iNTERNAHONAL JOURNAI. ()F LIPROSY^

Volume 67, Number 4Printed in the U.S.A.

(ISSN 0145-916X)

lsolation, Characterization, Molecular Cloning andAmplification of a Species-Specific M. leprae Antigeni

Carlos Y. Soto, Pedro A. Moreno, James T. Valencia, Maria M. Bernal,Fanny Guzman, Manuel E. Patarroyo, and Luis A. Murillo7

Leprosy is an infectious disease causedby the acid-alcohol-resistant bacillus Mv-cobacterium 1cm-cie. Severe socioeconomicand psychological implications are assoei-ated with this disease (".'') which is a seri-ous health problem in tropical and subtropi-cal areas. Leprosy exhibits a wide spectrumof clinicai forms and immunological mani-festations, ranging from lepromatous to tu-berculoid extremes. Lepromatous leprosy,in contrast to tuberculoid leprosy, presents asignificantly suppressed cellular immuneresponse to M. leprae and therefore thebacillus multiplies freely. Also, leproma-tous patients present high circulating anti-body titers against severa' M. leprae anti-gens (I•21).

Although leprosy can be treated withchemotherapy, knowledge of the bacillus'individual components is a prerequisite inthe search for molecules of potential im-munoprophylactic or immunodiagnosticvalue (''). Because M. leprae cannot yet becultured in vitro, recombinant DNA tech-nology must be used to study its genome'smolecular structure, as well as the functionof specific genes. Many proteins with anti-genic characteristics have been identifiedfrom the recombinant libraries constructedby Young, et al. (") and Clark-Curtiss, et al.(2), some of them being unique to the M.leprae organism ()2). Potential polymerasechain reaction (PCR)-based diagnostic

' Received for publication on 4 January 1999. Ac-cepted for publication in revised form on 6 August1999.

C. Y. Soto, B.Sc.; P. A. Moreno, B.Sc.; J. T. Va-lencia, M.D.; M. M. Bernal, B.Sc.; F. Guzman, B.Sc.;M. E. Patarroyo, M.D.; L. A. Murillo, M.D., Institutode Inmunologia, Hospital San Juan de Dios, Universi-dad Nacional de Colombia, Carrea 10 calle 1, Santa fede Bogota, Colombia.

Reprint requests to Dr. Murillo at the above addressor FAX: 57-1-280-3999.

methods have been developed, includingthe detection of genes coding for the 18-kDa, 36-kDa and 65-kDa proteins, 16rRNA, and repetitive element (REP) se-quences

With the aim of finding antigens havingrelevance to the immune response, the pres-ent study has focused ou the search forgenes and proteins exclusive to M. lepraeusing polyclonal sem obtained from pa-tients with active, untreated lepromatousleprosy. Identification of a M. leprae chro-mosomal fragment isolated from a genomiclibrary constructed with bacilli of humanorigin is reported here. This sequence alsohas partial homology to a hypothetical pro-tein from the M. tuberculosis MTCY9C4cosmid (4) and comprises an epitope whichis recomtized by both lepromatous leprosyand heáthy tuberculosis contacts and acutetuberculosis patients' sera.

MATERIALS AND METHODSBacterial strains and vectors. The fol-

lowing mycobacterial strains were obtainedfrom the ATCC and Trudeau MycobacterialCollection (TMC): M. africamun (ATCC25420), M. avium (TMC 25291), Al. bovis(ATCC 19210), M. bovis BCG (ATCC27291 Pasteur substrain), M. chelonaesubsp. abcessus (ATCC 19977, 11758), M.chelonae subsp. chelonae (ATCC 35752),M. diernhoferi (TMC 2301), M. flavescens(ATCC 14744), M. fortuitum (ATCC 6841),M. gastri (TMC 1456), M. gordonae(ATCC 14470), M. intracellulare (TMC13950), Al. kansasii (TMC 1204), Al. mar-inum (ATCC 927), M. microti (ATCC35781), Al. nonchromogenicum (TMC19530), M. peregrinum (TMC 1547), M.phlei (ATCC 11758), M. scrofidaceum(TMC 19981), M. simiae (TMC 1595), M.smegmatis (ATCC 14468), M. szu/gai

392

67, 4^Soto, et al.: Species-Specific M. leprae Antigen^393

(TMC 1201), M. terrae (TMC 1450), M.triviale (TMC 1453), M. tuberculosis(ATCC 27294 strain H37Rv), M. tuberculo-sis (ATCC 25177 strain H37Ra), M. vaccae(TMC 1526) and M. xenopi (TMC 1470).Escherichia coll strains Y1088, Y1089,Y1090 and JM101 and the Xgt11 bacterio-phage were obtained from Amersham,Amersham, U.K. The phagemid vectorBluescript M 13 (+/—) was purchased fromStratagene, La Jolla, California, U.S.A.

