INFECrION AND IMMUNITY, Apr. 1993, p. 1173-11790019-9567/93/041173-07$02.00/0Copyright X 1993, American Society for Microbiology

Cloning and Sequencing of the Gene for cx Antigen fromMycobacterium avium and Mapping of B-Cell EpitopesNAOYA OHARA,'* KAZUHIRO MATSUO,2 RYUJI YAMAGUCHI,2 AKIHIRO YAMAZAKI,2

HIROMICHI TASAKA,3 AND TAKESHI YAMADA'School of Dentistry, Nagasaki University, Sakamoto 1-7-1, Nagasaki City 852,1 Central Research

Laboratories, Ajinomoto Co., Inc., Suzuki-Cho 1-1, Kawasaki-ku, Kawasaki City 210,2 and HiroshimaUniversity School of Medicine, Kasumi 1-2-3, Minami-ku, Hiroshima City 734, 3 Japan

Received 21 August 1992/Accepted 6 January 1993

The complete nucleotide sequence of a antigen secreted from Mycobacterium avium (A-a) was determined.The gene encodes 330 amino acids, including 40 amino acids for the signal peptide, followed by 290 amino acidsfor the mature protein with a molecular mass of 30,811 Da. This is the first sequence of A-a. Comparisonsbetween A-a and a antigens ofMycobacterium leprae, Mycobacterium bovis BCG, and Mycobacterium kansasiishowed highly homologous regions which suggested a conserved functional domain and two less-homologousregions. Serological analysis of recombinant A-a, expressed by a series of deletion constructs, indicated thepossibility that A-a carries at least six B-cell epitopes. The three antigenic determinants were common toMycobacterium tuberculosis, M. kansasii, and M. avium. The results also suggested the possibility that there arethree species-specific epitopes.

A protein designated a antigen was first isolated from theculture filtrate of Mycobacterium tuberculosis (T-a) byYoneda and Fukui (36). Analogous antigens were studiedindependently by different investigators and given differentdesignations. MPB59, antigen 6, and BCG 85B are identicalto a antigen (15, 31). BCG 85 complex consists of threestructurally related components, A, B, and C (30, 32). BCG85A is identical to P32 (9). The~gene for a antigen was firstcloned by us from Mycobacterium bovis BCG (18) andMycobacterium kansasii (17). The analogous gene was re-cently cloned from Mycobacterium leprae (16, 28). Thegenes for the 85A component were cloned from M. tubercu-losis (4) and M. bovis BCG (10). The gene for the 85Ccomponent was cloned from M. tuberculosis (8).

Since antigen 85 complex is produced early in the growthphase (3) and in large amounts (20 to 30%), it may play animportant physiological role in immunopathology. Indeed,these proteins are a major stimulant of cellular and humoralimmunity (2, 8, 29, 31); binding to fibronectin (1, 21, 28) andthe induction of gamma interferon synthesis (13, 14) havebeen reported.The Mycobacterium avium-Mycobacterium intracellulare

complex was rarely identified as a cause of severe infectionsin humans until recently (6, 7, 33). However, it has recentlybeen recognized that the M. avium-M. intracellulare com-plex is a frequent opportunistic pathogen in patients withAIDS (5, 19). In the present study, cloning, sequencing, andB-cell epitope mapping of the gene encoding a antigensecreted from M. avium (A-a) were performed.

MATERIALS AND METHODSBacterial strains and plasmids. The bacterial strains and

plasmids used are presented in Table 1. M. avium ATCC15769 was grown in Sauton medium (24). Escherichia coliK-12 strain JM109 was used as a host for plasmids pUC18,pUC19, and their derivatives, and strain EQ192 was used asa host for plasmids pUR291, pUR278S, pUR289S, and their

* Corresponding author.

derivatives. E. coli JM109 and EQ192 were grown in theNZYM medium (1% NZ amine, 0.5% yeast extract, 0.5%NaCl, and 0.2% MgSO4. 7H20) (23).Media and reagents. NZYM medium and NZYM plates

containing 100 jig of ampicillin per ml were used for growingE. coli K-12 strains. All enzymes, pUC18 and pUC19vectors, the nick translation kit, the 7-deaza sequencing kit,and the DNA ligation kit were purchased from Takara ShuzoCo., Ltd. (Kyoto, Japan). [a- P]dCTP and the ECL direct

nucleic acid labelling and detection system were from Am-arsham Japan Co., Ltd. (Tokyo, Japan). Peroxidase-conju-gated swine anti-rabbit immunoglobulin was purchased fromDakopatts Co., Ltd. (Glostrup, Denmark).Amino acid sequencing. A-a was purified from M. avium as

described previously (25). About 30 ,ug of purified A-a wasanalyzed by a model 470A gas-phase protein sequencer(Applied Biosystems, Foster City, Calif.), and the N-termi-nal 18-amino-acid sequence was determined.DNA technology. Unless otherwise stated, standard pro-

cedures were used for the preparation and handling of DNA(23).

