Volume 68. Number 2Prinied in d)r U.S.A.

(ISSN 0148916X)

IN I ERNAlIONAI. JOURNAL OF LEPROSY

INTERNATIONALJOURNAL OF LEPROSY

and Other Mycobacterial Diseases

VOLUME 68, NUMBER 2^ .ZUNE 2000

Mycobacterium leprae Typing by Genomic Diversityand Global Distribution of Genotypes 1

Masanori Matsuoka, Shinji Maeda, Masanori Kai, Noboru Nakata, Gue-Tae Chae,Thomas P. Gillis, Kazuo Kobayashi, Shinzo Izumi, and Yoshiko Kashiwabara 2

Leprosy is a chronic infectious diseasecaused by Mycobacterium leprae and is stilla major health problem in the developingcountries of Asia, Latin America and Africa( 25 ). Global efforts to control leprosy by in-tensive chemotherapy have led to a signifi-cant decrease in the number of registeredpatients; however, new cases reported an-nually remain more than 0.5 million. The

' Received for publication on 28 March 2000. Ac-cepted for publication in rcvised form on II May2000.

M. Matsuoka, Ph.D.; S. Maeda Ph.D.; M. Kai,Ph.D.; N. Nakata, Ph.D.; K. Kobayashi, M.D., Ph.D.;Y. Kashiwabara, Ph.D., Leprosy Research Center, Na-tional Institute of infectious Discases, 4-2-1 Aobacho,Higashimurayama-shi, Tokyo 189-0002, Japan. G.-T.Chae, M.D., Ph.D., Institute of Hansen's Disease, Col-lege of Medicine, The Catholic University of Korea,505 Banpo-Done, Socho-ku, Seoul 137-701, Korea. T.P. Gillis, Ph.D., Molecular Biology Department, Labo-ratory Research Branch, GWL Hansen's Disease Cen-ter at Louisiana State University, P.O. Box 25072, Ba-ton Rouge, LA 70894, U.S.A. S. Izumi, M.D., Ph.D.,National Hospital Oshima Seishoen, 6034-1 Ajicho,Kidagun, Kagawa, Japan. Present address for Dr.Kobayashi: Department of Microbiology, Osaka CityUniversity Medical School, 1-4-3 Asahi-machi,Abono-ku, Osaka 545-8585, Japan.

Reprint rcqucsts to Dr. Kashiwabara at the aboveaddress or FAX 81-42-391-8212; e-mail:y-kashi nih.go.jp

successful elimination of leprosy will likelydepend upon a multifactorial approach, in-cluding appropriatc chemotherapy, im-munoprophylaxis and chemoprophylaxis.Criticai to the success of any eliminationstrategy will be the identification and re-moval of the natural source of transmissionand infection.

It has been difficult to identify sources ofinfection because of the protracted incuba-tion period preceding clinicai disease andbecause M. leprae remains uncultivable onartificial media. These two factors haveslowed our understanding of the route oftransmission of M. leprae and have contin-ued to confound efforts to design strategieseffective in eradicating the natural reservoirof M. leprae which could lead to the globalelimination of this disease. Strain-specificmarkers could provide the necessary toolsfor understanding this aspect of the epi-demiology of leprosy.

M. leprae appears to be minimally re-lated to other mycobacteria based on thecontent of guanine plus cytosine, genomesize C. "'), unique biochemical features,such as phenolic glycolipid-I (' 5) and thepresence of glycine in place of L-alanine inthe peptide portion of peptidoglycanComparative studies of M. leprae isolatesby total genomic hybridization have shownsigniticant homology, suggesting genetic

121

1 22^

International .lournal of Lepros_v^ 200O

similarity among them ( 2 ). Related studiesof the Al. leprae genome usine restrictionfragment-length polyn>orphisn> (RFLP)have demonst ated that Iess than 0.3Y( ofthe nucleotides differed among thegenon>es in isolates of Al. leprae from tli-verse origins, including leprosy patients, ar-madillos and n>angabey monkeys (').Williams, et al. reported that Al. lep rae iso-lates obtained fruiu geographically distinctarcas did not exhihit genotypic diversity byRFLP (' 7 ). de Wit and Klatser reported thatisolates of M. leprae from different sourceshad identical 16S-23S rDNA intergenicspacer regions (''), a documented source ofspecies and strain variability in some bacte-ria (' 1. '"). In contrast, Fsihi and Coledemonstrated variability associated with thepolA locus of Al. leprae among eight chro-mosomal DNA samples originating from Ju-dia, Africa and France ("). I-lowever, it wasnot olear whether diversity in the polA locuswas appliceble for typine of M. leprae.