M. leprae isolation. Bacilli were isolatedfrom lepromatous patients' lepromas fol-lowing the methodology previously de-scribed by Clark-Curtiss, et al.

Immunological analysis. On the basisof clinicai and histopathological criteria, anoutpatient from the Agua de Dio leprosycolony (Cundinamarca, Colombia) wasidentified. The patient was diagnosed assuffering from active lepromatous leprosyby routine bacteriological diagnostic tests,with the demonstration of acid-fast bacilli(AFB) in skin smears. The patient had notreceived multidrug therapy (MDT) at thetime. Two granis (wet weight) of lepromasand a blood sample were obtained. Thelepromas were frozen and transported forimmediate processing. The polyclonal an-tileprosy antiserum was pre-adsorbedagainst E. coli-Âgt1 1, as described by Sny-der, et al. (23), and an immunoblot using M.leprae sonicate as antigen (leprosin) wascarried out. Briefly, leprosin extract wasseparated by SDS-PAGE and transferredto a Hybond-N membrane (Amersham) asdescribed by Towbin, et al. (2(). The mem-brane was cut into two strips, each ofwhich was subsequently incubated with ei-ther antileprosy antiserum or a healthysubject's serum and detected with horse-radish peroxidase-conjugated anti-humanIgG (Promega Corporation, Madison, Wis-consin, U.S.A.) and H,0,-diaminobenzi-dine substrate (GIBCO BRL, Gaithers-burg, Maryland, U.S.A.).

Isolation of human and mycobacterialDNA. Human DNA was isolated from lym-phocytes and healthy skin cells by previ-ously described methods (14). M. lepraeDNA from human lepromas and other fast-and slow-growing mycobacterial DNAswere isolated as described previously (17).

M. leprae DNA isolated from armadillotissues, used in the Southern blot analysis,

were kindly provided by Dr. Patrick J.Brennan (Colorado State University, Col-lege of Medicine, Veterinary and Biomed-ical Sciences, Microbiology Department,Fort Collins, Colorado, U.S.A.).

Library construction and screening.An M. leprae genomic library was con-structed in the Xgt11 expression vector fol-lowing previously described methodology(17). The library was packaged in GigapackGold extracts (Stratagene) and amplified inE. coli Y1088. Antibody screening wasdone with a polyclonal antileprosy anti-serum (1:50 dilution) as previously de-scribed (12), using horseradish alkaline-phosphatase conjugated anti-IgG (Promega)and NBT-BCIP as substrates (IMMUNO-Select Kit; GIBCO BRL).

Restriction mapping, DNA sequenc-ing, and computer analysis. DNA was pu-rified from recombinant phage as previ-ously described by Manfioletti, et al. (13)•ML4- 1 insert in the Xgt11 bacteriophagetranscription direction was determined us-ing the Km/ I restriction enzyme (GIBCOBRL). The Xgt11 : ML4-1 clone insert wasexcised after digestion with Eco RI, as rec-ommended by manufacturer (GIBCOBRL), purified according to the Gene CleanII Kit protocol (Bio-101, Inc., La Jolla,California, U.S.A.) and cloned in phagemidBluescript SK+ II (Strategene). The result-ing construct was subsequently amplified inE. coli JM101; plasmid DNA was isolatedusing the maxi-prep methodology (1S) andrestriction mapping was done.

Unidirectional deletion generation wasperformed with the Erase-a-base system (18)in order to obtain the entire ML4-1 clone'ssequence. Single- and double-strand se-quencing (2") was done using a SequenaseV.2.0 kit (USB, Cleveland, Ohio, U.S.A.)and dye primers and dye terminators cyclesequencing, with Taq polymerase (Perkin-Elmer Applied Biosystems, Foster City,California, U.S.A.) on a 373 DNA sequencer(Perkin-Elmer Applied Biosystems). The re-action products were subjected to elec-trophoresis in 8% polyacrylamide gels, with7M urea.

Computer-assisted analysis was per-formed using GENEPRO (Genetics Com-puter Group, University of WisconsinBiotechnology Center, Madison, Wiscon-sin, U.S.A.), Staden (25) and Gene-Runner

394^ International Journal of Leprosy^ 1999

Version 3.0 copyright O 1994 (HastingsSoftware, Inc.) programs.

PCR primers and probe. The primersequences used in the PCR ampliticationcorrespond to nucleotides 114 to 131 and543 to 560. These were named LC1 andLC2 and the sequences are: GATCCT-GAACGAGAGGGA and GATCGACATT-GTCGGGAA, respectively. The oligonu-cleotides were synthesized in a Gene As-sembler equipment (Pharmacia, Upsala,Sweden), and puritied in a 20% polyacry-lamide gel (1").

