Microbial Pathogenesis 1992 ; 12 : 47-62

Molecular cloning and nucleotide sequence analysis of the gene encoding the immunoreactive Bruce//a abortus Hsp60 protein, BA60K R . Martin Roop ll,' * Michelle L . Price,' Bruce E . Dunn, 23 Stephen M . Boyle, 4 Nammalwar Sriranganathan 4 and Gerhardt G . Schurig 4 'Departments of Microbiology and Immunology, and 2Pathology, University of Arkansas for Medical Sciences, and 'Laboratory Service, John L . McClellan Memorial Veterans Hospital, Little Rock, Arkansas 72205, 'Department of Pathobiology, Veterinary Microbiology Research Laboratories, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, U .S .A . (Received July 16, 1991 ; accepted in revised form September 19, 1991)

Roop, R . M . (Depts of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, U .S .A .), M . L . Price, B . E . Dunn, S . M . Boyle, N . Sriranganathan and G . G . Schurig . Molecular cloning and nucleotide sequence analysis of the gene encoding the immunoreactive Brucella abortus Hsp60 protein, BA60K . Microbial Pathogenesis 1992 ; 12 : 47-62 . A recombinant 60 kDa Brucella abortus protein expressed in Escherichia coli was recognized in immunoblots by sera from mice experimentally infected with B . abortus and a dog experimentally infected with B . canis . Sera from humans and dogs with naturally acquired brucellosis also recognized this protein, which was designated BA60K . The gene encoding BA60K was localized within an 18 kb B . abortus genomic fragment and its direction of transcription determined by subcloning and maxicell analysis of selected restriction fragments . The nucleotide sequence of 1800 bases encompassing the predicted gene location was determined, revealing an open reading frame encoding a protein of 546 amino acids (predicted relative molecular mass of 57 515) . Solid phase micro-sequencing of BA60K eluted from twodimensional polyacrylamide gels confirmed the predicted amino acid sequence . Comparison of the predicted amino acid sequence of BA60K with a protein sequence database revealed that BA60K shares 67 .9% identity with the GroEL protein of E. coli, a member of the Hsp6O family of chaperonins . The immunodominant Hsp60 homologs from Legionella pneumophila, Chlamydia trachomatis and Mycobacterium tuberculosis were also found to share greater than 59% amino acid sequence identity with the BA60K protein . The identification of BA60K as a member of the Hsp60 family of chaperonins supports its role in stimulating a prominent host immune response during the course of Brucella infections . It also indicates that BA60K is an important candidate for studies aimed at identifying the antigens responsible for eliciting the protective immune response to brucellosis . Key words : Brucella ; brucellosis ; heat shock protein ; immune response ; recombinant Brucella antigen .

Introduction Bruce//a species are Gram-negative, facultative, intracellular bacterial pathogens which cause serious diseases in both humans and animals .' Live, attenuated vaccines have been effectively used to control brucellosis in food animals,' but our knowledge `Author to whom correspondence should be addressed at: Slot 511, University of Arkansas for Medical Sciences, 4301 W . Markham, Little Rock, Arkansas 72205-7199, U .S .A . 0382-4010/92/010047+16 $03 .00/0

© 1992 Academic Press Limited



48

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concerning protective immunity is incomplete . Both humoral and cellular immune responses are believed to be important . 3-10 Studies employing the murine model indicate that protective antibodies are directed predominantly against the 0-chain of 11,12 but the antigens responsible for inducing the bacterial lipopolysaccharide (LPS), protective cellular immunity have not yet been defined ." Recombinant, immunoreactive Brucella proteins are valuable tools for studying protective immunity to brucellosis . Large quantities of these proteins can be obtained from recombinant hosts, and they provide a means of analysing individual antigens without interference from contaminating, immunodominant Brucella LIPS ." Furthermore, genes encoding relevant immunogenic proteins can be used to construct replicating antigen delivery systems, such as those based on salmonellae 15 or vaccinia ." Immunization schemes employing both purified subunit preparations and live, recombinant antigen delivery systems should allow a comprehensive evaluation of the importance of specific Bruce//a proteins in eliciting protective immunity . Several immunologic studies employing recombinant Brucella proteins have been reported recently . 5 .17,18 This report describes the cloning and expression in Escherichia coil of a 60 kDa B . abortus protein, designated BA60K, which is immunoreactive with sera from hosts with both naturally acquired and experimentally induced brucellosis . Based on extensive amino acid sequence identity, BA60K has been identified as a member of the Hsp60 family of chaperonins . 19 In addition, based on the immunoreactivity of BA60K observed in this study, and the extensively documented immunogenic nature of the Hsp60 proteins in genera 1,21 we propose that BA60K will prove useful in studies designed to better define the antigenic specificity of the protective immune response to brucellosis .

