Cloning and characterization of an Eimeria acervulina sporozoite gene homologous to aspartyl proteinases

Molecular and Biochemical Parasitology, 62 (1993) 303 312 ~) 1993 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/93/$06.00

303

MOLBIO 02095

Cloning and characterization of an Eimeria acervulina sporozoite gene homologous to aspartyl proteinases a*

F a b r i c e L a u r e n t , C h r i s t i a n e B o u r d i e u a, M a u r i c e K a g a a, S t e f a n C h i l m o n c z y k a, Gizella Z g r z e b s k i b Pierre Y v o r U a n d Pierre P6ry a ~Unit~ de Virologie et Immunologie MolOculaires, 1NRA, 78352 Jouy-en-Josas, France; bZoologisches Institut der Universitdt Bonn, Poppelsdorfer Schloss, Bonn, Germany; and c Unit~ de Pathologie A viaire et Parasitologie, INRA, Tours-Nouzilly, France.

(Received 29 July 1993; accepted 3 October 1993)

A 2Zapl] cDNA library was constructed using m R N A from Eimeria acervulina sporulated oocysts and screened with monoclonal antibodies raised against Eimeria tenella sporulated oocysts. Monoclonal antibody N3CsBt2 identified a clone (6S2) potentially encoding an aspartyl proteinase since significant homology with cathepsin D, pepsin and renin proteinases was revealed by sequence comparisons. The 1500-bp c D N A fragment containing the coccidial gene was subcloned into pGEX-FA expression vector, leading to the production of an 80-kDa fusion protein (FA6S2) which was used to immunize rabbits. The anti-FA6S2 rabbit sera revealed a single 43-kDa protein present in Eimeria acervulina, Eimeria tenella, Eimeria maxima and Eimeriafaleiformis sporulated oocyst antigens. Indirect immunofluorescence and electron microscopy with mAb N3CsB~2 localized the putative aspartyl proteinase in the refractile bodies of Eimeria tenella sporozoites. Key words: Eimeria acervulina; Sporozoite; Aspartyl proteinase; Monoclonal antibody; cDNA library; Recombinant protein

Introduction

Eimerian parasites cause large financial losses in the poultry industry. The appearance of drug-resistant parasite strains suggests the need for vaccine development. Several live vaccines are now available in different countries [1]; of these, those containing attenuated parasite populations offer a greater degree of safety. Nevertheless, recombinant vaccines, rC~orresponding author. Tel.: 33 16 134 652 610; Fax: 33 16 134 652 621; E-mail: laurent(~biotec.Jouy.inra.fr. Abbreviations: SEaO, soluble E. acervulina oocyst antigen; SEtO, soluble E. tenella oocyst antigen; SEmO, soluble E. maxima oocyst antigen; TBSA, Tris-buffered saline complemented with bovine serum albumin; D-pEa, putative aspartyl proteinase of E. acervulina; mAb, monoclonal antibody. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession number Z 24676.

which should be easier to produce and administer industrially, will certainly represent a future alternative. The search for a protein vaccine candidate has turned towards the membranous proteins of extracellular phases of parasite development, which may be the target of different immune mechanisms inducing parasite destruction or the blocking of its penetration into host cells. Other antigens of interest are those expressed on the surface of infected cells, which can induce a cytotoxic phenomenon leading to the destruction of host cells or to activation of the killing mechanism in the host cell itself. Among the proteins already identified, little is known about their role in parasite development. One can hypothesize about the function of some coccidial proteins, because they have a conserved counterpart in better-studied organisms [24] or because they are localized in organelles such as rhoptries [5], micronemes [6] and dense granules [7].

304 In the present study, monoclonal antibodies (mAbs) were developed against E. tenella sporozoite antigens to screen a )~ZapII E. acervulina library in order to identify antigens common to both species. We report here the isolation of clones coding for a protein with homology to aspartyl proteinases which can be localized to the refractile bodies of sporozoites.