Shepard and McRae have observed thatthe growth rate of AI. leprae in mouse footpads varies among isolates and that the dif-ferences are stable ('4 ). Other studies havesupported these results although the basisfor phenotypes from the aspect of growthrate has not been elucidated. In ai) attemptto link growth rate with genotypic yaria-tion, we compared DNA sequentes of vari-ous genes including rpoT, a homoloe of themycobacterial principal sigma factor (' n.';).We found that a short repetitive region oftpo7' exhibited a sequente polymorphismamong isolates.

This report describes genetic diversity ofthe rpoT gene of M. lepra(' among 51 iso-lates obtained from 12 geographically dis-tinct arcas of the world and the distributionof genotypes.

MATERIALS AND METHODSSource of AI. leprae strains and prepa-

ration of genomic DNA. A total of 51 M.leprae isolates was used in this experiment.They were obtained from different arcas ofthe world, and included clinica) isolatesfrom a Biopsy specimen (BS) and those es-tablished by passages in nude mice (NM) orarmadillos (AL). The countries from whichAl. leprae isolates were obtained and thenumber of isolates tested are as follows:Bangladesh (3 BS), Brazil (3 BS), Haiti (1

BS), India (I AI.), Indonesia (4 I3S + 1 NM),Japanese Mainland (8 BS + 4 NM), JapanOkinawa (2 135 + 3 NM), Korea (I 113S),Nepal (1 I3S), Pakistan (1 13S), The Philip-pines (I BS + 1 AL) and Thailand (3 NM +1 AI,). Two of the AI. leprae isolates werefrom the nonhuman origins of a naturally in-fected armadillo (Louisiana, U.S.A: 1 AL)and a mmngabey monkey ( Nigeria: I AL).

Al. leprae was purified from tissues as de-scrihed ('"). Ext a:tion ol chrornosotnalDNA was carried out as previously de-scribed (`). Biopsy samples were honuoge-nized, and partially purified hacilli were dis-rupted with freezing and thawing followedby phenol extraction and ethanol precipita-tion. For the preparation of bacterial I)NAfrom Biopsy specimens enubedded in paraf-lin, thrse to Eive sections (5-pnm thick) weretreated by the method of de Wit, et a/. e).The skin-scraped materiais were suspendedin phosphate-bulfered salino (PI3S), puri-fied partially by centrifugation, and I)NAwas extracted aftcr freezing and thawing.

PCR and seduencing. PCR was carriedout usina Anupli "I'aq DNA polynuerase(Perkin-Elmer Applied Biosystems, Nor-walk, Connecticut, U.S.A.) in a 50 pl vol-ume containine 150 pg of genomic DNA or5 p1 of template DNA solution and I pM ofprimers with reagents and protocols sup-plied by the manufacturer. Primers A (5'-AAGCGTCGAI'ACAAAGGCACCGT-3')and B (5'-AGTAGCTTCGCCATCCTCG-GTTT-3') were used for amplification tospan the 300 bp fragment of the target re-gion of the rpoT gene. The amplificationwas carried out in a thermal cycler (AstechPC8OO: Astech Co., Fukuoka, Japan ) underthe conditions of 30 sec at 95°C, 2 min at60°C, and 4 min at 72°C for 30 cycles. Thefull length of the rpoT gene with up- anddownstream reeions was amplified withprimers C (5'-GCTGTCGGTCACGGC-TAI' 3') and D (5'-GAAAACCGCACCCC-GATGGT-3') under the conditions (>1 30 secat 95°C, 2 niin at 48°C, and 4 min at 72°Cfor 35 cycles. The short fraements, 9I bp or97 bp, containing the target region of rpoTwere obtained usineg primers E (5'-ATGCC-GAACCGGACCTCGACGTTGA-3') andF (5'-TCGTCTTCGAGGTCGTCGAGA-3') under the same conditions as for the 300bp fragment of the rpoT gene, except for 45cycles of the thermal reaction. The same se-