Results A B . abortus 2308 genomic library in pUC9 was used to transform E. coli DH5-a, and transformants producing B. abortus 30, 34, 60 and 66 kDa proteins were detected by colony blot enzyme-linked immunosorbent assay (ELISA) and Western blot analysis with B . abortus-specific hyperimmune goat serum . The 60 kDa recombinant protein [Fig . 1 (a)], designated BA60K, was subsequently found to be reactive in immunoblots with sera from mice experimentally infected with B . abortus [Fig . 1(b)], as well as with sera from goats and cattle experimentally infected with B . abortus, and a dog experimentally infected with B . canis (data not shown) . Further study showed antibodies reactive with BA60K were also present in the majority of the sera tested from humans (5/8) and dogs (7/9) with naturally acquired brucellosis . A representative immunoblot demonstrating the reactivity of serum from a dog with naturally acquired brucellosis with BA60K is shown in Fig . 1 (c) . Southern blot analysis confirmed that the gene encoding BA60K originated from the B . abortus genome, and that similar genes are also present in the genomic DNA of B . melitensis and B . canis (data not shown) . Restriction enzyme analysis of pBA21 6, the recombinant plasmid encoding BA60K, revealed the presence of an 18 kb insert . Subcloning of random sized Sau3A fragments generated from pBA216 into pUC19 led to the construction of pBA2161, which contained a 4 .5 kb insert encoding BA60K (Fig . 2) . Maxicell analysis of recombinant plasmids derived from pBA2161 (Fig . 2) further pinpointed the location of the gene encoding BA60K . Plasmid pBA2167, containing the 3 .0 kb Sma I/Pst I fragment encoded a truncated peptide of approximately 57 kDa in maxicells (Fig . 3, lane 4), while plasmid pBA2168, containing the 3 .0 kb Eco R I /Sal I fragment encoded the



0 M I

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50

R . M . Roop et al Sm

E

HH

HP I I

So I

4.5 kb

Sm I

pBA 2161 60 kDa

E I

HH II

HP II

pBA 2167

3.0 kb

57 kDa

HH II

E I

HP II

Sa I

3 .0 kb

pBA 2168 60 kDa

Fig . 2 . Restriction endonuclease map of the inserts from recombinant plasmids pBA2161, pBA2167 and pBA2168 . The size of the peptide expressed from these plasmids in maxicells is shown beneath the plasmid designation . The arrow indicates the direction of transcription of the gene encoding BA60K . Sm = Sma I ; E=EcoRI ;H=Hind III ; P=Pstl ; Sa=Sa/I .

60 kDa protein (Fig . 3, lane 5) . Thus it appeared that the majority of the gene encoding BA60K was located within the EcoR I/Pst I fragment common to pBA2167 and pBA21 68, with the remainder of the coding region lying in the sequence to the right of the Pst I site of pBA2168 as shown in Fig . 2 . These results also suggested that the gene encoding BA60K was transcribed in the direction indicated by the arrow in Fig . 2 . The nucleotide sequence of the first 1800 bases extending from the Eco R I site towards the Sal I site of pBA2168 was determined and an open reading frame with the appropriate coding capacity for BA60K was identified (Fig . 4) . Analysis of this

kDa 110-

84-

47-

a

b

c

d

e

Fig . 3 . Maxicell analysis of pBA216, pBA2161, pBA2167 and pBA2168 in E. co/i CSR603 . Lane a, CSR603/pUC19; lane b, CSR603/pBA216 ; lane c, CSR603/pBA2161 ; lane d, CSR603/pBA2167 ; lane e, CSR603/pBA2168 .



Immunoreactive Brucella Hsp60 10

51

20

30

40

50

60

70

GAATTCCATTCACAGTACAACATTTACAAATCTGACCGGGATATTCCC AGGAGAGTAAAATGGCTGCAAA CTTAAGGTAAGTGTCATGTTGTAAATGTTTAGACTGGCCCTATAAGGGTCCTCTCATTTTACCGACGTTT M A A K 80

90

100

110

120

130

140

AGACGTAAAATTCGGCCGCACTGCGCGCGAAAAGATGCTGCGCGGCGTCGATATCCTCGCTGACGCTGTTAA TCTGCATTTTAAGCCGGCGTGACGCGCGCTTTTCTACGACGCGCCGCAGCTATAGGAGCGACTGCGACAATT • V K F G R T A R E K M L R G V D I L A D A V K 150

160

170

180

190

200

210

GGTCACGCTCGGCCCGAAGGGCCGCAATGTCGTTATCGAGAAGTCCTTCGGCGCTCCGCGCATCACCAAG CCAGTGCGAGCCGGGCTTCCCGGCGTTACAGCAATAGCTCTTCAGGAAGCCGCGAGGCGCGTAGTGGTTC V T L G P K G R N V V I E K S F G A P R I T K 220

230

240

250

260

270

280

GACGGCGTTTCGGTCGCCAAGGAAGTCGAACTGGAAGACAAGTTTGAAAACATGGGCGCACAGATGCTGCG CTGCCGCAAAGCCAGCGGTTCCTTCAGCTTGACCTTCTGTTCAAACTTTTGTACCCGCGTGTCTACGACGC • G V S V A K E V E L E D K F E N M G A Q M L R 290