Preparation of probes. As nucleotide probes, two parts ofthe a antigen gene of M. bovis BCG were used. One was a0.7-kbp PstI fragment containing the N-terminal end of thegene (probe A). The other was a 0.3-kbp XhoI-PstI fragmentcontaining the C-terminal end of that gene (probe B) (18).For genomic Southern hybridization, probes were labelledby using an ECL direct nucleic acid labelling system asdescribed in the manufacturer's instructions. For colonyhybridization, approximately 1 ,ug of fragments was nicktranslated by using a nick translation kit and [a-32P]dCTP(3,00OCi/mmol) as described previously (18).Genomic Southern hybridization. M. avium DNA was

prepared as described previously (24). SphI digests of ap-proximately 1.0 ,ug of genomic DNA were loaded onto an0.8% agarose gel overnight at 15 mA and transferred toGeneScreen Plus (NEN Research Products, Boston, Mass.)for Southern blotting. Hybridizations were accomplished byusing an ECL direct nucleic acid labelling and detectionsystem as described in the manufacturer's instructions.

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TABLE 1. Bacterial strains and plasmids

Strains and plasmids Relevant properties Source or reference

StrainsM. avium ATCC 15769 Tasaka et al. (25)E. coli K-12JM109 recAl endA41 gyrA96 thi hsdRl7 supE44 relAl (lac-proAB)/F'[traD36 Sambrook et al. (23)

proAB+ lacI lacZ M15]EQ192 supF F' M15 lacI lacZ Yamaguchi et al. (34)

PlasmidspUC18 and pUC19 Apr Yanisch-Perron et al. (35)pAASp56 pUC19 recombinanta This studypAAKSpl pUC19 recombinant" This studypAABSpl pUC19 recombinant" This studypAAP08 pUC18 recombinant" This studypAAP200 pUC18 recombinant" This studypAAT5 pUC18 recombinant" This studypAAK2 pUC18 recombinant" This studypAA316 pUC18 recombinant" This studypAA519 pUC18 recombinante This studypAAS300 pUC18 recombinant" This studypAABR250 pUC18 recombinant" This studypAAESc800 pUC18 recombinant" This studypUR291 and pUR292 Apr Ruther and Muller-Hill (22)pUR278S and pUR289S Apr Yamaguchi et al. (34)pMAAl pUR289S recombinant" This studypMAA2 pUR278S recombinant" This studypMAA3 pUR291 recombinant" This studypMAA4 pUR291 recombinant" This studypMAAS pUR289S recombinant" This studypMAA6 pUR278S recombinant" This studypMAA7 pUR291 recombinant" This studypMAA8 pUR291 recombinant" This studypMAA9 pUR291 recombinant" This studypMAA10 pUR291 recombinant" This studypMAAll pUR292 recombinant" This studypMAA12 pUR292 recombinant" This studypMAA13 pUR278S recombinant" This studypMAA14 pUR278S recombinant" This studypMAA91 pMAA9 mutant This studypMAA92 pMAA9 mutant This studya Contains a 5.0-kb M. avium fragment comprising the a-antigen gene.bContains part of the a-antigen gene.

DNA cloning. The SphI fragnents with lengths of 5.6 kbpwere isolated from a preparative 0.8% agarose gel by usingDE81 papers, cloned into pUC19, and transformed into E.coli JM109 competent cells. Ampicillin-resistant transfor-mants were screened by the colony hybridization techniquewith nick-translated probes A and B.DNA sequencing. The sequencing strategy is represented

in Fig. 3. PstI fragments from the mycobacterial DNA insertwere subcloned into the PstI site of pUC18. In the sameway, KpnI fragments were subcloned into the KpnI site,Sau3AI fragments were subcloned into the BamHI site,TthHB8I fragments were subcloned into the AccI site,KpnI-SphI fragments were subcloned into the KpnI-SphIsite, and BamHI-SphI fragments were subcloned into theBamHI-SphI site of pUC18 or pUC19. The nucleotide se-quences of these subcloned fragments were determined bythe dideoxy chain termination method (12). Additionally,synthetic primers were used so that the complete sequencingwas done by using overlapping fragments on both strands.

