Molecular and Biochemical Parasitology, 55 (1992) 29-38 © 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00

29

MOLBIO 01801

Cloning and characterisation of members of a family of Babesia bigemina antigen genes containing repeated sequences M e r c y W. K u n g ' u a'b, Brian P. D a l r y m p l e a, Ian G. Wright a and Jennifer M. Peters a ~CS1RO, Long Pocket Laboratories, lndooroopilly, Australia; and bDepartment of Parasitology, University of Queensland, St. Lucia, Australia (Received 9 January 1992; accepted 19 May 1992)

Bovine polyclonal antisera to Babesia bigemina antigens separated by phenyl-Sepharose chromatography were used to screen a B. bigemina 2gtl I cDNA expression library. Eleven B. bigemina-specific cDNA clones were studied in detail. DNA sequencing of 2 representative clones identified open reading frames encoding polypeptides representing the carboxy-termini of 2 different proteins. Both polypeptides contained a related central motif of tandem repeats flanked by a highly conserved carboxy-terminal region, but the sequences preceding the repeats were not related. Hybridisation and restriction enzyme analysis of the cDNA clones indicated that they were derived from a family of at least nine related, but not identical genes. Four different members of the gene family have been isolated from a B. bigemina 2EMBL3 genomic library. The genes are not closely linked and they occur on the largest and smallest B. bigemina chromosomes resolved by pulsed field gel electrophoresis (PFGE). Antibodies raised against the native antigens and purified on recombinant fusion proteins bound to multiple proteins (50~70 kDa) in the original B. bigemina antigenic fractions. Key words: Babesia bigemina; Repetitive antigen; Gene family

Introduction

The protozoan parasites in the genus Babesia, phylum Apicomplexa [1], are major tick-borne pathogens of domestic animals [2,3]. Immunoprophylaxis against bovine babesiosis, caused by Babesia bovis and Babesia bigemina, is currently achieved by vaccination using attenuated live parasites [4-6]. However, Correspondence address: Brian P. Dalrymple, CSIRO, Long Pocket Laboratories, PMB No. 3, PO Indooroopilly 4068, Australia. Note; Nucleotide sequence data reported in this paper have been submitted to the GenBankT M data base with the accession numbers M81568 and M81569. Abbreviations: PFGE, pulsed field gel electrophoresis; CHEF, contour-clamped homogeneous electric field; TBE, Tris-borateEDTA; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

these vaccines have several disadvantages, including short shelf life, reversion to both virulence and transmissibility by the tick vector when passaged in intact animals, the possible transmission of other disease-causing organisms and the fact that about 5% of the vaccinated animals react severely to the vaccine [7,8]. To avoid these problems, recent research has been directed towards the identification of defined babesial antigens that could be used in recombinant vaccines. A number of B. bigemina-infected erythrocyte and merozoite surface antigens have been identified using monoclonal antibodies [9, 10]. Some of these antigens, purified to homogeneity, were shown to induce a degree of protection in cattle against B. bigemina challenge [11]. A recombinant form of one of the antigens was shown to elicit antibodies reactive with B. bigemina merozoite surface antigens [12]. We have recently described the

30

successful vaccination of cattle with B. bigemina antigens that initially bound to dextran sulphate and were subsequently fractionated using phenyl-Sepharose affinity chromatography [13]. Using antisera from the vaccinated animals, B. bigemina antigens of sizes 35, 51, 54, 62, and 68 kDa were identified [13]. This present study was undertaken to clone the protective antigen(s) involved. A family of B. bigemina genes encoding a family of repeat-containing proteins is described in this report.

Materials and Methods

Screening of B. bigemina cDNA and genomic libraries. Bovine polyclonal antisera to 2 previously reported B. bigemina phenyl-Sepharose-bound and unbound (PhB and PhUB) antigen fractions have been described previously [13]. These sera were used to screen more than 3 × 105 plaques from a B. bigemina (Q strain) 2gtl 1 cDNA expression library (gift from K.R. Gale) using standard techniques [14, 15]. More than 3×104 plaques from a B. bigemina (Q strain) 2EMBL3 genomic library (gift from C.M. Dimmock) were screened using the [~32p]dATP-labeUed Bbgl.1 probe described below. Preparation and analysis of DNA. B. bigemina genomic DNA was isolated by conventional means [14] from B. bigemina merozoites harvested from in vitro cultures (Q strain) or from infected calves (Townsville strain) [16]. DNA fragments purified using standard methods [14], were labelled with [~32p]dATP or digoxigenin-labelled deoxyuridine-triphosphate (DIG-dUTP, Boehringer Mannheim) using random priming [17]. Restriction enzyme analysis of the clones was carried out using various restriction endonucleases as recommended by the manufacturers. Southern transfer [18] of genomic DNA, cDNA and genomic clones and subsequent hybridisation with the appropriate probes were performed as described [14]. Hybridisations with the