300

310

320

330

340

350

CGAAGTGGCTTCCAAGACCAACGATACTGCCGGTGACGGCACCACGACCGCGACCGTTCTCGGTCAGGCC GCTTCACCGAAGGTTCTGGTTGCTATGACGGCCACTGCCGTGGTGCTGGCGCTGGCAAGAGCCAGTCCGG E V A S K T N D T A G D G T T T A T V L G Q A 360

370

380

390

400

410

420

ATCGTTCAGGAAGGCGCCAAGGCCGTTGCCGCTGGCATGAACCCGATGGACCTGAAGCGCGGCATCGAC TAGCAAGTCCTTCCGCGGTTCCGGCAACGGCGACCGTACTTGGGCTACCTGGACTTCGCGCCGTAGCTG I V Q E G A K A V A A G M N P M D L K R G I D 430

440

450

460

470

480

490

CTCGCTGTCAACGAAGTTGTGGCTGAGCTGCTGAAGAAGGCCAAAAAGATCAACACTTCGGAAGAAGTT GAGCGACAGTTGCTTCAACACCGACTCGACGACTTCTTCCGGTTTTTCTAGTTGTGAAGCCTTCTTCAA L A V N E V V A E L L K K A K K I N T S E E V 500

510

520

530

540

550

560

GCCCAGGTTGGCACCATCTCTGCCAACGGCGAAGCCGAAATCGGCAAGATGATCGCCGAAGCGATGCAG CGGGTCCAACCGTGGTAGAGACGGTTGCCGCTTCGGCTTTAGCCGTTCTACTAGCGGCTTCGCTACGTC A Q V G T I S A N G E A E I G K M I A E A M Q 570

580

590

600

610

620

630

AAGGTCGGCAACGAAGGCGTCATCACGGTTGAAGAAGCCAAGACCGCCGAAACCGAACTCGAAGTCGTCG TTCCAGCCGTTGCTTCCGCAGTAGTGCCAACTTCTTCGGTTCTGGCGGCTTTGGCTTGAGCTTCAGCAGC • V G N E G V I T V E E A K T A E T E L E V V 640

650

660

670

680

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700

AAGGCATGCAGTTCGACCGCGGCTACCTGTCGCCTTACTTCGTCACCAACCCTGAAAAGATGGTTGCTGA TTCCGTACGTCAAGCTGGCGCCGATGGACAGCGGAATGAAGCAGTGGTTGGGACTTTTCTACCAACGACT E G M Q F D R G Y L S P Y F V T N P E K M V A D 710

720

730

740

750

760

770

CCTCGAAGACGCCTACATCCTTCTGCACGAAAAGAAGCTCTCGAACCTTCAGGCTCTCCTGCCGGTTCTC GGAGCTTCTGCGGATGTAGGAAGACGTGCTTTTCTTCGAGAGCTTGGAAGTCCGAGAGGACGGCCAAGAG L E D A Y I L L H E K K L S N L Q A L L P V L Fig . 4 . Nucleotide sequence of the gene encoding BA60K and the predicted amino acid sequence of BA60K . The reading frame for BA60K begins at nucleotide 61 and the start codon, ATG, is underlined . A putative Shine-Delgarno sequence is underlined at bases 49-53 . Amino acid residues are denoted using the single letter code and the stop codon is designated by an asterisk . Inverted arrows at bases 1736-1742 and 1746-1752 denote regions of dyad symmetry defining a potential rho-independent transcription termination site .



52

R . M . Roop et al.