Construction of expression plasmids. To construct hybridplasmid pMAA1, the recombinant plasmid pAA316 wasdigested with Hinfl and PstI. The 218-bp Hinfl-PstI frag-ment was blunted and subcloned into the SmaI site of vectorpUR289S. To construct pMAA2, the recombinant plasmid

pAA5l9 was digested with HindIII-EcoRI. The 322-bp frag-ment was blunted and subcloned into the SmaI site ofpUR278S. Recombinant plasmid pMAA3 was constructedby subcloning the 0.8-kbp PstI-PstI fragment of pAAP08into the PstI site of pUR291. The hybrid plasmid pMAA4was constructed by subcloning the 374-bp BamHI-BamHIfragment from pAAP08 into the BamHI site of pUR291.The 214-bp PstI-KpnI fragment of pAAP08 was blunted andligated into the SmaI site of pUR289S, resulting in plasmidpMAA5. The 136-bp KpnI-BamHI fragment from pAAP08was blunted and ligated into the SmaI site of pUR278S,resulting in plasmid pMAA6. Recombinant plasmid pMAA7was constructed by subcloning the 451-bp BamHI-PstIfragment of pAAP08 into the BamHI-PstI site of pUR291.To construct plasmid pMAA8, the recombinant plasmidpAABSpl was digested with BamHI-HindIII. The 0.95-kbp fragment was subcloned into the BamHI-HindIII siteof pUR291. The 0.2-kbp BamHI-HindIII fragment frompAAP200 was subcloned into the BamHI-HindIII site ofpUR291, resulting in plasmid pMAA9. The 243-bp BamHI-RsaI fragment from pAAP08 was subcloned into the BamHI-HincII site of pUC18, giving rise to pAABR250. The result-ing plasmid was digested with BamHI-HindIII, generating a264-bp BamHI-HindIII fragment which was subcloned into

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the BamHI-HindIII site of pUR291, giving rise to pMAA10.Recombinant plasmid pMAA12 was constructed by sub-cloning the 161-bp Sau3AI-HindIII fragment of pAABR250into the BamHI-HindIII site of pUR292. The 800-bp EcoRI-ScaI fragment from pAAP08 was subcloned into theEcoRI-HincII site of pUC18, giving rise to pAAESc800.To construct plasmid pMAA13, the recombinant plasmidpAAES-c800 was digested with RsaI-HindIII. The 165-bpRsaI-HindIII fragment was subcloned into the SmaI-HindIIIsite of pUR278S. The 248-bp Sau3AI-PstI fragment frompAAP08 was subcloned into the BamHI-PstI site ofpUR292,resulting in plasmid pMAAll. The 40-bp ScaI-PstI fragmentfrom pAAP08 was subcloned into the SmaI-PstI site ofpUR278S, resulting in plasmid pMAA14.

Construction of pMAA9 mutants. Oligonucleotides weresynthesized automatically with a DNA synthesizer (model380A; Applied Biosystems) and purified by two cycles ofreverse-phase liquid chromatography. The sequences encod-ing 318-LeuGlnGlyThr LeuGlyAlaSerArgGlyGlyGlyGly-330were 5'-GGGCACGCTGGGCGCGTCCCGGGGCGGCGGCGGATA-3' for the upper strand and 5'-AGCITATCCGCCGCCGCCCCGGGACGCGCCCAGCGTGCCCTGCA-3' forthe lower strand, and those encoding 318-LeuGlnGlyThrLeuGlyAlaSer-325 were 5'-GGGCACGCTGGGCGCGTCCTA-3' for the upper strand and 5'-AGC`llAGGACGCGCCCAGCGTGCCCTGCA-3' for the lower strand. Those sequenceswere cloned into the PstI-HindIII site of the pUR291 vector.The resulting plasmids were named pMAA91 (the formersequences) and pMAA92 (the latter sequences), respec-tively.

Production of recombinants. The expression of lacZ-A-ahybrid genes from plasmids pMAAl-14 and pMAA91-92were carried out by transforming the plasmids into E. coliEQ192. The cells harboring plasmids were cultured in 10 mlofNZYM broth containing 50 ,ug of ampicillin per ml at 37°Cto an optical density at 600 nm of 0.6, isopropyl-p-D-thiogalactopyranoside was added at 1 mM, and an additional1.5 h of incubation followed. After cells were harvested andsonicated, the lysates were obtained by centrifugation. Theproduction of proteins was analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Gels containing 12.5% acrylamide were used. Western blot-ting (immunoblotting) was carried out as previously de-scribed (18).