[~32p]dATP-labelled probes were performed overnight in 5 x SSPE with 30% formamide or 3 x SSC with 50% formamide at 42°C. The blots were then washed for 3 x 2 0 rain in 2 x SSC at 42°C. The hybridisations with the DIG-dUTP-labelled probes were carried out overnight in 5 x SSPE with 50% formamide and the washings were carried out in 2 x SSPE containing 0.1% SDS for 15 min at room temperature, with a final washing in 1 x SSPE with 0.1% SDS for 5 min at 60°C. B. bigemina merozoites were purified using essentially the method described by Conrad et al. [16] for Theileria spp. Intact B. bigemina chromosomes, prepared as described by Van der Ploeg et al. [19] were separated by PFGE, using a CHEF-DR II apparatus (Bio-Rad). Electrophoresis conditions of 120 volts with 270 sec pulse-switching time for 46 hr in a 0.8% agarose gel in 0.5 x TBE (0.89 M Trisbase, 0.89M Boric acid and 20 mM EDTA) at 14°C were used. Saccharomyces cerevisiae or Schizosaccharomyces pombe chromosomes (Bio-Rad) were separated simultaneously for size estimation purposes. The separated DNA was transferred to nitrocellulose filters and hybridised with the [32p]dATP-labelled Bbgl.1 probe as described above.

DNA sequencing. The inserts of the 2gtll cDNA clones, Bbg 1.1 and Bbg 2.1, were subcloned into p G E M 7 Z f ( + ) (Promega Corp.) for double-stranded sequencing by the dideoxy chain-termination method [20] with T7 DNA polymerase or Klenow Pol I kits (Promega Corp.). Nested deletions of the Bbgl.1 clone were generated using Exonuclease III following the manufacturer's procedure (Erase-a-Base system, Promega Corp.). The insert in the Bbg 2.1 clone was sequenced in both directions from overlapping restriction enzyme fragments. The deduced amino acid sequences for the 2 clones were used to search the NBRF and translated GenBank databases using the MacVector 3.5 package (International Biotechnologies, Inc.). Western blot analysis of the native B. bigemina proteins. Proteins obtained in plate lysates of

31

recombinant 2gtl 1 Bbg 1.1 phage induced with isopropyl-//-D-thiogalactopyranoside (IPTG) were transferred to nitrocellulose filters [15]. The filters were used to affinity purify antibodies binding to the recombinant protein from the bovine antisera to the B. bigemina PhB and PhUB native antigens according to the method of Ozaki et al. [21]. The antisera were previously depleted of any antibodies to Escherichia coli as described by Ternynck and Avrameas [22]. The native antigens were electrophoresed by SDS-PAGE on 7.5% gels [23] and transferred onto nitrocellulose filters using a Trans-Blo.t cell (Bio-Rad) [24]. The blots were screened using bovine antisera

against native proteins purified on the recombinant B. bigemina antigens [21]. The blots were then reacted with goat anti-bovine IgG ( H + L ) - horse-radish peroxidase conjugate (Kirkegaard and Perry Laboratories). Luminol was used as the substrate and an X-ray film was then exposed to the blots [25]. In addition, native B. bigemina antigens were also separated on similar gels and probed with a bovine antiserum to a glutathione S-transferase (GST)-Bbg 1.1 fusion protein (Kung'u, Wright, Dalrymple, Goodger, Peters and Waltisbuhl, in preparation) and with an antiserum from an animal immunised with GST alone.

G.1

' B Hc (E) ! ........... L

Group 1

Bbg 1.1

(E) Bg

Bbg 1.2 (E) Bbg 1.3

,

Bg

Ba AHh(E)

I

I I

100 bp I J

I

" ' ' ~ ' " II .............................................JL~,,,

....,I

n J..

6bg 1.4

G.2 i

Group 2

N

I

(E) HcX

P

Ih(E) I

Bbg 2.1 Bbg 2.2

(E)

(E) Bbg 2.3 Bbg 2.4

(E) X kk..,.~,,,~,

Others

(E)

(E)

Bbg 3.1

(E)

I.,,,,~ (E)

Bbg 4.1

Bbg 5.1

I

..,

..... (E) ,~ k.~.~..~.."

A I

(E) I

Fig. 1. Restriction maps ofB. bigemina cDNA clones. Restriction sites; B, BamHI; Hc, HinclI; N, NciI; Bg, BglI; Ba, Ball; A, AccI; Hh, HhaI; X, Xbal and P, PvulI. The EcoR1 sites shown, (E) are in the linkers added during the construction of the library. The restriction fragments labelled G. 1 and G.2 represent the group-specific probes. The position of the stop codons of the open reading frames are indicated by arrowheads. The hatched areas represent the repeats, dashes indicate deletions and the unrelated sequences preceding the repeats in members of group 1 and 2 are indicated with 2 different patterns.