PCR analysis of the amplified prod-ucts. Amplitications by PCR were per-formed as described elsewhere ('"). Brietly,50 t1 reaction mixtures containing 100 ngof purified mycobacterial DNA, 125 gMdNTPs, 20 pmol of each primer, 5 gl 10xPCR buffer (100 mM Tris-HC1 [pH 8.31,500 mM KC1, 1.5 mM MgCl„ 0.01%gelatin) (Perkin-Elmer Cetus, Insiruments,Norwalk, Connecticut, U.S.A.) and 5 U Taqpolymerase were used. Each cycle was car-ried out for 1 min at 95°C, 1 min at 60°Cand 2 min at 72°C for a total of 30 cycles.Samples were stored at 4°C in the thermalcycler until removed for further analysis.

Southern blot and hybridization.DNAs from different mycobacteria were di-gested with Eco RI enzyme and run on 1%agarose gel as described elsewhere ("). TheDNAs were transferred onto Hybond-Nmembranes (Amersham) by the alkalinemethod and hybridized using the ML4-1clone's 585 bp Eco RI-Bom HI fragment asprobe, labeled with [32P] dCTP by randompriming, using the Megaprime DNA label-ing system (Amersham) to a specific activ-ity of 10 cpm/gg DNA. Hybridization wasperformed for 16 hr at 65°C in a solutioncontaining 1 x SSC ( 1 x SSC is 150 mMNaC1 and 15 mM sodium citrate, pH 7.0),0.2% bovine serum albumin, 0.2% Ficoll,0.2% polyvinylpyrrolidine (PVP), 0.5%SDS, and 20 vig/m1 salmon sperm DNA.The filter was gradually washed at 65°C us-ing SSC from 1 x to 0.1x and 0.5% SDS.PCR amplification products with LC1 andLC2 primers were blotted and hybridized asdescribed above.

Antigen preparation. Based on theML4-1 protein sequence, 32 nonoverlap-ping peptides, 20 residues in length, werechemically synthesized using the Solid

Phase Multiple Peptide Synthesis Tech-nique. Once synthesized, the peptides wereextracted with 10% acetic acid and purifiedby HPLC. Peptides were divided into sixgroups of tive peptides each and one groupof two peptides only. A dilution of 10vtg/mL in PBS was prepared from each pep-tide group. The synthetic peptide groups'antigenic capacity was tested by ELISA us-ing sera from leprosy patients (4 leproma-tous leprosy), tuberculosis patients (6 acute,6 under treatment, 3 post-treatment and 5healthy contacts), malaria patients (2 Plas-modium falciparum, 5 P. vivax and 3 co-in-fected with P. vivax and P. falciparum), andsera from human newborn subjects (18samples). The last group was used as a con-trol group in order to determine the cut-offvalues in the ELISA.

Enzyme-linked immunoassay (ELISA).For the ELISA, 1 pg per well of each pep-tide group was added to the Micro-ELISAplates (Nunc, Roskilde, Denmark). After 1hr of incubation at 37°C and a furtherovernight incubation of plates at 4°C, thewells were washed three times with a wash-ing solution containing 0.2 g potassiumchloride, 8 g sodium chloride, 0.2 g potas-sium monobasic phosphate, 1.15 g sodiumdibasic phosphate and 0.5% Tween-20 perliter, pH 7.4. Then 200 p1 5% skim milk inwashing solution (blocking solution) wasadded and the plates were incubated for I hrat 37°C. Wells were then washed threetimes and incubated for 1 hr at 37°C withsera diluted 1:200 or 1:400 (for peptide221-240). The wells were subsequentlywashed tive times with washing solutionand incubated for I hr at 37°C with anti-hu-man IgG peroxidase conjugate diluted1:10,000 and reaction was developed with100 gl of the chromogen substrate mixture

tetramethyl benzidine and hydro-gen peroxide) being added per well. Fifteenmin !ater the reaction was stopped with 100

1 M phosphoric acid per well, and theabsorbances were read at 450 nm.

RESULTSRecognition by antileprosy antibodies.

A leprosin protein extract was evaluated byimmunoblot with both human antileprosyantisera and normal human serum. Threepredominant bands of the M. leprae extractwith molecular masses of 24, 29 and 47.5

67, 4^Soto, et al.: Species-Specific M. leprae Antigen^395

kDa were strongly recognized in the West-em analysis by the antileprosy antisera(Fig. 1).

