Plasmodium yoelii: Cloning and Characterization of the Gene Encoding for the Mitochondrial Heat...

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Experimental Parasitology 93, 181–190 (1999)

Article ID expr.1999.4455, available online at http://www.idealibrary.com on

Plasmodium yoelii: Cloning and Characterization of the Gene Encoding forthe Mitochondrial Heat Shock Protein 601

Gloria I. Sanchez,*,†,2 Daniel J. Carucci,† John Sacci, Jr.,†,‡ James H. Resau,§William O. Rogers,† Nirbhay Kumar,* and Stephen L. Hoffman†

*Department of Molecular Microbiology and Immunology, School of Hygiene and Public Health,ia P‡DU.

, Fr

that is expressed in several of the developmental stages of P. yoelii.q 1999 Academic Press

Index Descriptors and Abbreviations: Plasmodium yoelii; Plasmo-

Johns Hopkins University, Baltimore, Maryland 21205 U.S.A.; †MalarCenter, 12300 Washington Avenue, Rockville, Maryland 20852 U.S.A.;and Immunology, University of Maryland, Baltimore, Maryland 21201§ABL-BRP, NCI–Frederick Cancer Research and Development Center

Sanchez, G. I., Carucci, D. J., Sacci, J., Jr., Resau, J. H., Rogers,W. O., Kumar, N., and Hoffman, S. L. 1999. Plasmodium yoelii:Cloning and characterization of the gene encoding for the mitochondrialheat shock protein 60. Experimental Parasitology 93, 181–190. Heatshock proteins are a highly conserved group of proteins required forthe correct folding, transport, and degradation of other proteins in vivo.The Hsp70, Hsp90, and Hsp60 families are among the most widelystudied families. Hsp60 is found in eubacteria, mitochondria, and chlo-roplasts, where, in cooperation with Hsp10, it participates in proteinfolding and translocation of proteins to the organelles. We have clonedand characterized the Hsp60 gene of Plasmodium yoelii (PyHsp60).PyHsp60 is a single-copy gene, located on chromosome 9, 10, or 11.The PyHsp60 cDNA sequence showed an open reading frame of 1737nucleotides that codes for a polypeptide of 579 amino acids, with93% amino acid identity to Plasmodium-falciparum Hsp60 (PfHsp60).Cloning and sequencing of a genomic PCR clone showed the presenceof a 201-bp intron, located 141 bp downstream of the ATG codon. Asingle, heat-inducible, 2.3-kb transcript was detected in Northern blots

of RNA isolated from blood stage parasites. Mouse antisera raisedagainst a DNA vaccine vector that expresses PyHsp60 recognizedsporozoites and liver- and blood-stage parasites by indirect fluorescentantibody test (IFAT). By Western blot, these antisera reacted with themycobacterial Hsp65 and recognized a protein of approximately 65kDa in P. yoelii sporozoites and P. falciparum blood stages. Theseresults show that PyHsp60 and PfHsp60 genes are homologous and

1The sequence data reported herein have been submitted to GenBankand assigned Accession Nos. AF103897 (genomic clone) andAF103898 (cDNA clone).

2To whom correspondence should be addressed. Fax: (301) 295-6171. E-mail: sanchezg@nmripo.nmri.nnmc.navy.mil.

0014-4894/99 $30.00 181Copyright q 1999 by Academic PressAll rights of reproduction in any form reserved.

rogram, Naval Medical Researchepartment of Microbiology

S.A.; andederick, Maryland 21702 U.S.A.

that of the PyHsp60 gene encodes a heat-inducible, intracellular protein

dium falciparum; protozoa; malaria; heat shock protein (Hsp); chaper-onin 60 (Cpn60); polymerase chain reaction (PCR); circumsporozoiteprotein (CSP); apical membrane antigen-1 (AMA-1); Plasmodium;mitochondrial chaperonin.

INTRODUCTION

Heat shock proteins (Hsps) are families of proteins ex-pressed in all prokaryotic and eukaryotic organisms whichplay a critical role in the development and adaptation ofcells to different environments (Hartl et al. 1994). They areconstitutively expressed as well as induced when cells areexposed to a variety of stressful conditions such as elevatedtemperature, nutrient deprivation, etc. They show a highdegree of sequence identity among species and function as

molecular chaperons. By recognizing structural elementsexposed in unfolded and partially denatured proteins, theystabilize the non-native conformation and facilitate their cor-rect folding (Hartl 1996). Additionally, Hsps located insidecellular organelles play critical roles in assisting transloca-tion of proteins from the cytosol into these organelles (Cheng

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et al. 1989). Despite their remarkable phylogenetic conserva-tion, Hsps are highly immunogenic and are considered animportant target of immune mechanisms that can provideprotection against several microorganisms or play a role inautoimmunity (Multhoff et al. 1998). Indeed, naked DNAvaccines encoding the mycobacterial Hsp65 gene (Lowrie etal. 1999) and a Histoplasma capsulatum Hsp60 recombinantprotein (Deepe et al. 1996) confer a high degree of protectionin mice against virulent Mycobacterium tuberculosis andlethal challenge of H. capsulatum.