Materials and Methods

Parasites and total oocyst antigen. Eimeria oocysts were cleaned according to the technique of Bontemps and Yvor6 [8] and were allowed to sporulate whilst being agitated in a 25°C water bath. E. acervulina oocyst antigen (SEaO, strain PAPa46), E. tenella oocyst antigen (SEtO, strain PAPt38), E. maxima oocyst antigen (SEmO, strain PAPml 1) and E. Jalciformis oocyst antigen (SEfO, strain PAPf65) were generated as described below. Oocysts were broken with glass beads, sonicated at 0 ° C in phosphate buffer saline (PBS) in the presence of protease inhibitors (250/~M N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), 135 #M N-~-p-tosyl-L-lysine chloromethyl ketone (TLCK), 1 mM phenylmethyl sulphonyl fluoride (PMSF) (Serva)), and centrifuged at 10 000 × g for 10 rain. The supernatant proteins were quantified with the Bio-Rad protein assay (Bio-Rad), stored at - 8 0 ° C and used after dilution for immunization, enzyme-linked immunofluorescent assay (ELISA) or western blotting. Hybridoma antibody production. MAbs were obtained by the fusion of SP2/O myeloma cells with mice splenocytes of three C3H/He (Jouy en Josas) 2-month-old females immunized intraperitoneally with 50 /~g SEtO antigen emulsified in incomplete Freund's adjuvant (IFA, Difco). After three repeated immunizations at an interval of 2 weeks, an intramuscular booster was performed two days before fusion with 80 #g of SEtO. The fusion was carried out according to the method of K6hler and Milstein [9]. Sporulated E. tenella oocyst specific antibody secreting hybrids were de-

tected by ELISA. MAbs isotype were determined by EL1SA using a streptavidin-Biotin mouse monoclonal kit (ZYMED).

Construction and immunological screening of cDNA library. When 80% E. acervulina oocysts were sporulated, they were washed and suspended in lysis buffer (50 mM Tris-HC1 pH 7.5/5 M guanidine isothiocyanate/ 10 mM EDTA), frozen in liquid nitrogen before being disrupted in a French press. /%Mercaptoethanol 0.1% (v/v) and Sarkosyl 0.5% (w/v) were added to the suspension, and cellular debris was pelleted by centrifugation at 10 000 × g for 30 rain. Total RNA was isolated as described by Chomczynski and Sacchi [10]. Selection of Poly A ~ RNA was achieved by 2 successive passages over oligo (dT)-cellulose columns (Pharmacia) and a 2ZaplI cDNA library was constructed according to the manufacturer's instructions (Stratagene). Plaques from the library were transferred to nitrocellulose, the filters blocked in Trisbuffered saline complemented with 3% (w/v) bovine serum albumin (TBSA) and immunoscreened with undiluted mAbs supernatants. Secondary rabbit anti-mouse antibody (Biosys) conjugated to alkaline phosphatase was diluted 1:2000 in TBSA. Binding of antibody to individual plaques was detected by using the substrate system NBT/BCIP according to the manufacturer's instructions (Gibco-BRL). Each positive phage was assessed through 3 rounds of immunoscreening and plaque purification before characterization and sequencing of the DNA inserts. DNA sequencing and sequence comparisons. cDNA containing pBluescript plasmids were derived from the 2ZapII phage using the in vivo excision procedure [11]. cDNA insert sizes were analyzed on 0.8% agarose gels, Double-stranded DNA insert sequencing was done first from each end using T3 and T7 dye primer sequencing kit (USB) according to the manufacturer's instructions. Two clones were selected and their inserts were subcloned into M13mpl8 with a shotgun strategy. DNA sequences were determined using -21 MI3

305

dye primer sequencing kit (USB) by the dideoxy chain termination technique [12] and subsequently loaded on a fluorescent 373A automated DNA sequencer (Applied Biosysterns). Sequencing data were analyzed using the Microgenie sequencing program (Beckman). Sequence comparisons were performed using the FASTA program [13] against the SwissProt database. The MOTIFS option from the UWGCG software [14] was used to identify sequence motifs by searching through proteins for the patterns defined in the PROSITE dictionary of protein sites and patterns.

Southern and northern blot analysis. E. acervulina sporulated oocysts were disrupted in a French press, total DNA was extracted, digested with various restriction enzymes (Boehringer), electrophoresed and transferred onto nitrocellulose (BAS 83, Schleicher and Schuell) according to standard procedures [15]. The blot was incubated in prehybridization buffer (50% deionized formamide/ 5 x SSC (1 x SSC is 150 mM NaC1/15 mM Na-citrate)/ 5 x Denhardt/100 pg m l - 1 calf thymus DNA) at 42 ° C for 2 h and hybridized in the same buffer with denatured 32p-labeled random primed EcoRI l KpnI restriction fragment of clone 6S2 (10 cpm ml - l ) for 12 h. Three washes were performed in 0.1 x SSC/ 0.1% sodium dodecyl sulfate (SDS) at 55°C. The blot was air dried and exposed to Kodak XAR film. Seven #g of poly(A) + RNA from sporulated E. acervulina oocysts were size-fractionated by electrophoresis on 1% agarose gel containing 10% (v/v) formaldehyde in 20 mM 3-(Nmorpholino)-propanesulphonic acid/ 5 mM Na-acetate, pH 7.0/ 1 mM EDTA (Mops) buffer pH 7.0 at 40 mA and blotted onto nylon membrane (Hybond-N +, Amersham) using 20 x SSC. Alkali fixation of the RNA to the membrane was obtained with 0.05 N NaOH. Prehybridization was performed for 1 h at 65°C in 0.25 M sodium phosphate pH 7.2/ 1% (w/v) non-fat dry milk/ 7% (w/v) SDS/ 10 -3 M EDTA buffer. The hybridization step was done in the same buffer with the denatured labeled probe (106 c p m m1-1) used in the

Southern blot for 12 h. The filter was washed and exposed as described above.