M. leprae isolation, genomic libraryconstruction and screening. Two grams oflepromas (wet weight) were analyzed andshown to contam n AFB of +2(1 to 10 bacilliin 10 observed fields) without cellular de-bris. DNA extraction yielded approxi-mately 500 ng of M. leprae bacilliDNA/mg of leproma and 100 pg/mg fromthe other mycobacteria. A genomic librarywas constructed containing 109 pfu/i_tgDNA amplified in E. coli Y1088 to a finaltiter of 10 pfu/ml. The library comprised86% recombinants as assessed by aO-galactosidase color assay, with insertsizes ranging from 1 to 7 Kbp.

Ten filters, representing a total of 20,000plaques, were screened with the human an-tileprosy antiserum from the leprosy patientfrom whom lepromas were isolated. In thefirst round of screening two positive cloneswere detected. These remained positivethrough three further rounds of consecutivepurification and were later determined to beidentical. This clone, named Xgt11:ML4-1,was analyzed by digestion with Eco RI andshown to contam n an insert approximately1.9 Kpb long.

Sequence analysis of the ML4-1 frag-ment. The ML4-1 clone restriction map al-lowed the identification of unique recogni-tion sites for Sal 1, Kpin I and Bam HI en-zymes within the insert. The orientation inthe Xgt11 : ML4-1 clone was determined tobe in the Eco RI —> Bani HI direction. TheML4-1 fragment's complete sequence wasobtained by a combined strategy of sub-cloning and re-ligations as described in theMateriais and Methods section. The M. lep-me fragment's sequence in the ML4-1clone is shown in Figure 2. The 1932 bp se-quence's G+C base composition was calcu-lated to be 63.5%, and one important openreading frame (ORF) was found. This ORFseems to code for a protein of 644 aminoacids (72.3 kDa) with a 61% identity(132/218) to an M. tuberculosis H37Rvcosmid nucleotide sequence MTCY 09C4(4)•

Southern blot and PCR. As shown inFigure 3, a positive 1932 bp signal WaS ob-tained in the M. leprae genome digestedwith the Eco RI enzyme but not in any of

FIG. 1. Antileprosy antiseruni and normal serumreactivity against an Al. lepra(' leprosin extract in aWestern blot assay. Leprosin extract (100 lag per lane)was diluted in Laemmli buffer containing 2.5%13-mer-captoethanol, boiled for 5 min at 100°C and loadedonto a 7.5% SDS-polyacrylamide gel. Proteins werethen transferred to a nitrocellulose membrane whichwas cut into strips and incubated in a 1:50 dilutionwith either the antileprosy antiserum (lane I) orhealthy subject (lane 2) sent. An anti-human IgGhorseradish-peroxidase conjugate was used as a sec-ond antibody. Molecular weight markers are indicated.

the other 24 mycobacterial genomes tested.Also, from PCR amplifications, using theLC1 and LC2 primers, it was possible todetect a specific 446 bp fragment from M.leprae DNA; no amplification product wasobserved using either human DNA or anyof the other 23 mycobacterial genomicDNA samples (Fig. 4), indicating the exclu-sive presence of the 446 bp fragment in theM. leprae genome.

ELISA peptide assays. Ali syntheticpeptides were tested by groups and tater on

396^ International fournal o f Lepmsy^ 1999

^ORF1-0. EFG^FDMR VTR VSE^

HpRI^TKP^FSD ESEN A 1, 14 A LO1 GAATTCGGTT TcGATATGCG CGTGACGCGc GTATcCGAGG ACCCCCGCAT CACCAAGCCG TTTTCCGACG AATCCTGCJ\ A T GCT CGA'r ;C Gcrcu",c

E KVD AIL NER DVRL TMG GEE TFVS^

IDD FES GEWN101 AAAAGGTGGA CGCGATCCTG AAGGAGAGGG ATGTGCGCCT GACCATGGGC GGCCAGCCCA CATTCGTGTC CATCGATGAT TTCGAATCGG GCGAATGGAA

LC1^TI) A^VOE^TKRE^<AI)

^ELI^14kLR^ERF^AEG^';FLH^OGQ201 CACCGATGCG GTCGGGCCGA CCAAGCGCGA AAAGGCCGAc AAGGIGATCC CGCGGCTGCG CGAG,:GTTrT GCGCCGGGTG GTT=GCA TTACGGGCAG

G KW YEGE SLP RWT^FSLY WRK DOE P IWVNED LIA

701 GGCAAGTGGT ATCCGGGCGA GAGCCTGCCG CGCTGGACGT TCTCGCTTTA TTGGCGCAAG GACGGCAAGC CCATCTGGGT cAATeCcGAC CTGATCGCGC

pENG KAD VKP EDAG KLL VAL AO EL GVE EEM VAEA401 CGGAGAACGG CAAGGCTGAC GTCAAGCCGG AAGATGCCGG AAAACTCCTC GTCGCGTTGG CGCAGGAGCT TGGCGTTGAA GAAGAGATGG TGGCGCCCGC