Plasmodium parasites have a complex life cycle alternat-ing between a poikilothermal invertebrate vector and awarm-blooded vertebrate host. It is not surprising that Hspsare abundantly expressed in these parasites (Kumar et al.1991), where they could play a significant role during theadaptation of the parasites to different environments. Genesof the Hsp70 (Bianco et al. 1986; Kumar et al. 1988), Hsp90(Bonnefoy et al. 1994; Su and Wellems, 1994), and Hsp60(Syin and Goldman, 1996; Das et al. 1997) families fromPlasmodium parasites have been cloned. Hsp70 has beenthe most extensively studied. It is expressed in all stages ofthe parasite life cycle (Kumar et al. 1993; Tsuji et al. 1994)and its expression is induced by high temperature duringthe erythrocytic stage (Kumar et al. 1991). Two differentgenes that code for proteins of the Hsp60 family have beencharacterized in Plasmodium falciparum. P. falciparum cpn60 (Pfcpn60) (Holloway et al. 1994) encodes a blood stageprotein of ,81.6 kDa with a 25–30% identity to proteinsof the Hsp60 family. The other gene, P. falciparum Hsp60(PfHsp60) (Syin and Goldman, 1996; Das et al. 1997), codesfor a protein of ,62.1 kDa and exhibits 54% identity tohuman Hsp60 but only 29% identity with Pfcpn60. Both

contain mitochondrial targeting signal sequences at their N-terminus, and PfHsp60 has been shown to be expressed in all parasite stages (Das et al. 1997). These proteins areimportant not only in the differentiation of the parasites butalso as attractive targets for protective immune responses.Here we describe the molecular characterization of a Plas-modium yoelii gene, the PyHsp60, that encodes a proteinwith 93% identity to PfHsp60.

MATERIALS AND METHODS

Molecular cloning of the PyHsp60 gene. The oligonucleotide prim-ers 60.1, corresponding to positions 339–369 of the PfHsp60 gene(GenBank U38963), and 60.2, corresponding to positions 893–924,were used to amplify a DNA fragment from P. yoelii genomic DNA

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(Fig. 1A and Fig. 4). The GeneAmp PCR reagent kit (Perkin–Elmer,Norwalk, CT) was used following the manufacturer’s instructions.Cycling conditions were one cycle at 948C for 5 min, followed by 25cycles of 948C for 30 s, 458C for 45 s, and 728C for 1 min. A PCRproduct with the expected size of 0.6 kb was cloned into the pCR-Script Amp SK(1) cloning vector (Stratagene, La Jolla, CA) andsequenced. The DNA of the cloned PCR fragment (Hsp60 probe, Fig.1A) was used to screen a P. yoelii (17NL) blood-stage ZAP ExpressEcoR-I/Xho-I cDNA library (commercially produced by Stratagene).Library amplification, titration, and in vivo excision and recirculariza-tion of the clones were done following the ZAP Express instructionmanual from Stratagene. Briefly, Escherichia coli XL1-blue MRF8strain was infected with serial dilutions of phage from the P. yoeliiblood-stage cDNA library. Plaques were transferred to nitrocellulosefilters (BA85, Schleicher & Schuell, Keene, NH) and hybridized withthe 32P-labeled Hsp60 probe. Hybridization conditions were 50% for-mamide, 103 Denhardt’s, 1% SDS, 53 SSC, and 100 mg/ml salmonsperm DNA at 428C overnight. Filters were washed at 508C for 1 hin 0.13 SSC/0.5% SDS and exposed to XAR films. Clones that stronglyhybridized with the probe were coinfected with ExAssist helper phageon XLOLR E. coli strain (Stratagene). PBK-CMV recombinantphagemid was purified using the QIAGEN plasmid mini kit (QIAGENInc., Chatsworth, CA).

Based upon the sequence obtained from the cDNA clone 60.6, theprimer Py60/58 containing the BamHI restriction site and the primerPy60/38 containing the BglII restriction site with the sequences of the58 and 38 ends of the coding region were used to amplify the PyHsp60gene from genomic P. yoelii DNA (non-lethal strain, clone 1.1). ThisPCR product (PyHsp60 gene) was cloned into pCR-Script Amp SK-(1) cloning vector (Stratagene) and completely sequenced.