Expression in E. coli and purification of fusion protein. EcoRI/KpnI restriction fragment of clone 6S2 was subcloned into pGEX-FA expression plasmid using standard protocols (pGEX-FA6S2) [15]. pGEX-FA plasmid is similar to pGEX3 plasmid but has an extended molecular cloning site [16]. DH5c~ Escherichia coli bacteria carrying pGEXFA6S2 were grown to an A600 nm of 0.5 in LB broth, induced with 2 mM isopropyl-/~-Dthiogalactopyranoside (IPTG, Boehringer) and incubated further for 1 h with vigorous shaking. Recombinant protein, present in an insoluble form in the bacterial lysate was extracted by a similar procedure to the one described by Valenzuela et al.[17] using 7.5 M guanidineHC1 solution. Protein renaturation steps were carried out by successive dialysis against decreasing urea concentration and finally dialysis against PBS solution. Soluble recombinant protein was purified by glutathione agarose chromatography [16]. Immunization of rabbit with recombinant protein. The purified recombinant protein FA6S2 (250 ~g) was injected subcutaneously with Complete Freund's Adjuvant (CFA, DIFCO) twice at 2-week intervals in 2 rabbits. Fifteen days later a final intramuscular immunization was performed, and after two weeks blood was collected. The same protocol of immunization was used to obtain anti-glutathione-S-transferase (anti-GST) rabbit sera. Polyacrylamide gel electrophoresis and western blot analysis. Oocyst antigens and recombinant antigen FA6S2 were analyzed by SDS polyacrylamide gel electrophoresis (PAGE) under reducing conditions [18], and either stained with Coomassie blue R 250 or blotted onto nitrocellulose for immunoassays essentially as in [19]. Free protein binding sites were blocked by 2 h incubation in TBSA. The blot was probed with rabbit anti-FA6S2 antiserum

306 i0 30 50 70 GGCTTCCCTTAAATTACTGA~CCACG~CAGCCAGGTAC~TGCGTTCCCTTCTGGTCGTG~CG~CT M R S L L V V A G L I o 90 Ii0 130 AGCTGGCT~AGTTCTTTCGCTCC~CCGATGC~GACATCGGTTTCTGAGTGAAACGTTGG~G~CCG

AGCSSMFAPTDARHRFLSETLEEP33 150 170 190 210 G~GATGT~TGCTG~GACGGCAGATCTTCACACAAATCTTTTACGCG~CCCCCCATGACGATC~ E D V M L K T A D L H T N L L R E P P M T I K L 5 7 230 250 270 TAGACAACAGATAC~GTTCACTGGCCTTGGCGAGCTGGTTTCACAGCTGATCGACCATCACACCAC~T D N R Y K F T G L G E L V S Q L I D H H T T M 8 0 290 310 330 350 GGG~GCGTTGGTTCCTCTGG~CGATGGCCCGGCAAAAGCT~TC~TTACCAC~CAGCCAGTATTTT G S V G S S G T M A R Q K L L N Y H N S Q Y F I o 3 370 390 410 GGCGAAATAAAGATCGG~CTCCCGGCAG~GATTCGTAGTTGTTTTTGACACTGGTTCCTCAAATCTGT L W127 430 450 470 490 GGGTTCCT~AGCGG~T~GAGAAAGGAGGATGCGCCCCCCATGAGAAATTCGACCCAAAGTATTCTAG V P A A E C E K G G C A P H E K F D P K Y S S I 5 o 510 530 550 CACATTTTCTCCCATACGGTCGCTGACTGGAGACCCAGCAGTCGCATTCATTC~TACGG~CTGGAGCA T F S P I R S L T G D P A V A F I Q Y G T G A I 7 3 570 590 610 630 TGCGTTCTTCG~TGGGTCGCGACATCGTGGAGATCGGCGGCATCAAAGT~CC~CCAGGC~TC~CC C V L R M G R D I V E I G G I K V P N Q A I G L I 9 7 650 670 690 TGGCAGTCG~G~TC~CTCATCCATTCGCTGACCTGCCTTTCGACGGGCT~TCGGCTTG~ATTCCC A V E E S T H P F A D L P F D G L V G L G F P 2 2 o 710 730 750 770 GGATGTGTCTGG~GAGGGACTTCCATC~GCGCACTTCCCATTGTTGACCAAATGGTT~GGAGAAA D V S G E E G L P S S A L P I V D Q M V K E K 2 4 3 790 810 830 GTTCTGGATCGAAATGTGTTCTCCGTCTATATGA~G~GACATC~CCGCCCCGGAGAGATTTCGTTTG V L D R N V F S V Y M S E D I N R P G E I S F G 2 7 7 850 870 890 910 GAGCAGCGGACCCGAAATATACTTTC~TGGGCACACACCT~GTGGTTCCCCGTCATCTCTCTAGACTA A A D P K Y T F A G H T P K W F P V I S L D Y 2 9 o 930 950 970 CT~GAAATTGGCCTACATGG~TGAAAATAAAC~AAAATCCTTTGGCGTATGTGAAAAACGCGGATGC W E I G L H G M K I N G K S F G V C E K R G C 3 1 3 990 i010 1030 1050 CGCGCAGCCGTGGA~CT~ATCCAGTTTGAT~CGGGACCATCATCTGTCATC~TCCCCTCATCAAAG R A A ii!i i!iiiiii li~liiilL I T G P S S V I N P L I K A337 1070 1090 1110 CACTC~CGTTGCTGAG~TTGCTCC~TCTTGG~CCCTGCC~CTCTCACATTTGTCCTAAAAGACAT