^

YED^PAE^WI IK^EGN^

LED^NVDP^SNS^

REI<^DEEE^RS 14501 CTACGAAGAT CCGGCGGAAT GGATCATCAA GGAGGGCAAT CTTCCCGACA ATGTCGATCC ATCCAACTCC AGACTCAAGG ATCCGGAAGA GCGCAGCCGC

LC2^ Bam Hl

IAR VEER GLT EPS GFVL EVO RwN SOAS GER WRS601 ATCGCCCGGG TTTTCGAGCG GGGCCTGACC GAGCCAAGCG GTTTTGTCCT GCCGGTGCAG CGCTGGAACA GCCAGGCCTC CGGCCCGCGC TGGCGCAGCG

E K8K TER GKL FLVP GOS PVG ORLE LGA LPH VOAS701 AAAAATGGAA GACGCGGCGC GGCAAGCTGT TTCTGGTGCC GGGGGATTCA CCCGTCGGAT ACCGGCTGCC GCTCGGCGCC CTGCCGCATG TTCCGGCCTC

E yP YIV PVDPSVD RGA LEDO APL ARE NELA VÃS801 GGAATATCCC TATATCGTGC CGGTCGATCC GTCGGTGGAT CGCGGGGCGC TGCCGGACCC GGCACCGCTG GCGCGTGAGA ACATCCTGGC GGTTGCCTCT

FTA DEGN 00E RVE QELG EIG GAV RTAI SVE ERD901 TTCACTGCTG ATGAAGGCAA CCAGCAGGAG CGGGTCGAGC AGGAACTGGG GGAAATCGGC GGGGCCGTGC GCACCGCCAT ATCGGTCGAG CCGCGCGACG

G ELO VEM PPV EALE DO L ELV AAAE AAA HEL GLPV1001 GGCGTCTCAG CGTGTTCATG CCGCCGGTGG AGGCGCTCGA GGACTATCTG GAACTGGTTG CGGCGGCGGA AGCTGCGGCG CACAAGCTCG GGCTGCCGGT

H IE^GYS^PPOD HRI^

NVI^RVAP^DPI) VIE VNIH^PAA1101 cCATATCCAA GGTTACAGCC CGCCGCAGGA TCACCGCATC AACGTCATCC GCGTTGCACC CGATCCCGGC GTGATCGAGG TCAATATCCA TCCGCCCGCC

N WR^DCVA TTT AI Y EEAR QTR LGA DK FM^IDO REIT1201 AACTGGCGCG ACTGCGTGGC AACGACAACG GCAATCTATG AGGAGGCCGG CCAGAcGCGG CTCGGTGCCG ACAAGTTCAT GATCGATGGC CC,CCATACCG.,..

G TGG GNH VVV GGET^

EAN SPE LRRP OLL ESL VLHW1301 GTAccGGCGG CGGCAACCAT GTGGTGGTCG GCGGCGAAAC ACCGGCCAAC AGCCCGTTCC TGCGCCGTCC TCATCTGCTC AAAAGCCTTG TCCTGCACTG

Kpn IQ RH^PSL^SOLE^SOL

^FIG^ETSQ APR

^IDE AHHD^sLY

1401 GCAGCGGCAT CCATCGCTGT CCTACCTGTT CTCCGGCCTG TTCATCGGGC CGACCAGCCA GGCACCTCGC ATCGACGAGG CGCGGCACCA CAC,TCTCTAC

E LE^IALS OVE RPG EGTE ELO WLV ORLE RNL LVD1501 GAGCTGGACA TTGCCCTCTC GCAGGTGCCC CGGCCGGGCG AGGGCACACC GCCTTTGCCC TGGCTGGTCG

ACCGGCTGTT CCGCAATCTT TTGGTGGATG

Sal^ TGN^TI) R^SEI^GIDE

^LES^PDG^RTGR

^LGL VEF^RGFE

1601 TCACCGGCAA CACGCATCGT TCGGAAATCT GCATeGACAA GTTGTTCTCG CCCCACGGTC CGACCGGCAG ACTCGGGCTG GTCGAGTTGC GCGGTTTCGA

NAR MOLA OQL LIR ALIA RFW NNP^IOGS^FVR1701 GATGCCGCCC AATGCGCGCA TGAGCCTTGc CCAGCAATTG CTGATCCGCG CGCTGATTGC CCGGTTCTGG AACAATCCGA TTGAGGGCAG TTTCGTCCGC

W GT ALHO RFM LPA YVWQ DFL DVL ADLR ENG EDF1801 TGGGGCACGG CGCTGCATGA CCGTTTCATG CTGCCGGCCT ATGTTTGGCA GGATTTCCTC GATGTTCTcG CCGATTTGCG CGAGAACGGC TTTGATTTCC