DNA preparation and Southern blot analysis. Leukocyte-free P.yoelii 17X (NL, clone 1.1)-infected red blood cells were obtained bypassing infected blood from CD-1 mice through a Sepacell R500leukocyte reduction filter (Fenwall, Baxter Healthcare Corp., Deerfield,IL). Red blood cells were lysed in ice-cold 1% acetic acid and parasitesdigested in 20 mM Tris (pH 7.6), 10 mM EDTA, 1% SDS, 200 mg/mlproteinase K at 568C for 24 h. Five micrograms of phenol:chloroform-extracted DNA was digested with 10 units of the desired restrictionenzyme at 378C for 2 h. Agarose gel-separated DNA fragments weretransferred to a Nytran 0.2mm membrane using the TurboBlotter trans-fer system (Schleicher & Schuell). Filters were hybridized with the32P-labeled and gel-purified PyHsp60 gene. The hybridization condi-tions were as described earlier.

DNA sequencing and analysis. DNA sequencing was performedwith the ABI PRISM dye terminator cycle sequencing Ready Reactionkit (Perkin–Elmer, Norwalk, CT), following kit instructions. Vector-and gene-specific primers that spanned the full-length sequence of theclone in both forward and reverse orientations were used (Fig.1A).

DNA sequences were analyzed using the Sequencher 3.0 program(Gene Codes, Corp., Ann Arbor, MI) and compared by submission fora search of homologous sequences using the BLAST algorithm

(Altschul et al. 1990) via the WWW-based Sequence Analysis Servicesat NCBI. Direct alignment of the PyHsp60 protein sequence to othereukaryotic and prokaryotic Hsp60 sequences was done using the soft-ware GeneWorks (IntelliGenetics, Mountain View, CA).

Heat induction and Northern blot analysis. Leukocyte-free P. yoelii17X (NL)-infected red blood cells were incubated at 25, 37, or 398Cfor 40 min. Total RNA was isolated with TRIzol LS reagent (LifeTechnologies, Gaithersburg, MD). Total RNA (5mg/lane) was separatedin a denaturing formadelhyde–1.4% agarose gel and transferred to a

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orranrs

FIG. 1. Schematic representation of the structure of the PyHsp60 g2 (nucleotides 546–2138). Open boxes represent 58 and 38 untranslat60.2(1101–1120), 60.3(90–105), 60.4(493– 510), 60. 5(652–669),60.10(1899–1915), and Py60/38(1921–1941). Nucleotide numbering camplification of the PyHsp60 gene from genomic P. yoelii DNA (B, lladder from Gibco BRL (Gaithersburg, MD) (B, lane 2). Relevant markein uppercase. 58 and 38 boundary junctions are in bold (C).

Nytran membrane. The membrane was first hybridized with a Plasmo-dium-specific 18S rRNA PCR probe generated with primers PB1 andPB2 (Briones et al. 1996) to ensure equal loading of RNA for eachtemperature. The blots were then stripped and hybridized with aPyHsp70 fragment (kindly provided by R. Hedstrom) and finally hy-bridized with the Hsp60 probe. The hybridization conditions were asdescribed above. Films were scanned and analysis was performed ona Macintosh Quadra 800 computer using the public domain NIH Imageprogram (developed at the U.S. National Institutes of Health and avail-

able from the Internet by anonymous ftp from zippy.nimh.nih.govor on floppy disk from the National Technical Information Service,Springfield, VA, Part No. PB95-500195GEI).

Production of antisera against PyHsp60 by DNA immunization,Western blotting, and IFA. Primers Py60/58 and Py60/38 (Fig. 1A)were used to amplify the PyHsp60 gene from cDNA clone 60.6. ThePCR product was cloned into pCR-Script Amp SK(1) cloning vector(Stratagene). The cloned PyHsp60 gene was completely sequencedand subcloned into the DNA vaccine plasmid VR1012 (Manthorpe et

e (A). Hatched boxes represent exon 1 (nucleotides 204–344) and exonregions. Locations of the primers are Py60/58(1–21), 60.1(538–559),.6(878–892), 60.7(1114–1132), 60.8(1478–1334), 60.9(1534–1549),esponds with the database entry with Accession No. AF103897. PCRe 1) or cDNA clone 60.6 (B, lane 3). Molecular weight markers: 1-kbare indicated on the left. The nucleotide sequence of the intron is shown

al. 1993). Female CD-1 mice, 5 to 8 weeks old, were immunized withVR1012/PyHsp60 plasmid. Each dose consisted of two im injections,in the tibialis anterior muscles of each leg, of 50 mg of DNA, in salinesolution. Mice were boosted three times and bled 2 weeks after thelast immunization. Sera obtained from the CD-1 mice before immuniza-tion, or from mice immunized with the VR1012 DNA vector alone,were included in the assays as negative controls. Monoclonal antibodiesNYS1 (Charoenvit et al. 1987) and NYLS3 (Charoenvit et al. 1995),which recognize sporozoites and liver and blood stages, were used as