Immunological techniques. Polyclonal antibodies raisedagainst A-a, a antigen of M. kansasii (K-a), and T-a wereprepared as previously described (26). The absorbed anti-A-a serum was prepared by absorption with the samevolume of partially purified T-a (26). Contaminating reactiv-ities to bacterial antigens were removed from sera by ab-sorption with bacterial cells lysed by boiling.Mapping of B-cell epitopes. The reactivities of unabsorbed

and absorbed anti-A-a sera with various LacZ-A-a fusionproteins and LacZ-mutated A-a fusion proteins were deter-mined by Western blot analysis (18). In the same way, thereactivities of anti-K-a and anti-T-a sera with pMAA2 andpMAA7-14 were deternined.

Nucleotide sequence accession number. The nucleotidesequence of the A-a gene has been entered at the EMBLdata library under accession number X63437.

RESULTSN-terminal amino acid sequence. The N-terminal amino

acid sequence of A-a is given in Fig. 1. The first 15 aminoacids were completely identical to the known N-terminal

5 10Phe X Arg Pro Gly Leu Pro Val Glu Tyr

Pro Tyr

15Leu Gln Val Pro Ser Ala Gly Met

FIG. 1. N-terminal amino acid sequence of A-a. The secondamino acid residue (indicated by X) could not be determined. Thethird and fifth residues were Arg or Pro and Gly or Tyr, respectively.

regions of the a antigens (antigen 85 complex B componentsof mycobacteria) of M. bovis BCG (18) and M. leprae (16,28) and of K-a (17). They were also identical to antigen 85complexA components ofM. tuberculosis or M. bovis BCG(4, 10) and antigen 85 complex C components of M. tuber-culosis (8).

Southern hybridization analysis. Genomic Southern hy-bridization was carried out with either probe A or probe B.We obtained three SphI-digested DNA fragments (5.6, 4.9,and 3.6 kbp) clearly hybridized with both probes A and B.Two additional weak bands (8.4 and 6.7 kbp) of lower opticaldensity were seen with probe A but not with probe B (Fig.2). The 5.6-kbp fragments which most strongly hybridizedwith both probes A and B were cloned and used for furtherstudy. The location of the a gene is shown with a restrictionmap in Fig. 3.

Nucleotide and amino acid sequencing. The 1,236-nucle-otide sequence and the deduced 330-amino-acid sequenceare shown in Fig. 4. The DNA sequence contained the990-bp open reading frame, starting with an ATG codon atpositions 1 to 3 and ending with a TAA codon at positions991 to 993. The N-terminal amino acid sequence of A-a (Fig.1) was found to be identical to that deduced from thenucleotide sequence beginning with the TTT codon at posi-tion 121. The DNA sequence for the first 40 codons wasexpected to encode a signal sequence, and the matureprotein must consist of 290 amino acids. Its theoreticalmolecular mass was calculated to be 30,811 Da. A putativeShine-Dalgarmo sequence AAGG (-11 to -8) was located 8

1 2

23.1 _

9.42_6.56-

4.36. ,

-WA

2.32..

2.032.

FIG. 2. Hybridization patterns with probes A (lane 1) and B (lane2). M. avium DNA was digested with SphI. The numbers at the leftindicate sizes of standard DNA fragments in kilobase pairs.

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1176 OHARA ET AL. INFECT. IMMUN.

S TH S S S TP

I 11 I 11I I

p A A S p 5 6 Is g n a IS 300 >316. >

Pr i-l - -->-r 1Q

B

TS K T SS ST S

1 antigen111 Iaz a nt i g e n

P TT

gen1e

_S300 P08.............

P r -4 ...........Pr i-5 KS p 1

K2-<-... BS p1Pr_-T5 Pr i-8

P r -7 <.................. P r i-3P r i-2

I P200l < l~~~~~~~~~~~~~~~0

1 00bp T 5

FIG. 3. DNA sequencing strategy. Arrows indicate direction and length of sequence determination. Solid or dotted arrows indicatesequencing with M13 or oligonucleotide primers, respectively. Restriction sites: B, BamHI; H, Hinfl; K, KpnI; P, PstI; S, Sau3AI; Sp, SphI;T, TthHB8I.