32

Results

Isolation and characterisation of the cDNA clones. Twenty-five positive clones were obtained during the screening of the B. bigemina 2gtl 1 c D N A expression library with the bovine polyclonal antisera to the 2 preparations of B bigemina proteins. All the clones were positive Bbg

I.I

Bbg

2.1

with both antisera. Eleven representative clones were analysed in detail. The inserts from 3 clones (designated Bbg 1.1, 1.3 and 5.1) hybridised to the inserts in all 11 clones. However, some cDNA inserts hybridised to the Bbg 1.1 and 1.3 probes with a much stronger signal than to the Bbg 5.1 probe, while the other clones hybridised approximate-

CTCAATCCTCATGTTGGTGGCCGTTCGGGAAGAAGAAGGCAAAGGTTAACTTTGGTGAAGTAGTGAGA

68

HinclI Bbg

I.I

.... GTTTCATGGGTATTAAAAGGAAGACCAACATTGGAGGTGTATTGGAAACCCTGGGTTACGGGGAGTCGAACAGGCGATCTTGGTTGAGTGGC

Bbg

2.1

GCCGTGGGGTATGATGATCTAGATTGTCTCGGTGGAGCCGGTAGTGCAAGTGCTCTCAAGGCTGTTAAGAATCTGAATCCGAATGTAGTGGATATC

Xba I Bbg

I.I

Bbg

2.1

92 164

BamH I *

CCAGATGTCACTGAGGCGTTGCAAAGTGTGAACCCAAACATGTTGTCCACACTCTACGAGCTTTCTGGATTCTGGGCTTTCTACGGATCCGGCAAA

188

GTCCACGACCTCTGTGGATTTGATGCTGTCTATGAATCGGAAAATCCAGAATCTGTACCAGCAGAAAAATCAGCTGAACCTGTAGCAGACGGATCG

260

A

1

A

Pvul I *

2

HincII

ACTGTGATCCCTGAAGCTGAGCCATCCGTTGAAGGTGAAGGCGAGCAG ........................ . .......... C..G..T,.A A 1 var

Bbg Bbg

i.I 2.1

TCCGTGATCCCCGAAGCTGAGCCAACCGTTGACGGTGAAGGTGAGCAG . . . . . T . . T . . . . . T . . . . . . . . A . . . . . . T .... A . . . . . G . . . . . A . . . ........... T.,G..C..A...T .......... C..G ..... A... A 2 vars HincII B A3 TCCTTGATCCCTGAAGATGAAAAATCCTTTGAAGGTGAAGGCGAGCAG TCCTTGATCCCTGAAGCTGAGCCATCCGTTGAAGGTGAAGGTGAGCAG ................................................................................................

Bbg Bbg

I.I 2.1

B A3 TCCTTGATCCCTGAAGATGAAAAATCCTTTGAAGGTGAAGGCGAACAG TCCTTGATCCCTGAAGCTGAGCCATCCGTTGAAGGTGAAGGTGAGCAG ................................................................................................

Bbg Bbg

i.i 2.1

TCCTTGATCCCTGAAGATGAAAAATCCTTTGAAGGTGAAGGTGAGCAG TCCTTGATCCCTGAAGATGAAAAATCCTTTGAAGGTGAAGGCGAGCAG ................................................................................................

Bbg Bbg

i.I 2.1

A4 TCTGTCATCCCTGAAGCTGAGCCATCCGTTGAAGGTGAAGGTGAACAG ................................................

A5 TCCATGATCCCCGAAGCTGAGCCAACCATTGAGGGTGAAGGCGAGCAG . . . G . . . . . . . . . . . . . C . . . . . . . . . T .... A . . . . . G . . T . . . . . . A 5 vat

A

A

Bbg Bbg

I.I 2.1

TCCGTGATCCCCGAAGTCGAACCGTCTGTGGAACCCGCCGGTGAAGAG CCTGTCATCCCGGAGGCTGAGCCATCCGTCGAGCCCGTGAAGCCTGAA ................ C...G..A ........................................................... T ............ A 6 var A 7 var NciI" Bql I

764 524

Bbg Bbg

I.I 2.1

GTCGATGACATTGAAAAACCTGTGAAGGTTGCGAAGGCTGCTAAAGTGGCAAGGTCTGTAAAGGCCGCCAAGAAGGCTGCCAAGAAGCTCGCCAAG .............. G .......................... A ........ G ....................... A ............ G,TT...CA

860 620

Bbg Bbg

i.I 2.1

AAAGCCCGCAAGAGGAAGGAAAGGAAACTGAAGAAGCAG•AGGAAGAACAGGCTCAGCAGGAATCCGCTGAGCAGTGAGTGGCAGTCCTACGA•TA ..... T...C.A.A...A,.G.A...G ........................... . ................ A..C ......... A..T ....... T

956 689

Bbg

.