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positive controls. For Western blotting, recombinant mycobacterialHsp65 (SPP-881, StressGene Biotechnologies Corp., Victoria, BC,Canada) and extracts of P. yoelii 17X (NL) sporozoites and P. falci-parum (3D7)-infected red blood cells were separated in a 4–20%Tris–glycine polyacrylamide gel (NOVEX, San Diego, CA) and blottedto Immobilon-P membrane (Millipore Corp., Bedford, MA). The West-ern-Light chemiluminescent detection system (Tropix Inc., Bedford,MA) was used for immunoblot detection of the proteins with the anti-PyHsp60 antisera. Immunofluorescence assays of air-dried sporozoites,

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air-dried infected red blood cells, and fixed infected liver cells werecarried out as described (Charoenvit et al. 1987). P. yoelii-infectedhepatocytes were counterstained with Evans blue (red) and DAPI(4 8, 6-diamino-2-phenylindole). A Zeiss (LSM 2) confocal laser-scanning microscope (CLSM) was used to examine antibody-stainedslides. Confocal analysis of the stained parasites was obtained bysectioning through the tissue with the CSLM in steps of 0.1–0.2 mm.The microscope was configured as follows: 25 mW Ar and He–Nelasers with 488, 514, and 543 maximum lines with Indec (Sungate,Capitalia, CA) software for image acquisition of x–y scans.

Pulsed-field gradient gel electrophoresis (PFGE). Leukocyte-freeP. yoelii 17X (NL)-infected erythrocytes were treated with 0.5% aceticacid and parasites washed several times in PBS. The parasites wereembedded in 0.5% Incert (FMC) agarose at approximately 5 3 108/ml, incubated in proteinase K sol (2 mg/ml proteinase K in 1% Sarkosyl,0.5 M EDTA) at 508C for 48 h, and stored in 50 mM EDTA at 48C. P.yoelii chromosomes were separated by pulsed-field gel electrophoresis

using a CHEF DRII (Bio-Rad) of parasite-embedded gel slices (approx-imately 1 mm thick) in 1% SeqPlaque agarose (FMC). The gel wasrun in two blocks: Block 1, 3 V cm, 200–400 s switch time, 1200 field angle for 60 h; and Block 2, 3 V cm, 600–1000 s switch time,1060 field angle for 32 h. Separated chromosomes were transferred toa Nytran membrane and the membrane was first hybridized with thePyHsp60 gene. The blot was then stripped and hybridized with thePyCSP gene purified from plasmid 1012/PyCSP (Sedegah et al. 1998)and finally hybridized with the PyAMA-1 gene (kindly provided byJ. Aguiar).

RESULTS

Cloning of the PyHsp60 gene. The sequence of the PCRfragment (,0.6 kb) amplified from P. yoelii genomic DNA,using primers 60.1 and 60.2, showed 95% sequence identityat the DNA level to the corresponding fragment of PfHsp60and only 45% to the corresponding fragment of the murineHsp60 gene (data not shown). By screening the P. yoeliiblood-stage cDNA library with this probe, we identified fivepositive clones. Enzyme digestion of the clones with EcoRI/XhoI revealed inserts ranging from 2.0 to 2.3 kb in sizewith an internal EcoRI site. The clone, 60.6, containing thelongest insert (2.3 kb) was sequenced. Southern hybridiza-tion with the PyHsp60 gene after digestion of the P. yoeliigenomic DNA with EcoRI, EcoRV, and HpaI generatedsingle fragments of ,7, ,13, and ,6 kb, respectively (Fig.

2, lanes 1, 2, and 3). Digestion with the enzyme SspI showedthat the probe recognized two fragments of approximately1.0 kb and 800 bp (Fig. 2, lane 4). Based upon the nucleotidesequence of the cDNA clone, the PyHsp60 gene is expectedto hybridize to two fragments of 976 and 578 bp whendigested with SspI. Recognition of the 800-bp SspI fragmentby the probe can be explained by the presence of a 201-bpintron. Confirmation of the presence of the intron was done

FIG. 2. Southern blotting of Plasmodium yoelii genomic DNA.Five micrograms of P. yoelii DNA was digested with the restrictionendonucleases EcoRI (lane 1), EcoRV (lane 2), HpaI (lane 3), andSspI (lane 4) and the blot was hybridized with the 32P-labeled PyHsp60gene. The molecular weight markers from Gibco BRL are indicatedon the left.

by PCR amplification with primers Py60/58 and Py60/38 of

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the PyHsp60 gene from both genomic DNA and cDNAclone 60.6. Figure 1B shows that a product approximately200 bp larger is amplified when genomic DNA is used astarget in the PCR. This analysis suggests that PyHsp60 ispresent as a single copy gene in the genome of P. yoelii andcontains an intron of 201 bp. The PyHsp60 gene hybridizedto a group of chromosomes that co-migrate in P. yoeliiparasites (Fig. 3, lanes 1 and 2). To locate the exact position