I IIIII IVGAC ATC GAC ATC GAC ATC GAC ATCGAC ATC GAC ATC GAC ATC GAC ATCGAC ATC GAC ATC GAC ATC GAC ATC

541- CCC- CCC-CCCPro Ile

AGCAGCAGCSer

GAGGAGGAGGlu

585GAC-GAC-GAC-AspAsp

GCCGCCGCCAla

GCCGCCGCCAla

Kyoto-1Hoshizuka-4Korea-1

GAAGAAGAA

Asp Ile Asp Ile Asp Ile Glu

GAAGAAGAAGAAGAAGlu

GCCGCCGCCGCCGCCAla

GCCGCCGCCGCCGCCAla

579GAC-GAC-GAC-GAC-GAC-Asp

68, 2^Matsuoka, et al.: M. leprae Gcnotvpes^ 123

rpoT

1^

1731 (1725)

ii

1^

II^

IIIGAC ATC GAC ATC GAC ATCGAC ATC GAC ATC GAC ATCGAC ATC GAC ATC GAC ATCGAC ATC GAC ATC GAC ATCGAC ATC GAC ATC GAC ATCAsp Ile Asp Ile Asp Ile

541WHO referente^-CCC AGC GAGThai-53^-CCC AGC GAGJapan(Okinawa) -ccc AGC GAGPHS-3(Mangabey) -ccc AGC GAGPHS-4(Armadillo) -ccc AGC GAG

Pro Ser Glu

Fio. I. Sequente alignment of DNA fragment corresponding from 541 to 585 (for 4 tandem repeats) or 541to 579 (for 3 tandem repeats) of the rpoT gene from M. leprae isolates. Sequences from seven isolates and thereferente (from Database, aecession no. UI5181) are shown. The nucleotide sequente data will appear ira theD1)13J/EMBL/Genl3ank nucleotide sequence databases with the accession numbers AB()19193 and A13019194.

quente information was obtained when EXTaq DNA polymerase (Takara Shuzo,Shiga, Japara), as 3'-5' exonuclease activity,was used for amplification.

DNA simples for sequencing were re-covered from agarose gel after elec-trophoresis using an Easy Trap DNA purili-cation kit (Takara Shuzo). Determination ofsequences was performed in both directionsusing a Thermo Sequenase kit (AmershamLife Science, Cleveland, Ohio, U.S.A.)with `'S-dCTP or a BigDye Terminator Cy-ele Sequencin`g FS Ready Reaction kit(Perkin-Elmer) in ara ABI Prism. 310 Ge-netic Analyzer (Perkin-Elmer). The nu-cleotide sequences obtained were analyzedusing the DNASIS computer program (Hi-tachi Software Engineering, Yokohama,Japan).

Agarose gel electrophoresis. For com-paring the differences in the repetitive re-gion of the tpoT gene, the short fragmentaof the target region were amplified, and re-action products, 97 and 91 base pairs, wereelectrophoresed in a 4% Meta Phort `t

agarose gel (FMC Bioproducts, Rockland,Maine, U.S.A.) using TBE buffer at 75 volts.

RESULTSSequence variation ira the rpoT gene of

M. leprae. DNA sequences of variousgenes involved in replication, transcriptionand translation, such as the ipoL the rpoA,the tuf, the polA and the I6S rRNA genes,were studied to determine whether DNApolymorphism exists in these genes thatmight explain growth rate differences in themouse foot pad. A single sequence variationira the tpoT gene was observed among iso-Iates but it did not appear to differentiatefast- and slow-growing phenotypes. Align-ment of the 300 bp target region of the rpoTgene showed that the number of tandem re-peats, composed of 6 bp repeats (GACATC),was different among the isolates. One `groupof isolates contained three tandem repeats;whereas a second group contained four re-peats ira the target region of rpoT (Fig. 1).Origins of M. leprae isolates from the lattergroup were limited to Japan (except Oki-

124^ International Joarnal o/ . Lepros.v^ 2000

97bp *91bp♦

♦ 50bp

1 2 3 4 5 6 7 8FIG. 2. Genotype detection hy eleclrophoresis in

aparou. Five p1 ot PCR products were electro-phoresed in 4(/ Meta I'hor 1 ' agarose and thcn stainedwith ethidium hrotnidc. Samples xvere: lane I, Kago-shima (Japan); lane 2, Kanazawa (Japan); lane 3, Ko-rea-I; lane 4, Thai-53; lane 5, Okinawa-I (Japan); lane6, PI IS-3 (mangabey monkey); lane 7. PIIS-4 (arma-dillo); and lane R, the DNA sim marker of 5U bp ladder.