CTCACCAGTTATTTGTCATTTCAAAATCTGGTGATGGCCACATTTATGTATTAACTTTTAATTTACATCATTGCTGTCGTAGAATGATGGTAGACA .... G . . C G C . . . . A .... C . T G . . . . . . . T . . . . . . . . . . . A A A . . . . . . G C G . . . . . . . . G C . . . A T . A A . . . - - . A . .... T .... - . - . . -

1052 779

Bbg Bbg

I.I 2.1

Bali HhaI Hhal GTC•TGAACTTCTA•ATAATGTTT•TAGTAGCG••ATATGGCGACTA•TTTGTTTTAA•GTCA•ACC•ATTCATATTATG•GATGAATG•GTTGTG

1148

Bbg

B

284 332 380

380

476

B

6

7

572

668 428

NciI

Bali

AccI

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

Bbg ~

TGG~GGC~GCGACe~GAT

Bbg

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

1168

Fig. 2. D N A sequence alignment of clones Bbg 1.1 and 2.1. The dots indicate conserved nucleotides, the nucleotides in bold result in change of the amino acid residue while nucleotides in plain text do not change the amino acid residues. The dashes indicate missing nucleotides in one sequence with respect to the other. The restriction enzyme sites are underlined (Bbg 2.1) or overlined (Bbg 1.1) and are labelled as shown. The repeats are labelled with bold italicised letters 'A' or 'B' depending on the type of variant of each repeat (see text) and the stop codons of the open reading frames are underlined. The internal restriction enzyme sites used to prepare the group-specific probes are indicated with asterisks.

33

ly equally to all 3 probes (data not shown). The differences in hybridisation signals suggested that not all of the clones were derived from the same gene. Detailed restriction enzyme analysis of the 11 cDNA clones identified several distinct groups of clones apparently derived from different m R N A molecules (Fig. 1). The sequences of cDNA clones representative of groups 1 (Bbg 1.1) and 2 (Bbg 2.1) were determined (Fig. 2). The translated DNA sequences identified open reading frames encoding polypeptides of calculated Mr 33 625 and 22 843 respectively, which were inframe with the vector fl-galactosidase protein. In searches of the protein and translated DNA sequence data bases, the B. bigemina protein sequences showed no significant homologies to known proteins.

and were followed by a carboxy-terminal region that contained a number of tetrapeptide repeats with the consensus sequence KAAK (Fig. 3). The sequence of the polypeptide encoded by the Bbg 2.1 insert had a similar organisation, but had a smaller number of repeats. However, the amino acid sequence preceding the repeats was not related to the equivalent region of Bbg 1.1, whereas the carboxy-terminal sequence was conserved between the 2 polypeptides (Fig. 3). In the repeats of the Bbg 1.1 polypeptide, the amino acid sequences SLIPEAEPSVEGEGEQ (A) and derivatives, and SLIPEDEKSFEGEGEQ (B) form the following pattern; ArA2-B-A3-B-A3-B-B-Aa-As-A6-A 7. The four 'B'repeats have only one nucleotide difference in 2 of the repeats (positions 425 and 518, Fig. 2), while the two 'A3' repeats are identical at the nucleotide level. The limited variation in the 'A3' and 'B' repeats suggest that thy may have been recently duplicated with both the 'BA 3' and the 'B' units amplified twice. The deduced Bbg 2.1 polypeptide does not contain any 'B' repeats and consists of variants of the

The cDNA clones encode regions with variant repeats. The deduced sequence of the Bbg 1.1 polypeptide contained 12 variable repeats of a 16 amino acid sequence. The repeated sequences were preceded by a non-repetitive sequence Bbg I. 1 Bbg 2.1

QS S C W W P F G K K K A K V N

16

Bbg I. 1 Bbg 2.1

........ FMG IKRKTN I G G V L E T L G Y G E S N R R S W L S G P D V T E A L Q S V N P N M L S TLYELS G F W A F Y G S G K FGEVV/~AVGYD D L D C L G G A G S A S A L K A V K N L N P N V V D IVHD L C G F D A V Y E SENP E SVPAE K SAEPVADG S

62 80

Bbg 1.1 Bbg 2.1 Bbg 2.1

TVIPEAEPSVEGEGEQ SVEGEGEQ

AI

A2

A I vat. B

A3

Bbg 1.1

SLIPEDEKSFEGEGEQ

Bbg i. 1 Bbg 2.1

SVIPEAEPSVEGEGEQ

A4

A2

SLIPEDEKSFEGEGEQ

S L I P E A E P S V E G E G E Q 126 120

B

SLIPEAEPS~GEGEQ

B

SLIPEDEKSFEGEGEQ

A5

S L I P E D E K S F E G E G E Q 190

A6

SMIPF~TIEGEGEQ SVIPF~TFEGEGEQ m

Bbg I. 1 Bbg 2.1

B

SVIPEAEPTVDGEGEQ S~FIPFA%EPTFEGEGEQ SVIPF2%EPS%q)GEGEQ A 2 varm.