LNVAEN+CSNLGTLPTLTFVLKDI36o 1130 1150 1170 1190 ATATGG~GGCTTGTAAACTTCAGCCTCG~CCCAGGGACTATGTAGT~GAGCTTGATGCGAGAGGA

YGRLVN+FSLEPRDYVVEELDARG383 1210 1230 1250 ~CCCT~C~CTGCGCA~TGGATTCATGGCTATGGACGTGCCAGCACCTCGAGGCCCCCTGTTCGTGC N P N N C A A G F M A M D V P A P R G P L F V L 4 0 7 1270 1290 1310 1330 TCGGAAATTCCTTCATCA~ATACTACAGTATTTTC~CCGCGATCACATGATGGTTGGATTCATGCG G N S F I R K Y Y S I F D R D H M M V G F M R 4 3 o 1350 1370 1390 GGCA~CCACG~GGCTCCGGACCGCTCATC~GGGGTATCCATCATCTGCGCCATCGGTGTCAGCGTCG A N H E G S G P L I K G Y P S S A P S V S A S 4 5 3 1410 1430 1450 1470 TGCCTTGTTGCA~CAGCGCTGCTGCATTCGCGCTATCCCTCTTTTAAACGTATTCCG~CAGTCTGCAT C L V A A S A A A F A L S L F * 1490 1510 GTGTG~GTT~GATGAGTGCCATCGAA~AAAAAAAAAAAA

Fig. 1. Nucleotide and corresponding amino acid sequence of the putative E. acervul&a aspartyl proteinase (D-pEa). Deduced amino acid residues (single letter code) are indicated below the nucleotide sequence. The > < symbol indicates the end of the signal sequence (residues 1 15). Putative active sites and ATP-binding site elements are shaded (in bold) and underlined respectively. Asparagine residues (N343, N366) with the + symbol denote possible N-glycosylation sites. The stop codon is indicated with the * symbol in amino acid position 469.

307 or rabbit anti-GST (dilution 1/100 in TBSA), then with affinity purified mouse anti-rabbit IgG alkaline phosphatase conjugate diluted 1/ 2000 (Biosys), and developed with nitro blue tetrazolium-bromo chloro indoyl phosphate (NBT-BCIP, Gibco, BRL) according to manufacturer's instructions. Localization of the protein with indirect immunofluorescence and electron microscopy assays. Excysted sporozoites of E. acervulina, E. tenella, E. maxima, E. falciformis, and E. papillata (strain Chobotar) were air dried onto multispot microscope slides (Dynatech) and fixed with cold acetone. Slides were incubated with N3CsB12 undiluted culture supernatant for 1 h at 37°C. After 3 washes in PBS, an additional 1 h incubation was performed with fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG (1/400 in PBS). Three washes in PBS were then performed and slides were mounted in an antifading solution (90% glycerol in PBS) and examined with an epifluorescent microscope. For immunoelectron microscopic studies, E. tenella sporozoites were fixed in 2% paraformaldehyde and 0.05% glutaraldehyde, dehydrated and embedded in LR White resin (London resin). Ultrathin sections were collected on copper grids and exposed to N3CsBI2 mAb supernatant, followed by gold-conjugated 10 nm goat anti-mouse IgG (BioCell) incubation. After staining with 4% uranyl acetate in 25% ethanol, the sections were examined using a Philips CM 12 electron microscope.