nawa) and Korea, while isolates with threetandem repeats were found in the OkinawaIslands, southern Asian countries such asBangladesh, India, Indonesia, Nepal, Pak-istan, The Philippines and Thailand, and theAmericas (Brazil and Haiti). In addition,two nonhuman isolates of M. leprae (ar-madi1lo and mangabey monkey) showedthe three tandem repeat genotype. •

Comparative analysis of the completerpoT DNA sequence (1725 bp coding for575 amino acids) among isolates of bothgenotypes showed that only the 6 bp tan-dem repeats were different. This analysisalso included up- and downstream regionsfor a total of 1900 bp. To confirm that thestrains used in this study were M. leprae,we identified the M. leprae-specilic frag-ment of the groEL gene by the method ofPlikaytis, et al. ( 22 ).

Genotype detection by electrophoresisin agarose gel. To simplify the detection ofthe two groups, we developed an assay ca-pable of classifying the two genotypes. Theassay was based 00 amplifying an approxi-mately 90 bp region containing the rpoTtarget region and analyzing the mobility ofresultant amplicons on 4% Meta Phor"Magarose. The DNA fragments obtained byPCR from two genotypes were clearly sep-arated as shown in Figure 2.

UISCUSSiON

'i'he n1o51 striking linding was the appar-ent linkage of the four tandem repeat geno-type between M. leprae strains isolated inJapan, excluding Okinawa, and thosestrains from Korea (Fig. 3A). The reasonfor this bias in distribution of the genotypewith the four tandem repeat is not elear atpresent. A similar bias in genotype-specilicdistribution has been reported in Ilelicobac-ter pv/ori within Korean and Japanesepopulations. Epidemiological studies on II.pvlori have indicated that almost ali isolatesfrom Korea and Japan are assoeiated withthe ca,1,' genotype; whereas in other paris ofthe world 30% to 40% of H. pvlori strainsare ca,t,' - (`' ". 21 ). In addition, H. pvloristrains isolated from Asia contei be sepa-rated from those of Caucasian origins onthe basis of the nucleotide sequence (L"). Ithas been postulated that an H. pvlori geno-type-specilic distribution is associated withhuman migration ( 7 ). Accordingly, it is rea-sonable to assume that the M. leprae st ain-specilic distribution that we observed maybe related to patterns of human migration.Similarly, predominance of a single geno-type was reported in M. trrhercuiosiss in EastAsian countries

The M. leprae strains found in Okinawawere unrelated to the strains in the rest ofthe Japanese islands (Fig. 3A). There is along history of concentrated interchangesbetween Korea and the main part of Japan,since the earlier migration of Mongolianpeople from Korea to Japan in the Yayoiand Kofun eras (300 BC to 600 AD). Sev-eral genetic markers have shown the doserelationship between populations in Koreaand most of Japan ( 20). In contrast, Okinawawas an independent country until the 19thcentury and had closer ties with southernAsian countries than with Japan.

The ubiquitous distribution of the threetandem repeat (Fig. 3B) suggests that fur-ther classification of this eenotype will benecessary to distinguish strains of M. lepraefor epidemiological studies. Fsihi and Colereported genomic variability in a polA locusamong five clinicai isolates and three ar-madillo-grown strains M. leprae ( 13 ). Weexamined this marker usine eight strains ofM. leprae (four strains with each r poT type)according to their method bui detected onlyone type of polA.

moo meã rw.,.rsie

y

♦ 150bp

f100bp

Matsuoka, et al.: M. Icprae Genelypes^ 125

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!.)0.1

co

4^r•-: ---"'•

I)^cL-.: =-a^Ci: ::'

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68, 2

126^ Internationul Jntrrnal n/'Lepros.v^ 2000

Despite efforts to detect genomic diver-sity aniong M. le/)rue isolates, clearly de-fine(' genetic polymorphism useful for epi-demiological purposes has not been re-ported. Our results demonstrate the geneticdiversity of M. leprae based upon differ-ences in the rpoT Lzene of 51 isolates.Strains of M. lepra(' isolated were dividedinto two genotypes and their distribution inthe world showed a striking bias, suggest-ing a relationship to the movement of theKorean and Japanese people. Further analy-sis of M. leprae strains is needed to explainthe hias seen in the global distribution aswell as to appreciate the utility associatedwith rpoT typing M. leprae for epidemio-logical survey.