A7

SVIPEVEPSVEPAGEE SVIPEAEPS~AGEE

5 vat.

A

P~FIPEAEPSVEP~FKPE 259 PVIPF2%EPSVEPg~KPE 168

6 var.

V D D I E K P ~ L A K F ~ R K L K K Q Q E E Q A Q ~ E S A E Q % q ) D I E I £ P ~ S R F ~ Q K K E K K QQES~H q

A

7 vat.

312 216

D

Potential amphiphilic~-helix Fig. 3. The alignment of the deduced amino acid sequence of the cDNA clones Bbg 1.1 and Bbg 2.1. Dashes were introduced to maximise alignment, amino acid identities are indicated in bold and the bold italicised letters 'A' or 'B' indicate the type of variant of each repeat. The arrow indicates a region of the sequences which has the potential to form an amphiphilic or-helix.

34

_4, ,-,. 6.11 3.59 2.81 1,95

B

m

~. ¢q.~.~ ~.~.~.~.~.~. i

i

i

i

i

i

i

i

i

i

i

i

¢,i

,-; ~ ; ,-; ,-; ¢,i ¢,i 4 4 o ~ ¢ , i

m

m

1.51 1.16 0.72 0.48 0.36

8.51

6.11 3.59 2.81 1.95 1.51

m

1.16 0.72 0 48

m

0.36

m

Fig. 4. Hybridisation of the group-specific DNA probes to the 2gtl I cDNA inserts. All the B. bigemina cDNA clones were digested with EeoR1 (E), EcoR1/Bgll (E/Bg) or EcoRi/Hincll (E/He) as shown and southern blots of these were probed with the DIG-dUTP-labelled Bbg 1,1 (A) and Bbg 2.1 (B) specific probes (G.I and G.2, see Fig. I) as described in materials and methods. Molecular size markers are indicated in kb.

EcoRI d i f f e r e n t f o r m s o f the ' A ' r e p e a t s ; A1-A2-A2As-A6-A7.

Group 2 contains at least 3 distinct genes. To establish the relationships between the nonrepeat sequences preceding the repeat regions, suitable group-specific probes were isolated from Bbg 1.1 and Bbg 2.1 (see Figs. 1 and 2) and labelled with the DIG-dUTP. Hybridisations with these probes confirmed the grouping of the c D N A clones in group 1 (Fig. 4). In addition to Bbg 2.1 and 2.2, the clones Bbg 2.3 and 2.4 also hybridised to the Bbg 2.1 probe despite their restriction site differences. Thus, group 2 appeared to contain c D N A clones from 3 closely related m R N A molecules. Four clones (Bbg 1.4, 3.1, 4.1 and 5.1) did not hybridise to either of the 2 probes, implying that these belonged to further groups. However, from the restriction enzyme analysis, the clone Bbg 1.4 appeared to be a member of group 1, the lack of hybridisation to the group 1-specific probe being explained by the short length of the clone (Fig. 1). All 4 clones in group 1 may have been derived from the same mRNA.

Q

T

HindIII

Q

T

23~ 9.4

:

6.7 4.4--

2.3

2

--"

Fig. 5. Hybridisation of B. bigemina genomic DNA from 2 different isolates with the Bbg 1.1 cDNA probe. The genomic DNA of 2 strains of B. bigemina, Q and T (Townsville) was digested with EcoRI or HindIII and analysed by southern hybridisation using the [c¢32p]dATPlabelled Bbg 1.I probe. Molecular size markers are as indicated in kb.

35

S c Bb

Bb 0

m

b3-2200 --

1600-.c 2.-

1125---

945"-

'c 1"

A

O

B

Fig. 6. Chromosomal assignment of the B. bigemina genes using PFGE. (A) Intact B. bigemina chromosomes (Bb) were separated by PFGE and visualised by ethidium bromide staining (A). A Southern blot of the chromosomes was hybridised with the [ct32pldATP-labelled Bbg 1.1 probe (B). Sizes of S. cerevisiae chromosomes (Sc) are indicated in kb. Origin is indicated 'o', band 3 is indicated 'b3' and chromosomes 1 and 2 are indicated ' c l ' and 'c2' respectively.