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FIG. 3. Chromosomal localization of PyHsp60. The resolved chro-mosomes were blotted and the same membrane was hybridized withdifferent probes. Ethidium bromide-stained gel showing separation ofP. yoelii chromosomes (lane 1) and the PyAMA-1 (lane 2), PyHsp60(lane 3), and PyCSP genes (lane 4).

of this group of chromosomes, the membrane was first hy-bridized with the PyAMA-1 gene, which is found on chromo-some 9 (Fig. 3, lane 3), and the PyCSP gene, which is foundon chromosome 4 (Fig. 3, lane 4), in several rodent malariaspecies (Janse et al. 1994). The PyHsp60 probe recognizedthe same group of chromosomes as did PyAMA-1. Thisgroup of chromosomes may consist of chromosomes 9, 10,and 11.

Sequence analysis. Analysis of the full sequence ofcDNA clone 60.6 showed that it contained an insert of 2303

bp with a single open reading frame of 1737 bp (Fig. 1A).The AT content of the 58 and 38 untranslated regions was82.7% and for the coding region 69.13%, which are similarto those found in P. falciparum genes (Weber 1988). Thesequence encodes a putative polypeptide of 579 amino acidswith a predicted molecular weight of 62,252. Analysis ofthe sequence of the PCR product amplified from genomicDNA showed that this gene contained an intron of 201 bp

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between positions 140 and 341 of the genomic sequence. 58and 38 splice junctions include the sequences AGG/tta. . . . .agg/GTG which are consistent with the consensussequences for splicing sites of eukaryotic organisms (Mount1982) (Fig. 1C). A WWW-based database search using theBLAST program at NCBI identified marked similarity withthe Hsp60 protein of P. falciparum and, to a somewhat lesserextent, with several proteins of the Hsp60 family.

Molecular chaperons occur ubiquitously and they havean essential function in promoting the ATP-dependent fold-ing of proteins under both normal and stress conditions(Hartl 1996). They are highly conserved and the minimumidentity that is observed between any two Hsp60 sequencesis about 40% over their entire length (Gupta, 1995). Thosethat are imported into the mitochondria contain a typical N-terminal cleavable targeting peptide that consists of a 20-to60-amino-acid sequence rich in basic amino acids (Horwich1990). Very often mitochondrial signal peptides of differentproteins do not share primary amino acid sequence similarity,and three distinct cleavage motifs have been recognized inthem (von Heijne 1992). Alignment of the PyHsp60 se-quence with other Hsp60 sequences revealed a high aminoacid identity with the homologous Hsp60 of P. falciparum(93%), mouse (52%), Trypanosoma cruzi (49%), and Helico-bacter pylori (48%) (Fig. 4). Comparing the N-terminusregion of PyHsp60 and PfHsp60, we concluded that althoughthey only share 60% identity, they both contain the featurescommon to most mitochondrial targeting peptides. In bothproteins the targeting sequences are very rich in basic aminoacids, particularly arginine and lysine. More importantly,they share a sequence of 4 amino acids which contains aputative cleavage-site motif with arginine at position 22from the cleavage site (Fig. 4). As with most Hsp genes,PyHsp60 has the conserved GGM sequence at its C-terminalend. The structural or functional significance of this motifis currently not known.

Northern blot analysis, heat induction, and stage-specificexpression of PyHsp60. Total RNA (5 mg/lane), purifiedfrom leukocyte-free P. yoelii blood-stage parasites andtreated for 40 min at 25, 37, and 398C, were separated in a1.4% formaldehyde gel and transferred to a Nytran mem-brane. Hybridization of the membrane with an 18S rRNA

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probe showed that approximately equal amounts of RNAwere loaded in each lane (Fig. 5C). Hybridization with thePyHsp60 probe showed a twofold increase in a single ,2.3-kb transcript after shifting the parasites from 25 to 378C anda two to three fold increase after shifting from 37 to 398C(Fig. 5A). Sera obtained from mice immunized with theVR1012/PyHsp60 clone when used in Western blot reactedwith the recombinant mycobacterial Hsp65 protein (Fig. 6,

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FIG. 5. Northern blot analysis of heat induction of the Py Hsp60gene. Five micrograms of total RNA isolated from blood-stage parasitesthat were previously exposed for 40 min at 25, 37, or 398C was loadedin each lane. Membrane was hybridized with PyHsp60 (A), PyHsp70(B), and Py 18S rRNA probes (C).