780 790 800 810 820 830 840 GAAGCTGTCGTCCAGACCTCCAAGCCGCTTCTCATCATTGCTGAAGACGTCGAAGGCGAAGCTCTTGCAA CTTCGACAGCAGGTCTGGAGGTTCGGCGAAGAGTAGTAACGACTTCTGCAGCTTCCGCTTCGAGAACGTT E A V V Q T S K P L L I I A E D V E G E A L A 850 860 870 880 890 900 910 CGCTCGTCGTCAACAAGCTGCGCGGCGGCCTGAAGATTGCTGCCGTCAAGGCTCCGGGCTTCGGCGATCG GCGAGCAGCAGTTGTTCGACGCGCCGCCGGACTTCTAACGACGGCAGTTCCGAGGCCCGAAGCCGCTAGC T L V V N K L R G G L K I A A V K A P G F G D R 920 930 940 950 960 970 980 CCGCAAGGCCATGCTCGAAGACATCGCGATCCTCACTGGCGGTCAGGTCATCTCCGAAGACCTCGGCATC GGCGTTCCGGTACGAGCTTCTGTAGCGCTAGGAGTGACCGCCAGTCCAGTAGAGGCTTCTGGAGCCGTAG R K A M L E D I A I L T G G Q V I S E D L G I 990 1000 1010 1020 1030 1040 1050 AAGCTTGAAAGCGTTACGCTCGACATGCTGGGCCGCGCCAAGAAGGTTTCGATCTCCAAGGAAAACACGA TTCGAACTTTCGCAATGCGAGCTGTACGACCCGGCGCGGTTCTTCCAAAGCTAGAGGTTCCTTTTGTGCT K L E S V T L D M L G R A K K V S I S K E N T 1060 1070 1080 1090 1100 1120 1130 CGATCGTTGACGGTGCAGGCCAGAAGGCCGAAATCGACGCTCGCGTTGGCCAGATCAAGCAGCAGATCGA GCTAGCAACTGCCACGTCCGGTCTTCCGGCTTTAGCTGCGAGCGCAACCGGTCTAGTTCGTCGTCTAGCT T I V D G A G Q K A E I D A R V G Q I K Q Q I E 1130 1140 1150 1160 1170 1180 1190 AGAAACCACTTCGGACTACGACCGTGAAAAGCTTCAGGAACGTCTTGCCAAGCTCGCTGGCGGCGTTGCC TCTTTGGTGAAGCCTGATGCTGGCACTTTTCGAAGTCCTTGCAGAACGGTTCGAGCGACCGCCGCAACGG • T T S D Y D R E K L Q E R L A K L A G G V A 1200 1210 1220 1230 1240 1250 1260 GTGATCCGCGTCGGCGGTGCAACGGAAGTTGAAGTGAAGGAAAAGAAGGACCGCGTTGACGACGCCCTGA CACTAGGCGCAGCCGCCACGTTGCCTTCAACTTCACTTCCTTTTCTTCCTGGCGCAACTGCTGCGGGACT V I R V G G A T E V E V K E K K D R V D D A L 1270 1280 1290 1300 1310 1320 1330 ACGCAACCCGCGCTGCGGTTGAAGAAGGTATCGTTGCCGGCGGCGGCACCGCCCTGCTCCGCGCTTCGAC TGCGTTGGGCGCGACGCCAACTTCTTCCATAGCAACGGCCGCCGCCGTGGCGGGACGAGGCGCGAAGCTG N A T R A A V E E G I V A G G G T A L L R A S T 1340 1350 1360 1370 1380 1390 1400 CAAGATCACCGCAAAGGGTGTGAATGCCGACCAGGAAGCTGGCATCAACATCGTTCGTCGCGCGATCCAG GTTCTAGTGGCGTTTCCCACACTTACGGCTGGTCCTTCGACCGTAGTTGTAGCAAGCAGCGCGCTAGGTC • I T A K G V N A D Q E A G I N I V R R A I Q 1410 1420 1430 1440 1450 1460 1470 GCTCCGGCCCGCCAGATCACGACCAATGCCGGTGAAGAAGCTTCGGTAATCGTTGGCAAGATCCTCGAAA CGAGGCCGGGCGGTCTAGTGCTGGTTACGGCCACTTCTTCGAAGCCATTAGCAACCGTTCTAGGAGCTTT A P A R Q I T T N A G E E A S V I V G K I L E 1480 1490 1500 1510 1520 1530 1540 ACACGTCCGAAACCTTCGGCTACAACACGGCCAATGGCGAATATGGCGACCTGATCTCGCTCGGCATTGT TGTGCAGGCTTTGGAAGCCGATGTTGTGCCGGTTACCGCTTATACCGCTGGACTAGAGCGAGCCGTAACA N T S E T F G Y N T A N G E Y G D L I S L G I V 1550 1560 1570 1580 1590 1600 1610 TGACCCGGTCAAGGTTGTCCGCACGGCTCTGCAGAACGCAGCTTCGGTTGCCGGCCTGCTGATCACGACG ACTGGGCCAGTTCCAACAGGCGTGCCGAGACGTCTTGCGTCGAAGCCAACGGCCGGACGACTAGTGCTGC • P V K V V R T A L Q N A A S V A G L L I T T 1620 1630 1640 1650 1660 1670 1680 GAAGCAATGATCGCCGAACTGCCGAAGAAGGACGCAGCTCCGGCTGGCATGCCTGGCGGTATGGGTGGCA CTTCGTTACTAGCGGCTTGACGGCTTCTTCCTGCGTCGAGGCCGACCGTACGGACCGCCATACCCACCGT E A M I A E L P K K D A A P A G M P G G M G G

Fig . 4 . Continued.



Immunoreactive Brucella Hsp60

53

1690 1700 1710 1720 1730 1740 1750 TGGGCGGCATGGACTTCTAAGAAGTCCATAAAGCTCGCCCATGAACTAAATGCCTGCGCCGCAAAGCGGC ACCCGCCGTACCTGAAGATTCTTCAGGTATTTCGAGCGGGTACTTGATTTACGGACGCGGCGTTTCGCCG M G G M D F * > 1760 1770 1780 1790 1800 GCGGGGGCGGCATGGACTTCCAAGAAGTCCATGCCACCGCAGGACAGGCG CGCCCCCGCCGTACCTGAAGGTTCTTCAGGTACGGTGGCGTCCTGTCCGC Fig . 4 .