Results

Isolation and characterization of clone 6S2. A fusion was carried out with the aim of developing hybridomas producing mAbs raised against E. tenella and to screen the E. aeervulina cDNA library. One mAb (N3C~B12) reactive with SEtO and SEaO antigens in ELISA was cloned by limiting dilution. MAb N3CsB~2 is an IgG1 isotype. Hybridoma supernatant N3CsB12 was used to screen the

amplified 2ZapII expression library and allowed the identification of 23 positive phage. After in vivo subcloning, DNA insert sizes were analyzed on agarose gel and clustered into 3 groups of 1500 bp, 1300 bp and 850 bp. All these inserts had similarities in restriction enzyme mapping and cross-hybridized to each other. One representative clone from each group (clone 6S2, clone 16B1, clone 14B3) was sequenced at its extremities, confirming that the three clones were derived from the same gene. Clones 14B3 and 6S2 were sequenced and were fully homologous in their common part. Four additional bases (ATCG) were present before the poly(A) sequence in clone 16B1 with regard to clone 6S2. The complete cDNA sequence (Fig. 1) revealed an open reading frame of 475 amino acids. The predicted polypeptide starting at the first methionine is 468 amino acids long with an expected size of 51 kDa. To check if the complete mRNA had been cloned, a northern blot was performed using the 1500-bp 32p_ labeled insert of clone 6S2. A single band of 1.5 kb corresponding to the full-length mRNA was revealed (Fig. 2A). The protein encoded by clone 6S2 shows strong homology to aspartyl proteinases. Sequence comparison was performed against the SwissProt database with the FASTA program. This study revealed significant amino acid identity between the protein encoded by clone 6S2 and several aspartyl proteinases such as human cathepsin E (36% in 409 amino acids) [20], chicken pepsinogen (34% in 302 amino acids) [21] and mouse renin (36% in 386 amino acids) [22] suggesting that the protein could be an aspartyl proteinase. In the aminoacid sequence, the first methionine is followed by an hydrophobic sequence with characteristics of a signal peptide. Since aspartyl proteinases are generally synthesized with a signal peptide, we presume that the first ATG encodes the initiating methionine. The most likely cleavage site of the signal peptide is between S~5 and FI6 according to Von Heijne [23]. Two potential N-glycosylation sites are

308

A

N)) involving aspartic amino acid residues Dl20 and D318 (Fig. 1), but also revealed the presence of a potential ATP binding site (consensus pattern: (A,G)-X(4)-G-K-(S,T)) (Fig. 1). Partial aminoacid sequence alignments with chicken cathepsin D [24], chicken pepsinogen [21], human cathepsin E [20] and mouse renin [22] are shown in Fig. 3.

B 23,0

9,5_ 7,54,4-

l

9,4 -6,6-4,4--

2,4-

2,3--

1,4-

2,0

-

0,20,5 -Fig. 2. (A) Northern blot analysis of the D-pEa messenger. Seven #g of poly(A)* RNA from (80%) sporulated E. acervulina oocysts were electrophoresed on a 1% agarose formaldehyde gel, transferred to Hybond N - membrane and probed with 32p-labeled EcoRI/Kpnl fragment of clone 6S2. RNA molecular weight markers (in kb) are indicated on the left. (B) Southern blot analysis of the D-pEa gene: 2 /~g of genomic DNA was digested each time with 25 U of restricuon enzymes, electrophoresed on a 0.7% (w/v) agarose gel, transferred to nitrocellulose and probed with 32p-labeled EcoRI/Kpnl fragment of clone 6S2. DNA molecular weight markers (in kb) are indicated on the left.

present at residues N343 and N366. A computer search with the MOTIFS program led to the

identification of the two well conserved aspartyl proteinase active sites (consensus pattern: (L,I,V,F,A)-D-T-G-(S,T,A)-(S,T,A,

The putative E. acervulina aspartyl proteinase (D-pEa) is encoded by a single gene. In order to determine the copy number of the D-pEa gene, a Southern blot was performed using the 1500-bp 32p labeled insert of clone 6S2. The hybridization pattern shown in Fig. 2B suggests that DpEa gene is single copy and do not contain an intron. The D-pEa is' localized in the refractile bodies of the sporozoite and seems to be conserved in several Eimeria species. Immunofluorescence studies performed on acetone-fixed E. acervulina, E. tenella, E. maxima, E. falciformis and E. papillata sporozoites using mAb N3CsB~2 localize the protein in the refractile bodies of such species (Fig. 4). This location was confirmed by cytologic observations of samples of E. tenella sporozoites (Fig. 4). In order to know the molecular weight of the mature enzyme and to search for its presence in other parasite development stages, western blots were performed. As

95

D-pEa: ch-cD: hu-cE: ch-pe: mo-re:

134

LLNYHNSQYFGEIKIGTPGRRFVVVFDTGSSNLWVPAIEE-E -K--MDA--Y---G

.... PQK-T

-I--LDME---T-S

.... PQN-T-I

...........