B. bigemina contains at least 9 members of the gene family. The clone Bbg 1.1, which contained the repeat sequences and the common carboxy-terminal domain, was hybridised to EcoRI or HindIII-digested B. bigemina genomic DNA obtained from 2.different isolates (Fig. 5). The hybridisation of the probe to at least 9 bands in the B. bigemina genomic DNA for each of the EcoRI and the HindIII digests in both of the strains indicated the presence of a multiple gene family, containing at least 9 members. The weaker signals obtained with the B. bigemina strain T reflected the lower concentration of DNA in the sample used. The group 1 and 2-specific probes (see Figs. 1, 2) were hybridised to B. bigemina genomic DNA digested with EcoRI or HindIII. The group 1 probe hybridised to a 6.0-kb EcoRI and an 8.8-kb HindIII band,

while the group 2 probe hybridised to an 8.8kb and a 15-kb EcoRI band and an 8.8-kb HindIII band (data not shown). Two B. bigemina chromosomes of estimated sizes 1070 and 980 kb were resolved under the PFGE conditions used (Fig. 6A). In addition, a band that was > 3000 kb (band 3) was present. This band have may contained more than one chromosome. A blot of the chromosomes was probed with the Bbg 1.1 clone. The Bbg 1.I probe hybridised to the smallest chromosome and also to band 3 (Fig. 6B).

Isolation and eharacterisation of genomic clones. When the B. bigemina genomic library was screened with the Bbg 1.1 cDNA insert, 8 clones were obtained. Restriction enzyme analysis of these clones demonstrated the presence of 4 different members of the family of genes (Fig. 7). The genomic clones (Gc 1, 4, and 8) containing the Bbg 1.1 gene were identified by restriction enzyme analysis and hybridisation with the group 1-specific probe (data not shown). The group 2-specific probe did not hybridise to any of the genomic clones (data not shown). The 3 other groups of genomic clones therefore represented other members of the family of genes. Identification of a family of native proteins in the size range 50-70 kDa. The Bbg 1.1 recombinant B. bigemina protein was used to affinity purify antibodies from bovine antisera against the native PhB and PhUB fractions. On western blots of the native proteins reacted with these antisera, multiple bands in the range 50-70 kDa (p50-p70) were obtained (Fig. 8A and data not shown). Similar results were obtained when whole native B. bigemina protein was reacted with an antiserum to a recombinant Bbgl.I-GST fusion protein (Fig. 8B). An antiserum from an animal immunised with GST alone gave no reaction with the B. bigemina proteins (Fig. 8B).

Discussion A family of 9 or more related, but-not

36

Gc.l,

4,

H

8 E

I

E

B

Gc.

H

S

X

B

9, 7

224 -E

S

E

E

B

A

E

Y

R

G

1 0 9 --

B

71-109 -71-45-Gc. 10, S

11

BS S

BE

45-28-18--

HB B

E

18 -~

Gc. 12 B

ii~¸ ~

Fig. 8. Western blot analysis of native and recombinant B. S

S

B

Fig. 7. Restriction maps of the B. bigemina 2EMBL3 genomic clones. The restriction sites shown are S, Sall; E,EcoRI; B, BamHI and H, Hind III. The hatched areas indicate the shortest restriction fragments to which the [~32p]dATP-labelled Bbgl.l probe hybridised.

identical, B. bigemina genes has been identified and partially characterised in this study. The genes are located on 2 of the 3 B. bigemina chromosomes which were resolved by PFGE. At least several members of the family of genes encode proteins with variants of a common carboxy-terminal region. On the basis of the region preceding the repeats, the family can be divided into 3 or more groups of related genes. Group 1 contains one gene, while group 2 contains at least 3 distinct genes. The relationships among 3 further cDNA clones and 3 groups of genomic clones have not yet been resolved. The different members of the family of genes exhibit variation in the copy number and sequence of the repeat region both between and within the groups with homologous amino-terminal domains. The observation of differences in hybridisation intensities

bigemina antigens. Western blots of native antigens [PhB (X) and PhUB (Y), see text] reacted with the antisera (PhUB) affinity-purified with the B. bigemina Bbg 1.1 recombinant protein (A). Western blots of whole B. bigemina antigens (B) were reacted with antiserum to a Bbgl.1 GST recombinant antigen (R) and antiserum to GST (G). Molecular size markers are shown in kDa.

of different probes to the different cDNA clones was the product of the variation in number of the repeats and the distinct preceding sequences. The repeat regions of the B. bigemina genes characterised here appear to be evolving by duplication, followed by other genetic changes as has been postulated for other proteins containing repetitive sequences [26-29]. An independent process of partial gene duplication and recombination presumably led to the observed association of the conserved carboxyterminal region and the variable repeat region with the unrelated amino-terminal regions in different members of the family. The sizes of the native B. bigemina proteins detected with antibodies purified on the recombinant protein correspond to those of proteins originally detected in the PhUB fraction (51, 54, 62 and 68 kDa) with antisera from animals immunised with the PhUB fraction [13]. The antisera from animals