nucleotides upstream of the initiation codon ATG (positions1 to 3). The sequences TCGACA (-77 to -72) and TAAATT(-53 to -48), which closely resembled the -10 and -35regions for E. coli promoters, were observed with 18-bpspacing. Promoter-like sequences and the Shine-Dalgarnosequence were identical with those of M. leprae (16, 28).Inverted remnants of transcriptional termination were seen.As Fig. 5 shows, alignments of the deduced amino acid

sequence of A-a with the amino acid sequences of previ-ously described mycobacterial a antigens (B component ofantigen 85 complex) exhibited 86, 82, and 81% identity withK-al and the ac antigens of M. bovis BCG and M. leprae,respectively. The A-a sequence also exhibited 64 and 72%homology with the A and C components of antigen 85complex of M. bovis BCG or M. tuberculosis. There wereextensive areas of amino acid sequence which were con-served throughout all four a antigens. However, there wereseveral regions where A-ae showed divergence from the othera antigens, most notably the carboxyl terminus (Gly-320 toGly-330) and interior region (Ser-147 to Asp-181).Mapping of B-cell epitopes on A-a. To localize the B-cell

epitopes of A-a, a series of deletion constructs were pro-duced from pAASp56 by using restriction enzymes whichallowed expression of A-ao sequences in transformed E. colicells (Fig. 6). The resultant recombinants were analyzed bySDS-PAGE and Western blotting with both unabsorbed andabsorbed sera raised against the purified A-a, and the resultsare presented in Fig. 7. The unabsorbed serum detected thefusion proteins coded by plasmids pMAA2-5 and pMAA7-9(Fig. 8), whereas the absorbed serum detected only thefusion proteins coded by plasmids pMAA3 and pMAA7-9(Fig. 9). These results indicated that the M. avium proteincarries at least two epitopes specific for M. avium in theGly-169 to Gln-319 and Leu-318 to Gly-330 amino acidsequences. Both anti-K-a and anti-T-at sera detected thefusion proteins coded by plasmids pMAA2, pMAA7, andpMAA8 but not that of pMAA9 (data not shown). Theseresults indicated that A-ao protein also carries at least twoB-cell epitopes in the Leu-51 to Tyr-123 and Gly-169 toGln-319 amino acid sequences. Further study of the regionbetween Gly-169 and Gln-319 was carried out. Both anti-K-aand anti-T-a sera detected the fusion proteins coded by

CGCCCGACAGCTAGCGGGCGGCGAAATTGCGAGGTGAAAACCTGC

GAGACGAAAAGAGATGCGAAACCGAGACTCGTTCGCGTCCCAATCGACATTTGACGGACT-35

CACACGGTAAATTCCTGTGAGACCACGATCACATGCGACCACATCCGACAAGGGCAGGT-10 SD

10 20 30 40 50 60ATGACAGATCTGAGCGAGAAGGTCCGGGCCTGGGGGCGCCGGCTTTTGGTCGGCGCAGCGMetThrAspLeuSerGluLysValArgAlaTrpGlyArgArgLeuLeuValGlyAlaAla

70 80 90 100 110 120GCGGCTGTAACCCTGCCGGGCCTGATCGGTCTTGCCGGCGGCGCGGCGACCGCGAATGCGAlaAlaValThrLeuProGlyLeul leGlyLeuAlaGlyGlyAlaAlaThrAlaAsnAla

130 140 150 160 170 180TTTTCGCGTCCGGGCCTGCCCGTCGAGTACCTGCAGGTGCCCTCCGCCGGAATGGGCCGCPheSerArgProGlyLeuProValGluTyrLeuGlnValProSerAlaGlyMetGlyArg

190 200 210 220 230 240GACATCAAGGTCCAGTTCCAGAGCGGCGGCAACGGCTCCCCCGCGGTGTATCTGCTGGACAspI leLysValGlnPheGlnSerGlyGlyAsnGlySerProAlaValTyrLeuLeuAsp

250 260 270 280 290 300GGCCTGCGGGCTCAGGACGACTACAACGGCTGGGACATCAACACCCCGGCCTTCGAGTGGGlyLeuArgAlaGlnAspAspTyrAsnGlyTrpAspI leAsnThrProAlaPheGluTrp

310 320 330 340 350 360TACTACCAGTCCGGCCTGTCGGTGATCATGCCCGTCGGCGGACAGTCCAGCTTCTACGCGTyrTyrGlnSerGlyLeuSerVal I leMetProValGlyGlyGlnSerSerPheTyrAla

370 380 390 400 410 420GACTGGTACCAGCCCGCGTGCGGCAAGGCCGGTTGCTCCACCTATAAGTGGGAGACCTTCAspTrpTyrGlnProAlaCysGlyLysAlaGlyCysSerThrTyrLysTrpGluThrPhe

430 440 450 460 470 480CTGACCAGCGAGCTGCCGTCGTACCTGGCCTCCAACAAGGGTGTGAAGCGCACCGGCAACLeuThrSerGluLeuProSerTyrLeuAlaSerAsnLysGlyValLysArgThrGlyAsn