Continued.

open reading frame with the PCGene program predicted that it coded for a peptide consisting of 546 amino acids, with a relative molecular mass of 57 515 and an isoelectric point of 4 .84 . This corresponded well with the results of analysis of BA60K by two dimensional (2-D) gel electrophoresis (Fig . 5) . This latter procedure also allowed the separation of BA60K from a protein of similar molecular weight (protein 3, Fig . 5) present in the host E. coil cell, which was not possible with standard SDSPAGE . Presumably due to blockage of BA60K at its N-terminus, a limited amount of N-terminal amino acid sequence data was obtained by direct microsequencing of BA60K cut out of 2-D gels and blotted onto Immobilon (Millipore, Bedford, Massachusetts) paper . However, the amino acid sequence obtained, X-X-X-Asp-ValLys-Phe-Gly-X-Thr-Ala-, matched exactly with residues 5-9 and 11 and 12 of the predicted amino acid sequence for BA60K (Fig . 4) . The predicted amino acid sequence for BA60K was compared with the SWISSPROT Protein Sequence databank, revealing a high level of sequence identity with members of the Hsp60 family of chaperonins, including 67 .9% identity with the GroEL

IEF-) Acidic

Basic

976645-

29 -

Fig . 5 . Silver stained two-dimensional gels of maxicell preparations of (a) CSR603/pUC19, and (b) CSR603/pBA2161 . The pH gradient extended from 4 .5 (left side of gel) to 6 .8 (right side of gel) under the conditions used . Numbers 1-8 indicate individual proteins readily identified in both preparations for orientation . BA60K [in brackets ; Fig . 5(b)], was not observed in the host maxicell preparation [brackets ;

Fig . 5(a)] .



54

R . M . Roop et al.

protein of E. co/i 21 (Fig . 6) . The amino acid sequence for BA60K was aligned with the corresponding published sequences for GroEL and homologous Hsp60 proteins from other pathogenic bacteria using the PCGene program (Fig . 6) and the following relationships were observed : 66 .3% identity with the HtpB protein of Legione//a pneumophila ; 22 61 .4% identity with the Chlamydia trachomatis HypB protein ;" and 59 .3% identity with the immunodominant 65 kDa protein of Mycobacterium tuberculosis." In addition, BA60K also showed 54% identity in a 546 amino acid

10

BA60K groEL Lp HtpB M - 65 kDa Ct HypB

M A A K D A . E T . V A . N

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40 R N V V I E . . . . L D . . . . L . . . . . L . . H . . . D

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BA60K groEL Lp HtpB M - 65 kDa Ct HypB

Q M L V V E L V V

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BA60K groEL Lp HtpB M - 65 kDa Ct HypB

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BA60K groEL Lp HtpB M - 65 kDa Ct HypB

BA60K groEL Lp HtpB M - 65 kDa Ct HypB

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BA60K groEL Lp HtpB M - 65 kDa Ct HypB

I L V . A .

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70 N M G A . . K I . . .

90 T A G D G T T T A T V L G A . . A A V . . A K . . A

110 A K A V A A G M N P M D L K R G L H . . . . . . . . L R N . . . . A . L G L R N . T . . A .

130 I D L A V N E V V A E L K T A A . E . K L A T K K E K E K T E T . K K V . D Q I E A A Q E

D N N N K

K S F G A P . . . . . Y . K W . S .

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BA60K groEL Lp HtpB M - 65 kDa Ct HypB

V . . L .

L K Q . K

K A A . .

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150 V A Q V G T I S A N G E A I . . T S D E I S D E I A T A A . * D Q I A N D .

A K K I S V P C S P C E V S P V

140 N T S D K D E . Q H

S . . K H

E K K . K

160 E I G K M I A E T V . . L A . A I S . D L . . N L

Fig . 6 . Comparison of the predicted amino acid sequence of BA60K with the published sequences of other Hsp60 homologues . The single letter code is used for denoting amino acids. Dots indicate identity between corresponding amino acid residues and asterisks indicate gaps inserted by the alignment program to maximize identity between sequences . groEL, the groEL protein of E. coli ; 21 Lp HtpB, the HtpB protein of Legionella pneumophila ; 22 M-65 kDa, the 65 kDa immunodominant protein of Mycobacterium tuberculosis ; 24 Ct HypB, the HypB protein of Chlamydia trachomatis . 23



Immunoreactive Brucella Hsp60

BA60K groEL Lp HtpB M -65 kDa Ct HypB

55

170 180 A M Q K V G N E G V I T V E E A K . D G K . . D G . . D . . . . . . S K N . S 190 E V V D . . Y . . . L T

E . . .

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D

.

.

.

.

BA60K groEL Lp HtpB M- 65 kDa Ct HypB

M G . Q Q

V A S E E

A V C . C

BA60K groEL Lp HtpB M- 65 kDa Ct HypB

L P . S . .

BA60K groEL Lp HtpB M- 65 kDa Ct HypB

K T N N

T A G L G L . F G F

BA60K groEL Lp HtpB M- 65 kDa Ct HypB BA60K groEL Lp HtpB M- 65 kDa Ct HypB

E . . . Q

240 A V . . G . K . Q .

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260 E A L A T L V V N A . . . . . . . . . . . . . . . S . . . . . . . . .

K T N . R

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. I V . V . . . . I V . V C . . . T F . S V A . . . F R V C . .

BA60K groEL Lp HtpB M- 65 kDa Ct HypB

S N T T .