37

vaccinated with the PhB fraction only detected a 68-kDa protein in the 50-70-kDa size range. However, since both sera bound to the same cDNA clones, phenyl-Sepharose did not efficiently purify the antigens from the original B. bigemina dextran sulphate-bound fraction. The 35-kDa doublet antigen, the major band detected in the PhB fraction with the antisera from animals immunised with the PhB fraction, was not cloned. A family of related B. bigemina merozoite surface proteins in the size range 46-68 kDa, has been identified with a number of monoclonal antibodies [10]. It is possible that the family of B. bigemina proteins encoded by the genes characterised in this study may represent members of this group of proteins. Proteins containing repeated regions have been proposed to be involved in immune evasion [30-33]. However, such a large number of related genes have not been identified in other Apicomplexan parasites such as Plasmodium falciparum, where most such gene sequences appear to be allelic variants [26, 31]. Trypanosomes, on the other hand do contain substantially larger families of related genes [34]. However, only one gene is expressed by an individual at any one time, although different individuals in the population may be expressing different copies of the gene. The large number of genes ( > 9) and the similarities in the patterns from the 2 independent isolates of B. bigemina suggests that it is most unlikely that all of these genes represent allelic variants. Rather, that one strain of parasites contains multiple genes. Many, if not all, members of the family of B. bigemina genes in this study appear to be expressed by members of the population of parasites within the one host. A number of B. bigemina proteins, p50-p70, were detected with antibodies purified on the recombinant protein. However, we cannot distinguish between the expression of only one protein by any individual parasite cell with different cells expressing different proteins, and the expression of most of the proteins by most members of the population. The suggested role of parasite repeat antigens in immune evasion, rather than in

the induction of protective immunity [30-33], raises an important question as to the nature of antibodies raised against the B. bigemina p50p70 family of proteins. The efficacy, or otherwise, of a cloned member of this family of proteins in the induction of protective immunity in cattle to B. bigemina is being investigated.

Acknowledgements The authors are grateful to Dr. K.R. Gale and Ms. C. Dimmock for the gift of B. bigemina cDNA and genomic libraries, to Mr. K. Rode-Bramanis for his assistance, to Dr. B.V. Goodger for reading the manuscript and to the Australian International Development Assistance Bureau for financial assistance.

References 1 Levine, N.D., Corliss, J.O., Cox, F.E.G., Deroux, G., Grain, J., Honigberg, B.M., Leedale, G.F., Loeblich, III, A.R., Lom, J., Lynn, D., Merinfeld, E.G., Page, F.C., Poljansky, G., Sprague, V., Vavra, J. and Wallace, F.G. (1980) A newly revised classification of the Protozoa. J. Protozool. 27, 37 58. 2 Riek, R.F. (1968) Babesiosis. In: Infectious Blood Diseases of Man and Animals, Vol. II (Weinmann, D. and Ristic, M., eds.), pp. 219-268, Academic Press, New York. 3 McCosker, P.J. (1981) The global importance of babesiosis. In: Babesiosis (Ristic M. and Kreier J.P., eds.), pp. 1-24, Academic Press, New York. 4 Callow, L.L. and Mellors, L.T. (1966) A new vaccine for Babesia argentina infection prepared in splenectomised calves. Aust. Vet. J. 42, 464465. 5 Dalgliesh, R.J., Callow, L.L. Mellors, L.T. and McGregor, W, (1981) Development of a highFy infective Babesia bigemina vaccine of reduced virulence. Aust. Vet. J. 57, 8-11. 6 Wright, I.G. (1990) Immunodiagnosis of and immunoprophylaxis against the haemoparasites Babesia sp. and Anaplasma sp. in domestic animals. Rev. Sci. Tech. Off. Int. Epizoot. 9, 345-356. 7 Callow, L.L., Mellors, L.T. and McGregor W. (1979) Reduction in virulence of Babesia bovis due to rapid passage in splenectomized cattle. Int. J. Parasitol. 9, 333-338. 8 Wright, I.G. and Riddles P.W. (1989) Biotechnology in tick-borne diseases: present status, future perspectives. In: Biotechnology for Livestock Production, pp. 325340. FAO/Plenum, New York. 9 McElwain, T.F., Perryman, L.E., Davis, W.C., and