490 500 510 520 530 540GCCGCAGTCGGCATCTCGATGTCCGGATCCTCGGCGATGATCCTGGCCGTCAACCATCCCAlaAlaValGlyIleSerMetSerGlySerSerAlaMetIleLeuAlaValAsnHisPro

550 560 570 580 590 600GACCAATTCATCTATGCCGGATCGCTCTCGGCCCTGCTCGACCCGTCCCAGGGCATGGGGAspGlnPheI leTyrAlaGlySerLeuSerAlaLeuLeuAspProSerGlnGlyMetGly

610 620 630 640 650 660CCGTCGCTGATCGGTCTGGCGATGGGTGACGCCGGCGGCTACAAGGCCGACGCCATGTGGProSerLeuI leGlyLeuAlaMetGlyAspAlaGlyGlyTyrLysAlaAspAlaMetTrp

670 600 690 700 710 720GGCCCGTCCAGCGACCCGGCCTGGCAGCGCAACGACCCGAGCCTGCACATCCCCGAGCTGGlyProSerSerAspProAlaTrpGlnArgAsnAspProSerLeuHisI leProGluLeu

730 740 750 760 770 780GTCGGCCACAACACCCGGCTGTGGCTGTACTGCGGTAACGGGACACCGTCGGAGCTGGGTValGlyHisAsnThrArgLeuTrpLeuTyrCysGlyAsnGlyThrProSerGluLeuGly

790 800 810 820 830 840GGCGCCAACATGCCCGCCGAGTTCCTGGAGAACTTCGTGCGCAGCAGCAACCTGAAGTTCGlyAlaAsnMetProAlaGluPheLeuGluAsnPheValArgSerSerAsnLeuLysPhe

850 860 870 880 890 900CAGGACGCCTACAACGGCGCCGGCGGCCACAACGCCGTGTTCAACTTCAACGCCAACGGAGlnAspAlaTyrAsnGlyAlaGlyGlyHisAsnAlaValPheAsnPheAsnAlaAsnGly

910 920 930 940 95U 960ACGCACAGCTGGGAGTACTGGGGAGCCCAGCTCAACGCCATGAAGCCCGACCTGCAGGGCThrHisSerTrpGluTyrTrpGlyAlaGlnLeuAsnAlaMetLysProAspLeuGlnGly

970 980 990ACCCTGGGCGCGTCCCCGGGCGGCGGCGGATAACCCCTCGACCGCTCGCGGCCACTACTGThrLeuGlyAlaSerProGlyGI-GlyGly***

CfCTCGACGATGACACCGTCGCGGCGCAGTAGTGCTGTTGCGGCCCCGACCG_- t

FIG. 4. Nucleotide and deduced amino acid sequences of A-ao.The potential promoter region and the Shine-Dalgarno sequence areunderlined. Arrows indicate palindromes.

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SEQUENCING OF GENE FOR a ANTIGEN FROM M. AVIUM 1177

Ma.awn HTDLSE[VRAWGRRLLVGAAAAVTLPGLIGLAGGAATANAFSRPGLPVEALQAPSAGNGR 60M.be .1.V.G.1.... T-...T...5ES...At.bovisBCG ...V.1.... .. .. G..G PS...M. ki,sa ... 1. ..AA ..G.I A...

M.BAian DIKVQFQSGGNGSPAVYLLDGLRAQDDYNGWDINTPAFEWYYQSGLSVIHPVGGQSSFYA 120M.as T .. .S SM. boWsBCG ........... PIV. sM. kisaW S D S

A. avium DWYQPACGKAGCSTYKVETFLTSELPSYLASN[GVIRTGNAAVGISNSGSSAMILASNHP 180M.bAe ... S.. .T.SA.RS. S..S.V..L. L..L AS..M.boviBCG ...S.. .Q L QW.SA.RA..P..S..I.L..A. AY.M.k&aW ...S T.QV.SA.RS..P..S. A. . L..S.Y.

M. avian DQFIYAGSLSALLDPSQGHGPSLIGLAHGDAGGYKADAHWGPSSDPAWQRSDPSLHIPEL 240M.are .. S.. .............. AD PP.. I.QAG.M. bovbs BCG .. AD.. E TQQA ..

M._ks Q. H.SD.

M. aviuw SGANTRLALYCGNGTPSELGGANNPAEFLENFVRSSNLKFQDAYNGAGGHNAFNFPNANG 300M.biore AN. .h V. T.. HG .. . .D.M.bovsBCG . .. I...4 KP. PP..M. khlsaUi AN. I. V.. A. LD .