BA60K groEL Lp HtpB M- 65 kDa Ct HypB

.

.

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300 I A I L T G G Q V I S . . T . . . . T . . .

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K D

270 L I M I I

280 290 G F G D R R K A M L E D Q . Q . Q . . . . . . . . . . . . . E D L G . E I . . E I . . E V . . E . .

Q A L R E M R E M

250 S K P L L I I A E D V E G G . G R G . S . E S G R . I T A S A

BA60K groEL Lp HtpB M- 65 kDa Ct HypB

210 E K . T Q N . R . T

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D L E D A Y I L L H E K K L S N L E . A D I . I S P F . E H P F . . V D V S I V . P . . . V S S . V T V V . . L V I Y D I G I

L . . . .

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200 Q F D R G Y L S P Y F V T N P . . . . I N K . . . . . . I I N . Q R . . K . I G . . . . D A . N . N . . S . . A . . .

Q K K .

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350 360 370 I K Q Q I E E T T S D Y D R E K L Q E R L A K R A V . R A . M . . . . V . . R Q E . . N S D . K . . . D S S . K Fig . 6 . Continued.



R . M . Roop et a/.

56

BA60K groEL Lp HtpB M- 65 kDa Ct HypB

380 L A G G V A V I R V G G A K A . K . . A . K A . A . S . A .

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E . . . .

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. . V Q

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BA60K groEL Lp HtpB M- 65 kDa Ct HypB

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Fig . 6 . Continued.

overlap with the human mitochondrial P1 protein, 25 a eukaryotic Hsp60 protein (data not shown) . As expected from their extensive amino acid sequence identity, BA60K and the other bacterial Hsp60 proteins which were examined exhibited immunologic cross-reactivity . Polyclonal sera raised against the C. trachomatis HypB protein and the 65 kDa protein from Mycobacterium bovis, which has the identical amino acid sequence as the homologous protein in M . tubercuiosis, 24 reacted strongly with BA60K



Immunoreactive Brucella Hsp60

57

kDa 10680-

49-

a

b

c

d

e

f

Fig . 7 . Reaction of recombinant and host E. coli whole cell preparations and B . abortus RB51 cell lysate in Western blots with C. trachomatis HypB specific (lanes a-c) and Mycobacterium bovis 65 kDa specific (lanes d-f) serum . Lanes a and d, E. co/i DH5-a/pUC19 ; lanes b and e, DH5-a/pBA2161 ; lanes c and f, B. abortus RB51 cell lysate .

in immunoblots (Fig . 7) . These sera also reacted with a 60 kDa protein in the E. co/i host cell, which is presumed to be GroEL . Discussion The identification of BA60K as an Hsp60 homologue supports a potential role for this protein in the immune response to brucellosis . The immunogenic nature of many bacterial Hsp60 proteins is well established, 20 and the presence of antibodies reactive with BA60K in sera from both naturally and experimentally infected hosts demonstrated in this study indicates that this particular Hsp60 protein elicits an active immune response in many animals during Brucella infection . These findings correlate well with previous reports describing significant antibody reactivity with Brucella proteins similar in size to BA60K in sera from humans infected with B. melitensis, 2S dogs infected with B . canis and B . abortus, 27 goats infected with B . melitensis, 2S sheep infected with B . ovis 29 and mice and goats experimentally infected with B . abortus (R . M . Roop II, unpublished data) . Although bacterial Hsp60 proteins are generally very immunogenic, the biological relevance of the immune responses directed towards these antigens is unclear ." Both protective20 and pathologic 30-33 functions for these responses have been proposed, and this is currently a very active area of investigation . With respect to brucellosis, experimentally infected mice 13 and cattle 10 develop cellular immune responses directed against a variety of Brucella protein antigens, few of which have been identified . Furthermore, immunopathologic reactions with apparent reactivity toward Brucella .35 cellular antigens are occasionally associated with natural infection S7,11 or vaccination Therefore, an examination of the role of BA60K in the immune response to brucellosis appears to be warranted . The recombinant form of BA60K and the gene which encodes this protein will be useful for examining the possible roles of BA60K in the immune response to brucellosis . Overexpression of BA60K in E. co/i provides a convenient source for the isolation and purification of this protein . It also eliminates the problems associated with LPS 1 .3' The data obtained from the contamination of isolated native Brucella proteins .' 2-D gel analysis of BA60K described in this report provides important biochemical

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characteristics which can be used to initiate isolation and purification procedures . In addition, the gene encoding BA60K can be used to construct live, recombinant antigen delivery systems . In this regard, preliminary studies indicate that BA60K is well expressed in attenuated strains of Salmonella typhimurium (S . M . Boyle, unpublished data) . Therefore, the availability of both subunit and live, recombinant immunogens should provide the basis for a comprehensive analysis of the role of BA60K in the immune response to brucellosis . Information gathered from such studies will not only be useful for gaining a better understanding of the immune response to brucellosis, but will probably also contribute to our general knowledge concerning the role of Hsp60 proteins in bacterial infections .