RFEW^FEAOLEE1901 GCCCGGAATG GTTCGAGGCC CAGCTGGAAT TC

FIG. 2. Nucleotide sequence of ML4-1 genomic fragment and amino-acid sequences deduced from main ORF.ORF I and the Bom HI, Kim I and Sal 1 restriction sites are shown. Localization of LC I and LC2 primers are in-dicated.

individually. Peptides belonging to groupthree showed a marked pattern differencebetween distinct sera tested, as seen in TheTable. Peptide corresponding to position221-240 was predominantly recognized ei-ther by ali lepromatous leprosy patients orhealthy tuberculosis contact sera examined.Surprisingly, this peptide had a cut off pointhigher than the rest of the peptides, evenworking at a serum dilution of 1:400. Also,a Chow and Fasman analysis showed thatthis peptide has a predominant alpha helixand a hydrophilic pattern, indicating its po-tential antigenic capacity.

DISCUSSION

The identification and characterization ofM. leprae protective antigenic determinantsis important for two reasons. First, it is rel-evant for the identification of species-spe-cific sequences for the development of im-proved diagnostic methods and, secondly,for the ultimately more important goal ofdeveloping an effective vaccine (3")• In thisstudy, a recombinant DNA expression li-brary with M. lepme DNA isolated fromhuman lepromas was constructed, and itwas screened using human serum from a

67, 4^Soto, et al.: Species-Specific M. leprae Antigen^397

3^4^5^6^"^8^9 111^II^12 13 14^15 16 r^IN

1.9 Um)

19 20 21 22 23 24 25 26 27 28 29 30

4— 445

Fin. 3. Southern hybridization using ML4 - 1 Eco RI-Bani 1-11 585 bp fragment as a probe. ChromosomalDNAs from different mycobacteria were digested with Eco RI and hybridized with MIA - 1 Eco RI-Bam HI 585bp probe. Lanes: 1)M. avium, 2) M. bons, 3) M. bons BCG, 4)M. clzelonae subsp. abs., 5) M. chelonae subsp.(he., 6) M. diernhoferi, 7) M. fiavescens, 8) M. formitum, 9) MWM, I()) M. intracellulare, 12) M. kansasii, 13)

M. leprae, 14) M. mar u no, 15) M. nonchromogenicion, 16)M. peregrinum, 17) M. phiei, 18)M. scrofidaccum,19) M. simiac, 20) M. smegmatis, 21) M. szulgai, 22) M. ferroe, 23) M. triviale, 24) MWM, 25) M. tuberculosis1-137Rv, 26) M. vaccae, 27) M. xenopi, 28) human lymphocytes, 29) human skin, 20) positive control (446 bpPCR product).

lepromatous leprosy patient. A Âgt11 re-combinant clone was subsequently isolatedand sequenced, having a 1.932 bp with onemain open reading frame coding for a 72.3-kDa protein. Also, three predominant bandsof the M. lepme extract with molecularmasses of 24, 29 and 47.5 kDa werestrongly recognized in the Western blot

analysis by the antileprosy antisera whichcould be breakdown products of the puta-tive 72-kDa protein (Fig. 1).

The human-derived recombinant DNAlibrary described in this article may help toanswer some of the many outstanding ques-tions regarding M. lepme infection, such asthe role of polymorphism in drug resis-

445 bp

16^17^18^19^20^21^22^

23 24 25^26^

27 28 29 30

398^ International Journal Leprosy^ 1999

1^2^3^4^5^6^7^8^9^10^11^12^13^14^15

445 bp

FIG. 4. Hybridization of PCR amplification products using LC1 and LC2 pritners. Lanes: 1) I. ofricammi,2)M. avium, 3) M. bons, 4)M. bons BCG, 5) M. chelmme, 6)M. (liernheferi, 7) M. ferwitum, 8) MWM, 9) M.gastri, 10) M. intracellulare, 11) M. kansavii, 12) AI. leprae, 13) AI. marimmt, 14) AI. micro(,, 15) M.non(hr( megenicum, 16) AI. peregriman, 17) AI. phlei, 18) M. scrofiduceum, 19) M. smegmatis, 20) AI. szolgoi,21)M. terrae, 22) M. iriviale, 23) MWM. 24)M. mberculo.sis H37Ry, 25)M. tubereulosis 1137Ra, 26)M. xenopi,27) burilai) lymphocytes DNA, 28) hunian skin DNA, 29) positive control (pBS:ML4-1) and 30) negative con-trol (without DNA).

tance. Polymorphism has been found in dif-ferem geographical and host isolates' chro-mosomal DNA (3), but all recombinantDNA clones isolated to date have been ob-tained from genomic libraries constructedwith M. leprae grown in armadillo 33)•The levei of polymorphism present in hu-man-derived isolates is, therefore, com-pletely unknown and the use of recombi-nant libraries derived from leprosy patientswould allow the detection of alterations at

the genomic levei associated with resis-tance and, in addition, could provide probesfor drug-resistance markers.