M. avitan THSWEYWGAQLNAMKPDLQGTLGASPGGGG* 330M. swe . ................... NT.H.V.RS.-*M.bovisCG Y...G. SS.. .G-----*M. kwasi YC.. .. AS. R-----*

FIG. 5. Comparison of amino acid sequences of a antigens ofmycobacteria. Identical residues are represented by dots, and gapsthat had to be introduced to maximize sequence alignment areindicated by dashes. The arrow indicates the beginning of themature A-a protein.

plasmids pMAA10 (Gly-169 to Tyr-250) and pMAA13 (Gly-252 to Gln-305) but not that of pMAA12 (Ile-204 to Tyr-250).The conclusion was drawn that there were at least twoepitopes common to M. kansasii and M. tuberculosis in theGly-169 to Leu-203 and Gly-252 to Gln-305 amino acidsequences. Although both unabsorbed and absorbed seradetected the fusion proteins coded by plasmids pMAA10 andpMAA13, the extent of the reaction with absorbed serum

K

pAASp56 ~f

pAAKSplpAABSp1/pMAA8pAAP0 8/pMAA3pAAP200/pMAA9pAAT5pAAK 2pAA31 6pAA519/pMAA2pAAS300pMAA 1pMAA4pMAA 5pMAA 6pMAA 7pAABR250/pMAA10pMAAllpMAA12pM AA 13pM AA 14

was less than that with unabsorbed serum. The plasmidpMAA12 (Ile-204 to Tyr-250) did not react with both sera.All of these data combined with the conclusion describedabove suggested that each region might contain one moreepitope unrelated to M. kansasii and M. tuberculosis.

DISCUSSION

Three closely related immunodominant antigens, 85A, -B,and -C, with molecular masses of 32, 30, and 33 kDa,respectively, are known in M. bovis BCG and M. tubercu-losis. DNA analysis revealed that these antigens constitute agene family. Among them, antigen 85B corresponds to the aantigen. To clone the gene encoding A-a, Southern hybrid-ization analysis was performed with two kinds of probes.The probe DNA encoding the C-terminal end of the matureprotein produced three bands, while the probe containingDNA for the N-terminal end of the mature protein producedfive hybridization bands. This suggested that the antigen 85complex of M. avium might consist of more than three, andprobably five, structurally related components. We havebeen unable to identify more than three bands with otherspecies of mycobacteria (i.e., M. bovis BCG, M. kansasii,M. intracellulare, M. szulgai, M. simiae, M. scrofulaceum,and M. mannum) examined to date. However, the consid-erations described above for M. avium do not seem to beexceptional. The finding that the 27-kDa MTP51 proteinfrom M. tuberculosis immunologically cross-reacted with allthree components of the antigen 85 complex and that theN-terminal amino acid sequences of MTP51 and T-ot showed60% homology (20) suggested that M. tuberculosis mightcontain more than three components of antigen 85 complex.The deduced N-terminal amino acid sequence of A-a was

R R BTTHSS STP TSRK TSSS

1111 11 II

11 111111

Signal

R ScPTT

Il1 iSp

Mature protein

II

500bpFIG. 6. Physical maps of recombinant plasmids. The open brackets of pAASp56 represent vector DNA. The position of the A-a gene is

indicated by a solid bar. Transcription of the gene is from left to right. Restriction sites: B, BamHI; H, Hinfl; K, Kpnl; P, PstI; R, RsaI; S,Sau3AI; Sc, ScaI; Sp, SphI; T, TthHB8I.

I -I

I I

H

aI

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1178 OHARA ET AL.

S S RP RSRK RSB SS R ScP

I1 IVIIII I11 40 330signal mature protein

lacZ 1 52...... --22 123

51 319- .................-51 170

51 123-.-.-

123 170

169 319----------------------- mms

169 330------------------------------------.------------------------ --

318 330-- ---------- -------- ------------ _''''''''''''''''

169 250

204 319........................................... -

204 250l ---

252 305..............-.........M

3073 19

Reactiv ty with anti-A- a serum

unabsorbed absorbed

pMAA 1 - -

pMAA2 + -

pMAA 3 + +

pMAA 4 +

pMAA 5 +

pMAA6 -

pMAA 7 +

pMAA 8 +

pMAA9 +

pMAA 10 +

pMAA 11 +

pMAA 12 -

pMAA 13 +

pMAA 14 -

+++++

+

1 2 3 4 5 6 7 8 9 10 11

9;4-67..