SVH-H SVY-T

MT--MDAS-Y-T-S

.... QQD-S-I

...........

SIY-K

-T--L

.... PQT-K-I

...........

STK-S

....

Y---G

310

D-pEa: ch-cD: hu-cE: ch-pe: mo-re:

.............

331

KRGCRAAVDTGSSLITGPSSVI

391

422

GFMAMDVPAPRGPLFVLGNSFIRKYYSIFDRD

-G--E-I

....

T ......

SE--Q-I

....

T .......

TKEV

FFT-Q-I

....

T--LVM-QGAY

EE--EVV

......

DK-

F-SA-T-SL

--...---.-.---..--..__..__.. --EN-GT-TEL-EQWI--DV---E--V

.... .... A

--,..-...-.---..--..---,.__.___. ALH---I-P-T--VW---AT

.... F-TE---H

Fig. 3. Partial alignment of amino acid sequences of the D-pEa, chicken cathepsin D (ch-cD) [24], chicken pepsinogen A (chpe) [21], human cathepsin E precursor (hu-cE) [20] and mouse renin precursor (mo-re) [22]. Dashes in the lower four sequences indicate identity with the amino acids in D-pEa. Possible aspartic residues involved in the active-site are in bold.

309

F

Fig. 4. Indirect immunofluorescence staining of acetone fixed sporozoites of E. acervulina (A), E. tenella (T), E. maxima (M), E. Jbk'(fbrmis (F) and E. papillata (P) with undiluted mAb N3CsBI2 supernatant. Immunogold labeling (10 nm gold particules) with mAb N3CsBI2 of ultrathin sections of E. tenella sporozoites. Refractile body (RB) labeling on cross (1) and longitudinal sections (2) is observed. The bar represents 10 mm in panels A, T, M, F, P and 1 mm in panels 1 and 2.

N3CsBI2 was unable to recognize the putative aspartyl proteinase in western blots under reducing or non reducing conditions (data not shown), rabbit antisera raised against the fusion protein FA6S2 were produced, These sera are able to recognize under reducing conditions a 43-kDa protein on a P A G E in SEaO, SEtO, SEmO and SEfO mAb

antigens (Fig. 5) and two bands of 46 kDa and 43 kDa in a soluble extract of unsporulated oocyst of E. acervulina. As mAb N 3 C s B 1 2 was negative in western blot experiments, the specificity of the m A b for the D-pEa was confirmed by ELISA using FA6S2 fusion protein and GST recombinant protein as negative control (data not shown).

310

1 200 92

-

69

-

46

- - ~

30

--

21 14

2

3

~'

~," t,'

4

5

6

---

Fig. 5. Immunoblot analysis of the putative proteinase on oocyst antigen of four Eimeria spp. Lane 1, 3 SEaO, lane 4 SEtO, lane 5 SEmO and lane 6 SEfO antigens. Lane 2, soluble extract of unsporulated E. acervulina oocyst. All the nitrocellulose strips were incubated with the rabbit anti-FA6S2 antisera except for the negative control incubated with the rabbit anti-GST (lane 3). Molecular weight markers (in kDa) are indicated on the left.

Discussion

Screening of an E. acervulina c D N A expression library by an heterologous mAb was carried out with the aim of obtaining conserved antigens, which could be used as vaccine candidates against several species of coccidia. One of the clones (clone 6S2) identified by N3CsB12 mAb codes for a protein (D-pEa) with strong homologies to aspartyl proteinases like cathepsin D, cathepsin E, pepsinogen A and renin. Besides the two aspartyl proteinase motifs clearly identified in the sequence involving the aspartic amino acids D~20 and D3~s, a potential ATP binding site was revealed by the MOTIFS program. ATP has been shown to activate bovine cathepsin D [25-26] and to stabilize human cathepsin E [27]. However, this ATP binding site is not found in the sequence of other aspartyl proteinases, and the presence of such a motif is not sufficient to assert that ATP has an