Materials and methods Bacterial strains . Brucella abortus strains 2308 and RB51, 37 an LPS 0-chain deficient mutant derived from strain 2308, were obtained from the culture collection of one of the authors (G .G .S .) . Working cultures were grown for 48 h at 37°C with 5% C0 2 on trypticase soy broth supplemented with 1 .5% agar (TSBA) and stored at 4°C, with monthly transfer . Stock cultures were stored in skimmed milk at -80°C . Escherichia coli DH5-a [supE44 A/acUl69 (080/acZ AM1 5) hsdRl 7 recAl endA1 gyrA96 thi-1 re/Al] was obtained from Bethesda Research Laboratories, Bethesda, Maryland . E. co/i CSR603 [recAl uvrA6 phr-1 ] 38 was obtained from Jack Crawford, McClellan Memorial Veterans Hospital, Little Rock, Arkansas . Working cultures of E. co/i strains were grown on LB agar 39 overnight at 37°C and stored at 4°C, with monthly transfer . Stock cultures were stored in LB broth supplemented with 25% glycerol at -80°C . Sera . Bruce//a abortus-specific hyperimmune serum was prepared in the following manner . B . abortus RB51 was grown on TSBA for 48 h at 37°C with 5% CO2 and growth from one plate was harvested into 10 ml 10 mm Tris-HCI, pH 8 .0 . Bacterial cells were killed by adding an equal volume of acetone and stirring overnight at room temperature . Killed cells were washed three times in 10 mm Tris-HCI, pH 8 .0, resuspended in 5 ml of 10 mm Tris-HCI, pH 8 .0 and lysed by sonicating at 60% full power [Sonic Dismembrator, model 300, 3/8"(9 .5 mm) titanium tip; Artek Systems, Farmingdale, New York] in 30 s bursts with 30 s cooling between bursts . A male, mixed breed goat was immunized by subcutaneous injection of 0 .5 ml of cell lysate mixed with an equal volume of Freund's Complete Adjuvant (Sigma Chemicals, St Louis, Missouri) . Nineteen days later, the goat was given a second subcutaneous injection using the same dose of lysate and adjuvant . Ten days following the second injection, blood was collected from the jugular vein, the serum removed and stored at -80°C . Blood from female C3H/He mice experimentally infected with B . abortus RB51 and challenged 6 weeks later with strain 2308 37 was collected by cardiac puncture at necropsy 3 weeks postchallenge, and serum removed and stored at -80°C . Sera from infected humans were obtained from Harold Russell, Respiratory Diseases Branch, Centers for Disease Control, Atlanta, Georgia, and were classified as positive based upon reactions in a microagglutination assay ." Sera from experimentally and naturally infected dogs were obtained from Leland Carmichael, College of Veterinary Medicine, Cornell University, Ithaca, New York . Paul Nicoletti, College of Veterinary Medicine, University of Florida, Gainesville, Florida, also provided sera from naturally infected dogs. Canine sera were considered positive based upon reactions in standard tube agglutination assays and/or their reactions in agarose gel immunodiffusion assays ." For the purposes of this study, natural infection was defined as the presence of clinical signs indicative of brucellosis along with positive serology as described above . In some, but not all cases, positive serology was confirmed by positive blood cultures . Human and canine sera were filter-sterilized and stored at 4°C . Hyperimmune rabbit sera specific for the C . trachomatis HypB and M. bovis BCG 65 kDa proteins42 were obtained from Patrik Bavoil, University of Rochester, Rochester, New York, and stored at -80°C . E. co/i-specific antibodies were removed from large volumes of sera (> 15 ml) by absorption with both whole cells and cell lysates of E. co/i DH5-a/pUC9 or DH5-a/pUC19 . 43 E. co/ispecific antibodies were removed from smaller volumes of sera using the following procedure . E. coil DH5-a/pUC19 was grown in LB broth supplemented with 1 mg/ml ampicillin (LB1 Oa) overnight at 37°C with shaking (175 rpm), harvested by centrifugation, resuspended in 1/20