30-

FIG. 9. Immunoblot analysis of A-a fusion proteins expressed inE. coli reacted with absorbed anti-A-a serum. Lanes: 1, sizemarkers; 2, pUR291 (control); 3, pMAA1; 4, pMAA2; 5, pMAA3; 6,pMAA4; 7, pMAA5; 8, pMAA6; 9, pMAA7; 10, pMAA8; 11,pMAA9. Numbers at the left indicate molecular mass (in kilodal-tons).

- 0.5 kb

FIG. 7. Physical maps of recombinant plasmids expressing var-ious regions of the A-a gene and reactivity of unabsorbed andabsorbed anti-A-a sera to proteins expressed by these plasmids.Transcription of the gene is from left to right. (Left) =, lacZ partof recombinant DNA; M, signal peptide part of A-a gene; _,mature protein part of A-a gene. The numbers indicate the aminoacid residues in the A-a protein sequence which delimit the dele-tions. (Right) The reactivities of unabsorbed and absorbed anti-A-asera to total protein from induced EQ192 cells harboring variousplasmids, established by Western blot analysis. Restriction sites: B,BamHI; K, KpnI; P, PstI; R, RsaI; S, Sau3AI; Sc, ScaI.

identical to that of A-a purified from the culture filtrate ofM.avium. A comparison of the amino acids of A-ca revealed ahigher sequence similarity to the at antigen (B component ofantigen 85 complex) from M. bovis BCG (82%) (18) than tothe A and C components from M. tuberculosis or M. bovisBCG (64 and 72%) (4, 8, 10). The A components from M.tuberculosis and from M. bovis BCG were identical exceptfor a silent single nucleotide change at position 1023 (10).Promoter-like sequences and the Shine-Dalgarno sequencewere absolutely identical with those of M. leprae. Howevercompared with other a-antigen species, fewer homologieswere seen at Ser-147 to Asp-181 and Gly-320 to Gly-330.On the basis of antiserum reactivity patterns with the

fusion proteins, the putative epitopes were mapped at sixdistinct regions. The amino acid regions Leu-51 to Tyr-123,Gly-169 to Leu-203, and Gly-252 to Glu-305 contained at

1 2 3 4 5 6 7 8 9 10 11

94-67 -

43-

30-

FIG. 8. Immunoblot analysis of A-a fusion proteins expressed inE. coli reacted with unabsorbed anti-A-a serum. Lanes: 1, sizemarkers; 2, pUR291 (control); 3, pMAA1; 4, pMAA2; 5, pMAA3; 6,pMAA4; 7, pMAA5; 8, pMAA6; 9, pMAA7; 10, pMAA8; 11,pMAA9. Numbers at the left indicate molecular mass (in kilodaltons).

least three epitopes which were common to M. tuberculosis,M. kansasii, and M. avium. Also, at least three epitopesunrelated to T-a were mapped on the Gly-169 to Leu-203,Gly-252 to Glu-305, and Leu-318 to Gly-330 amino acidsequences. The ax antigen is a cross-reacting antigen which iswidely distributed in mycobacteria. The cross-reactivitieswere previously examined by the agar gel diffusion tech-nique in detail (25-27). T-a and a antigen fromM. bovis BCGwere serologically identical, whereas the other mycobacteria(M. avium-M. intracellulare complex, M. scrofulaceum, M.gordonae, M. szulgai, M. kansasii, and M. marinum) pos-sessed common antigenic determinants and species-specificantigenic determinants. From these considerations, we candraw the conclusion that A-a may possess three species-specific epitopes. The carboxyl-terminal region, which wasprobably a species-specific epitope, has a Pro residue atposition 326, which predicted occasional involvement of a0-tum potential in an antigenic site (11). Pro-326 was re-placed with positively charged Arg (pMAA91), and thereactivity with the anti-A-ao serum was examined. The ex-pressed protein with the amino acid substitution resulted inlittle loss of recognition by the anti-A-a serum. A deletionmutant, which encoded 318-LeuGlnGlyThrLeuGlyAlaSer-325, was constructed (pMAA92) and expressed. The ex-pressed protein containing these amino acids resulted in norecognition of the anti-A-a serum. These results suggestedthat Gly-327 to Gly-330 required for the recognition by theserum.To our knowledge, this is the first report on the epitope

mapping of a component of mycobacterial antigen 85 com-plex. The precise mapping of B-cell and T-cell epitopes withsynthetic peptides is under way. It is hoped that thesestudies will be valuable in furthering the understanding ofprotective and pathologic immunities as well as in diagnosisand vaccine development.

ACKNOWLEDGMENTS

This work was partly supported by grants from the SasakawaMemorial Health Foundation, the Oyama Health Foundation, andthe U.S.-Japan Cooperative Medical Science Program.We thank K. Kobayashi for his valuable advice and discussions.

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