lane 3) and recognized a protein of approximately 65 kDain P. yoelii sporozoites (Fig. 6, lane 1) and P. falciparum-infected red blood cells (Fig. 6, lane 2). Sera obtained frommice immunized with VR1012 DNA alone did not show

reaction in this assay. IFA of blood stages with the controlsera (pre-immune sera) as well as sera from mice immunized

located in chromosome 10 or 11 (A. P. Waters, personalcommunication). These observations and the evidence thatDISCUSSION

the location of homologous genes among the chromosomesof different malaria species is conserved (Carlton et al. 1994)led us to speculate that the PyHsp60 gene, as with its homo-We have isolated and characterized a cDNA clone from

a blood-stage P. yoelii cDNA library that codes for the P. logue, the PfHsp60 gene (Syin and Goldman 1996), may be

CLONING AND CHARACTERIZATION OF HEAT SHOCK PROTEIN 6

with the VR1012 DNA vector were negative. Sera from miceimmunized with the VR1012/PyHsp60 plasmid exhibitedreactivity against sporozoites (Fig. 7a) and liver (Fig. 7b)and blood stages (Fig. 7c) of P. yoelii parasites. Confocalmicroscopy analysis revealed a distinct punctate intracellularpattern in the different stages of parasite development.

yoelii Hsp60 protein, a mitochondrial chaperonin protein.Based on the deduced amino acid sequence, PyHsp60 shows

FIG. 4. Alignment of the deduced protein sequence of the PyHsp60 g(Pf, U389630), mouse (Mo, X550230), and H. pylori (Hp, X738400). ShaRXXS indicates the cleavage-site motif of the mitochondrial targeting signare underlined.

FIG. 6. Western blot analysis of anti-PyHsp60 mice antisera. P.yoelii sporozoites (2 3 106 lane 1), 1 3 106 P. falciparum (3D7)schizonts (lane 2), and 200 ng of purified recombinant mycobacterialHsp65 (lane 3) were separated in polyacrylamide gel and blotted toImmobilon-P membrane. Anti-PyHsp60 antiserum at 1:100 dilutionwas used for immunodetection. The standards for molecular weightprotein (SeeBlue of NOVEX) are indicated on the left.

93% identity with PfHsp60 and 52% identity with mouseHsp60 at the amino acid level. These results also clearlyshow that the sequence described here is the homologueof PfHsp60 and not of the previously described Pfcpn60(Holloway et al. 1994), with which it shares only 24% aminoacid identity. Our studies indicated that the Hsp60 gene waslocated on chromosome 9, 10, or 11. Further analysis of thepatterns of PyHsp60 hybridization to blots of P. berghei, P.chabaudi, and P. vinckei suggests that the PyHsp60 gene is

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localized on chromosome 10. PyHsp60 is transcribed as asingle 2.3-kb transcript from P. yoelii blood-stage parasites.

ene (Py) with Hsp60 homologs of T. cruzi (Tc, X67473), P. falciparumded boxes indicate residues that are identical between the five proteins.al. Residues corresponding to primers used to amplify the Hsp60 probe

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are very prominent antigens and the target of humoral andcellular immune responses in a wide spectrum of diseases.Hsp60 plays a very important role in the development ofpathogenic and protective immune responses (Zugel and

Research and Development Command Work Units STO F6.

FIG. 7. Confocal microscopy showing the immunofluorescencereactivity of anti-PyHsp60 mice antisera with Plasmodium yoelii para-site stages. (a) Sporozoite, (b) 44-h liver stage, (c) schizont.

A temperature shift from 25 to 378C and from 37 to 398Cproduced a twofold and two to threefold induction, respec-tively. The level of heat induction was comparable to apreviously characterized heat-inducible gene in Plasmodium(Kumar et al. 1991). Regulation of transcription as well asof heat induction of Hsp varies in different parasite species,

and expression is developmentally induced in many organ-isms (Maresca and Carratu 1992). Different molecular mech-anisms that account for differential expression of Hsp geneshave been identified (Hausler and Clayton 1996). In P. falci-parum, PfHsp70 is abundantly expressed in ring and tropho-zoite stages but is down-regulated in schizonts even afterheat shock (Sanchez and Kumar, unpublished). As indicatedby the stage-specific expression of different antigens, gene

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expression in malaria parasites is highly controlled (Hor-rocks et al. 1998). Hsp genes, an evolutionary conservedfamily of proteins, offer the opportunity to study and com-pare the regulatory mechanisms of gene expression. Serafrom mice immunized with VR1012/PyHsp60 recognizedP. yoelii sporozoites and liver and blood stages. Recently Daset al. (1997) raised antisera against a recombinant PfHsp60.These antisera recognized P. falciparum sporozoites and P.falciparum blood-stage and P. yoelii liver-stage parasites byIFA and located PfHsp60 in the mitochondrion. All theseresults establish that mitochondrial Hsp60 from malaria para-sites is expressed in these stages of development and confirmthat PfHsp60 and PyHsp60 are cross-reacting antigens. Hsps

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Kaufmann 1999). The cloning and characterization ofPyHsp60 will permit us to explore in vivo whether or notPlasmodium Hsp60 is the target of protective immuneresponses.