SUMMARYThe genetic diversity and related global

distribution of 51 Mveobacteriunt lepraeisolates were studied. Isolates were ob-tained from leprosy patients from 12 geo-graphically distinct regions of the worldand two were obtained from nonhumansources. Polymerase chain reaction (PCR)followed by DNA sequencing was per-formed targeting the rpoT gene of M. lep-me. Isolates were classified roto two groupsbased on the number of tandem repeatscomposed of 6 base pairs in the rpoT gene.Isolates from Japan (except Okinawa) andKorea belonged to one group, while thosefrom Southeast Asian countries, Brazil,Haiti and Okinawa in Japan belonged to asecond genotype. M. leprae obtained fromtoo nonhuman sources (an armadillo and amangabey monkey) revealed the lattergenotype. These results demonstrate the ge-netic diversity of M. leprae and the relatedgenotype-specific distribution in the world.

RESUMENSe estudiú la diversidad genética y la distrihucicín

global de 51 aislados de Mveobucteriunr leprae. Losaislados se obtuvieron de pacientes con lepra de 12 re-giones geográficas distintas dei mundo y de dosfuentes no humanas. El an:ílisis se hizo por la reacciónen cadena de la polimerasa (PCR) y por secuenciacióndei DNA de la región correspondiente al gene rpo T deM. leprae. Los aislados se clasilicaron en dos gruposcoo base en el número de "secuencias tandem" com-puestas por 6 pares de bases en el gene rpo T. Los ais-lados de Japim (excepto Okinawa) y Korea corre-spondieron a un genotipo, n ientras que los aislados delos países dei sudeste asiático, Brazil, I laití, y Okinawa

en Japún, correspondieron a un segundo genotipo. Losaislados de M. lepra' obtenidos de dos fuentes no hu-manas (un armadillo y un mono mangabey) nostraroncl sugundo genotipo. Estos resultados muestran la di-versidad genética de M. /eproe y la distribuciún de losgenotips de M. leprae en el inundo.

RÉSUMILa diversité génétique et la distribution géo-

graphique globale correspondante de 51 isolais de Mv-cnbucteriwn lepra(' furent étudices. Les isolats furentobtenus à partir ele patients lépreux provenant ele 12 ré-gions geographiquentent distinctes et deux isolaisfurent obtenus de source non-huntaine. La réaction depolymerase en chain (PCR) suivie par le séquençagedes produits obtenus fut réalisée sur le gene rpoT deAl. /epruc. Les isolais furent classés en deux groupesbasés sur le nombre de motifs répétés 2 par 2 (tandemrepeats) composés de 6 paires de bases au seio du gènerpoT. Les isolats du Japon 1excepté Okinawa) et Coréeappartenaicnt à un groupe, alors que ceux provenant deI'Asie du Sud-Est, du Brézil, d'l laïti ct d'Okinawa auJapon appartenaient au second genotype. Al. lepra('provenant des deux sources non hum nines (un tatou àneuf bandos et un signe mangabey) revélèrent cedernier génotype. Ces résultats clémontrent la diversitégénétique de Al. leprae et la distribution mondiale re-liée au génotypes spéciliquement étudiés ici.

Acknowledgment. We thank Drs. Eiji Nagao(National Sanatorium Oshima Seishoen, Kagawa,Japan), Kunihiro Kinjoh (National Sanatorium Oki-nawa Airakuen, Okinawa, Japan), Masako Namisato(National Sanatorium Tania Zenshoen, Tokyo, Japan,Masantichi Goto (National Sanatorium 1ioshizukaKeiaien, Kagoshima, Japon), Atsushi Hosokawa(School of Medicine, University of the Ryukyus, Oki-nawa, Japan), Akiko Obara (Medical College, KyotoUniversity, Kyoto, Japan), M. M. Bari (Leprosy Con-trol Institute and Hospital, Dhaka, 13ang1adesh), L. E.Takaoka (Sociedate Filantropica 1lum:uitas, Brazil)for supplying clinica) samples and Dr. M. J. Colstonfor supplying armadillo-grown M. leprae of an Indianisolate. We also express thanks to Mr. Kunio Kawatsufor preparing sections.'l'his work was supported by aHealth Science Research Grani of Research 011 Emerg-ing and Re-emerging Infectious Diseases, OhyamaHealth Foundation. Sasakawa Memorial Ilealth Foun-dation, and the U.S.-Japan Cooperative Mcdical Sci-ence Program.

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