38 McGuire, T.C. (1987) Antibodies define multiple proteins with epitopes exposed on the surface of live Babesia bigemina merozoites. J. Immunol. 138, 2298 2304. 10 Figueroa, J.V., Buening, G.M., Kinden, D.A. and Green, T.J. (1990) Identification of common surface antigens among Babesia bigemina isolates by using monoclonal antibodies. Parasitol. 100, 161-175. 11 McElwain, T.F., Perryman, L.E, Musoke, A.J. and McGuire, T.C. (1991) Molecular characterization and immunogenicity of neutralization- sensitive Babesia bigemina merozoite surface proteins. Mol. Biochem. Parasitol. 47, 213 222. 12 Mishra, V.S., Stephens, E.B., Dame, J.B., Perryman, L.E., McGuire, T.C. and McElwain, T.F. (1991) lmmunogenicity and sequence analysis of recombinant p58: a neutralization-sensitive, antigenically conserved Babesia bigemina merozoite surface protein. Mol. Biochem. Parasitol. 47, 207 212. 13 Kung'u, M.W., Goodger, B.V., Bushell, G., Wright, I.G. and Waltisbuhl, D.J. (In Press) Vaccination of cattle with dextran sulphate-binding Babesia bigemina antigens. Int. J. Parasitol. 14 Maniatis, T. Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 15 Huynh, T.V., Young, R.A. and Davis, R.W. (1985) Constructing and screening cDNA libraries in ),gtl 0 and )~gtl 1. In: DNA Cloning. A Practical Approach, Vol. l (Glover, D.M., ed.), pp 49-78. IRL Press, Oxford. 16 Conrad, P.A., Iams, K., Brown, W.C, Sohanpal, B. and ole-MoiYoi, O.K. (1987) DNA probes detect genomic diversity in Theileria parva stocks. Mol. Biochem. Parasitol. 25, 213-226. 17 Feinberg, A.P. and Vogelstein, B. (1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 613. 18 Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol., 98, 503 517. 19 Van der Ploeg, L.H.T., Schwartz, D.C., Cantor, C.R. and Borst, P. (1984) Antigenic variation in Trypanosoma brueei analyzed by electrophoretic separation of chromosome-sized DNA molecules. Cell 37, 77 84. 20 Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463 5467. 21 Ozaki, L.S., Mattei, D., Jendoubi, M., Druihle, P., Blisnick, T., Guillotte, M., Puijalon, O. and Da Silva, L.P. (1986) Plaque antibody selection: rapid immuno-

logical analysis of a large number of recombinant phage clones positive to sera raised against Plasmodium .falciparum antigens. J. lmmunol. Methods 89, 213 219. 22 Ternynck, T. and Avrameas, S. (1976) Polymerization and immobilization of proteins using ethylchloro formate and glutaraldehyde. Scand. J. lmmunol. Suppl. 3, 29-35. 23 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 685. 24 Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc. Natl. Acad. Sci. USA 76, 4350 4354. 25 Schneppenheim, R., Plendl, H. and Budde, U. (1988) Luminography: an alternative assay for detection of von Willebrand factor multimers. Thromb. Haemostasis 60, 133 136. 26 Fenton, B., Clark, J.T., Khan, C.M.A., Robinson, J.V., Wa[liker, D., Ridley, R., Scaife J.G. and McBride, J.S. (1991) Structural and antigenic polymorphism of the 35 to 48-kilodalton merozoite surface antigen (MSA-2) of the malaria parasite Plasmodium falciparum. Mol. Cell. Biol. I l, 963 971. 27 Wellems, T.E. and Howard, R.J. (1986) Homologous genes encode two distinct histidine-rich proteins in a cloned isolate of Plasmodium falciparum. Proc. Natl. Acad. Sci. USA 83, 6065-6069. 28 Colomer-Gould, V. and Enea, V. (1990) Plasmodium yoelii nigeriensis circumsporozoite gene structure and its implications for the evolution of the repeat regions. Mol. Biochem. Parasitol. 43, 51-58. 29 Galinski, M.R., Arnot, D.E., Cochrane, A.H., Barnwell, J.W., Nussenzweig, R.S. and Enea, V. (1987) The circumsporozoite gene of the Plasmodium cynomolgi complex. Cell 48, 311 319. 30 Anders, R.F. (1986) Multiple cross-reactivities amongst antigens of Plasmodium falciparum impair the development of protective immunity against malaria. Parasite Immunol. 8, 529 539. 31 Kemp, D.J, Cowman, A.F. and Walliker, D. (1990) Genetic diversity in Plasmodium Jalciparum. Adv. Parasitol. 29, 75 149. 32 Hyde, J.E. (1990) Molecular Parasitology. Open University Press, Milton Keynes, U.K. 33 Schofield, L. (1991) On the function of repetitive domains in protein antigens of Plasmodium and other eukaryotic parasites. Parasitol. Today 7, 99-105. 34 Cross, G.A.M. (1990) Cellular and genetic aspects of antigenic variation in Trypanosomes. Annu. Rev. Immunol. 8, 83 110.