ACKNOWLEDGMENTS

We thank Drs. B. Kim Lee Sim (Entremed Inc.) and Sanjai Kumar(Naval Medical Research Center) for the mRNA for the P. yoelii cDNAlibrary; Dr. Joao Aguiar (Naval Medical Research Center) for PyAMA-1 DNA; Dr. Richard Hedstrom (Naval Medical Research Center) forguidance and technical advice and the PyHsp70 probe and Dr. AndrewP. Waters (Leiden University) for helpful discussions about the chromo-somal location of the PyHsp60 gene. We are also grateful to JamesPedersen for technical assistance with sequencing and R. Wallace andA. Belmonte for P. yoelii, blood-stage infections, and sporozoites.G.I.S. was a recipient of a UNDP/World Bank/WHO/TDR researchtraining grant and received financial aid from the U.S. Navy and theDepartment of Molecular Microbiology and Immunology, School ofPublic Health, Johns Hopkins University. N.K. is the recipient ofNIH Grant AI31589. This work was supported by Naval Medical

161102A0101.S13.BFX and STO F 6.2622787A0101.870.EXF. Theexperiments reported here were conducted according to the principlesset forth in the Guide for the Care and Use of Laboratory Animals,Institute of Laboratory Animal Resources, National Research Council(Department of Health and Human Services, National Institutes ofHealth Publication No. 86-23). The views expressed in this article arethose of the authors and do not necessarily reflect the official policyor position of the Department of the Navy, Department of Defense, orthe U.S. Government.

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REFERENCES

Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J.1990. Basic local alignment search tool. Journal of Molecular Biol-ogy 215, 403–410.

Bianco, A. E., Favaloro, J. M., Burkot, T. R., Culvenor, J. G., Crewther,P. E., Brown, G. V., Anders, R. F., Coppel, R. L., and Kemp, D.J. 1986. A repetitive antigen of Plasmodium falciparum that ishomologous to heat shock protein 70 of Drosophila melanogaster.Proceedings of the National Academy of Sciences of the UnitedStates of America 83, 8713–8717.

Bonnefoy, S., Attal, G., Langsley, G., Tekaia, F., and Mercereau Puija-lon, O. 1994. Molecular characterization of the heat shock protein90 gene of the human malaria parasite Plasmodium falciparum.Molecular and Biochemical Parasitology 67, 157–170.

Briones, M. R., Tsuji, M., and Nussenzweig, V. 1996. The large differ-ence in infectivity for mice of Plasmodium berghei and Plasmodiumyoelii sporozoites cannot be correlated with their ability to enter intohepatocytes. Molecular and Biochemical Parasitology 77, 7–17.

Carlton, J. M., Vinkenoog, R., Waters, A. P., and Walliker, D. 1998.Gene synteny in species of Plasmodium. Molecular and BiochemicalParasitology 93, 285–294.

Charoenvit, Y., Leef, M. L., Yuan, L. F., Sedegah, M., and Beaudoin,R. L. 1987. Characterization of Plasmodium yoelii monoclonal anti-bodies directed against stage-specific sporozoite antigens. Infectionand Immunity 55, 604–608.

Charoenvit, Y., Mellouk, S., Sedegah, M., Toyoshima, T., Leef, M. F.,De la Vega, P., Beaudoin, R. L., Aikawa, M., Fallarme, V., andHoffman, S. L. 1995. Plasmodium yoelii: 17-kDa hepatic and eryth-rocytic stage protein is the target of an inhibitory monoclonal anti-body. Experimental Parasitology 80, 419–429.

Cheng, M. Y., Hartl, F. U., Martin, J., Pollock, R. A., Kalousek, F.,Neupert, W., Hallberg, E. M., Hallberg, R. L., and Horwich, A.L. 1989. Mitochondrial heat-shock protein hsp60 is essential forassembly of proteins imported into yeast mitochondria. Nature337, 620–625.

Das, A., Syin, C., Fujioka, H., Zheng, H., Goldman, N., Aikawa, M.,and Kumar, N. 1997. Molecular characterization and ultrastructurallocalization of Plasmodium falciparum Hsp 60. Molecular and Bio-chemical Parasitology 88, 95–104.

Deepe, G. S., Jr., Gibbons, R., Brunner, G. D., and Gomez, F. J. 1996.A protective domain of heat-shock protein 60 from Histoplasmacapsulatum. Journal of Infectious Diseases 174, 828–834.

Gupta, R. S. 1995. Evolution of the chaperonin families (Hsp60, Hsp10

and Tcp-1) of proteins and the origin of eukaryotic cells. MolecularMicrobiology 15, 1–11.

Hartl, F. U., Hlodan, R., and Langer, T. 1994. Molecular chaperons inprotein folding: The art of avoiding sticky situations. Trends inBiochemical Sciences 19, 20–25.

