise the EGFreceptor

recogni-tion site (13). We recognize that only five of six consensus

cysteinesinthis motif are apparent in WI- I and that the

align-mentoftheseconservedresidues isnotprecise when compared

totheEGF consensus sequence.However, thespacing of con-served residuestypicallyvaries in EGF-likedomains so that at leastthreetypesofconsensus sequences havebeendescribed (13). When comparedtoother molecules

containing

EGF-like domains, WI-I shows thebest fit with the repeatsof lymph node homing receptor (26), uromodulin (27),

nidogen

(28),

andfibulin,each of which governs diverse cell-cellor

cell-ma-trix interactions.

This observation,

taken together with the

close proximityofthe EGF repeat and multipleinvasin-like

domains, wouldsupportthe notion that the

carboxy

terminus

of surface exposed WI-I may promote adhesion of

Blasto-myces yeasts tohost cellsorextracellularmatrixproteins. Pre-liminaryexperiments indeed have shown thatWI-I functions

asan

adhesion-promoting

protein and

mediates

the

binding

of

yeasts tomammalian cells.

Study

and further clarificationof

the role of WI-I in these interactive events may

provide

a clearerpictureof the molecularpathogenesisofblastomycosis.

Acknowledgments

Wethank Robert DeMars and the members of his laboratory,

espe-ciallyStephanieCeman and Jean Petersen, fortechnical advice;John Marx and JamesStephens fortechnicalassistance;themedical media

staffattheWilliam S.MiddletonMemorialVeteransHospital for pho-tography andillustration;andEmilieEbel,Department ofPediatrics, University of Wisconsin HospitalandClinics, for preparationof the

manuscript.

The work was supported by National Institutes ofHealth grants

AI-00905andAI-31479, andby the VeteransAdministrationMedical

ResearchService.

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