Synthetic peptides derived from the en-tire sequence reveal an important epitope(amino acid position 221-240) recognizedby sera obtained from lepromatous leprosy,from healthy contacts and from acute tuber-culosis patients. By comparison, 6 out of 20amino acids are identical in the putative M.tuherculosis protein from the MTCY9C4

67, 4^Seio, et al.: Species -Specific M. leprae Antigo'^399

THE TABU. ELISA from group 3 individual peptides."

PeptideNo. positive sera tested for each patiem group-'

A C E G

201-220 0/4 0/5 0/6 0/6 0/3 0/5 0.125221-240 4/4 1/5 3/6 1/6 0/3 4/5 0.244241-260 1/4 0/5 0/6 0/6 0/3 0/5 0.149261-280 1/4 1/5 0/6 1/6 0/3 0/5 0.089281-300 1/4 1/5 0/6 0/6 0/3 0/5 0.115

'Patient grottps: A = Lepromatous leprosy; B = mataria; C = acme tuberculosis; D = mbereulosis under trem-mem; E = post-treatmem tiffieretdosis; E = hcalthy contacts of tuberculosist G = cut-off point.

cosmid, where two tryptophan, two argi-nine, one glycine and une serine are pre-served and could be implicated in the cross-reactivity between these mycobacterial spe-cies. Since hydrophilicity has beencorrelated with antigenicity ("), it is alsopossible that this region is the recognitionsite for the antileprosy antiserum antibod-ies. On the other hand, leprosy diagnosis isbased on clinicai and histopathological cri-teria, which means that it could be delayedby 2 or 3 years' post-infection. The lowmultiplication rate of the bacilli permits theroutine identification of acid-fast bacillionly in late post-infection stages. Moreover,IgG antibody presence against phenolicglycolipid-I (PGL-I) has been shown tohave low sensitivity ("). Thus, it is neces-sary to develop methodologies for routinediagnosis to facilitate the initiation ofchemotherapy and immunotherapy duringthe disease's early stages. The use of PCRfor infectious diseases' diagnosis is a pow-erful tool (`'), and many tests for the specificPCR identification of M. leprae exist(1". " 2`)..u). A new M. leprae species-spe-cific antigen is identified in this articlethrough the use of PCR and hybridizationstudies using different mycobacterialstrains. Although some nucleotide sequencehomology with an M. tuberculosis cosmidwas observed, there was not a positive sig-nal either by Southern blot or PCR analysis,probably due to the stringency conditionsused in both experiments and because the61% nucleotide sequence homology is lo-cated in the 3' end of the ML4-1 clone andthe sequence analyzed in the present studyis located in the 5' end. Therefore, thisunique genomic fragment could be pro-posed as a future candidate in the develop-

ment of a potentially specific diagnosticmethod for the detection of Al. leprae infec-tion.

SUMMARYA polyclonal serum sample from a lepro-

matous leprosy (LL) patient, which pre-sented a specific recognition pattern for lep-rosin, was used to screen a Mycobacteriumleprae genomic library constructed withDNA isolated from human lepromas. Oneclone, designated ML4-1, which expresseda specific antigenic determinant of AI. lep-ra(' as part of a I3-galactosidase fusion pro-tein, was isolated. The 1.932 bp M. leprae-derived genomic fragment was sequenced,and it had an incomplete open-readingframe shown to code for a 644 amino-acidpolypeptide (72.3 kDa). Some partial nu-cleotide homology to the Al. tuberculosisMTCY9C4 cosmid and the M. lepraeB1913 cosmid were found. Southern blotassays using the 584 bp Eco RI -Bani Hlfragment excised from the ML4-I clone re-vealed that this sequence is present only inthe Al. leprae genome and not in the 24 dif-ferent mycobacterial DNA tested. Twooligonucleotides based on the genomic se-quence were also synthesized and used asamplifiers for a polymerase chain reaction(PCR) test, giving a positive signal exclu-sively in AI. leprae DNA. Furthermore, 32sequential synthetic peptides, 20 amino-acids long, spanning the entire protein cor-responding to the hypothetical ML4-1 clonesequence, were synthesized and evaluatedby ELISA. A peptide included in the221-240 region was significantly recog-nized by either lepromatous leprosy orhealthy tuberculosis contact patient sent.Thus, PCR amplification of this fra,gment,

400^ International finava' of Leprosy^ 1999

along with the recognition of its protein se-quence by leprosy patient sera, could be auseful tool for a potential diagnosticmethod in the detection of M. leprae infec-tion in the future.