Hartl, F. U. 1996. Molecular chaperons in cellular protein folding.Nature 381, 571–579.

Hausler, T., and Clayton, C. 1996. Post-transcriptional control of hsp70

189

mRNA in Trypanosoma brucei. Molecular and Biochemical Parasi-tology 76, 57–71.

Holloway, S. P., Min, W., and Inselburg, J. W. 1994. Isolation andcharacterization of a chaperonin-60 gene of the human malaria para-site Plasmodium falciparum. Molecular and Biochemical Parasitol-ogy 64, 25–32.

Horrocks, P., Dechering, M., and Lanzer, M. 1998. Control of geneexpression in Plasmodium falciparum. Molecular and BiochemicalParasitology 95, 171–181.

Horwich, A. 1990. Protein import into mitochondria and peroxisomes.Current Opinions of Cell Biology 2, 625–633

Janse, C. J., Carlton, J. M., Walliker, D., and Waters, A. P. 1994.Conserved location of genes on polymorphic chromosomes of fourspecies of malaria parasites. Molecular and Biochemical Parasitol-ogy 68, 285–296.

Kumar, N., Syin, C. A., Carter, R., Quakyi, I., and Miller, L. H. 1988.Plasmodium falciparum gene encoding a protein similar to the 78-kDa rat glucose-regulated stress protein. Proceedings of the NationalAcademy of Sciences of the United States of America 85, 6277–6281.

Kumar, N., Koski, G., Harada, M., Aikawa, M., and Zheng, H. 1991.Induction and localization of Plasmodium falciparum stress proteinsrelated to the heat shock protein 70 family. Molecular and Biochemi-cal Parasitology 48, 47–58.

Kumar, N., Nagasawa, H., Sacci, J. B., Jr., Sina, B. J., Aikawa, M.,Atkinson, C., Uparanukraw, P., Kubiak, L. B., Azad, A. F., andHollingdale, M. R. 1993. Expression of members of the heat-shockprotein 70 family in the exoerythrocytic stages of Plasmodium ber-ghei and Plasmodium falciparum. Parasitology Research 79,109–113.

Lowrie, D. B., Tascon, R. E., Bonato, V. L. D., Lima, V. M. F., Faccioli,L. H., Stavropoulos, E., Colston, M. J., Hewinson, R. G., Moellling,K., and Silva, C. L. 1999. Therapy of tuberculosis in mice by DNAvaccination. Nature 400, 269–271.

Manthorpe, M., Cornefert Jensen, F., Hartikka, J., Felgner, J., Rundell,A., Margalith, M., and Dwarki, V. 1993. Gene therapy by intramuscu-lar injection of plasmid DNA: Studies on firefly luciferase geneexpression in mice. Human Gene Therapy 4, 419–431.

Maresca, B., and Carratu, L. 1992. The biology of the heat shockresponse in parasites. Parasitology Today 8, 260–266.

Mount, S. M. A catalogue of splice junction sequences. Nucleic AcidsResearch 10, 459–472.

Multhoff, G., Botzler, C., and Issels, R. 1998. The role of heat shockproteins in the stimulation of an immune response. Biological Chem-istry 379, 295–300.

Sedegah, M., Jones, T. R., Kaur, M., Hedstrom, R., Hobart, P., Tine,J. A., and Hoffman, S. L. 1998. Boosting with recombinant vacciniaincreases immunogenicity and protective efficacy of malaria DNAvaccine. Proceedings of the National Academy of Sciences of theUnited States of America 95, 7648–7653.

Su, X. Z., and Wellems, T. E. 1994. Sequence, transcript characteriza-tion and polymorphisms of a Plasmodium falciparum gene belongingto the heat-shock protein (HSP) 90 family. Gene 151, 225–230.

Page 10

190

Syin, C., and Goldman, N. D. 1996. Cloning of a Plasmodium falci-parum gene related to the human 60-kDa heat shock protein. Molecu-lar and Biochemical Parasitology 79, 13–19.

Tsuji, M., Mattei, D., Nussenzweig, R. S., Eichinger, D., and Zavala,F. 1994. Demonstration of heat-shock protein 70 in the sporozoitestage of malaria parasites. Parasitology Research 80, 16–21.

von Heijne, G. 1992. Cleavage-site motifs in protein targeting se-quences. Genetic Engineering N.Y. 14, 1–11.

SANCHEZ ET AL.

Weber, J. L. 1988. Molecular biology of malaria parasites. ExperimentalParasitology 66, 143–170.

Zugel, U., and Kaufmann, S. H. E. 1999. Role of heat shock proteinsin protection from and pathogenesis of infectious diseases. ClinicalMicrobiology Reviews 12, 19–39.

Received 18 March 1999; accepted with revision 12 August 1999