Immunoreactive Brucella Hsp60

59

the original volume of 0 .5 M NaCl, 0 .02 M Tris, pH 7 .5 (TBS) and lysed by sonication . Two millilitres of cell lysate was added to 10 ml of serum diluted 1 :40 in TBS and incubated in a glass petri dish on a rotary shaker for 1 h at room temperature . The mixture was then centrifuged at 100000xg for 1 h at 4°C, the supernatant passed through a 0 .2 urn syringe filter (Nalge, Rochester, New York) and stored at 4°C . DNA isolation . Bruce//a species were grown on TSBA plates and high molecular mass chromosomal DNA isolated following the procedures described by Johnson . 44 Cells were washed with chloroform prior to lysis to allow for effective separation of high molecular mass DNA from proteins during subsequent phenol/chloroform extractions . Cloning procedures. B . abortus 2308 chromosomal DNA was partially digested with Sau 3A and fragments ranging in size from approximately 1-22 kb were cloned into the Bam H I site of pUC9 using procedures outlined by Sambrook et al. 39 The resulting recombinant plasmid bank was used to transform competent E. co/i DH5-CC, 45 and transformants detected by plating onto LB agar39 supplemented with 100 pg/ml ampicillin, 1 mm IPTG and 50 µg/ml Bluo-Gal . Expression of B . abortus antigens in E. coli clones was detected using a modification of a previously described colony blot ELISA 46 with B . abortus-specific hyperimmune goat serum (1 :1000 dilution in TBS) serving as the primary antibody . Colonies reacting with the B. abortusspecific hyperimmune serum were picked to fresh LB agar plates supplemented with 100 pg/ml ampicillin (LBa), grown overnight at 37°C, subcultured in LBa broth and stored at -80°C in 25% glycerol . Plasmid DNA was isolated from LB10a broth cultures of the recombinant and host E. coli 3 Prime, West strains using the alkaline lysis procedure . 39 pZ523 spin columns (5 Prime Chester, Pennsylvania) were used for large scale plasmid isolations . Plasmids were digested with restriction endonucleases and subjected to electrophoresis in gels containing 0 .6-0 .8% agarose . 39 For mapping of restriction sites and subcloning, individual fragments were cut out of agarose gels and purified using the GeneClean system (Bio 101, LaJolla, California) . SDS-PAGE and Western blotting. Whole cell preparations from recombinant and host E. co/i cultures grown overnight in LB1 Oa broth, crude B . abortus antigens, 37 and the B . abortus RB51 cell lysate were subjected to SDS-PAGE 47 on 10% acrylamide gels, transferred to nitrocellulose and Western blot analysis performed ." The dilutions of the primary antibodies used were as follows : B . abortus-specific hyperimmune goat serum, 1 :500 ; sera from experimentally infected mice, 1 :100 ; serum from the experimentally infected dog, 1 :500; sera from naturally infected humans and dogs, 1 :40; rabbit hyperimmune serum specific for the M . bovis 65 kDa and C. trachomatis HypB proteins, 1 :1000 . Secondary antibodies were used at the following concentrations : rabbit anti-goat, goat anti-human, goat anti-dog and goat anti-rabbit, 1 :1000 ; and goat anti-mouse, 1 :800 . All dilutions were made in TBS . Secondary antibodies were peroxidase-conjugated and IgG (heavy and light chains) specific and were obtained either from Sigma Chemicals, St Louis, Missouri, or Organon Teknika Cappell, Westchester, Pennsylvania . Subc/oning . Plasmids from immunoreactive clones were digested with the appropriate restriction enzymes, and the resulting fragments separated by agarose gel electrophoresis . Selected restriction fragments were cut out of gels, extracted and ligated into pUC1 8 or pUC1 9 as described above . These recombinant plasmids were used to transform E. coli DH5-x, and the transformants tested for expression of Brucella antigens by SDS-PAGE and Western blotting as described above . Maxice/l analysis . Recombinant and vector plasmids were also used to transform E. co/i CSR603 and maxicell analysis was performed ." Maxicell preparations pulse-labeled with 35 Smethionine were subjected to SDS-PAGE and Western blotting as described above . Gels were subjected to autoradiography to identify the proteins produced from each plasmid . 2-dimensional gel electrophoresis and N-terminal amino acid sequence determination . 35 Slabeled maxicell preparations of E. coli CSR603 containing pBA2161 were subjected to 2-D gel electrophoresis with isoelectric focusing in the first dimension (pl = 4 .5-6 .8), followed by SDS-PAGE in the second dimension ." Gels were stained with silver, 50 dried, then subjected to autoradiography to identify plasmid-encoded proteins . Maxicell preparations of CSR603 containing pUC19 were used to identify vector-encoded proteins . Unlabeled maxicell preparations of CSR603/pBA2161 were then subjected to 2-D gel electrophoresis, lightly stained



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with Coomassie Blue and spots corresponding to BA60K were cut out of gels . BA60K was transferred from the acrylamide cutouts to Immobilon paper (Millipore, Bedford, Massachusetts) by electroblotting and subjected to solid phase microsequencing to determine the N-terminal acid sequence ." DNA sequencing . Dideoxy sequencing was performed directly from the pUC plasmids with M13 primers, or after subcloning into the pBC vectors (Stratagene, LaJolla, California) with T3 and T7 primers, using the method described by Sanger et al." Oligonucleotide primers were generated as needed within the determined sequence to produce overlapping fragments and for sequencing of the complementary strand in the opposite orientation . Nested deletions were also generated in subcloned fragments in the pBC vectors ."

Amino acid sequence analysis. The predicted amino acid sequence for BA60K was compared with protein sequences deposited in the SWISS-PROT Protein Sequence data and other published amino acid sequences using the PC/GENE program (Intelligenetics, Mountain View, California) .

The authors thank Robert Sigh and Jan Simmers for excellent technical assistance . This work was supported by Public Health Service grant Al-28867 from the National Institutes of Health and an Institutional Biomedical Research Support grant from the College of Medicine, University of Arkansas for Medical Sciences to R .M .R . II, by grant no . 85-CRCR-1 -1848 from the United States Department of Agriculture to G .G .S ., S .M .B . and N .S ., and by the Medical Research Service of the Veterans Administration .

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