action on D-pEa. This point therefore, needs further investigation. D-pEa gene is single copy as suggested by Southern blot, and the 1500 bases of the full length corresponding messenger in northern blot confirms that a near complete transcript was cloned. In order to obtain recombinant protein, the insert coding for the complete DpEa was subcloned into pGEX-FA expression vector. Unfortunately, the recombinant protein was insoluble in the absence of guanidineHC1 treatment. After renaturation steps, GST fusion protein could be purified on glutathione agarose chromatography and used to immunize rabbits. The rabbit sera recognized a single 43-kDa band on western blots performed with the sporulated oocyst antigen of all the species tested. In contrast, mAb N3CsB12 was negative. This mAb positive on FA6S2 recombinant protein on ELISA appears therefore, to recognize a conformational epitope lost after SDS treatment. The 43-kDa molecular weight probably corresponds to the active form of the enzyme, and the presence of a single band in sporulated oocyst antigen and two bands in unsporulated oocyst antigen indicates a faster processing of the proprotein in sporulated oocysts. Indeed, aspartyl proteinases are synthesized as a preproenzyme and are autocatalytically processed in an acidic environment [28]. By immunofluorescence microscopy the mAb N3C8B12 appears to bind to the refractile bodies of acetone fixed sporozoites of eimerian species infecting chickens (E. acervlina, E. tenella, E. maxima) and mice (E. falciformis, E. papillata). This location was confirmed by immuno-electron microscopy in E. tenella sporozoites. Thus the refractile bodies seem to be the final location of the putative proteinase. The pH inside this organelle is not known. To date, the occurrence of lysosomes or lysosome-like structures in Eimeria spp. has not been reported. The signal peptide could be necessary for the targeting of the D-pEa into the refractile bodies. Recently, a protein with similarity with pyridine nucleotide transhydrogenase from Escherichia coli was identified in the refractile bodies of E.

311

acervulina and E. tenella sporozoites [29], but the functions of these organelles are still unknown. Sera collected from E. acervulina infected chickens were unable to recognize FA6S2 recombinant protein or a band corresponding to the molecular weight of the proteinase in western blot (data not shown). This absence of recognition is probably due to the intracellular location of the proteinase. Even if this protein is presented poorly or not at all to the immune system it represents an interesting target, because its specific inhibition might block parasite cell invasion [30], or growth. Pepstatin is a specific inhibitor of aspartyl proteinase, and it reduces significantly the number of intracellular sporozoites of E. tenella in primary chicken kidney culture cells [31]. This shows that aspartyl proteinases may play an important role in the parasite biology. Changes in number, shape and location [32] of the refractile bodies appear shortly after the invasion of the host cell by the sporozoite. Further investigations on the presence and location of the D-pEa in the subsequent stages of parasite development have to be performed. Characterization of D-pEa, present in organelles as yet poorly studied will be of great interest in the understanding of sporozoite physiology.

Acknowledgements The authors would like to thank Gordon Langsley (Institut Pasteur, France) for critical reviewing of the manuscript and helpful discussion. We also acknowledge Bill Shobotar (Andrews Univ., USA) for the gift of E. papillata oocysts.

References 1 Shirley, M.W. (1993) Live vaccines for the control of coccidiosis. VIth International Coccidiosis Conference (Barta, J.R. and Fernando, M.A. eds.) Guelph, Canada. pp. 61 72. 2 Robertson, N.P., Reese, R.T., Henson, J.M. and Speer, C.A. (1988) Heat shock-like polypeptides of the sporozoites and merozoites of Eimeria bovis. J.