RESUMENSe utilizo un suem policlonal de un paciente con

lepra lepromatosa (1,1,) con un patrón de re-conOciiiilento especifico de la leprosina, para analizaruna biblioteca genóni ca de Myeobacterium lepra('

construida con el DNA aislado de lepromas humanos.Se aisló una dona, designada ML4- I , la cual expresóun determinante antigénico específico de M. Ieprae

como parte de una proteina de fusión con beta-galac-tosidasa. El fragmento genómico secuenciado de 1.932pares de bases (pb) de M. leprae tuvo un marco de lec-tura abierto incompleto que codificó para un polipép-tido de 644 aminoácidos (72.3 kDa) y mostro homolo-gias parciales con las secuencias de los cósmudosMTCY9C4 de M. fabereijosis y B1913 de M. leprac.

Los análisis de Southern blot usando el fragmento de584 pb Eco RI-Bani III obtenido de la dona ML4- Ireveló que esta secuencia sólo está presente en clgenoma de M. /eprue y no eu cl DNA de 24 diferentesmicobacterias probadas. También se sintetizaron dosoligonucleótidos hasados en la secuencia genómica 5,se usaron como iniciadores eu la prueba de la reaccióneu cadmia de la polimerasa (PCR). Sólo se obtuvoseital positiva con el DNA de M. leprae. Además, sesintetizaron 32 péptidos sintéticos secuenciales de 20aminoácidos de logitud, que abarcaron la j)roteinacompleta correspondicnte a la secuencia hipotética dela clona ML4- 1. Estos péptidos fueron evaluados porELISA. Un péptido incluido eu la región 221-240 fuereconocido de manera significativa por los sueros depacientes con lepra lepromatosa o tuberculosis per nopor los sueros de contados sanos. Asi, la amplificaciónpor PCR de este fragmento y el reconocimiento de susecuencia proteica por los sueros de los pacientes conlepra, podrian servir como herramientas de diagnósticopara detectar la infección por M. lepra(' en el futuro.

RÉSUMÉUn éclmantiilon de sérum polyclonal provenant d'un

patient soultrant de lèpre lépromateuse et qui exhihamtune reaction spécifique à la léprosine, fut utilisé pourtester une hibliothèque génomique construite à partird'ADN provenant de lépromes humains. II fut isolé undone, appelé ML4- 1. qui exprimait un déterminantantigénique spécifique de M. leprae fusionné à uneprotéine de (3-galactosidasc. Le fragment de génomedérivé de M. leprae, mesurant 1932 paires de bases, futséquencé: ii montrait un cadre de lecture ouvert in-complct codant mi polypeptide de 644 acides aminés(72.3 kilodaltons). Une homologie partielle deséquence fut trouvée avec le cosmide MTCY9C4dérivé de M. tuberculosis et le costnide B1913 dérivéde M. Ieprae. Une analyse par Southern blot, utmlisant

un fragment de M1,4-1 mesurant 584 paires de bases,obtenti par digestion avec les enzymcs de restrictionEco RI et Bani 1-11, a dénmontré que cette séquence n'é-tait rencontrée que dans le génome de M. /eproe et nonpas dans l'ADN de 24 atares mycobactéries testées.Deux oligonucléotides Invent égalernent synthétisés etutilisés pour amplilier le fragment MI.4-1, par la réac-tion de polymérase en chaine (PCR), mie donnant un ré-subia positif qu'en présence d'ADN de M. /eprae. Deplus, 32 oligopeptides artificieis de 20 acides aminésde long., qui mis bout à bout représentent la protéinehypothétique correspondant à la séquence du doneMI.4-1, furent synthétisés et évalués par ELISA. Unpeptide, inclu dans la région 221-240 de Ia protéinehypothétique, fut significativement reconnu par lessérums issus de patients lépromateux ou des personnesen contact avec des patients tuberculeux. Ainsi, l'am-plification par PCR de ce fragment génomique et la re-connaissance par le sérum de patients atteints de lèprede sa fraction protéique correspondante, pourrait êtreun outils intéressant et une méthode diagnostique po-tentielle pour Ia future détection de l'infection par M.

leprae.

Acknowledgment. We gratefully acknowledgeDoctors José Fernando Velásquez and Amoldo Bar-bosa for obtaining the 'eliminas and the blood sam-ples; Gloria Ampara Mejia for maintaining the myco-bacterial strain collection and for assistance with thebacilloscopies; Constanza Cardenas for technicztl as-sistance and flernan Flurtado, Ph.D., Director, Neuro-science Laboratory, Instituto Nacional de Salud, forhaving provided us with untreated lepromatous leprosypatients. This work was supported by the ColombianMinistry of I-lealth and the German Leprosy Relief As-sociation.

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