Parasitol., 74, 1004-1008. 3 Abrahamsen, M.S., Clark, T.G., Mascolo, P., Speer, C.A. and White, M.W. (1993) Developmental gene expression in Eimeria boris. Mol. Biochem. Parasitol. 57, 1 14. 4 Clarke, L.E., Tomley, F.M., Wisher, M.H., Foulds, I.J. and Boursnell, M.E.G. (1990) Regions of an Eimeria tenella antigen contain sequences which are conserved in circumsporozoite proteins from Plasmodium spp. and which are related to the thrombospondin gene family. Mol. Biochem. Parasitol. 41, 269 280. 5 Jenkins, M.C., Lillehoj, H.S., Barta, J.R., Danforth, H.D. and Strohlein, D.A. (1990) Eimeria acervulina: Cloning of a cDNA encoding an immunogenic region of several related merozoite surface and rhoptry proteins. Exp. Parasitol. 70, 353-362. 6 Tomley, F.M., Clarke, L.E., Kawazoe, U., Dijkema, R. and Kok, J.J. (1991) Sequence of the gene encoding an immunodominant microneme protein of E. tenella. Mol. Biochem. Parasitol. 49, 277 288. 7 Lecordier, L., Mercier, C., Torpier, G., Tourvieille, B., Darcy, F., Liu, J.L., Maes, P., Tartar, A., Capron, A. and Cesbron-Delauw, M.F. (1993) Molecular structure of a Toxoplasma gondii dense granule (GRA 5) associated with the parasitophorous vacuole menbrane. Mol. Biochem. Parasitol. 59, 143 154. 8 Bontemps, M. and Yvor+, P. (1974) Technique de purification des suspensions de sporozoites d'Eimeria sur colonne de fibres synthetiques. Ann. Rech. Vet. 5, 109 113. 9 K61er, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495497. 10 Chomczynski, P. and Sacchi, N. (1987) Single step method of RNA isolation by acid guanidium thiocyahate-phenol-chloroform extraction. Anal. Biochem. 162, 156 159. 11 Short, J.M., Fernandez, J.M., Sorge, J.A. and Huse, W.D. 0988) ,:~ ZAP: A bacteriophage with in vivo excision properties. Nucleic Acids Res. 16, 7583 7600. 12 Sanger, F., Coulsen, A.R., Barrel, B.G., Smith, A.J.H. and Roe, B. (1980) Cloning in single stranded bacteriophage as an aid to rapid sequencing. J. Mol. Biol. 143, 161 178. 13 Pearson, W.R. and Lipman, D. (1988) Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. 85, 2444-2448. 14 Devereux, J., Haeberli, P. and Smithies, O. (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12, 387 395. 15 Maniatis, T., Fritsch, E.F and Sambrook, J. (1992) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 16 Smith, D.B., and Johnson, K.S. (1988) Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67, 31 40. 17 Valenzuela, D., Weber, H. and Weissmann, C. (1985) Is sequence conservation in interferons due to selection for functional proteins?. Nature 313, 698 700. 18 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 685. 19 Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacryla-

312

20 21 22 23 24

25

26

mide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. 76, 4350~4354. Azuma, T., Pals, G., Mohandas, T.K., Couvreur, J.M. and Taggart, R.T. (1989) Human gastric cathepsin E. J. Biol. Chem. 264, 16748-16753. Baudys, M. and Kostka, V. (1983) Covalent structure of chicken pepsinogen. Eur. J. Biochem. 136, 89 99. Kim, W., Murakami, K. and Nakayama, K. (1989) Nucleotide sequence of a cDNA coding for mouse Ren 1 preprorenin. Nucleic Acids Res. 17, 9480. Von Heijne, G. (1986) A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14, 4683 4690. Retzek, H., Steyrer, E., Sanders, E.J., Nimpf, J. and Schneider, W.J. (1992) Molecular cloning and functional characterization of chicken cathepsin D, a key enzyme for yolk formation. DNA and Cell Biol. 11, 661 672. Pillai, S., Botti, R. and Zull, J.E. (1983) ATP activation of parathyroid hormone cleavage catalyzed by cathepsin D from bovine kidney. J. Biol. Chem. 258, 9724-9728. Watabe, S., Terada, A., Ikeda, T., Kouyama, H., Taguchi, S. and Yago, N. (1979) Polyphosphate anions increase the activity of bovine spleen cathepsin D.

Biochem. Biophys. Res. Commun. 89, 1161 1167. 27 Thomas, D.J., Richards, A.D., Jupp, R.A., Ueno, E., Yamamoto, K., Samloff, I.M., Dunn, B.M. and Kay, J. (1989) Stabilisation of cathepsin E by ATP. FEBS Lett. 243, 145-148. 28 Tang, J. and Wong, R.N.S. (1987) Evolution in the structure and function of aspartic proteases. J. Cell. Biochem. 33, 53 63. 29 Vermeulen, A.N., Kok, J.J., Van den Boogaart, P., Dijkema, R. and Claessens, J.A.J. (1993) Eimeria refractile body proteins contain two potentially functional characteristics: Transhydrogenase and carbohydrate transport. FEMS Microbiol. Lett. II0, 223-230. 30 Adams, J.H. and Bushell, G.R. (1988) The effect of protease inhibitors on Eimeria vermi/brmis invasion of cultured cells, int. J. Parasitol. 18, 683-685. 31 Fuller, A.L. and McDougald, L.R. (1990) Reduction in cell entry of Eimeria tenella (coccidia) sporozoites by protease inhibitors, and partial characterization of proteolytic activity associated with intact sporozoites and merozoites. J. Parasitol. 76, 464467. 32 Fayer, R. (1969) Refractile body changes in sporozoites of poultry coccidia in cell culture. Proc. Helminthol. Soc. Washington, 36, 224 231.